MEDRAP II MEDITERRANEAN ARTEMIA, TRAINING COURSE … · • 71Lipid composition of cysts of the brine shrimp Artemia sp. from Spanish populations. By J. C. Navarro, F. Amat and J

MEDRAP II

RAB/89/005-RER/87/000

FIELD DOCUMENT

94/33

United Nations Development Programme

Food and Agricultura Organisation of the United Nations

MEDITERRANEAN ARTEMIA, TRAINING COURSE AND SITE SURVEY

Tunisia and Libya, May 25-30 1994

Edited by MEDRAP II Regional Center Tunis - Tunisia

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise, without the prior permission of the copyright owner. Applications for such permission, with a statement of the purpose and extent of the reproduction, should be addressed to the Director, Publications Division, Food and Agriculture Organisation of the United Nations, Viale delle Terme di Caracalla, 00100 Rome, Italy.

Preparation of this Document

This document is one of a series of documents prepared during the course of the Project identified in the title page. The conclusions and recommendations given were considered appropriate at the time it was prepared. They may be modified in the light of further knowledge gained at subsequent stages of the Project.

The designations employed and the presentation of the material in this document do not imply the expression of any opinion whatsoever on the part of the Food and Agriculture Organisation of the United Nations concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.

The opinions expressed by the Authors in this document are not necessarily those of FAO or the Governments of the participating contries.

This document was edited by Hassen AKROUT and Mohieddine BELKHIR in collaboration with Othman BEJI and Neila KAFFEL, the revision was made by Michel LAMBŒUF.

Abstract

The Training course on Sites surveys on Mediterranean Artemia was organised from 25 to 30 May in Tunisia and Libya.

The objective was to update participants with biology, ecology and production of artemia.

Better exchange between participants of Mediterranean coutries was facilitated, this should be contiinued and cooperation reinforced. The production of artemia could contribute to food security in the region in the long term, a complete inventory of the possibilities and a development of technical capabilities is required.

Acknowledgements

The Editor would like to thank the Tunisian and Libyan authorities, for their support to the organisation of this activity. Thanks are also addressed to the participants from Member/Associated countries who accepted to contribute to the workshop.

Note from the reviser

The revision and publication of this document could only be done a long time after the closure of the project. This has led to some difficulties in finalising the documents and implementing corrections, because authors and contributors as well as some of the original material or files were no longer available.

Therefore contributions from participants and session papers annexed to most of the documents were left in their original form. No language corrections were introduced, the content was not modified and left under their respective authors' responsibility.

Considering the above, we hope that the reader will understand that a standard of publication could not e maintained on a level as high as we would have liked it to be.

CONTENTS • Agenda 1

• List of participants 5

• Report 9

• COMMUNICATIONS 13, 15

• L'Artemia en Tunisie, By Néji Aloui

17

• Potentiel d' Artemia en Tunisie, By B. Missaoui and N. Abdelkader

25

• Artemia production case study in Egypt, By S. Ghoneim

29

• Geografical distribution of Artemia in the Jamahiriya, By M. O. Magsodi

31

• Situation de L' Artemia au Liban, By M. A. Abi Saad

33

• Sur L'Artemia Marocaine, By M. H. Amane

33

• Artemia statement in Turkey, By N. Gokgoz

33

• Sur L'Artemia Algérienne, By I. Samia

35

• Les salines, sebkhas, chotts et l'Artemia en Tunisie 35 By M. S. Romdhane

• Artemia production case study in Tunisia, By M. Belkhir

37

• Artemia production in Egypt, By M.A.S.D. Ahmed

39

• The cosmopolitan brine shrimp By G. V. Stappen and P. Sorgeloos

41

• Morphological characterisation of adult Artemia from different geographical origin in the Mediterranean populations By F. Hontoria and F. Amat

47

• Further characterisation of two Artemia populations from Northern Greece biometry, hatching characteristics, caloric content and fatty acid profiles. By T. Abatzopoulos, G. Karamanlidis, P. Léger and P. Sorgeloos

57

• Lipid composition of cysts of the brine shrimp Artemia sp. from Spanish populations. By J. C. Navarro, F. Amat and J. R. Sargent

71

• International Study on Artemia IX. Lipid level, energy content and fatty acid compostion of the cysts and newly hatched nauplii from five geographicial strains of Artemia S. By Paul Schauer, D. Michael Johns, Charles E. Olney, and Kenneth. L. Simpson USA

81

• International Study on Artemia*. XXVI. Food Value of Nauplii from Referenc Artemia Cysts and Four Geographical Collections of Artemia for Mud Crab Larvae By C.R. Seidel, D.M. Johns, P.S. Schauer and CE. Olney

91

• Overview genetics of mediterranean bisexual Artemia. By E. J. S. Pilla

954

• The nutritional value of Artemia By P. Leger, D. A. Bengtson, P. Sorgeloos, K.L. Simpson and A. D. Beek

107

• Improved larviculture outputs of marine Fish shrimp and prawn By P. Sorgeloos and PH. Léger

125

• The use of brine shrimp Artemia in biological management of solar saltworks By W. Tackaert and P. Sorgeloos

141

• Semi Intensive culturing in fertilised ponds By W. Tackaert and P. Sorgeloos

151

AGENDA

First Part - TUNISIAWednesday 25 May

- Opening session - Welcome addresses - Election of officials

9:00

- Adoption of the agenda - Artemia basic support of aquaculture (historical aspects, main areas of production, world production potentialities) By Patrick Sorgeloos (Belgium) - Artemia in the Mediterranean basin (needs and potential production By G. Van Stappen (Belgium)

10:00

- Discussion 11:15 - Coffee break

11:30 - Visit to Mègrine saltworks: field observations, presentation of ecological characteristics By N. Aloui (Tunisia)

13:30 - Lunch 15:30 - Biology and ecology of Mediterranean Artemia:

• Artemia populations of the Eastern Mediterranean basin Th. Abatzopoulos (Greece)

• Genetics of Mediterranean bisexual Artemia By Th. Abatzopoulos (Greece)

• Genetics of Mediterranean bisexual Artemia By John A. Pilla (UK) and F. Amat (Spain)

- Discussion - MEDRAP members' experiences on Artemia By respective delegates

17:00

- Discussion

Thursday 26 May

7:00 - Departure to Monastir 8:30 - Visit of “Aquaculture Tunisienne” marine fish farm in Hergla: Scope on

Artemia use in marine hatchery. By M.S. Zine el Abidine (Tunisia) 10:30 - Visit of Sahlin saltworks: field observations; presentation of ecological

caracteristics of the saltlake By Mrs A. Medhioub (Tunisia)

12:30 Visit of Centre National d'Aquaculture in Monastir (CNA): The CNA experience in larvae nutrition using Artemia as basic support in marine hatching. By Mrs. A. Medhioub (Tunisia)

13:30 - Lunch in Monastir 15:00 - Departure of Sfax

17:00 - Visit Sfax saltworks: fiel observations; presentation ecological characteristics of the saltlake By Khemakhem (Tunisia)

Second Part - LYBIAFriday 27 May

6:00 - Departure to Tripoli 11:00 - Visit of Abi Kamash salt lake: field observations; presentation of ecological

characteristics By M. Omar Maksoudi (Libya) 14:00 - Arrival to Tripoli 14:30 - Lunch 18:00 - Departure of SIRTE (from Marine Biology Research Centre) 23:00 - Arrival to SIRTE

Saturday 28 May

- Opening session in Lybia - Welcome speeches

9:00

- Election of officials 10:00 - Tea break 10:30 - State of the art with regard to Artemia strain selection, naupliar manipulation

and use in aquaculture By P. Sorgeloos (Belgium) - Artemia model systems: * USA case study. By p. Sorgeloos * Vietnam case study. By G. Van Stappen * Tunisia case study. By M. Belkhir * Egypt case study. By S. Ghoneim

11:00

Discussion 13:00 - Lunch

- Mediterranean Artemia production perspectives: * Developsment strategies * Constraints (socio-economic aspects) * Integration with salt production, * Biological problems, * Production and utilisation

14:00

- Discussion

16:00 - Round table and general discussion on the workshop report: proposals for conclusions and recommendations

17:30 - Visit of Great Man Made River Project 19:30 - Back to resident place

19:45 - Reporting: Conclusions and Recommendations 21:30 - Adjournement 22:00 - Dinner

Sunday 29 May

7:00 - Departure from Sirte to Tripoli 11:00 - Visit to Aïn Kaam aquaculture farm (Khoms) 12:00 - Visit to Lebda archeological site 13 : 00 - Lunch 15 : 30 - Back to Tripoli and Lunch. 15 : 00 - Visit to Marine Biology Research Centre 17 : 00 - Arrival to Tripoli

Monday 30 May

6 : 00 - Departure from Tripoli to Tunisia

LIST OF PARTICIPANTS

MRS. IZEM SAMIA ALGER Port, Algeria C/O Mr. Zenasni Phone :213.2.71.213.940

ALGERIA

Fax :213.2.71.21.42

MR. GEORGE GEORGIOU Biologist Department of Fisheries-Ministry of Agriculture Natural Resources and Environment 13, Aeolou Str. Nicosia, Cyprus Phone :357.2.30.35.26

CYPRUS

Fax :357.2.36.59.55

MR. AHMED SALAH EL DEEN AHMED Specialist of Marine Hatchery General Authority for Fish Resources Development 4, Tayaran Str. Nasr City - Cairo Phone :202.2620 118

EGYPT

Fax :202.2620 117

MRS. MARIE ABI SAAB ABOUD Principle Researecher/Plankton Marine Research centre. PO BOX 123 Jounieh

LEBANON

Phone :961.9.918 857

Mr. MOHAMED OMAR MAGSODI Marine Biology Research Centre P.O Box 30 830 - Tajoura Phone :218.21.59.0001/3

LIBYA

Fax :218.21.69.0002

MR. HAMID AMANE Manager of live food Société MAROST BP 4 NADOR Phone :212.6.60.68.32

MOROCCO

Fax :212.6.60.68.16

Mr. ALOUI NEJI Researcher Aquaculture Institut National Scientifique et Technique d'Océanographie et de Pêche - 2025 Salammbo Phone :216.1.730 420/548

TUNISIA

Fax :216.1.732 622

MRS. NEVIN GOKGOZ Vice Head-Live Food Production Unit Marine Fish Hatchery - Ministry of Agriculture and Rural Affairs, Beymalek. Central Aquaculture and Development Centre. PO Box 61 07570 Kole/Antalya Phone :90.242.871.4089/4090

TURKEY

Fax :90.424.871.5053

MR. G. VAN STAPPEN Research Assistant Laboratory of Aquaculture & Artemia Reference Centre - Rozier 44/9000 Ghent, BELGIUM Phone :32.9.26.43.754 Fax :32.9.26.41.93 MR. PATRICK SORGELOOS MR. JAMES CLEGG Ghent, BELGIUM Phone :32.9.26.43.754 Fax :32.9.22.37.326 MR. SAMIR GHONEIM Head of Fish Research Centre & Manager of Artemia Project - Suez Canal University, Isamailia-EGYPT Phone :2. 64 321 916 Fax :20 64 321 916 MR. FRANCISCO AMAT Investigator (Sr) C.S.I.C Instituto de Acuicultura - Torre de la sal 12595 Ribera de Cabanes - Castellon SPAIN Phone :34 64 31 95 00/31 96 28

Lecturers

Fax :34 64 31 95 09

MR. ERNANI PILLA Geneticist / Senior Researcher University of Wales, Swansea Singleton Park, Swansea Wales) U.K. - SAZ 8 PP Phone :44 792 295 383 Fax :44 792 295 447 MR. THEODORE ABATZOPOULOS

Senior Lecturer / Geneticist

Aristotle University of Thessaloniki Faculty of Sciences, Depart. of Genetics, Dev. & Mol. Biol. Aristotle Univ. of Thessaloniki - G 54006 GREECE MR. M. S. ROMDHANE INAT, TUNISIA Phone :216.1.280.950 Fax :216.1.799.391 MR. JAMES CLEGG BODEGA Marine Laboratory University of Californai, Davis - USA Phone :1.707.875.2211 Fax :1.707.875.2009 MR. KENETH K. SIMPOSON Food Sci. Nutrition Univ. R. I University of Rhode Island - USA Phone :1.401.792.2977 Fax :1.401.792.4017 MRS. A. MEDHIOUB MRS. N.MISSAOUI Ministère de l'Agriculture, Direction Générale de la pêche et de l'Aquaculture. TUNISIE Phone :216.1.891 993 Fax :216.1.891 993 MR. M. KHEMAKHEM MR. M.S.ZINE EL ABIDINE Phone :216.3.248 188 Fax :216.3.248 187

MR. HASSAN AKROUT MR. MOHEIDDINE BELKHIR MR. DENIS LACROIX Phone :216.1.784.979

MEDRAP II

Faes :216.1.793.962

REPORT In the framework of its activities, MEDRAP II organised a Training Course and Sites Survey on Mediterranean Artemia, from 25 to 30 May, 1994 in Tunisia and Libya. This activity was prepared by experts from Algeria, Tunisia and Belgium met on 7 and 8 October 1993, at the Artemia Reference Centre, in Ghent University.

The objectives of this activity, was to update participants with the biology, ecology and production potential of Artemia in and outside the Mediterranean basin. Participants would be urged to learn how to explore the potential of Artemia developments in their own country and to set up integrated systems for commercial production of salt, Artemia, fish and shrimp.

Having been participating to the Mediterranean Artemia Training Course and Site survey, MEDRAP members delegates from Algeria, Cyprus, Egypt, Lebanon, Turkey, Morocco, Libya and Tunisia. These participants were selected upon those who have good background and practical experience on Artemia biology and ecology and upon researchers or engineers involved partly or totally in Artemia studying productions activities.

Eminent experts on Artemia from Belgium, Egypt, Greece, Spain, Tunisia, United Kingdom and USA were invited to present conferences on Artemia and enrich the debates through their experience in the relevant field.

The first part of the Mediterranean Training Course and Site Survey started in Tunis - Tunisia. It was organised in concertation with the Tunisian Fishery and Aquaculture General Directory, while the second part ended in Sirte - Libya and sponsored by the Libyan Marine Biology Research Centre.

On the way from Tunis to Sirte a study tour allowed the participants to visit several potential areas for Artemia and to perform demonstrations and practical works related to its ecology and exploitation.

The first session held in Tunis was opened by Mr. H. Akrout, the project coordinator, who welcomed all participants and proceeded to the election of Training Course Officials as following :

Chairman : Mr. Samir Ghoneim (Egypt) Vice Chairperson : Mrs. Nevin Gokgoz (Turkey) Rapporteurs : Mr. George Georgiou (Cyprus) : Mr. Belkhir Mohiedine (MEDRAP II).

The relevant proposed activities agenda was discussed and adopted.

The official session was opened while in Sirte under his Excellency the Minister of Fisheries in Libya. The Lybia Sub-regional coordinator welcomed the participants and enumurated benefits that Libyan scientific and technical staff particularly got from MEDRAP activities. He presented then, the guidelines and main activities that Lybia proposed to enhance and to develop aquaculture sector, and finally stressed the presents for more cooperation in order to promote activities related to Artemia production and uses.

The project coordinator, Mr. Hassen Akrout prsented his congratulations to the Libyan authorities for hosting the Training Course and Site Survey on Mediterranean Artemia and to his Excellency the Minister of Fisheries for chairing this meeting. He notified that

Libya was and still playing an efficient role at national and sub-regional scales through their coordinators. Mr. Akrout thanked the UNDP representative for all efforts he did in organising this activity in Libya and through him, he also addressed the FAO for its support and contribution to MEDRAP activities. Lastly, Mr. Akrout addressed all participants and invited them to forget the long bus way travel wishing to them full success in their works and very good stay in Libya.

Thenafter, the UNDP representative and his Excellency, the Minister of Fisheries in Libya took, successfuly the floor. They each addressed the participants for attending the training course and contributing efficiently to the success of its works. They expressed their full satisfactory with the achievement so far of the project which contributed in the implementation for a permanent cooperation, by creating four networks which were already initiated and called the SIPAM (system of Information for the Promotion of Aquaculture in the Mediterranean), the TEGAM (Technology of Aquaculture in the Mediterranean), the SELAM (Socio-economic and Legal Aspects of Aquaculture in the Mediterranean) and the EAM (Evironment and Aquaculture in the Mediterranean). They also highlighted the need to consolidate cooperation between north and south, by handling collaborative research in specific activity in the field of cooperation. Artemia activity would be an excellent example to more exchange of experience between the mediterranean countries and to enhance sustainable human development.

The conferences given by the invited experts, Mr. P. Sorgeloos, Mr. F. Amat, Mr. F. Hontoria, Mr. S. Pilla, Mr. V. Stappen, showed that a lot of scientific and technical works having been done on Artemia and results are tremendous and conspicuous; conferences appended, allowed to rise hereun-der, the main groups of accurate subjects related to the promotion of Artemia production, beyond it the aquaculture and the larvae culture in particular :

1 State of the art on Artemia as basic support to aquaculture (biological and ecological characteristics. Main areas of production and potentialities. Practical requirements for culturists and enrichment of Artemia in larvae culture.

2 Statement of mediterranean Artemia and perspective of development {Artemia population.Genetics of bisexual Artemia. Model of quality evaluation. Artemia strain selection. Naupliar manipulation and use in aquaculture. Integration of Artemia with salt production and constraints.

3 Artemia commercial model system (presentation of case study in USA, Vietnam, Egypt and Tunisia).

The study tour which was of a big interest allowed the participants to have field observations and to learn about the ecological characteristics of Artemia in their natural biotopes either in Tunisia when visiting the saltworks of Megrine (north part) of Sahline (central part) and of Sfax (south part) or in Libya when visiting the salt lake of Abi Kamech;

During the discussion where having field visits, it was pointed out that the geographical distribution of Artemia is covering the whole mediterranean basin and that could be of a big potentiality of Artemia production now technically possible to be reached.

In order to improve Artemia productivity in saltworks, Prof. Sorgeloos emphasised the link between this productivity and the management of saltlakes, he highlighted the need to mainly conduct the following activities :

• to deepen the pond when technically possible, in order to increase its water depth;

• to apply fertilisation to enhance phytoplankton growth and macroalgae the growth of which should be inhibited if not avoided;

• to improve Artemia strain. It was pointed out that Artemia franciscana is a very prolific species even at high salinities (up to 310 %o) while Artemia tunisiana is less prolific and it can only withstand salinities of up to 210 %o. Also, quality of the final product should be taken into consideration (cysts and biomass) and not only the quantity.

Beside the saltworks, participants enjoyed visiting the facilities of the marine fish farming called "Aquaculture Tunisienne de Hergla" in Tunisia and the ones in Libya of Aïn kaab aquaculture farm at Khoms. They also have a scope on Artemia use in marine fish hatchery (in Tunisia) and in integrated fish farm of Aïn Kaab which is concerned by both the fresh water culture and the marine water shrimp culture.

On the way going from Tunis (Tunisia) to Sirte (Libya) the participants visited the facilities of two research centers and get informed about the existing skills and kind of research programmes going on Artemia and other species in the "Centre National d'Aquaculture de Monastir" in Tunisia and in the "Marine Biology Research Centre" of Tajoura, in Libya. There is need to indicate that the study tour allowed to learn on some economical aspects, when visiting the Man Made River Project in Sirte (Libya), and the industrial complex in Abi Kamech saltlake and also on touristical ones when visiting archeological sites mainly El Jem in Tunisia and Lebda in Libya. .

Despite the presence of Artemia and several areas of relatively high potentiality of Artemia natural production, most of the participating countries to the training course seemed to indicate that Artemia population still remain unidentified and needed more cooperation to go further in having food skills and more knowledge in order to exploit benefically the local Artemia.

Indeed, the delegates national reports related to Artemia activities in Tunisia, Morocco, Lybia, Egypt, Lebanon, Turkey and Algeria showed that Artemia still beeing a big concern in their aquaculture structure and that needs more works and studies before getting satisfied in producing qualitatively and quantitatively their respective local Artemia.

Conclusions and Recommendations Considering :

a - MEDRAP Project has contributed to more exchange of experience between mediterranean countries.

b - Artemia production activities are a source of creation of jobs, where women should participate actively in development of this matter.

c - The activity on Artemia would be more productive in the context of marine resources, hence Artemia activity should consider this ultimate objective as an important requirement.

d - The production of Artemia is a part of achieving sustainable food security of the region.

e - The production of this activity required development of competence and advanced scientific technologic capacity which would be required only through

human development resources and beyond it in the achievement of sustainable human development.

f - The participants insisted to training and also to promotion of the private sector role.

g - The participants insisted on the consolidation of cooperation between mediterranean countries on Artemia activities, which is an excellent example of consolidating relations between North and South.

The participants expressed satisfactory with the achievement so far of the MEDRAP Project which lead to landing national project and more important effort in training of national competence; and they recommend :

1. More exchange in the future through seminars, consultancies, study tours and especially collaborative research.

2. The identification of existing national resources and the possible areas of Artemia production.

3. The compilation and the treatment of information on local Artemia strains in collaboration and through SIPAM.

4. The set up a demonstration project on Artemia permitting the use for extension and advanced training purposes through TECAM.

5. The identification of the present Artemia requirements and perform commercial feasibility studies possible new market outlets.

COMMUNICATIONS L'ARTEMIA EN TUNISIE

BY NÉJIALOUI

POTENTIEL D'ARTEMIA EN TUNISIE BY B. M1SSA0UI AND N ABDELKADER

ARTEMIA PRODUCTION CASE STUDY IN EGYPT BY S. GHONEIM

GEOGRAFICAL DISTRIBUTION OF ARTEMIA IN THE JAMAHIRIYA BY M. O. MAGSODI

SITUATION DE L'ARTEMIA AU LIBAN BY M. A. ABISAAD

SUR L'ARTEMIA MAROCAINE BY M.H.AMANE ARTEMIA STATEMENT IN TURKEY BY N. GOKGOZ

SUR L'ARTEMIA ALGÉRIENNE BY I SAMIA

LES SALINES, SEBKHAS, CHOTTS ET L'ARTEMIA EN TUNISIE BY M. S. ROMDHANE

ARTEMIA PRODUCTION CASE STUDY IN TUNISIA BY M. BELKHIR

ThE COSMOPOLITAN BRINE SHRIMP BY G.V. STAPPEN AND P. SORGELOOS

MORPHOLOGICAL CHARACTERISATION OF ADULT ARTEMIA FROM DIFFERENT GEOGRAPHICAL ORIGIN IN THE

MEDITERRANEAN POPULATIONS BY F. HONTORIA AND F. AMAT

FURTHER CHARACTERISATION OF TWO ARTEMIA POPULATIONS FROM NORTHERN GREECE : BIOMETRY, HATCHING CHARACTERISTICS, CALORIC

CONTENT AND FATTY ACID PROFILES. BY T. ABATZOPOULOS, G. KARAMANLIDIS, P. LÉGER AND P. SORGELOOS

LIPID COMPOSITION OF CYSTS OF THE BRINE SHRIMP ARTEMIA SP. FROM SPANISH POPULATIONS

BY J. C. NAVARRO, F. AMAT AND J. R. SARGENT

INTERNATIONAL STUDY ON ARTEMIA IX. LIPID LEVEL, ENERGY CONTENT AND FATTY ACID

COMPOSTION OF THE CYSTS AND NEWLY HATCHED NAUPLII FROM FIVE GEOGRAPHICIAL STRAINS OF ARTEMIA

BY PAUL S. SCHAUER, D. MICHAEL JOHNS, CHARLES E. OLNEY, AND KENNETH, L. SIMPSON USA

INTERNATIONAL STUDY ON ARTEMIA*. XXVI. FOOD VALUE OF NAUPLII FROM REFERENCE ARTEMIA CYSTS AND FOUR GEOGRAPHICAL

COLLECTIONS OF ARTEMIA FOR MUD CRAB LARVAE BY C.R.SEIDEL,D.M. JOHNS, P.S. SCHAUER AND C.E. OLNEY

OVERVIEW GENETICS OF MEDITERRANEAN BISEXUAL ARTEMIA BY E.J.S.PILLA

THE NUTRITIONAL VALUE OF ARTEMIA BY P. LEGER, D. A. BENGTSON, P. SORGELOOS, K.L. SIMPSON AND A. D. BEEK

IMPROVED LARVICULTURE OUTPUTS OF MARINE FISH SHRIMP AND PRAWN BY P. SORGELOOS AND PH. LÉGER

THE USE OF BRINE SHRIMP ARTEMIA IN BIOLOGICAL MANAGEMENT OF SOLAR SALT-WORKS

BY W. TACKAERT AND P. SORGELOOS

SEMI INTENSIVE CULTURING IN FERTILISED PONDS BY W. TACKAERT AND R SORGELOOS

L' ARTEMIA EN TUNISIE

PAR NÉJI ALOUI

1- INTRODUCTION

Le succès d'une culture en masse de poissons et de crustacés depend surtout de la disponibilité d'une nourriture abondante et adequate pour les jeunes stades.

Très vite, il s'est avèré que la culture en masse du zooplancton, qui constitue la nourriture naturelle pour les stades larvaires, n'était pas écoriomiquement réalisable.

La découverte par les chercheurs que les larves d'Artemia constituaient une excellente source de nourriture pour les jeunes alevins, représentant une percée importante dans le développement de l'aquaculture.

Cette nourriture vivante peut en effet être produite facilement àpartir d'oeufs trouvés sur les berges des lacs sales.

En Tunisie, les recherches sur l'Artemia viennent de voir le jour et ceci après avoir pris connaissan-ce de la nécessité de cet animal pour les activités auquacoles.

II- BIOLOGIE DE L'ARTEMIA

L'Artemia est un animal aquatique, qui fait partie de la classe des crustacés, il appartient àla sub-classe des Branchiopodes et àl'ordre des Anostraca.

L"Artemia vit dans les eaux salées et hypersalées et peut s'adapter facilement àdes variations de sali-nité, allant de 70% à 300%. Les populations d'Artemia rencontrées dans la nature peuvent être, soit bisexuées, soit parthénogénétiques.

La reproduction est soit ovovivipare (production de nauplii), soit ovipare (production d'oeufs). Dans ce second cas, la femelle pont des oeufs appelés oeufs durables ou cystes de l'embryon peut rester vivant à l'intérieur de la coque pendant des années quand l'oeuf est bien conservé contre l'humidité. Ces oeufs, une fois incubés dans l'eau de mer préparée aux conditions d'incubation, se gonflent, attei-gnent le maximum de leur diamètre qui varie entre 100 et 300 um et donnent naissance àdes nau-plii dont la taille est de l'ordre de 400 um. Le nauplius passe par plusieurs stades de développement pour arriver au stade adulte (0,8 à 2 cm).

III- REPARTITION DE L'ARTEMIA DANS LE MONDE

Les habitats d' Artemia sont distribués partout dans le monde à l'exception de l'Antartique. Actuellement, on compte plus de 300 biotopes d' Artemia (Fig.l) (SORGELOOS et al, 1986).

D'après cet auteur, 80 % des populations étudiées dans l'hémisphère occidental sont bisexuées, alors que 70% de celles de l'hémisphère oriental sont parthéinogènétiques.

Les biotopes naturels d'Artemia sont limitées aux milieux où les concentrations ioniques sont assez élevées, ce qui exclut les prédateurs vu les conditions très difficiles. C'est ainsi que les populations naturelles d' Artemia sont observées soit dans les eaux athalassohalines. Les sites thalassohalins sont des eaux de mer consentrées en Nacl, alors que les eaux athalassohalines sont caractérisées par une composition ionique qui diffère beaucoup de l'eau de mer, telle que les eaux riches en carbonates ou en potassium ou en sulfates (PERSONNE et SORGELOOS, 1980).

IV - REPARTITION DE L'ARTEMIA EN TUNISIE (Fig.2) L'existence d'Artemia en Tunisie a été noté pour la premiere fois par (SEURAT, 1921) dans le Chott Ariana. Après cette date, ce petit crustacé a été mentionné par (HELDT, 1926) dans les anciens ports de Carthage et par (GAUTHIER, 1928) àSebklet Sidi-El-Hani.

Recemment, une premiere etude prospective a été effectuée par UNSTOP; cette etude a montre que l'Artemia est présente en Tunisie, elle vit dans les milieux hypersalés qui sont très nombreux et qui peuvent être classes en deux categories : les étangs athalassohalins et les étangs thalassohalins. Les premiers sont continentaux et alimentés par l'eau des oueds et des pluies, alors que les seconds sont en communication directe avec la mer et la composition chimique de leur eau est surtout chlorée.

4.1. Les étangs athalassohalins

4.1.1. Sebket Ariana

Cet étang se trouve dans la banlieue nord de Tunis et est limité au Sud par la Soukra, l'Est par la zone de Rraoued, au Nord par Sidi-Amor-Bou-Khtioua et à1'Ouest par Bourj-Touil. La superficie de cet étang est d'environ 1000 ha. C'est une zone maraicageuse, la salinité de l'eau varie selon léepoque de l'année et selon les endroits.

Des prospections ont été effectuées par L'INSTOP dans les parties Sud (Soukra) et Est (Raoued). Elles ont permis de récolter des oeufs d' Artemia dans la zone de la Soukra, mais une bonne partie de ces oeufs étaient vides et morts.

4.1.2. Sebket- Sidi - El - Hani Située au centre du pays, d'une superficie dis fois plus grande que le Sebklet Ariana, la Sebket Sidi - El - Hani et le village de Khniss; àl'Est, elle longe la route de Sousse- Sfax sur trente Kilometres; au Sud par, la région de Souassi et àl'Ouest par la piste reliant Kairouan àZmala de Souassi sur tren-te cinq Kilometres.

Des prospections ont été faites par UNSTOP; elles ont révelé l'existence de l'Artemia dans la partie nord du Sebka.

Figure 1 : Réparation de l'Artemia dans le monde (d'après SORGELOS et al., 1986)

Figure 2 : Réparation de l'Artemia en Tunisie

• Salinés * Sebkhas 1 - Saline de Mégrine 2 - Saline de Bekalta 3 - Saline de Sfax 4 - Sebkhet Ariana 5 - Sebkhet Sidi El - Hani 6 - Sebkhet El- Moknine

4.1.3. Sebket - El - Moknine

Cette sebka se trouve dans la région de Mahdia, elle a une superficie de 3.500 a 4.000 ha et elle est limi-tée au Nord par Moknine, àl'est par la route de Bekalta àMahdia sur une distance de sept Kilometres au sud par la-route Mahdia - Sidi - Bannour et àl'Quest par la route Moknine - Sidi Bannour.

Les prospections effectuées par l'INSTOP ont révélé l'existence de l'Artemia dans l'eau de la Sebka, alors que les cystes sont presque inexistants.

4.2 Les étangs thalassohalins. Ces étangs sont des salins, en communication directe avec la mer et servant a la fabrication du sel. Ils sont constitués par un ensemble de bassins qui communiquent les uns avec les autres. L'eau arrive de la mer soit par pompage, soit par gravité et stagne dans les premiers bassins où la densité de l'eau est de 3 a 8° baumé; de ces bassins l'eau passe dans un autre bassin appelé pièce maitresse (10° a 15° Be); celle-ci passe par la suite dans le bassin de reserve où elle atteint une densité de 23° Be et elle servira alors àalimenter les tables salines (25° Be) où se fait la fabrication du sel.

Parmi ces milieux salins, il y a lieu de citer: la saline de Sfax, la saline de Bekalta et la saline de Megrine.

4.2.1. La saline de Sfax Cette saline appartient àla campagnie COTUSAL, entreprise semi-étatique; elle a une superficie de 1485 ha. Les prospections effectuées par UNSTOP ont révélé la presence de l'Artemia dans certains bassins de la saline.

4.2.2. La saline de Bakalta

C'est une saline privée, située dans la région de Bekalta àSidi El Baghdadi, elle a une superficie d'environ 120 ha. L'eau passe de la mer dans les partenements par gravité puis fait tout son circuit pour arriver dans les cristallisoirs pour la fabrication du sel.

Les prospections effectuées par l'INSTOP ont révélé la présence de l'Artemia dans cette saline.

4.2.3. La saline de Megrine

Cette saline appartient àla compagnie COTUSAL; elle a une superficie de 1000 ha.

Les prospections effectuées par TINSTOP ont révélé la présence de YArtemia dans quelques bassins de la saline.

4.3. Conclusions des prospections

Les prospections menées par UNSTOP ont permis de révéler les faits suivants :

a) l'Artemia est répandue en Tunisie, elle existe dans les milieux athalassohalins et thalassohins;

b) L'accession et la manipulation de l'Artemia dans les milieux athalassohalins est très difficile voir même impossible dans la plupart des cas;

c) Les oeufs d'Artemia se trouvant dans les milieux athalassohalins sont de mauvaise qualité;

d) L'accession et la manipulation de l'Artemia dans les milieux thalassohalins est plus facile que dans les milieux athalassohalins;

e) Les oeufs d' Artemia se trouvant dans les milieux thalassohalins sont de bonne qualité

V - TRAVAUX EFFECTUES A côté de l'étude prospective, paragraphe IV; d'autres etudes ont été effectuées dont nous citons :

− L'étude effectuée par l'INSTOP portant sur l'Artemia dans la saline de Megrine.

Les résultats de cette etude ont permis de dégager les faits suivants :

a) Les oeufs d'Artemia récoltés sur les berges des bassins contenaient une grande quantité d'oeufs vides par rapport aux oeufs récoltés directement dans l'eau;

b) le pourcentage d'éclosion ainsi que l'éfficacité d'éclosion sont plus élevés pour les oeufs prélevés directement dans l'eau (70,2% et 188.267 n/g de cystes sec) que ceux provenant des berges (40% et 126.933 n/g de cystes sec);

c) sur le plan quantitatif et durant toute la durée de l'étude, la quantité d'oeufs d'Artemia récoltée était de 4 kg de poids sec.

− L'étude effectuée par (VAN BALLAER et al., 1987) et qui a traité des echantillons d'oeufs d' Artemia en provenance des sebkas et des salines. Sur ces échantillons, des analyses relatives à l'identification de l'espèce tunisienne, au sex-ratio et aux caractères biomètriques des oeufs et des nauplii ont été effectuées.

Les résultats de ces analyses ont permis de dégager les faits suivants :

a) Les oeufs d' Artemia en provenance des milieux salins sont de bonne qualité;

b) Les oeufs d'Artemia en provenance des sebkas sont de mauvaise qualité;

c) Les croisements génétiques ont permis d'identifier la souche tunisienne : il s'agit d' Artemia tunisiana;

d) Le sex-ratio est de 1,0 pour la souche de sfax, de 1,5 pour la souche de Bekalta et de 1,38 pour la souche de Megrine;

e) Le diamètre de l'oeuf est de 258,8 um pour la souche de Megrine, de 251,6 um pour la souche de Bekalta et de 235,4 um pour la souche de Sfax;

f) La longueur du nauplius est de 467,7 um pour la souche de Megrine, de 482,3 um pour la souche de Bekalta et de 422,2 um pour la souche de Sfax.

− L'étude effectuée par (KHEMAKHEM, 1988) portant sur l'Artemia dans la saline de Sfax. Ce travail a traité l'hydrologie de la saline d'une part et la biologie de l'Artemia qui y habite d'autre part.

L'hydrologie des bassins représentant tous les circuits de la saline a permis de reveler des fluctuations de certains paramètres physicochimiques, liées aux conditions climatiques d'une part et au sys-tème de fonctionnement de la saline d'autre part.

L'étude de l'Artemia a révélé que l'abondance et la distribution saisonnière d' Artemia sont liées aux conditions du milieu. La population d'Artemia dans cette saline est bisexuée. Après fécondation, les femelles peuvent se reproduire soit par ovoviviparité, soit par oviparité avec emission dans le premier cas de nauplii et dans le deuxième cas

d' oeufs. Sur le plan quantitatif des oeufs d'Artemia et durant toute la durée de l'étude, l'auteur a récolté une quantité très faible.

En conclusion de tous ces travaux, nous pouvons dire que dans les sebkhas, on a découvert l'exis-tance de l'Artemia en très faible densité et de faibles quantités de cystes dispersés sur les berges par l'action du vent. L'étude de la qualité de ces cystes a montré un très faible taux d'éclosion du au fait que les cystes sont exposés aux changements climatiques et se trouvent alors hydratés et deshydra-tés plusieurs fois, ce qui diminue la viabilité de l'embryon àl'intérieur du cyste et diminue par consé-quent le taux d'éclosion de ces derniers. Dans les salines, le cas se présente différement, c'est àdire que les cystes présentent moins de risques de déterioration et de mortalité, étant donné qu'ils restent baigner dans le milieu salin, par consequent le taux d'éclosion reste élevé.

VI - RECHERCHES ACTUELLES

Compte tenu de ce qui precede, nous menons actuellement des recherches relatives a la bio-écolo-gie de l'Artemia dans les salines, àl'utilisation des oeufs d' Artemia récoltés de ces salines, au sevra-ge et a la maîtrise de l'élevage de l'Artemia en milieu contrôlé.

Les résultats des recherches actuelles ont permis de mettre en évidence les faits suivants:

6.1. Aspect bio-écologique (population de Megrine).(Fig.3) - La souche est bisexuée;

- dominance des jeunes stades fin de l'hiver - début du printemps;

- l'éclosion des oeufs a lieu en automne et fin de l'hiver;

- la souche de Mégrine est tantôt ovovivipare, tantôt ovipare;

- le pourcentage des femelles ovipares s'accroit avec l'arrivée de l'été;

- la production en oeufs est de 20 kg (poids sec).

6.2. Aspect utilisation en milieu d'élevage

L'étude relative àl'utilisation des oeufs d'Artemia en provenance de la saline de Mégrine a donné de bons résultats aussi bien au niveau de la croissance que pour le taux de survie des larves.

6. 3 Aspect élevage de I'Artemia àl'échelle du laboratoire

L'élevage de l'Artemia àl'échelle du laboratoire est maîtrisé.

VII - PRESPECTIVES

− poursuivre les etudes biologiques et écologiques relatives àl'Artemia dans les milieux thalassohalins;

− élever l'Artemia dans des structures plus grandes afin de s'assurer de la maîtrise de son élevage;

− mener des expérimentations sur le larvaire (experiences de sevrage).

− chercher un substitut de l'Artemia

Figure p 25 à reduire

Fig. 3 Salines de mergrine

POTENTIEL D' ARTEMIA EN TUNISIE

PAR B. MISSAOUI &N. ABDELKADER

INTRODUCTION L'activité aquacole a commence en Tunisie depuis les années 60 avec l'implantation de la premiere station aquacole "Elevage de coquillage" au nord du pays. Ce n'est qu'au cours du Vléme Plan (1982-86) que les essais de reproduction de poissons ont commencè au niveau de l'écloserie de Ghar El Melh permettant de dégager pour la première fois le problème d'alimentation larvaire.

C'est ainsi qu'un programme de recherche a été mis au point pour étudier les potentialités d'exploitation de l'Artémia en Tunisie.

PROSPECTION

Les prospections (B. Abdelkader, 1985) faites durant les années 1982 et 1983 signalent l'existence de rArtémia dans les Chotts, Sebkas et Salins suivants (Chott El Jerid, Sebkhra de l'Ariana, Sebkha de Kourzia, Sebka de Moknine, Sebka de Sidi El Heni. Salins de : Megrine, Sahline, Bekalta et Sfax) (voir carte).

1- Chotts et Sebkhas : Ce sont des étangs continentaux n'ayant pas de communications avec la mer et qui sont alimentés par l'eau des oueds et des pluies.

Selon l'époque de l'année, ces étangs peuvent être couverts d'eau en hiver ou asséchés en été, et la salinité peut varier de quelques grammes pour mille àplus que 200%o.

Les cystes se présentent sous forme de frange de couleur brunatre sur les rives de l'étang. II est àsignaler que ces étangs présentent des difficultés d'exploitation résultant de ;

- La faible hauteur de la colonne d'eau sur ces étangs,

- Les difficultés d'accs àces étangs,

- L'assèchement de ces plans d'eaux pendant l'été.

2 - Salins : II s'agit de bassins communiquants alimentés par l'eau marine; La salinité dans ces bassins varie entre 40%o et plus de 200%o.

Les salins prospectés appartiennent en majorité a une compagnie semi étatique COTUSAL.

Les prospections dans ces salins ont révélé la présence d'Artémia vivante ainsi que des cystes viables.

Contrairement aux sebkas, les salins sont plus faciles à expoiter en raison de :

- l'existence d'une plus importante hauteur de la colonne d'eau dans des bassins,

- la facilité d'accés aux bassins d'artemia

- la possibilité de gestion de la biomasse d'Artémia dans les bassins de faible salinité.

RESULTATS DES CAMPAGNES DE PROSPECTION : La quantité d'artemia existante dans les salins est faible et ne peut faire l'objet d'une exploitation rentable. En effet il s'agit d'une récolte de quelque dizaines de Kg dans le salin de Sfax. L'examen des échantillons de cystes d'Artémia tunisienne a été effectué conjointement par des tech-niciens tunisiens et beiges.

Les résultats révèlent l'existence de l'espèce bisexuelle Artemia tunisiana ainsi que l'espèce Artemia pathenogenetica répandue surtout dans le salin de Mégrine.

La qualité de cystes récoltés en Tunisie varie d'un site à un autre. Les cystes récoltés dans le salin de Sfax sont de bonne qualité (taille des cystes décapsule < 220 pm; et présentent un bon taux d'éclo-sion; et bonne composition en acide gras polynisaturé).

Taille des cystes MEGRINE BEKALTA SFAX

Cystes non traités 258,8 251,6 235,4

Cystes décapsulés 234,1 228,0 215,1

Taille des cystes MEGRINE BEKALTA SFAX Taux d'Eclosion MEGRINE BEKALTA SFAX Pourcentage (%) 60,5 83,2 84,8

Bien que ces cystes soient de haute qualité pour leur utilisation en aquaculture, elles se trouvent peu résistantes aux facteurs environnants et présentant une faible productivité.

ESSAIS D'INOCULATION : Au vue des résultats des prospections et dans le cadre d'un projet de cooperation avec 'TArtémia Reference Center", il nous a paru judicieux d'inoculer une espèce étrangères d'Artémia plus productive et plus résistante que l'espèce locale Artemia tunisiana. C'est le salin de Mégrine qui a été choi-si pour réaliser cette experience d'inoculation.

Les essais ont été faits avec la souche de "Maccau" et celle de "Great Salt Lake" en comparaison avec la souche tunisienne.

D'autres essais ont été faits àplus grande échelle au salin de Sahline moyennant une fertilisation par des déchets de volaille.

Les résultats de ces deux operations n'étaient pas concluants pour plusieurs raisons

- les operations de fertilisation et d'inoculation n'étaient pas bien programmées.

- suivi irrégulié de cette experience,

- faible hauteur de la colonne d'eau dans les salins.

- faible productivité de l'eau dans le salin de Sahline

PERSPECTIVES DE DEVELOPPEMENT

Pour une bonne exploitation de l'Artémia dans les salins tunisiens, une etude a été élaborée dans le cadre du Plan Directeur de l' Aquaculture.

Dans ce travail des recommandations et des suggestions ont été proposées dans le but de la production d'Artémia en Tunisie. II a été propose de procéder en 2 étapes :

1- 1ère Etape : Vérifier l'hypothèse qu'une souche plus performante donne de plus grandes productions (biomasse et cystes) et une meilleure qualité du produit.

Cette verification se fait a travers des inoculations.

L'espèce àinoculer doit être performante (A. fransiscana) et résistante a des températue élevées (A.fransiscana produite au Vietnam).

Des suggestions ont été faites pour le choix de l'endroit d'inoculation, (Mégrine bassins d'éva-poration n° 6 ou Sfax Canal a 150g/I).

2 - 2ème Etape : En fonction des résultats de l'expérience d'inoculation procéder a Intégration de production d'Artémia avec le sel.

Cette action nécessite :

− des aménagements supplémentaires pour augmenter la hauteur de la colonne d'eau dans les circuit de 130 et 200 g/l :

− un programme d'élimination de prédateurs,

− un programme de fertilisation des bassins avec l'urée et le phosphate d'ammonium.

− un programme d'inoculations,

− récolte, conditionnement et traitement des cystes et de la biomasse,

− contrôle de la qualité du produit.

Production attendue = 150 T/an.

Estimation des revenus = 375.000 US$/an

Artemia dans les chotts et sebkhas de Tunisie

Estimation des revenues = 375.000 US$/an

ARTEMIA PRODUCTION CASE STUDY IN EGYPT

BY S.GHONEIM

Survey for natural artemia available in Egypt: the occurrence of artemia were recorded in different sites. Most of theses sites are man made salines located on the Mediterranean coast, while two sites are located in inlands areas

The coastal sites are : Port-Fouad, Balteim, El-Max and Bourg EL-Arab salinas. The inland sites : Malaha El-Salfate in Wady E-Natroun and Fayom salina beside Qaroun lake. Bisexual artemia are found in Malaha El-Salfate (in Wady El-Natroun) port Fouad Salina and Balteim Salina.

Cross breeding tests performed and revealed that artemia from Wady El-Natroun is belonging to artemia tunisiana, while the recent available artemia franciscana. However, parthenogenetic artemia is found in Fayom salina. Artemia from wady El-Natroun possessed the smallest cyst diameter while artemia from Fayom salina possessed the largest cyst diameter.

Artemia franciscana has been inoculated in Port Fouad salina, and it is proved that the production artemia is technically and economically feasible.

GEOGRAPHICAL DISTRIBUTION OF ARTEMIA IN THE JAMAHIRIYA

BY M. O. MAGSODI

The importance of Artemia increased remarkably in the last years in most countries of the world. This importance stems from expanding its use as life food for many species of fine fish and crustacean as well as the development of its use for larvatic stages of the same species especially after the spread of aquaculture on a large scale.

In the Jamahiriya three sites have been known located for the existence of Artemia, two of which are coastal areas and the third is in the desert south Libya as follows :

1- El Kuwaim sebka in the central region in Brega region; this sebkha is located at a distance of about 770 km from Tripoli.

2- Abukamash sebka, this coastal sebka is located at a distance of about 150 km West of Tripoli, in Abukamash area near the tunsian boarders.

3- Lakes located in the Southern, it can be said that the existence of Artemia in the Jamahiriya and common use as food for human beeings started before now and the importance of this creature as food for fish and crustacean is increasing with farming activities.

Therefore, there is need to point out that:

• Just now, no classification and to identification of Artemia have been done in Libya.

• No produce of Artemia in Libya.

• A few cysts collected from the different sites to run experiments and get some studies going on.

SITUATION DE L'ARTEMIA AU LIBAN

BY M.A. ABI SAAD

L'absence d'une intense activité aquacole au Liban fait que les besoins en Artemia ne sont pas du tout ressentis et que son utilisation se fait seulement en petite quantité dans la nourriture du poisson d'or-nementation.

D'autre part, eu égard a l'inexistance des lacs sales types sebkha, les populations d'Artémia ne sont pas rencontrées dans les eaux Libanaises. Toutefois, la presence au Liban des marais salants aban-donnés pourraient inciter ày établir une activité d'élevage d' Artémia àpetite échelle et qui serait inté-grée àcelle de la protection du sel.

SUR L'ARTÉMIA MAROCAINE

PAR M.H. AMANE

La production naturelle d'artémia au Maroc est trés faible; il s'agit d'une souche parthénogenetique (artemia parthenogenetica) qu'on rencontre dans les marais salants au nord du Maroc du cote de la Méditerranée et àl'ouest sur les cotes atlantiques.

Deux grandes sociétés d'aquaculture implantées au Maroc depuis 1986 consomment des grandes quantités d'artémia (5 tones/an).

Cependant, aucune décision visant la production des cystes d'artémia n'a été a ce jour envisagée sur le plan public ou privé.

ARTEMIA STATEMENT IN TURKEY

BYN.GOKGOZ

There is no production of artemia in Turkey which imported yearly about 400 tonnes of dry artemia cysts for larvae culture from Belgium and other countries. While natural production is represented by the Artemia parthenogenetica met in the only salt lake existing in Turkey and quality and size seem to be well adapted in feeding larvae, the presence of that Artemia is not too great.

SUR I'ARTÉMIA ALGÉRIENNE

PAR I. SAMIA

L'étude prospective dans certains sites favorables àla production naturelle de l'Artemia fut établie en 1988 en Algérie. Elle revela la présence d'une population d'Artémia composée de larves, de cystes et d'adultes bisexuels dans la saline d'Arzew.

L'étude biologique et systèmatique ont permis d'identifier l'espèce comme appartenant au genre Artemia tunisiana.

La saline d'Arzew assure une production de 87 000 T de sels par an soit 65% de la production nationale.

La souche d' Artémia d'Arzew est d'une qualité comparable a celle des souches qui sont commercia-lisées de nos jours (San Francisco Bay et Great salt lake).

La collecte de l'Artemia produite naturellement se fait facilement dans la saline d'Arzew et l'Algérie pense dans l'avenir y entamer la culture de cette artémia.

LES SALINES, SEBKHAS, CHOTTS ET L'ARTEMIA EN TUNISIE

BY M.S. ROMDHANE

La Tunisie, avec ses 1300 km de côtes présente, àpart les lagunes côtières, trois catégories de plan d'eau salées : les salines, les sebkhas et les chotts.

Les salines Tunisiennes sont réparties en 2 groupes, les grandes salines avec une grande superficie et une grande capacité de production, et les petites salines moins équipées et d'activités irrégulières.

Les sebkhas et les chotts couvrent environ 1 million ha et leurs superficies trés variables; I' Artémia est observée dans les sebkhas d'Ariana, de Komzia, de Moknine, de Bekalta, de Sidi El Hani et d'El Melah, ainsi que dans 8 chotts El Jerid, El fjaj et El Gharsa.

L'Artémia présente en Tunisie est identifiée comme souche bisexuelle d'espèce Artemia tunisiana. L'artémia parthénogénica est observée seulement dans les salines de Mégrine.

ARTEMIA PRODUCTION CASE STUDY IN TUNISIA

Presented byMr.Belkhir

• Aquaculture activities in Tunisia was enhanced in large scale since 1982 and still being supported to now by the government.

• During the period 1982 - 86 trials on fish artificial reproduction showed for the first time several problems in the fish larvae stage.

• Then a research program was conceived; it was dealing particularly with the Artemia investigation in the field studies of Artemia characteristics in lakes;

• That program was held in the framework of cooperation between the scientific institution (INS-TOP) in Tunisia and Ghent University.

• Results were very useful and indicated relative and spontaneous richness of Artemia within salt water and some coastal waters.

• Ecological and biological characteristics of Artemia were presented to the participants during the first session of the Artemia training course.

• Briefly there is need to remind that the artemia cysts found in the south of Tunisia seemed to be better than the northern ones.

• Results indicated that Artemia is the bisexuel called : Artemia tunisiana; and other one which is the Artemia parthenogenica was found essentially in the northern part of Tunisia.

• From these conclusions, a project was foreseen to develop inoculation trials using the Maccau strains and the great salt lake one.

• Activities were held in a very roughly and preliminary way using several fertilisers

• Unfortunately, this project fell down and obtained results, even if they were interesting they could not be very indicative because we notify a. lack in the efficiency and the regularity of the investigation enterprised during the preliminary phase.

• Therefore specialised institutions are according actually an important interest to Artemia exploitation and the guideline of the forthcoming activities on Artemia are conceived in the framework of the national matter plan for the development of aquaculture :

- to develop a more performant strain which gives greater productivity and better products quality for that, Artemia franciscana and the resistant specy to temperature such as the Artemia fran-ciscana produced in Vietnam will be inoculated and tried for production.

- in the second phase, effort will be undertaken to integrate Artemia exploitation and production within the salt production activities.

• Before undertaking any pilote activities the following points should be taken into consideration :

- the conception of ponds management, salt waters and their expoitation;

- set up a methodolgy for reducing predators;

- control and follow up of cysts production in term of quality and quantity.

• The foreseen production is : 150 tones/year.

• The foreseen flow back is : 375 000 US $ / year.

ARTEMIA PRODUCTION IN EGYPT

BY M.A.S.D.AHMED

• Artemia was used for the first time in Egypt in Macrobrachium feeding at Saft Khaled hatchery.

• following the studies on artemia done by the university of Suez Canal a commercial Unit of Artemia production was implemented at El Nasr Salines company

• the production of Artemia salina cysts and Artemia franciscana reached 500 kg per year covering a total area of 100 ha

• culture conditions are : 20-30° C, and 2000 Lux

THE COSMOPOLITAN BRINE SHRIMP

BY GILBERT VAN STAPPENAND PATRICK SORGELOOS

A tiny crustacean, adapted to living in highly saline waters, has become the most widely used live food item in aquaculture. Improved harvesting and processing techniques, as well as culture of the organism itself have helped make the Artemia virtually indispensable to the industry.

Salt has, always been very precious to man, not only because of its dietary value, but also as an indispensable tool for preserving food. In ancient times salt was a means of payment. This explains the etymology of the word salary, used in so many languages. Modern industry is using sodium chloride salt as a basic ingredient for numerous chemical processes and consumes more than 90% of the annual production of more than 200 million mt.

All over the world local populations have developed methods for solar salt production, ie the extraction of salt from seawater. Management techniques were developed to maximise evaporation, to allow sequential precipitation of caronates and sulphates so as to eventually collect the sodium chloride.

What man didn't realize, until very recent times, was the fact that these very salty waters, often considered lifeless, were the habitat of a remarkable invertebrate organism, the 1-cm long brine shrimp Artemia. Its high food value for aquaculture and aquarium pet organisms was soon appreciated and in less than two decades commercial harvests reached over 1 000 mt of cysts and over 10000 mt of biomass annually. Worldwide sales in 1992 of Artemia cysts and biomass are estimated at over US$50 million.

The selective cosmopolitan

The small crustacean Artemia is found all over the world in a wide array of hypersaline biotopes, eg coastal salt pan, inland salt lakes, and chloride, sulphate. and carbonate waters. The only factors limiting its presence are the salinity has to be sufficiently high (mostly above 100 g/1 total salts) to exclude the presence of predators (fish and arthropods), and the water temperature, that must allow development and reproduction. Depending on these factors, populations are seasonal or are present on a year-round basis.

In an optimal environment the habitat is colonised at an astonishing rate : mature females, reproducing ovoviviparously, can produce 200 - 300 free-swimming nauplii every four days. These nauplii grow into adults in less than two weeks. In a combination of adverse conditions like oxygen stress, high salinity, changes in temperature, food limitation, etc the reproductive mode is switched to ovi-parity : fertilised eggs develop into a late gastrula, at which moment the metabolism is reversiby interrupted and each individual embryo, now called a cyst, is enveloped by a protective shell. Mother Artemia release these ametabolic cysts which float at the water surface, are aggregated by the wind, and eventually accumulate in a dehydrated form on the shores.

Since the Artemia species has no means of active dispersal, its natural occurence is determined by the factors of wind and migrating water fowl acting upon the dry cysts ; hence its geographic distribution shows a discontinuous pattern. The ecological diversity of these isolated biotopes and the genetic flexibility of the species have led to the evolution of more than 350 geographical strains. As could be expected with the

exploration of new habitats of Artemia (eg in Central Asia and China), the list is still growing.

An adult male Artemia approximately one cm long

Artemia strains Strain Characteriastion is a crucial item in Artemia study, enjoying, however, a dubious reputation among Artemia specialists. Indeed, as more and more natural habitats of this organism were discovered in the late 1970s and 1980s, it soon appeared that all these different populations were displaying a considerable variability in all kinds of characteristics.

This necessitated elaborate morphological, biometrical, and genetic research, making Artemia, the favourite of tenacious taxonomists. subdivision of the genus into species, occurence of pathenoge-neticand bisexual strains, and generally the entire problem of genetic flexibility and speciation are, however, not merely discussions to be carried out in the confines of ivory towers: practice proves that these strain differences have implications in the field of aquaculture that can hardly be underestimated on one hand, that provide unique opportunities for later selection work on the other.

Tapping nature's resources

The discovery and study of sevral brine shrimp habitats resulted in a better knowledge of the complex Artemia biology. At the same time, the commercial exploitation of these biotopes has definitely and probably definitely rebalanced the very uneven situation of the 1960s and early 1970s, when a few US companies exploited the hudge Artemia resources of Great Salt Lake, Utah, and San Francisco Bay, California. They owned the world monopoly on cysts and eventually increased prices to levels almost unbearable for many developing countries.

Presently a large part of the cyst market is still supplied by harvests from location, the Great Salt Lake. This situation makes the market still extremely vulnerable to eventual climatological and/or ecological evolutions in this lake.

An Artemia couple in precopulation An Artemia cysts hatching

A seabass (Lates calcarifer) Havesting newly-hatched Artemia

Larvae eating enriched Artemia nauplii nauplii in a shrimp hatchery in Ecuador

Interest in the commercial harvesting of other Artemia biotopes is, however, steadily growing, brought about by local import restrictions and/or the inreasing demand for Artemia products as a valuable source of live feed in aquaculture. The quality of Artemia differs from strain to strain and from location as a result of genotypic variation (ie cyst size, cyst hatching characteristics, caloric content and fatty acid composition of the nauplii) determine if a particuler cyst product is suitable for use in commercial hatcheries of fish or shrimp as a larval food source. As a result, it is imperative to determine the nutritional quality of the adult artemia and /or its cysts for specific aquaculture purposes prior to considering commercial use of natural Artemia biotopes. Techniques for cyst and biomass harvesting, storing and processing, once a matter of trial and error, are now well established and guarantee, if properly applied, a high quality product.

Maximum sustainable yields of cysts and biomass are influenced by the population dynamics of the local Artemia resource, the determination of maximal harvesting rates is, however, complicated by the heterogenous distribution of Artemia, which makes accurate sampling and consequently, precise population estimates very difficult. The recruitement rate of the population may be high in ponds where the dominant reproduction mode is ovoviviparity, and low in cyst-production ponds. Predation by waterbirds is another factor to be taken into consideration. Natural recruitement can eventually be increased by introduction of a more productive strain. In most Artemia habitats population densities are very low as a result of food limitation due to low nutrient contents of the intake waters. Consequently eutrophication of coastal waters by human activity, though reviled by many, is not unwelcome for those involved in the Artemia Business.

An aerial view of large scale cysts harvesting at the

Great Solt Lake, Utah, USA Artemia cysts harvesting from an

inoculated saltpond in Chachoengsao, Thailand

Artemia cum grano salts Over the centuries, hundreds and thousends of hectares of salt pans have been constructed all over the world in tropical and subtropical areas, for so-called solar salt making. Seawater contains salts of every chemical element, in at least trace amounts. Solar salt is normally produced by pumping seawater from one evaporation pond into another, allowing carbonates and gypsum to precipitate, and finally draining NaCl-saturated brine or pickle into crystalliser ponds where sodium chloride precipitates. Before all NaCl has crystalled, the mother liquor, now called bittern, has to be drained off to reduce contamination of the sodium chloride with bromides and other salts that begin to precipitate at these elevated salinities. The techniques of solar salt production thus involves fractional crystallisation of the salts in different ponds to obtain sodium chloride in the purest form possible, eg up to 99.7% on a dry weight basis.

The quantity and quality of salt produced is largely determined by the hydrological activity in a solar salt operation. Algal blooms are generally beneficial since they ensure increased solar heat absorption, resulting in faster evaporation and inceased yields of salt. But if they are not metabolised in time (ie eaten by Artemia), algal excretion and decomposition products, such as dissolved carbohy-dates, act as chemical traps and consequently prevent early precipitation of gypsum which will contaminate the sodium chloride in the crystallisers and reduce salt quality. Furthermore, such organic impurities as algal agglomerates, wich turn black on oxydation, may contaminate the salt and reduce the size of the cristals and, hence, the salt quality. In the worst situation, high water viscosities may completely inhibit salt crystal formation and precipitation.

Artemia gives a helping and by controlling algal blooms and by providing essential nutrients from Artemia metabolites and/or decaying animals as substrate for the proliferation of halobacterium in the crystallisation ponds. High concentrations of these red halophilic bacteria promote heat absorption, thereby accelerating evaporation, and reduce the concentration of dissolved organics. Lower viscosity levels promote the formation of larger salt crystals and, thereby, improve salt quality.

As said before, dispersal of Artemia cysts is a purely passive matter; in many situation the salt farmer cannot rely on this opportunistic dispersal method. In saltworks with short water retention times in the evaporation ponds, a rapid dillution may wash away the

existing population; excessive rainfall may eliminate the population or reduce it to such a level that it cannot cope with the algal blooms. Moreover, some saltworks may be completely isolated from natural sources of Artemia dis-persal. In other cases the autochthonous strain has poor productivity and the population remains too small to control the algae. In these cases the salt producer has to proceed with the controlled introduction (inoculation) of another strain, better production chatracteristics. However, only general guidelines can be formulated, since each situation has its own requirements: water retention time, food concentration, water temperature and salinity and the fluctuation of these parameters over the year are all factors to be considered when selecting a suitable Artemia strain.

Last but not least, proper Artemia management should lead not only to improved salt production but also provide opportunities for the harvesting of the valuable by-product Artemia, in the form of cysts and biomass. In the large solar saltworks (with individual evaporation ponds of tens to hundreds of hectares) Artemia production yields are limited and can only be influenced by inoculation strategies (eg strain, nauplius density, location and frequency of inoculation). However, harvests of up to 20 kg (dry weight) cysts per ha, or 2000 kg (wet weight) adult biomass per ha per month are achieved in tropical/subtropical regions in Southeast Asia and Central and South America in small artisanal saltworks (mostly operated on a seasonal basis) that are intensively managed for artemia production; ie pond depths are increased, and the low salinity ponds are fertilised twice or more a week with cor-ganic (eg chiken manure, waste of monosodium glutamate processing) and/or inorganic nutrients (eg urea, triple superphosphate). As has been proven in Vietnam, for example this type of integrated, salt, Artemia and shrimp culture, eventually alternated with rice and shrimp in the rainy season, offers interesting socio-economic perspectives for those countries where solar salt production has or is becoming a marginal undertaking.

Drying Artemia cysts in Macau, Brazil Artemia can be used as a carrier for nutrients or chemotherapeutics

Artemia: polyvalent and versatile

Among the live diets used in larviculture of marine as well as freshwater fish and crustaceans, brine shrimp (Artemia) nauplii constitute the most widely used food item. Annually over 1000 mt of dry Artemia cysts are marketed worldwide for on-site hatching of the nauplii. Although the use of these cysts appears to be most simple, considerable progress has been made in the past decade in improving and increasing its value as larval diet, eg selection of the most appropriate strains and batches, naw techniques for cyst desinfection and decapsulation, nauplius hatching, enrichment and cold storage.

Using particulate or emulsified products, rich in highly unsaturated fatty acids, the nutritional quality of the Artemia can be further tailored to suit the predators' requirements by bioencapsulating specific amounts of these products in the Artemia metanauplii. Application of this method of bioen-capsulation, also called Artemia enrichment or boosting, has had a major impact on improved larvi-culture outputs, not only in terms of survival, growth and success of metamorphosis of many species of fish and crustaceans, but also with regard to their quality eg reduced incidence of malformations, improved pigmentation and stress resistance. The same bioencapsulation method is now being developed for oral delivery of vitamins, chenlotherapeutics and vaccines.

Harvested Artemia biomass may be fed live to the predators, or frozen, freeze-dried or ensiled for later use, or made into a flake diet, with relatively little loss of nutritional composition. Live Artemia biomass is apparently a good food both for the growth of penaeidshrimp juveniles in nursery ponds and for the maturation of adult broodstock.

In the very extended saltworks locatedalong the Bohai Bay, PR China, in fact the largest solar salt producing area in the world, huge quantities (up to severalthounsand mt per year) of adult Artemia biomass are harvested and used in local hatcheries, grow-out and maturation facilities of the chine-se white shrimp, Penaeus chinensis. Throughout their grow-out cycle, shrimp are fed Artemia biomass at a rate of about 15000 kg/ha, supplemented with artificial feeds, chopped fish and mollusc flesh; a quite unique situation -definitely the chinese way- made possible by the abundant availability of Artemia biomass at very low cost from the local salt ponds.

MORPHOLOGICAL CHARACTERIZATION OF ADULT ARTEMIA (CRUSTACEA, BRANCHIOPODA) FROM DIFFERENT GEOGRAPHICAL ORIGIN. MEDITERRANEAN POPULATIONS

BY F. HONTORIA and F. AMAT

ABSTRACT. Twelve morphological parameters have been measured in Artemia individuals belonging to 27 different populations located around the Western Mediterranean basin. An analysis, through multiva-riate discriminant procedures, allows us to establish relationships among different populations. The three different types of population studied (bisexual diploid, parthenogenetic diploid and partheno-genetic tetraploid) are thoroughly characterized by their morphological characteristics. This simple method is shown to be useful in grouping different populations and to have predictive value in assigning new populations to the groups previously analyzed,

INTRODUCTION

The use of Artemia as food for marine fish and crustacean larvae in aquaculture has caused a considerable interest in this organism in the last decades. The variation shown by different populations in many of their biological processes has introduced the need for reliable methods for characterization, in order to understand better the relationships between populations of this complex of species, and to some extent to be able to predict some of their features.

It has only recently been demonstrated that more than one species exist in the genus Artemia (Barigozzi, 1972; 1974; Clark and Bowen, 1976; Bowen et al, 1978). It has been proposed that the specific name A. salina Leach, 1819, formerly used be abandoned, until systematics of this group can be completely understood (Persoone et al., 1980). The present investigation was in mind.

Previous work on the morphology of Artemia (Gilchrist, 1960) has shown that Artemia individuals undergo morphological changes according to the environmental conditions. Furthermore, this study states that wide differences among populations and between males and females can be found, even when the animals were cultured in the same medium, thus demonstrating intrinsic influences as well as environmental ones. Similar results were reported by Amat (1979, 1980) after a complete morphological study on 22 different Mediterranean populations, and comparing these results to those from a population coming from San Francisco (Califronia). He concludes that the variation in these characters observed among Artemia males and females from different populations allows one to classify the different main types of Artemia, but it is difficult to distinguish among populations of the same type. In a recent work Varo (1988) has studied three parthenogenetic diploid populations (two from the Canary Islands and one from the Eastern Spanish coast). Although, she could not clearly distinguish these populations (pertaining to the some type), as Amat (1979, 1980) predicts, she found that the most discriminant variables studied are : length of the furca; diameter of eyes; width of the head; length of the first antenna; and the distance between the eyes.

In the present paper variation of different morphological parameters measured on Artemia females from different populations coming from the Iberian Peninsula and adjacent areas is studied. Multivariate discriminant analysis has been used in order to maximize the differences among groups of observations and thus to obtain an increase

in the sensitivity of the morphological studies, which use only direct observation of the data. Thus, allowing some discrimination among the populations of the same type.

METHOD Studied populations

We have considered 27 populations, most of them (20) coming from the Iberian Peninsula. The remainder were obtained from cysts collected in the Balearic and Canary Islands, Morocco, France and Italy, and, therefore, very closely related to the former. All of them can be clustered in three different types of populations, which are the most frequently found around the Mediterranean basin : zygogenetic diploid (bisexual); parthenogenetic diploid; and parthenogenetic tetraploid. The different population origins, the abbreviations utilized from now on, the population type to which they belong, and the number of individuals studied in each case are shown in Table I. Figure 1 shows the geographical location of each population.

Culture conditions

All populations studied were raised under standardized conditions in order to minimize the strong environmental influences dispalyed by the body form in Artemia.

Cysts belonging to each population were allowed to hatch in sea water (38%o salinity) at 28°C under constant aeration and lighting. The nauplii were transferred to 11 containers with the bottom closed by a piece of 60 µm plankton net. These containers were then suspended in groups of 10 in bigger reservoirs (150 1) filled with seawater (salinity 30-32%o), thus all sharing the same culture medium. The water was maintained at 25°C, under summer natural photoperiod (16 h light : 8 h dark), and supplied with moderate aeration from the bottom, this sets the oxygen concentration to saturation. The culture densities were never allowed above 50 animals 1-1. Cultures were fed live unicellular algae (Tetraselmis succia) at an approximate density of 100 000 cells ml-1 (ad libitum). The medium was completely renewed twice per week with fresh seawater and microalgae cultures. It did not seem necessary to control the ammonia level because the volume, density and renewal conditions described the adult state (this was conspicuous when females had their ovisac developed), after 15-30 days in tetraploid, different samplings were performed in order to measure them.

Table I. Studied populations, abreviation utilized (numbers in parentheses identify populations in Figure I), number of observations (n), and type of population to wich they belong, T, parthenogene-tic tetraploid; d; B; bisexual diploid

Type Population Abbreviation n T Alcochete (Portugal) ALT (2) 30 T Bujaraloz (Spain) BUJ (19) 27 T Comacchio (Italy) COM (22) 40 T Delta of Ebro River (Spain) DTE (15) 20 T Guerande (France) GND (21) 33 T Larache (Morocco) LAT (6) 33 T Margherita di Savoia (Italy) MSV (23) 33 T Medinaceli (Spain) MED (18) 24 T Saelices (Spain) SAE (17) 27 T Sanlùcar (Spain) SLT (3) 29 T Tierzo (Spain) TZO (16) 24 D Bonmati(Spain) BNM (12) 27 D Bras de Port (Spain) BPP (11) 24 D Calpe(Spain) CAL (13) 27 D Cabo de Gata (Spain) CGT (7) 31 D Gerri de la sal (Spain) GER (20) 21 D Janubio (Spain) JAN (1) 24 D Larache (Morocco) LAR (6) 27 D La Mata (Spain) LMT (9) 17 D San Fernando (Spain) SFP (5) 25 . D San lùcar (Spain) SLC (3) 27 B Bras de Port (Spain) BPB (11) 27 B Ibiza (Spain) IBZ (14) 21 B Salinera Española (Spain) SEB (10) 24 B San fernando (Spain) SFR (5) 27 B San Felix (Spain) SFX (4) 24 B San Pedro del Pinatar (Spain) SPP (8) 30

Fig, 1. Geographical location of the studied populations. The populations that numbers identify are given in table 1. Parthenogenetic tetraploid (*); Parthenogenetic diploid (+); bisexual (o).

Measuring individuals

From each population a random sample was taken. Animals were anesthetized with some droplets of water saturated with chloroform and females, always more than 20 (except LMT, which underwent a high mortality in culture) were separated from the rest. The following morphological parameters were quantified in each female : total length; abdominal length; width of third abdominal segment; width of the ovisac; length of furca; number of setae inserted on each branch of the furca; width of head; maximal diameter and distance between compound eyes; length of first antenna; and the ratio abdominal length x 100/total length. Figure 2 illustrates these above mentioned body measures. In all cases, a similar number of individuals for each length interval was included in order not to bias results through the sampling. Preadult individuals were considered as well.

Statistical analysis

The 12 morphological variables, measured in all individuals (Table I), were used to establish relationships among these populations through discriminant analysis. This multivariate procedure provides a series of variables (Z1, Z2,...), which are linear functions of the morphological variables studied, with the form Zn = X1+ X2+... (where are the calculated discriminant coefficients and Xs the variables being considered). They maximize the separation among different groups of observations defined a priori (Anderson, 1984). Thus, the first discriminant function is the equation of a line cutting across the intermixed cluster of points representing the different observations. This function is constructed in such a way that the different predefined groups will evaluate it as differently as possible. Obviously, this will not be accomplished if the nubmer of groups is high, and subsequent discriminant functions will be needed. Two analyses were carried out: first, all observations were grouped by the type of population (bisexual diploid, parthenogenetic diploid and parthenogenetic tetraploid); in the second analysis, the separation criterion was the origin of the population. These analyses have been

performed using a backward stepwise procedure that allows removing the different variables out of the model separately and ranking them for their relative importance in discriminating Artemia populations. Nevertheless, all described variables were kept in the model. These calculations have been performed with the help of the statistical package Statgraphics v. 3. 0 (Statistical Graphics Corp., Rockville, MD) run on an IBM AT personal computer.

RESULTS In Table II, the results obtained when the type of population was used as a separation factor are displayed. The two functions found give 100% separation, and both are statistically highly significant (P<0.001). Morphological characteristics allow a clear differentiation among the three groups consiered (Table II, group centroids). The morphological characteristics that most significantly contribute to the discrimiation among the three groups are : lengh of first antenna, width of head and those related to the form and size of the head, the ratio abdominal length/total length in form of percentage and the width of ovisac and abdomen (Table II).

Results of the second analysis (factor of separation is population of origin) are shown in Table III and Figure 3. In this case, 12 discriminant functions are needed in order to separate thoroughly the 27 populations, but the first five of them give a cumulative separation percentage of 90.66 (the four discriminant functions shown in Table III give a 85,87% cummulative separation). The first eight functions calculated are highly statistically significant (P<0.001), the ninth is also significant (P<0.05) and the last three are not significant. The morphological characteristics that most signi-fiantly contribute to separate the groups in this case are : distance between eyes, eye diameter, length of the first antenna and all variables related to the shape and size of the head and the length of the furca (Table III).

For clarity, the first two or three discriminant functions solved in the mean points of each group (cen-troids) instead of individualized observations are shown in Figures 3 and 4. Centroids can be considered representative of each group because of the significance levels. Figure 3 shows that the cen-troids of the populations belonging to the same type tend to be grouped. Although the goal of the discriminant analysis is to separate thoroughly the different groups of observations considered, the relative distance between centroids in a multidimensional space (12 dimensions in this case) allows us to draw a very precise idea of the mophological relationships among different populations. Figure 4 shows the same data as Figure 3, with the addition of the third discriminant function. The increase in amount of information allows us to be more precise describing the different morphological relationships. This representation reveals some hidden separations in Figure 3. For example, CAL (13) appears in Figure 4 as markedly different from other parthenogenetic diploid populations, while in Figure 3 it seems more alike to them.

Table II. Results of the discriminant analysis on the morphometric variables measured in Artemia females from 27 different locations, and grouped by the type of population to which they belong. Non-standardized coefficients of the resulting discriminant functions. F-value contrasting the decrease of separation among groups when each variable is removed from the model. Group cen-troids were solved for the discriminant functions obtained.

Discriminant function 1 - 2 - F-value Total length 0.51 0.35 1.2 Abdominal length 1.37 -0.08 4.2 Width of ovisac -0.67 1.89 44.6 Abdominal width 8.92 1.56 37.5 Length of furca 0.92 7.16 21.8 No. setea left branch 0.01 0.13 7.9 No. setea right branch 0.03 0.01 0.7 Width of head -5.15 12.33 47.1 Distance between eyes -1.59 -7.18 33.2 Diameter of eyes -23.13 -0.46 23.8 Length of 1st antenna 7.05 -3.51 107.8 % length abdomen/total length -0.38 0.04 44.9 Constant 18.99 -1.92 Group centroids Parthenogenetic diploid 1.67 0.68 Parthenogenetic tetraploid -0.42 -1.31 Bisexual -1.87 1.60

Fig. 2. Shematic illustration of adult female Artemia showing the different body measurements utilised. (A) Total length; (B) abdominal length; (C) width of ovisac; (D) width of third abdominal segment; (E) length of furca; (F) width of head; (G) length of first antenna; (H) maximal diameter of complex eye; (I) distance between complex eyes.

Table III. Results of the discriminant analysis on the morphometric variables measured in Artemia females from 27 different locations, and grouped by its origin. Non-standardized coefficients of the first four resulting discriminant functions. F-value contrasting of separation among groups when each variable is removed from the model.

Discriminant functions 1 2 3 4. F-value

Total length -0.40 0.02 -0.59 -1.73 11.6

Abdominal length 0.68 0.06 -1.94 2.98 9.1

Width of ovisac -0.29 1.60 0.69 -1.38 8.4

Abdominal width 6.11 1.53 2.57 17.69 11.8

Length of furca 6.22 8.11 -6.44 -14.95 18.1

No. setae left branch 0.07 0.18 0.07 0.16 3.7

No. setae right branch 0.05 0.04 0.01 0.13 1.6

Width of head -8.28 6.77 5.99 0.46 12.6

Distance between eyes 2.47 -8.13 8.79 -0.66 19.2

Diameter of eyes -26.49 1.66 22.32 -8.43 35.8

Length of first antenna 5.93 -5.07 -6.10 -1.54 27.3

% length abdomen/total length -0.41 0.20 0.11 -0.03 12.0

Constant 20.16 -8.15 -9.82 0.93 _

Fig. 3. Group centroids solved for the first two functions result ing from the disriminant analysis when using the origin of each individual as separation factor. Bisexual (O); parthenogenetic diploid (+), parthenogenetic tetraploid (*).

Fig.4. Group centroids solved for the first three functions result ing from the disriminant analysis when using the origin of each individual as separation factor. Bisexual (O); parthenogenetic diploid (+), parthenogenetic tetraploid (*).

DISCUSSION This method clearly allows us to differentiate the morphological patterns that characterize at least some different Artemia species. In some cases, it is possible to discern between varieties having a sufficiently, important characteristic, such as the different ploidies in parthenogenetic populations (Table II). It is not possible to assign a morphological pattern to every population studied with the parameters utilized here, in disagreement with previous work described in the introduction (Gilchrist, 1960). However, this fact should be considered as a validation of the methodology more than a defect, since some of these populations probably have a high genetic closeness. For example, the third discriminant function from the second analysis, makes it possible to discern between some populations that previously seemed closely related (Figure 4). This is the case of LMT (9) and SLT (3). In addition, bigger differences in each of the three main groups can be seen with this procedure.

Among diploid parthenogenetic populations, CAL (13), GER (20), JAN(l) and LMT (9) seem clearly different from the remaining populations of this type (Figure 4). For CAL (13), the observations of Barigazzi and Baratelli (1982) about the presence of triploid nauplii among the more frequent diploid could account for some morophological differences. However, these fingings do not agree with Amat (1979, 1980), who could not differentiate both forms through direct observation of individuals; with Abreu-Grobois and Beardmore (1982), who do not find triploid mitosis in the preparations of nauplii from this population; and with Varo (1988), who did not get triploid mitosis in her preparations, nor can separate CAL (13) from JAN (1) through a discriminant analysis using similar variables to ours.

The population of GER (20) has not been studied very extensively, but it holds two characteristics which can be of interest in this context. It is located at a higher latitude, which has forced this population to develop in different climate conditions and to adapt to

colder temperatures. This, as stated above, has a very strong influence on the morphology of Artemia, The other characteristic is that the salterns that this population inhabits are considerably far from the sea. This possibly means a different ionic composition of the brine. Such an environmental characteristic is known to cause Artemia populations living under it to have remarkable differences in their biology. This is the case of some inland athalassic lakes in North America that hold some interesting Artemia populations: Mono Lake, California; Great Salt Lake, Utah; Chaplin Lake, Canada; etc. (Lenz, 1980; 1984).

In the case of JAN (1) and LMT (9), an explanation is more difficult, since the characteristics of the ecosystems where they live are very similar to those of the other populations studied which display a very close morphological pattern among them.

Bisexual populations show a more homogenous distribution, without remarkable differences among them, this is probably due to the closeness of some of them, making easier an exchange of genetic information through fertile interbreeding. This situation is not possible among parthenogens even in closely located populations.

Tetraploid parthenogenetic populations display greater variability than populations in the two former groups. Nevertheless, there is a group of four populations (DTE -15), ALT -2-, SAE - 17- and TZO -16-) that are closely related, and MED (18) and GND (21) farther from this nucleus, but still rather morphologically similar to them. There are two more different groups : BUJ (19) and MSV (23); and COM (22), LAT (6) and SLT (3). In this case, the possible explanations are not easy to draw, since they share none of the above mentioned characteristics (geographical closeness, environmental or physical traits of their ecosystem, etc.). The only reasonable facts are the similarity between SLT (3) and LAT (6), which are populations located in very close places, on both sides of the Gibraltar Straits; and its separation from more northern populations such as BUJ (19) and MSV (23).

Studying the standardized coefficients of all discriminant functions from these two analyses (data not presented here) and from others performed on different populations as well, and from the results of the stepwise analyses, it is possible to determine which variables have more influence on discriminating different populations. Generally, these variables are the ratio abdominal length/total lengh, which agrees with Gilchrist (1960) and Amat (1979, 1980), but not with Varo (1988); the form of the head (distance between compound eyes, eye diameter and head width), and the length of the first antenna, which are very variable among populations as stated by the above mentioned authors.

The main difficulty in using this technique of characterization is the need for raising an adult population under standardized conditions. It is, thus, not a rapid methodology. In spite of this, it is very simple, since only a few measurements are required instead of more sophisticated, complex or expensive methods.

The discriminant analysis carried out in this study has an important predictive value, in addition to the clustering purposes used here. Thus, the discriminant functions calculated allow one to classify new data sets into the different groups utilized in this study. In addition to this, since standardized culture techniques have to be used, the phenotypic variation is likely to reflect genetic variability; consequently, morphological data could be used through discriminant analysis to study relatedness among Artemia populations. This extent is currently being developed in our laboratory through the morphological study of 37 Spanish populations. These morphological databases as well as other data concerning American populations, by means of the discriminant analysis,

offer a powerful tool in order to accomplish a better understanding of the taxonomy of the genus Artemia; particularly, when these results and genetic studies can be contrasted together.

ACKNOWLEDGEMENTS The authors would like to acknowlege Dr John H. Crowe and Dr Petra H.Lenz for their helpful reviewing of the manuscript and M.Nieves Sanz for her assistance in the measurements. This work has been supported by the grants AC83/0002 funded by the CAICYT and MAR88/0224 by the CICYT. F. Hontoria has been granted by the Exma. Diputacion Provincial de Castellon.

REFERENCES ABREU-GROBOIS, F.A. and BEARDMORE, J.A. (1982) Genetic differentiation and speciation of the brine shrimp Artemia. In Barigozzi, C. (ed.). Mechanisms of Speciation. Alan Liss Inc., New York, pp. 345-376.

AMAT, F. (1979) Diferenciacion y distribucion de las poblaciones de Artemia (Crustaceo Branquiopodo) de Espana. Ph.D. thesis. Universidad de Barcelona, Spain.

AMAT, F. (1980) Diferenciacion y distribucion de las poblaciones de Artemia (Crustaceo, Branquiopodo) de Espana. I. Analisis morfologico; Estudio alométricos referidos al crecimiento y a la forma. Inv. Pesq., 44,217-240.

ANDERSON, T.W. (1984) An Introduction to Multivariate Statistical Analysis, 2nd edn.J.Wiley & Sons, New York.

BARIGOZZI, C. (1972) Problems of speciation in the genus Artemia. In Battaglia, B. (ed.). Proc. Fifth European Marine Biology Symposium. Piccini Editore, Padua, Italy, pp.61-66.

Barigozzi, C. (1974) Artemia : a survey of its significance in genetic problems. Evol. Biol, 7, 221-252.

BARIGOZZI, C. and BARATELLI, L. (1982) New data on chromosome number of the genus Artemia.

ACADEMIA NAZIONALE DEI LINCEI. Rendiconti della classe di Scienze fisich, matematiche e naturali, 73, 139-143.

BOWEN, ST., DURKIN, J.P., STERLING, G. and CLARK, L.S. (1978) Artemia hemoglobins : genetic variation in parthenogenetic and zygogenetic populations. Biol. Bull., 155, 273-287.

FURTHER CHARACTERISATION OF TWO ARTEMIA POPULATIONS FROM NORTHERN GREECE: BIOMETRY, HATCHING CHARACTERISTICS, CALORIC CONTENT AND FATTY ACID PROFILES

Theodore ABATZOPOULOS,GherdA KARAMANLIDIS,

Philippe LEGER & Patrick SORGELOOS

ABSTRACT

Cysts of parthenogenetic Artemia strains collected from the Citros and M. Embolon saltworks in Northern Greece are evaluated for their potential use in aquaculture. The following characterizations were performed: cyst and naupliar biometrics, cyst hatching characteristics, fatty acid profile of the nauplii, caloric content of nauplii stored at 25° C and in a refrigerator (4 - 8° C). The above evaluation reveals that the two Artemia strains studied exhibit good qualities for use in aquaculture, espe-cially in culturing fresh-water species. The biometrical analysis of cysts, nauplii and adults shows a high degree of similarity with other parthenogenetic strains from various geographical sources, but especially with tetraploid Artemia from Spain. The Greek Artemia strains cannot be considered as 'sources' for aquacultural uses unless proper management of the saltworks is assured.

INTRODUCTION

Since the brine shrimp Artemia is of high economic importance, it is essential to analyse the 'commercial' characteristics of various populations with regard to their potential for application in aquaculture. In previous studies, two Northern Greek Artemia populations found in Citros, Piera and M. Embolon, Thessaloniki, have been characterized as to their mode of reproduction and ploidy. They were found to be parthenogenetic and tetraploid (Abatzopoulos et al, 1986). Data from studies on DNA reassociation Kinetics and on thirteen enzymatic systems of these two populations indicate that they belong to one species (Abatzopoulos et al., 1987).

In this paper we report on the biometrical characteristics of cysts, nauplii and adults, on the hatching characteristics of cysts, i.e. hatching rate, hatching percentage, hatching efficiency and hatching output, on the changes in dry weight and caloric content of instar-I nauplii maintained at room temperature and cold storage conditions, and on the fatty acid profiles of instar-I nauplii.

MATERIALS AND METHODS

The two populations of Northern Greek Artemia evaluated are from the saltworks of Citros, Pieria (C strain), and from the saltworks of M. Embolon, Thessaloniki (ME strain). Cysts were collected in 1983, processed according to Sorgeloos et al. (1978), and stored in sealed plastic bags at -25° C, for future use.

Biometry

Hydrated cysts, and instar-I nauplii were measured according to the technique of Vanhaecke & Sorgeloos (1980).

Adult brine shrimps were anesthetized in chlorofrom-saturated seawater and measured under a dissection microscope using a Leitz micrometer. The following measurements were performed : total length, abdominal length, maximal width of brood pouch, width of third abdomianal segment, width of head, length of first antenna, maximal diameter of complex eye, and distance between complex eyes (Amat, 1980). Measurements were

performed on animals - sample size = 50 animals-originating from wild cysts and cultured under standard laboratory conditions (yeast as food, temperature of 25 + 1° C, and seawater of 35%o salinity). The data were statistically treated by analysis of co-variance (Sokal & Rohlf, 1981).

Hatching characteristics

Hatching analyses were carried out according to Vanhaecke & Sorgeloos (1983). Hatching efficiency, hatching percentage, hatching rate and hatching output were analysed following rate and hatching output were analysed following Sorgeloos et al. (1978), Bruggeman et al. (1980). Vanhaecke & Sorgeloos (1982), and Vanhaecke & Sorgeloos (1983), respectively.

Caloric content

The hatching of the cysts and the separation and collection of the nauplii (instar-I to instar-III) were carried out according to the procedure described in Vanhaecke e al. (1983).

The samples of nauplii were dried in an incubator at 55°C for 48 hrs. For each naupliar stage studied, four to six replicate samples, of approximately 10 mg dry material were burned totally in pure oxygen and their caloric content was measured in a Phillipson microbomb calorimeter.

Fatty acid profiles

Fatty acids were determined on freshly hatched nauplii of both strains by capillary gas chromatography.

Prior to analysis, samples were homogenized in an ultrasonic homogenizer (Sonifier B12) and total lipids were extracted according to the method.of Bligh & Dyer (1959). Saponification and methyla-tion were performed as described by Shauer & Simpson (1978). Fatty acid methylesters were injected in a capillary column (25 m fused silica; inner diameter; 0.32 mm; liquide phase : SILAR 10C, film thickness : 0.3um) installed in a Carlo Erba Mega 2350 gas chromatograph.

Operating conditions were as follows: oncolumn injector, carrier gas : hydrogen, flow rate : 2 ml/ min, F.I.D. detection, oven temperature programme : 105° C to 150°C at 10°C/min and 150° C to 200° C at 5°C/min. Peak identification and quantification was done with a caliberated plotter-integrator (Hewlett-Packard 3390 A) and reference standards.

Results are expressed in area- percent composition of total fatty acids and mg fatty acid methyles-ters per gram dry weight.

RESULTS Biometry

We counted 223 000 + 1563 cysts g-1 and 222 900 + 2322 cysts g-1 in the C and the ME strains, respectively.

The results of the measurements for the calculation of the mean diameter and the mean volume of the cysts are presented in Table 1.

Table I. Mean diameter and cyst volume of greek strains (harvested from the wild of from standard cultures at 35%o salinity).

Mean diameter Strains

(µm) (sd)

Mean volume (106 µm3)

Number of cysts measured

Cytros (wild): 260.19 ±0.031 9.20 500

(from culture): 270.13 ±0.048 10.30 500

M. Embolon (wild): 264.73 ± 0.038 9.62 500

(from culture): 279.36 ± 0.077 11.48 500

The size frequency distribution of cysts (Fig.l) appears to be similar between the two strains, although a slight increase in cyst diameter is observed in cysts produced in the laboratory. Figure 2 shows the size distribution of cysts from teh C strain in two different havests ot the same year, which seems remarkably stable.

There are no significant differences between the naupliar length and the size frequency distribution of the nauplii from the C and ME strains (Table 2, Fig. 3). As shown in Fig. 4, ovoviviparous nauplii are significantly larger than those hatched from cysts and show a different size distribution.

Table 3 summarizes the mean values for the various variables measured on adult brine shrimps, while Fig. 5 (a -g) prsents the correlation between these variables and total individual length within the same population as well as between populations. Analysis of co-variance showed that the regression lines in each case can be replaced by a single regression line and therefore, the values from the two populations for any variable can be considered as values derived from one strain only.

Hatching quality

Artemia cysts collected in the C and the ME saltworks and analysed following standard pocedures appear to have a good hatching quality. The C strain presents slightly better hatching characteristics than the ME (Table 4). Hatching for ME and C cysts ranges between 84-88% (narrow range) and most nauplii occur within 25 hrs of incubation. Figure 6 presents the hatching curves of C and ME cysts.

Fig. 1 Size frequency distribution of cysts from the tow Northern Greek strains :

: ME (wild population), : ME (from culture), : C (Wild population), : C from culture

Fig. 2 Size frequency ditribution of cysts from Citros saltworks during June ( ) and September ( )

Fig. 3 Size frequency ditribution of nauplii (instar-I) hatched from cysts : : ME strain, : C strain

Fig. 4. Size distribution of oviparous and ovoviviparous nauplii from the Greek strains. Oviparous; ME; O, C; ; Ovoviviparous: ME: , C:

Fig. 5. parameters (B-H) versus total length (A) in the C strain (A) and the ME (A) Artemia cultured under standard conditions (see also Table 3)

Table 3. Mean values for various parameters of adult Artemia shrimps cultured under standard laboratory conditions.

Citros strain (mm) M. Embolon strain (mm)

a 12.428 (+0.0829) 12.192 (+0.09)

b 6.550 (+0.0468) 6.435 (+0.049)

c 1.862 (+0.0218) 1.803 (+0.023)

d 0.573 (+0.0056) 0.563 (+0.056)

e 0.828 (+0.0102) 0.809 (+0.011)

f 1.147(0.015) 1.121 (+0.011)

g 0.304 (+0.0045) 0.293 (+0.002)

h 1.628 (+0.0164) 1.626 (+0.019)

a : Total length, b : abdominal length, c: maximal width of brood pouch, d: width of 3rd abdominal segment, e: width of head, f: length of 1st antenna, g: maximal diameter of complex eye, and, h: dis-tance between complex eyes.

Table 4. Hatching characteristics of cysts from Citros and M. Embolon strains (Greece).

Citros M. Emblon

Hatching Percentage

hatching medium 35 ppt 90.5 86

Hatching efficiency 198000 188000

(nauplii/g cysts)

Hatching rate characteristicsa

T0 16 17

T10 17.5 19

T90 25 28

TS 7.5 9

Hatching output 601 590

(mg/dry biomass/g cysts) a Values refer to time-lapses (in hours) from incubation until the appearence of the first nauplii (T0) or the moment by which 10% (T10) and 90% (T90) of the hatching efficiency has been reached TS

=T90-T10;TS is a measure for hatching synchrony.

Individual dry weight and caloric content

The naupliar (instar-I) dry weight, the ash content and the individual energy content are remarkably similr in the two strains as can be seen from the data which are summarized in Table 5. Figure 7 shows the energy consumption in early larval stages in correlation with time under normal culture temperatue (25° C + 1° C), and under cold storage of live nauplii in a refrigerator at 6° + 2° C.

Fatty acid profiles

The analyses of lipids extracted from freshly hatched nauplii (Table 6) - following the procedure of gas chromatography - show that the fatty acid profiles of C and ME material are very similar to each other. Eicosapentaenoic acid (20 : 5 3) is present and can be considered as barely sufficient for cul-turing marine species (Léger et al., 1986) while the other essential fatty acid 22 : 6 3 is found in detectable amounts only in the C strain. Also, the C strain contains high levels of linoleic acid 18 : 2 6.

Fig. 6. Hatching curves for the cysts from Citros (1) and M. Embolon (2).

Table 5. Individual dry weight, ash content and energy content of instar-I nauplii from two N. Greek population.

Strain Individua dry weight) (in mg)

Ash content (in % of dry weight)

Energy content Kcal/g

joules/g Individual energy content (in joules)

Citros

Hatching medium 35 ppt 3.04 (± 0.29) 4.44 5.5299 (± 0.05) 23050 (± 200) 0.070072

Hatching medium 5 ppt 5.9517 (± 0.12) 24810 (± 500) -

M. Embolon

Hatching medium 35 ppt 3.14 (± 0.32) 4.56 5.3447 (± 0.11) 22320 (± 450) 0.070084

Hatching medium 5 ppt - - 5.5226 (± 0.06) 23040 (± 250) -

Table 6. Composition of fatty acid methyl esters (F.A.M.E.) of artemia nauplii hatched from cysts collected in the saltworks of Citros (C) and M. Embolon (ME) Greek, 1983).

Area % mg/g DW .F.A.M.E. C ME C ME

14:0 2.0 1.3 3.1 1.5 14:1 2.6 1.6 4.1 1.8 ?* 0.9 . 1.4 - 15:0 0.5 0.7 0.8 0.8 15:1 0.7 1.1 1.1 1.2 16:0 11.7 12.6 18.4 13.8 16:1ω7 11.9- 17.0 18.7 18.8 17:0 1.0 1.3 1.6 1.5 16:3ω4 3.2 3.3 5.0 3.6 18:0 4.4 4.4 6.9 4.8 18:1ω9 25.4 14.9 40.0 16.4 18:lω7 — 20.5 — 22.6 18:2ω6 11.1 6.6 17.4 7.2 20:1 0.8 1.0 1.3 1.1 18:3ω3 13.2 4.8 20.7 5.2 18:3ω6 0.2 _ 0.3 — 18:4ω3 1.2 0.8 1.9 0.8 20:3ω3 0.2 — 0.3 — 20:4ω3 1.0 2.3 1.6 2.6 22:1 0.4 1.0 0.6 1.1 20:5ω3 3.4 2.7 5.3 3.0 20:3ω3 0.9 2.1 1.4 2.3 20:5ω3 0.2 _ 0.3 — 20:6ω3 0.5 — 0.9 —

* Not identifiable.

Fig. 7. Energy content of c C (●) and ME (O) nauplii stored at two temperatures (1) 25°C (+- 1°C) (2) 6°C (+- 2°C)

DISCUSSION

The two Greek Artemia strains examined, exhibit no differences in cyst diameters and resemble those of Spanish parthenogenetic populations (Amat, 1980; Vanhaecke & Sorgeloos, 1980; léger et al, 1986); they are significantly larger than bisexual Artemia-a typical phenomenon for parthenogenetic tetraploid strains but they must be considered quite small among the parthenogenetic and tetra-ploid strains (Vanhaecke & Sorgeloos, 1980). The increase in cyst diameter which was observed in cysts produced in standard laboratory cultures (Table 1) might be due to optimal culture conditions-such as food abundance - and lower metabolic costs.

The size frequency distributions of cysts from the Greek strains are very similar for the different batches (Figs 1 and 2) and again they resemble strains from Spain (Amat, 1980).

The results of the hatching characteristics from Greek Artemia cysts and their comparison to other strains reveal that these Greek Artemia are of high quality (Sorgeloos et al., 1986). The small differences in hatching rate and hatching efficiency (Table 4) between the ME and C strain may be due to the exposure of the cysts to suboptimal conditions - before harvesting - which can result in mortality of some embryos (Sorgeloos et al., 1976, Vanhaecke & Sorgeloos, 1983; Van Ballaer et al., 1987). The hatching percentage can be slightly improved by incubation of cysts in a 5 ppt hatching medium, where the energy consumption during hatching data for the Greek Artemia Strains are close to those obtained from French Artemia (Lavalduc) (Vanhaecke & Sorgeloos, 1983). It is interesting to note that the hatching synchrony-expressed by Ts (Table 4) - for the ME and C cysts ranges between 7-9 hrs and therefore it is easy to harvest only instar-I nauplii which bear the highest energy content (Table 5) (Benijts et al., 1976).

The hatching outputs for our strains are among the highest mentioned (Vanhaecke & Sorgeloos, 1983). Of course, the high hatching outputs do not necessarily assure success in using these products as larval food sources because other criteria should be taken into account as well, i.e. the size of newly hatched nauplii (Beck et al., 1980; Vanhaecke & Sorgeloos 1980), the energy content and the fatty acid profile (Schauer et al., 1980). Should be taken into account, as well, i.e. the size of newly hatched nauplii

(Beck et al., 1980; Vanhaecke & Sorgeloos 1980), the energy content and the fatty acid profile (Schauer et al., 1980).

The naupliar size of ME and C Artemia approximates the size range of Spanish parthenogenetic tetraploid strains (Amat, 1980) and can be considered relatively large - typical for parthenogenetic strains (Vanhaecke, 1983) - (Vanhaecke & Sorgeloos, 1980; Léger et al., 1986). Among parthenogenetic tetraploid Artemia the naupliar size of Greek Artemia is certainly one of the smallest, while the naupliar dry weight is quite high and similar to the Artemia strains from Lavalduc (France) and Tiensin (China) (Sorgeloos et al., 1983; Léger et al.,1986). The above combination can be beneficial as long as the size of the nauplius does not interfere with the ingestion mechanism of the predator (Sorgeloos et al.,1983). Since the ME and C nauplii are larger than 480 um, they are good for use with predators where Artemia size is not critical, such as freshwater fishes; they are, however, of less value for marine fish and shrimp larvae where they can be used only in later stages (Léger et al., 1986, 1987).

The individual energy content and individual dry weight of newly hatched nauplii appear to be very similar for the ME and C strains. The nonsignificant differences in energy content, on an ash-free dry weight basis, might be due to slightly different metablolic rates during pre-larval development in the uterus, caused by varying environmental conditions.

The energy content can be increased by 3-8% when cysts are hatched in 5 ppt, which is to be expected since less energy is needed for the emergence of the nauplius in a lower external osmotic pressure (Clegg, 1964).

Variation in growth rates of unfed nauplii which results in energy and dry weight losses can be avoided by storing instar-I nauplii at low temperatures (Léger et al., 1983). Nauplii from the Greek strains hatched at 5 ppt and maintained at 25°C lose 17-20% of their initial caloric content after 40 hrs, while the same nauplii stored 6 + 2° C lose only 7% after nearly 80 hrs (Fig.7).

The comparison of our data on energy content with the results from other parthenogenetic strains of different geographical origin reveals great similarities (Vanhaecke et al., 1983; léger et al., 1986). It is interesting to mention that the drops in energy contents between instar-I and instar-II and III nauplii which have been reported for other Artemia strains (Vanhaecke et al., 1983) appear to be faster than those observed for our material.

In general terms, the fatty acid profiles for these Greek Artemia agree with a 'normal' composition (Léger et al., 1986). The 18 : 2ω6 (linoleic acid) content, however, is slightly higher in the C strain than in most other strains; its levels are similar to those that have been reported for Artemia from Colombia, the Bahamas and the Phillipines (Léger et al., 1986).

The levels of the essential fatty acids (E.F.A.) 18 : 3ω3 (linolenic acid) and 20 : 5ω3 may not be sufficient to secure good survival and growth of marine larval organisms (Léger et al., 1986), Our Artemia strains appear to be better food for fresh water organisms which demand high 18 : 3ω3 levels and low 20 : 5co3 content, and in that respect the Greek strains belong to the so-called 'fresh-water type' Artemia.

It is interesting to note that the C strain contains detectable levels of another essential fatty acid, 22 : 6ω3, which is rarely found in Artemia nauplii and is known to promote survival and growth especially in crab cultures (Levins & Sulkin, 1984; Léger et al, 1987).

The slight variation observed in the levels of E.F.A. and especially in 20 : 5ω3 between the two Greek Artemia strains can be due to small differences in food conditions in Artemia ponds; this has been demonstrated in culture experiments where it has been found that Artemia cysts reflect different 20 :5 0)3 Levels of the diet available for the parental population (Léger et al., 1987). Variability in E.F.A. content is particularly great in strains produced in solar saltworks which are characterized by completely different food composition in the various ponds. Thus, it can be deduced that the food conditions in the two Greek solar saltworks are very similar.

The analysis of all morphometric parameters examined in adult brine shrimps revealed that the two Greek populations exhibit considerable similarities with parthenogenetic tetraploid populations from the Iberian peninsula (Amat, 1980) and support the view, we expressed elsewhere, that they belong to the same strain (Abatzopoulos et al., 1986, 1987).

CONCLUSIONS This study of two N. Greek Artemia populations has shown that their 'commercial' characteristics are quite good. The size of cysts and nauplii are among the smallest known for parthenogenetic tetra-ploid populations, and their nutritional composition is acceptable for use, mainly with freshwater organisms, because their high energy content could result in better and faster growth rates, since less energy will be spent by the predators in hunting and food uptake.

As they stand, the data on the fatty acid profiles of the strains studies are acceptable for possible practical use. However, since the content in 20 : 5ω3 may fluctuate significantly within each strain both between harvests of cysts of the same and/or different years (Van Ballaer et al., 1987) we cannot be certain as to the usefulness of the N. Greek cysts for particular aquacultural demands, unless culture tests are performed with specific predators (Sorgeloos et al., 1983). Surely, the appropriate enrichment (Léger et al., 1986, 1987) will improve the quality of this material.

Finally, the biometrical data, the hatching criteria and the nutritional value of the Greek Artemia reveal their good quality for possible commercial use, under the condition, of course, that the solar saltworks' ecosystem is properly managed.

ACKNOWLEDGEMENTS We thank Professor C . D. Kastristsis for help with this manuscript, and for useful discussions throughout this study. Thanks are also due to Drs. M Lazaridou and G. Stamou for allowing us to use their facilities. This work was supported in part by grants from the Greek Ministry of Agriculture and the Greek Ministry of Industry, Research and Technology. PS is a senior scientist with the Belgian National Science Foundation (NFWO).

REFERENCES ABATZOPOULOS, TH. J., C. D. KASTRISTSIS & C.D. TRIANTAPHYLLIDIS, 1986. A study of Karyotypes and heterochromatic associations in Artemia, with special reference to two N. Greek populations. Genetica 71 : 3-10.

ABATZOPOULOS, TH. J., CD. TRIANTAPHYLLIDIS & CD. KASTRITSIS, 1987. Preliminary studies on some Artemia populations from northern Greece. In: Sorgeloos, P., Bengtson, D.A., Decleir, W. & Jaspers, E. (eds). Artemia research and its applications, 1: Universa Press, Wetteren (Belgium): 107-114.

AMAT, D.F., 1980. Differentiation in Artemia strains from Spain. In: Persoone, G., Sorgeloos, P., Roels, O.. & Jaspers, E. (eds).The brine shrimp Artemia, 1. Universa Press, Wetteren (Belgium): 19-39.

BECK, A. D., D.A. BENGTSON & W.H.HOWELL, 1980. International study on Artemia. V.Nutritional value of five geographical strains of Artemia: effects on survival and growth of larval Atlantic silverside Menidia menidia. In: Persoone, G., Sorgeloos, P., Roels, D. & Jaspers, E. (eds). The brine shrimp Artemia, 3. Universa Press, Wetteren (Belgium): 249-259.

BENIJTS, F.,E. VANVOORDEN & P. SORGELOOS, 1976. Changes in the biochemical composition of the early larval stages of the brine shrimp Artemia salina L. In: Persoone, G. & Jaspers, E. (eds). Proc. 10th Europ, Symp. on Marine Biology, 1. Universa Press, Wetteren (Belgium): 1-9. Bligh, E.G. & W. J. Dyer, 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-917.

BRUGGEMAN, E., P. SORGELOOS & P. VANHAECKE, 1980. Improvements in decapsulation technique of Artemia cysts. In: Persoone, G., Sorgeloos, P., Roels; O. & Jaspers, E. (eds). The brine shrimp Artemia, 3. Universa Press, Wetteren (Belgium): 261-269.

CLEGG, J.S., 1964. The control of emergence and metabolism by external osmotic pressure and the role of free glycerol in developing cysts of Artemia salina. J. Exp. Biol. 41: 879-892.

LÉGER, PH., D.A. BENGTSON, K.L. SIMPSON & P. SORGELOOS, 1986. The use and nutritional value of Artemia as a food source. Oceanogr. Mar. Biol. Ann. Rev. 24: 521-623.

LÉGER, PH., E. NAESSENS-FOUCQUAERT & P. SORGELOOS, 1987. International study on Artemia, xxxv. Techniques to manipulate the fatty acid profile in Artemia nauplii, and the effect on its nutritional effectiveness for the marine crustacean Mysidopsis bahia (M.). In: Sorgeloos, P., Bengtson, D.A., Decleir, W. & Jaspers, E. (eds). Artemia research and its applications, 3. Universa Press, Wetteren (Belgium): 409-424.

LÉGER, PH., P. VANHAECKE & P. SORGELOOS, 1983. International Study on Artemia. XXIV. Cold storage of live Artemia nauplii from various geographical sources: potentials and limits in aquaculture. Aquacult. Engineering 2: 69-78.

LIPID COMPOSITION OF CYSTS OF THE BRINE SHRIMP ARTEMIA SP. FROM SPANISH POPULATIONS

J.C.NAVRRO, f. AMAT AND J.R. SARGENT

ABSTRACT

The lipid class composition of Artemia cysts from several Spanish populations was analysed by high-performance thin-layer chromatography (HP-TLC). A total of 11 lipid classes was detected, of which six were phospholipids and five neutral lipids. The chromatograms were quantified by single-wave-lengh scanning densitometry. In general, triacylglycerides were the major component, comprising = 50% of the total lipids, cholesterol accounted for 10% and cholesterol esters fluctuated from 3 to 10%. Phospholipids, mainly phosphatidylcholine and phosphatidylethanolaminc in a ratio 2 : 1, accounted for 10-20% of the total lipids. Pigements and free fatty acids were found to be very variable.

INTRODUCTION

Fatty acid compositions of Artemia have been frequently studied. Artemia nauplii can be deficient in certain essential fatty acids necessary for fish and crustacean marine larvae (Watanabe et al., 1978, 1983). Determination of fatty acid composition is important to assess the nutritional quality of a source of Artemia. However, specific lipid classes can also be important in fish and crustacean nutrition (Kanazawa et al.,1971; D'abramo et al., 1981), but information on the lipid class composition of Artemia is relatively scarce. Some analysis have been carried out in adults (Enzler et al., 1974; Gallagher & Brown. 1975; Sasaki & Capuzzo, 1984), Whereas other analysis have been reported for nauplii, either as information complementary to the main aim of the work (Seikai et al., 1987; Fox, 1990; Reza Ahmadi et al., 1990; Webster & Lowell, 1990), or as part of a broaader biochemical characterisation of the lipovitellin of a single strain (De Chaffoy & Kondo, 1980; De Chaffoy et al., 1980). Rudneva & Shchepkina (1990) have recently reported some values for the polar lipids, tri-acylglycerides and cholesterol of Artemia cysts.

The aim of this work was to define and quantify the main classes of Artemia cysts and to analyse their variability using from several Artemia populations of the Iberian peninsula.


Cysts from the following coastal and inland populations of Artemia were analysed: Coastal:

(1) La Trinidad: parthenogenetic tetraploid strain of the La Trinided salines, Ebro river delta, Tarragona. (2) La Mata: parthenogenetic diploid strain of the Laguna de la Mata, Torrevicja, Alicante. (3) Bonmati: mixture of parthenogenetic diploid and bisexual strains from the Salina de Bonmati, Alicante. (4) Los Hermanos: bisexual diploid strain from the Los Hermanos saline, San Fernando, Cadiz. Inland: (5) Olmeda: parthenogenetic tetraploid strain from Siguenza, Guadalajara. (6) Imon : parthenogenetic totraploid strain Siguenza, Guadalajara. (7) Medinaceli: parthenogenetic tetraploid strain from Medinaceli, Soria. (8) Anana : parthenogenetic tetraploid strain from Anana, Alava. (9) Rolda : parthenogenetic tetraploid strain from Naval, Huesca.

(10) Petrola : parthenogenetic tetraploid strain inhabiting sulphate waters of the Petrola lagoon, Albacete. (11) Gerri: parthenogenetic diploid strain from Gerri, Lérida. The locations of these popualtions are shown in Fig. 1.

The cyst samples used in this study were personally harvested. Once harvested from the shore of the salines, cysts were kept in saturated brine transported to the laboratory where they were cleaned by differential flotation in freshwater and saturated brine as described elsewhere (Sorgeloos et al., 1977; Amat, 1985; Navarro, 1985). The cysts were then dried and stored under nitrogen in tightly sealed containers prior to analyses.

Prior to lipid extraction, cyst samples were hydrated in seawater (salinity 34 g/1-1) until the cysts were observed to be spherical under a dissecting microscope. They were then decapsulated according to the procedures described in Bruggeman et al. (1979). After a thorough wash in distilled water, the cysts were blotted over soft paper tissue and divided into six aliquots, three of which were dried for 24 h at 110°C to calculate the moisture content, the remaining three being used for lipid extraction. Preliminary studies showed no differences between the lipid classes of decapsulated and whole cysts. Decapsulated cysts were routinely analysed here because their lipids are easier to extract. Lipids were extracted using the method of Folch et al. (1957), quantified gravimetrically using an analytical balance (Mettler H64) and stored at -20°C at a concentration of 10 mg. mg.-1 in chloroform : methanol (2:1, v:v) containing 0.01% (w : v) of butylated hydroxytoluene (BHT) as antioxi-dant. Nitrogen was flushed through the vial before being scaled. Extractions were carried out in triplicate.

HPTLC (10 X 10 cm) plates of silica gel 60 (E. Merck, Darmstadt, FRG) were pre-run as described in Olsen & Henderson (1989) and activited for 30 min at 110°C. Five separate 1. 5- ul lipid samples were applied to each plate with a micro-syringe. A sixth sample containing only BHT and the solvent was also applied as a sovent blank.

Plates were run at room temperature following the double development procedures described in Olsen & Henderson (1989). The first solvent system developed the polar lipids to a distance of 5 cm from the origin. The neutral lipids were developed in the second solvent system up to 0.5 cm of the top of the plate. The polar system was a mixtue of methyl acetate: isopropanol: chloroform : metha-nol: 0.25% KCI (12.5 : 12.5 : 4.5 by vol.); the neutral system was : hexane : diethyl either: acetic acid (42.5 : 7.5 : 0.75 by vol.).

The individual lipid classes were detected after charring (110°C for 20 min) plates sprayed with 3% cupric acetate in 8% phosphoric acid (Fewster et al., 1969). Quantification of the resulting chroma-tograms was performed on a Shimadzu dual wavelength TLC scanner CS-9000 linked to a Shimadzu data recorder DR-13. The background created the solvent fronts was also quantified from the blank run and subtracted from those zones coinciding with the solvents fronts. Individual lipid classes were identified by comparison with known standards.

The means of the lipid classes of the coastal and inland cysts were compared with a Student test (Sokal & Rohlf, 1981), after aresine-transforming the data (Zar, 1984).

Fig. 1. Location of sampling areas : 1, La Trinidad; 2, LaMata; 3, Bonmati; 4, Los Hermanos; 5, Olmeda; 6, Imon; 7, Medinaccli; 8, afiana; 9, Rolda; 10, Petrola; 11, Gerrl

RESULTS A total of 11 lipid classes was consistently detected, consisting of six phospholipids: sphingomyclin (SM), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid/ cardiolipid (PA/CL) and phosphatidylethanolamine (PE): and five neutral lipids: pigments, cholesterol (CHOL), free fatty acids (FFA). triacylglycerides (TAG) and cholesterol-esters (CE). Tables I and II summarize the results. The analytical method empolyed did not separate PA and CL. In some samples trace amounts (<0.1%) of lyso-phosphatidylcholine were noticed (Los Hamanos) and in other samples (Imon, Rolda, Petrola), SM could not be quantified because it nigrated very close to PC.

Table 1 : Lipid classes, polar (PL), natural (NL) and total lipids (TL)of cysts from coastal Artemia population. Data for individual lipids are expressed as % of total lipids. Data for total lipids are expressed as % of dry weight off cysts. All data are x ± SD values. Other abbreviations are defined in text.

Populations Bonmati La Mata La Trinidad Los HermanosSM 0.12±0.02 0.31±0.06 0.22±0.03 0.37±0.03PC 6.61±0.41 8.01±0.16 12.38±0.15 5.54±0.34PS 0.18±0.03 0.24±0.04 0.56±0.04 0.35±0.03PI 1.37±0.06 1.48±0.09 2.57±0.11 1.16±0.09PA/CL 0.61 ±0.03 0.72±0.02 1.50±0.08 0.50±0.01PE 4.04±0.16 2.26±0.15 6.32±0.28 3.14±0.11Pigments 2.30±0.22 10.63 ± 0.08 1.66±0.30 3.82±0.06CHOL 10.21±0.08 9.90±0.35 12.54±0.93 12.30±0.24FFA 7.05±0.14 4.98±1.11 8.63±0.77 16.92±0.53TAG 60.61±0.87 56.34±1.76 50.83 ± 1.02 48.92±0.49CE 6.91±0.51 4.73±0.52 2.87±0.53 6.98±0.31PL 12.92±0.60 13.41±0.31 23.48±0.64 11.06 ±0.61NL 87.08±0.060 86.59±0.31 76.52 ±0.64 88.94±0.61TL 19.42±0.14 17.80±0.52 11.48±0.68 15.57±0.82

The sterol ester zone of Artemia, named here as cholesterol esters (CE), has been reported as cholesterol esters by other authors (Seikai et al., 1987; Liou & Simpson 1989; Rudneva & Shchepkina, 1990). However, the technique used here did not allow us to identify the esterified sterol present. The same applies for the sterols, identified as cholesterol by Teshima & Kanazawa (1971) and named here as this compound (CHOL). It should be noted that hydrocarbons, reported to be present in Artemia in trace amounts by Seikai et al. (1987), co-migrate with CE in the solvent systems used here.

The neutral lipids accounted for 75-89% of the total lipids and exhibited a substantial variation from 9.5% in Rolda to 19.4% in Bonmati.

Fig. 2 is a graphic representation of the mean values obtained after pooling the data of the different lipid classes. The least abundant lipid classes were SM, PS and PA whereas TAG was the most abundant accounting for = 50% of the total lipids. Most lipid classes, particularly FFA and pigments, were found to be highly variable among samples. CHOL on the other hand, was very constant at = 10% of total lipids. Despite the extensive variations, some patterns can be identified, particularly among the phospholipids. Thus, SM accounted for < 0,5% of total lipids, the ratio PC: PE was consistently = 2 : 1, PI comprised 7-10% of total phospholipids, PA together with CL accounted for 5-6% of total phospholipids and PS constituted 2-3% of total phospholipids.

With the exception of CHOL, the neutral lipids showed a less fixed pattern CE was found to be more abundant (P<0.0021) among the cysts of the inland (x = 11.1%, SD = 2.2%) than the coastal populations (x = 5.4%, SD = 2 %). The remaining lipid classes did not show significant (P<0.05) differences.

Table II: Lipid classes, polar (PL), neutral (NL) and total lipids (TL) of cysts from inland Artemia populations. Data for individual lipids are expressed as % of total lipids. Data for total lipids are expressed as % of dry weight of cysts. All data are x ± SD values. ± Not detected. Other abbreviations are defined in text.

Populations

Olmeda Medinaceli Gerri Ariana Imon Rolda Petrola

SM 0.30 ± 0.05 0.36 ± 0.07 0.34± 0.04 0.28±0.11 * * *

PC 8.55 ± 0.29 9.68 ± 0.05 8.48 ± 0.63 13.02±t 0.70 13.78±0.46 12.44 ± 0.55 12.94 ± 0.30

PS 0.22 ± 0.01 0.35 ± 0.06 0.37 ± 0.08 0.71 ± 0.04 0.69±0.18 0.72±0.15 0.46 ± 0.05

PI 1.45 ± 0.19 1.40±0.09 1.07±0.00 2.21 ± 0.05 2.00 ± 0.19 2.09± 0.11 1.63 ± 0.19

PA/CL 1.03 ± 0.01 0.97 ±t 0.03 0.58 ± 0.03 1.36±0.16 1.39 ± 0.31 1.44±0.07 1.04±0.07

PE 4.49 ± 0.20 4.68 ±0.06 3.15 ± 0.18 5.58 ± 0.40 6.75 ±0.25 5.68 ± 0.27 5.85 ± 0.06

Pigments 6.63 ± 0.38 1.03 ±0.03 1.57±0.19 0.68 ± 0.16 2.73±0.14 1.35 ± 0.06 0.43 ± 0.01

CHOL 11.34±0.09 8.81 ± 0.89 10.45 ± 0.86 12.48 ± 0.27 13.56± 1.05 10.88 ± 0.24 12.02 ± 0.44

FFA 20.61 ± 0.14 0.53 ± 0.05 2.25 ± 0.68 3.95 ± 0.67 3.15 ± 0.18 2.08 ± 0.25 3.14±0.17

TAG 37.49 ± 0.88 63.84 ± 0.76 59.54 ± 0.72 46.73 ± 1.09 43.45 ± 1.46 53.16 ± 0.72 50.24 ± 0.57

CE 8.01 ± 0.69 8.41 ± 0.10 12.72±0.15 13.48 ± 0.94 12.50 ± 0.22 10.17±0.14 12.26±0.14

PL 15.92 ± 0.84 17.45 ± 0.14 17.45 ± 0.14 13.79± 1.07 22.68 ± 1.75 24.61 ± 0.50 22.36 ± 1.02

NL 84.08 ± 0.84 82.55 ± 0.14 82.55 ± 0.14 86.21 ± 1.07 77.32 ± 1.75 75.39 ± 0.50 77.64 ± 1.02

TL 12.23 ± 0.56 12.72 ± 0.30 16.70 ± 0.42 13.49 ± 0.20 9.63 ± 0.30 9.52±0.18 11.07±0.42

Fig 2. Graphic representation of x values of polar lipids, neutral lipids, and different lipid classes detected in cysts of Artemia population. Error bars are SD values

DISCUSSION The total lipid contents of Artemia cysts reported in this study agree with data from other authors (Schauer et al. 1980; Leger et al. 1987) and the proportion of polar/neutral lipids, although subject to high variability, agrees with values reported by other authors for Artemia nauplii (Scikai et al, 1987; Webster & Lovell, 1990) and cysts (Rudneva & Shchepkina, 1990). However, values of polar lipids of = 50% of total lipids have been reported for some nauplii (Fox, 1990), mainly due to very high values of PE (24.3%). Higher values of all the phospholipid classes were reported by Sasaki & Capuzzo (1984) in cultured adults of a Brazilian strain, using the latroscan technique (TLC-flame ionization detector).

Rudneva & Shchepkina (1990) found values of CHOL in Artemia cysts ranging between 2.2 and 10.6% of total lipids, whereas the CE varied between 2.9 and 12.1%. The content of free CHOL and CE in Artemia nauplii has also been studied by Liou & Simpson (1989) who found much lower values than those reported in this work . Scikai et al. (1987) found even lower values of CHOL, but the total lipid content of their nauplii (5-6%) is very low compared with the values reported in the literature (Leger et al., 1987) raising doubts about the efficiency of their lipid extractions.

The differences in the CHOL content of Artemia lipids reported in the literature may be explained either by the use of different techniques for the detection and quantification of the compound, or by the analyses having been performed on different life history stages. The latter explanation is ques-tionalbe (Navarro et al., 1991). The former, however, does have validity in that, using the methodology here reported, some interference of the CHOL zone with zones of monoacylglycerides and dia-cylglycerides can be expected (Olsen & Henderson, 1989). However, the latter two lipid classes, if present, generally occur in very small amounts in marine zooplankton (Olsen & Henderson, 1989).

The difference found between the CE contents of the coastal and inland populations suggests a phe-notypic influence. The pigments of Artemia cysts, identified by Czeezuga (1971, 1980) mainly as a mixture of astaxanthin and B- carotene and by Soejima et al. (1987) as a mixture of echineone (0.5-5.5%) and canthaxanthin (94.5-99.5%), are Known to be strongly influenced by the diet This could account for the high variability in pigment levels found among the different populations analysed in the present work.

Some cyst samples in this study presented high levels of FFA (particularly, Olmeda and Los Hermanos). In general, FFA do not exist naturally in substantial amounts in biological samples because of their market affinity for many proteins resulting in an inhibitory action on many enzyme activities. Where FFA are reported as major constituents they are usually artefacts due to cell damage (Gurr & James, 1980), with polar lipid classes likely to be the dominant sources of FFA because they are the most suceptible to hydrolysis by lipases (Sasaki & Capuzzo, 1984). From the data reported in this study, it can be deduced that the FFA are more inversely correlated with the TAG (r =-0.5936; p<0.0542) than with the polar lipids (r = -0.4137; p<0.2059). A possible explanation for this could be that fatty acids are mobilized from the TAG as a source of energy for cellular development and that, in those samples where FFA are prominent, hydration of the cysts prior to the decapsulations has triggered TAG metabolism. The fact that lyso-phosphatidylcholine was detected in a population (Los Hermanos) with low levels of polar lipids and, in particular, PC points to limited phospholipid hydrolysis (Sasaki & Capuzzo, 1984).

In conclusion, the lipid classes of the cysts from the different Artemia populations showed substantial variability, but some patterns can be established. From the point of view of gross nutritional value of the cyst lipid, TAG is the major component at = 50% of the lipids. CHOL and CE account for 10 and 3-10%, respectively. Pigment levels can be very variable and are clearly subjected to phe-notypic influence. Phospholipids, mostly PC and PE in a ratio of 2 : 1, account for = 17% of total lipids.

ACKNOWLEDGEMENTS Antonio Rodriguez kindly provided the Los Hermanos sample. J.C. Navarro is a Post-Doctoral Fellow of the «Plan para el Perfeccionamiento de Doctores y Tecnologos» from the Spanish Education and Science Ministry. Special thanks are given to all colleagues who helped in the sampling of Artemia cysts and to R. J. Henderson, M.V. Bell, J.G. Bell and R. Wilson who kindly answered J.C. Navarro's everyday questions.

REFERENCES AMAT, F, 1985. Utilization de Artemia en acuicultura. Inf. Tecn. Inst. Inv. Pesq., Vol. 128-129, pp 1-59. Bruggeman, E.,M. Bacza-Mesa, E.Bossuyt & P. Sorgeloos, 1979. Improvements in the decapsulation of Artemia cysts. In, Cultivation of fish fry and its live food, edited by E.Styezynska-Jurewiez et al., Eur.

MARICULT. SOC. SPEC. PUBL., No. 4, pp. 309-315.

CZEEZUGA, B., 1971. Studies on the carotenoids in Artemia salina L. egg. Comp. Biochem. Physiol., Vol. 40B, pp. 47-52.

CZEEZUGA, B., 1980. Carotenoid content in Artemia salina L. eggs and vitality of the young specimens of this crustaccan. In, The brine shrimp Artemia. Physiology, biochemistry. Molecular biology, edited by G. Persoone et al., Universa Press, Wetteren, Belgium, 607 pp.

D'ABRAMO, L.R., C. E. BORDNER, D.E. CONKLIN & N. A. BAUM, 1981. Essentiality of dietary phosphatidylcholine for the survival of juvenile lobsters. J. Nutr., Vol. 11, pp. 425-431.

DE CHAFFOY, D.,J HEIR L. MOENS & M. KONDO, 1980. Artemia lipovitellin. In. The brine shrimp Artemia. Physiology, biochemistry, molecular biology, edited by G. Persoone et al., Universa Press, Wetteren, Belgium, pp. 379-394.

DE CHAFFOY, D. & M. KONDO, 1980. Lipovitellin from the crustacean, Artemia salina. J. Biol. Chem., Vol. 225. pp. 6727-6733.

ENZLER, L.,V SMITH, J.S. LIN & H.S. OLCOTT, 1974. The lipids of Mono lake, California, brine shrimp {Artemia salina). J. Agr. Food Chem., Vol. 22, pp. 330-331.

FEWSTER, M.E., B.J. BURNS & J.F. MEAD, 1969. Quantitative densitiometric thin layer chro-matography of lipids using copper acetate reagent. J. Chromatogr., Vol. 43, pp. 120-126.

FOLCH, J., N. LESS & G.H. SLOANE-STANLEY, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. Vol. 226. pp. 497-509.

FOX, C. 1991. Studies on polyunsaturated fatty acid nutrition in the larvae of a marine fish - the herring. Clupea harengus L. Ph. D. thesis. University of Stirling, 196 pp.

GALLAGHER, M. & W. D. BROWN, 1975. Composition of San Francisco Bay brine shrimp (Artemia salina). J. Agric. Food Chem., Vol. 23. pp. 630-632.

Gurr, M. I. & A. T.James, 1980. Lipid biochemistry : an introduction. Chapman and Hall, London, 247 pp.

INTERNATIONAL STUDY ON ARTEMIA IX. LIPID LEVEL, ENERGY CONTENT AND FATTY ACID COMPOSTION OF THE CYSTS AND NEWLY HATCHED NAUPLII FROM FIVE GEOGRAPHICAL STRAINS OF ARTEMIA

BY Paul S. SCHAUER, D. Michael JOHNS,Charles E OLNEY, and Kenneth L SIMPSON

USA

ABSTRACT.

Artemia cysts and newly hatched nauplii from Australia, Brazil, Italy and the United States (California and Utah) were analyzed for their total lipid level total fatty acid level and composition, and their energy content in an effort to evaluate their lipid nutritional value as diets of marine organisms. Results are compared to biological data from a nutrtional evaluation of these five Artemia strains on various marine organisms.

The total lipid, fatty acid methyl ester and energy levels of all strains appeared to be adequate to promote good growth and survival of the marine organisms. The fatty acid spectrum of the cysts and nauplii were nearly identical, indicating that the cyst shell contains little fatty acid-type lipids. However, significant differences were found in the fatty acid compostion between the various strains.

Artemia were classified into two groups based on their oo3 polyunsaturated fatty acid composition. The major difference between the two major difference between the two groups was that one group contained predominantly 18 ; 3oo3 and 18:4oo3, while the other group contained chiefly 20 : 5oo3. Considering the importance of 20 : 5 oo3 to marine organism nutrition, the Australia, Italy, Brazil, and San Francisco Bay 321 strains which contained the higher level of this fatty acid would provide the best nutrition of the five strains; The San Pablo Bay 1628, San Francisco Bay 313 and Utah strains would probably be of less nutritional value due to the low level of 20: 5oo3 and/or the excess amount of 18 : 3oo3. It is possible, however, that there is an interaction between an essential fatty acid (20 : 5oo3) deficiency and a dietary contaminant. This possibility is discussed with reference to biological results obtained when these five Artemia strains were fed to three different marine organisms.

INTRODUCTION

Artemia (brine shrimp) is a widely used food source in laboratory and commercial rearing of many marine organisms. Several new geographical Artemia strains have recently become available and a subsequent need has developed for their analysis as potential supplements or replacements for the present commercial sources.

An important determinant of the overall nutritional value of any food stuff is its lipid content. Triglyceride-type lipids are a major source of a diet's metabolizable energy and are directly linked to the growth of the consumer organism (Pandian, 1975). The dietary fatty acid composition ultimately determines the fatty acid composition of the structural phospholipids (castell et al 1972 ab; Norred and Wade, 1972). Phospholipids are functionally active in maintaining proper membrane fluidity and cellular transport mechanisms. Recent research has demonstrated that the ω3 polyunsa-turated fatty acids (PUFA) are required for lobsters (Castell and Covey, 1976), prawns (Guary et al., 1976; Kanazawa et al., 1979) and for several marine fish, including plaice (Owen et al., 1972), red sea bream (Yone and Fujii, 1975 a) and turbot (Cowey et al, 1976).

The purpose of this research was to determine the total lipid content, fatty acid composition and energy content of cysts and newly- hatched nauplii of Artemia from five geographical regions.


Sources and culturing of Artemia

Deshydrated cysts from five geogrphical locations were provided by the Artemia Reference Center (Ghent, Belgium). The origin of these cysts were Shark Bay, Australia (World Ocean, lot no; 113); Macau, Brazil (Companhia Industrial do Rio Grande do Norte, CIRNE-Brand, harvested 1978); Margherita di Savoia, Italy (harvested 1977);Great Salt Lake, Utah, USA (harvested 1977); San Pablo Bay, California, USA (Living World, San Francisco Bay Brand, Inc., lot no. 1628) and two samples from San Francisco Bay, California, USA (San Francisco Bay Brand, Inc., lot no. 313/3006 and lot no. 321995). The latter three Californiari samples will be referred to by their respective full or abbreviated lot numbers : SP 1628, SF 313 and SF 321.

Stage one Artemia nauplii were hatched from cysts incubated at 25°C in 30%o filtered (0.45 urn) seawater for a specific period of time that was dependent on the particular strain (Johns et al, 1980).

Samples were either held at-20° C until analysis or dried at 60°C to a constant weigh for determination of the energy content.

LIPID EXTRACTION AND ANALYSIS

Total lipids

Cysts were ground in mortar and pestle and extracted twice with chloroform/methanol/water (20 ml/40 ml/16 ml) (Bligh and Dyer, 1989). The remaining solids were repeatedly extracted in acetone until the supernatant locked pigmentation. The lipid in the acetone solution was transferred to petroleum either (PE) and along with the chloroform fraction from the Bligh and Dyer extraction was evaporated to dryness at 30° C. Total lipid weight was determined gravimetrically after which the lipids were dissolved in benzene and stored at -20°C until analysis. Total lipid weight of the nauplii was determined in a similar manner. Lipid weights are presented as mg lipid/g dry weight sample.

FATTY ACIDS

The fatty acid composition of each sample was determined by gas-liquid chromatography on two independent columns as described by Schauer and Simpson (1978). Results are presented here as fatty acid methyl esters (FAME) weight percent of total lipid. Quantification of total FAME weights were mad by co-injecting the FAME 20 : 2ω6 as an internal standard and are presented as mg FAME/ g lipid of the dry weight samples.

DETERMINATION OF ENERGY CONTENT Energy content of newly hatched nauplii was determined using wet oxidation in the presence of an acid-dichromate mixture (Maciolik, 1962). These values are reported as Joule/ gram ash-free dry weight.

DATA ANALYSIS

One-way analysis of variance was computed for total lipids, FAME and energy content. If significant differences (P<0.05) were found, a Student-Neuman-Kuels posterior

comparison was used to determine where the difference lay (Snedecor and Gochran, 1967).

RESULTS The Australian cysts' lipid level was statistically greater than the other four strains while the means of the Utah, SP 1628 and Brazilian strains were greater than that of the Italian cyst strain (Talbe I). The nauplii lipid levels, however, were all statistically different from one another except for the SF 321 and SP 1928 strains. The highest level of lipid in the nauplii was found in the Utah strain, and in descending order strains were ranked as follows; Brazil, Australia, SF 313, SP 1628, SF 321 and Italy.

Table I : The amount of total lipid and fatty acid methyl esters in the cysts and newly hatched nauplii of five strains of Artemia

Artemia sources

Australia Brazil SF 313 SF 321 SP 1628 Italy Utah

mg total lipids2

Cysts 157a±8 134b±8 .N.A-3 N.A. 134b±22 91.0c±6 136b± 1

Nauplii 185C±9 202b ± 8 174d±4 I59e±13 160e±3 156f±2 224a± 14

mg fatty acid methyl esters4

Cysts 510b±18 502b ± 12 N.A. N.A. 573b±39 704a ± 34 490b± 19

Nauplii 751ab± 41 854a±26 716a,b±30 602b± 15 711a±58 739a± 12 742a,b± 101 Values within each row which bear the same letter are note significantly different at P < 0.05. 2 Per gram dry weight of sample. 3 N.A. = not analyzed. 4 Per gram lipid. Although the total lipid level of the Italian strain was the lowest in both cysts and nauplii, it contained significantly greater levels of FAME (mg/g lipid) than the other statistically similar cyst strains. The FAME levels for the nauplii of the Brazil, Australia, Utah, Italy, SP 1628 and SF 313 strains were all statistically similar as were the Australian, Utah, SF 313 and SF 321 nauplii, but the Brazilian, Italian and SP 1628 strains were significantly greater than the SF 321 strain.

The fatty acid composition of cysts and nauplii is presented in Table II and Table III, respectively. The relative proportion of fatty acids in cysts and nauplii remain the same, indicating that the fatty acid content of the chorion is small.

Table II : Fatty acid compostion of whole cysts of five strains of Artenia

FAME Aust. Brazil SP 1628 Italy Utah

14 : 0 1.80 2.04 0.65 1.79 1.20

14 : 1 2.11 1.03 2.88 3.55 1.94

15 : 0 1.02 0.95 0.22 0.14 -----1

16 : 0 15.1116.35 9.80 14.15 12.39

16 : 1 10.66 12.88 6.49 13.05 6.00

16 2co7 0.58 1.68 1.67 2.04 1.68

16 : 3ω417 :lω8

3.98 3.93 2.69 3.47 1.47

18 : 0 2.66 2.34 2.38 3.31 3.55

18 : Iω9 26.71 33.50 27.43 26.05 28.03

18 : 2ω6 6.229.17 5.30, 7.08 5.58

18 : 3ω3 13.19 4.39 31.85 6.24 28.16

18 : 4ω3 4.41 0.97 5.15 1.55 3.52

20 : Iω9 0.27 0.49 0.46 0.31 0.21

20 : 2ω6/ω9 0.08 0.29 0.15 0.62 0.16

20 : 3ω6 0.77 2.30 0.04 1.35 0.27

20 : 5ω3 9.32 8.35 1.66 12.61 3.23

22 : 6ω3 0.26 0.11 trace ----1 trace 1 No value (—) means the fatty acid was not found

The major fatty acids in all Artemia strains tested were 16:0, 16:1, and 18:10)9. In addition, levels of 18 :3ω3 and/ or 20 :5ω3 were substantial in various strains. Docosahexaenoic acid (22 : 6ω3) was found in only small amounts in the Australian and Brazilian Artemia nauplii. The Artemia strains were divided into two groups based on their most predominant long chain ω3 PUFA. Strains that contained mostly 18 : 3ω3 and 18 : 4ω3 included the SF 313, SP 1628, and Utah strains, while the group that contained predominantly 20 : 5ω3 included SF 321, Italian and Brazilian strains. The Australian strain contained substantial amounts of both 18 : 3ω3 and 20 : 5ω3 and therefore could be included in either of the two groups.

TABLE III: Fatty acid composition of newly hatched Artemia

FAME Aust. Brazil SF 313 SF 321 SP 1628 Italy Utah

14 : 0 1.34 1.57 0.99 1.57 0.43 1.53 0.93

14 :1 2.23 0.81 1.27 0.74 2.26 3.30 1.45

15 : 0 1.34 0.67 0.16 0.58 0.25 0.11 0.11

15 :1 0.15 0.24 0.20 0.13 0.46 0.54 0.37

16: 0 13.45 5.42 10.33 12.13 7.79 15.23 11.78

16: 1 9.97 10.79 13.27 19.52 5.24 10.38 5.64

16:2 ω7 ---1 ---1 ---1 ---1 1 .51 2 94 ---1

16 :3ω4/17:ω8 3.87 3.88 2.09 2.32 2.44 3.28 2.90

18 :0 3.07 2.79 6.83 2.90 3.08 3.17 4.07

18 :Iω9 28.23 35.86 26.97 31.20 29.15 29.05 28.58

18 :2ω6 5.78 9.59 9.35 3.69 4.60 6.79 4.60

18:3ω3 14.77 4.87 17.33 5.16 33.59 6.35 31.46

20:4ω3 4.37 0.96 3.26 1.28 4.88 1.01 3.10

20:Iω9 0.37 0.52 0.41 0.35 0.35 0.42 0.37

20:2ω6/ω9 0.12 0.06 0.06 ---1 0.24 0.20 0.09

20: 3ω6 0.79 2.76 1.01 , 2.23 0.05 1.47 0.48

20:3ω3/20:4ω6 ---1 ---1 1.48 2.69 1.48 ---1 ---1

22:5ω3 10.50 8.98 4.06 12.44 1.68 13.63 3.55

22:6ω3 0.26 0.06 ---1 ---1 ---1 ---1 ---11 No value (-----) means fatty acid was note found.

Energy content for the geographical strains is presented in Table IV. Using the same statistical tests as in the lipid and FAME evaluations, differences in energy content between the various strains are significant (P<0.05). The Australian strain contained the most energy with 2.50 x 104 J/g ash-free dry weight (5.961 Kcal/g); the Italian strain contained the least with 2.24 x 104 J/g ash-free dry weight (5.370 Kcal/g).

TABLE IV : The energy content1,2 (X104) of five strains of Artemia nauplii

Australia Brazil SP 1628 Italy Utah

2.50a+0.16 2.35a,b+0.04 2.35a,b+0.11 2.24b+0.06 2.34a,b+0.8 1 J/g ash-free dry weight (ash content 5.4% dry weight).

2 Values which bear the same letter subscript are not significantly at P<0.05.

DISCUSSION

Major differences were found in the lipid level, FAME levels, fatty acid composition and the energy content of the cysts and nauplii ot the five geographically different strains. These differences could have been caused by variations in the genetic make-up or the previous dietary history of the parental stock which produced the cysts. Clark and Bowen (1976) have isolated six separate species from 27 different geographical strains of Artemia and Bowen et al. (1978) have shown a genetic variation in the hemolymph proteins of various strains. More recently, Abreu-Grobois and Beardmore (1980) found evidence for speciation between populations of Artemia and Seidel et al. (1980) showed a variation in the total protein electrophoretic patterns of the same five strains analyzed here.

The total FAME analysis provided an indication of the type of lipids associated with the various strains. A disproportionately high level of FAME for the Italian and a low value for the Utah strains indicated the two extremes. The Italian strain must have contained a higher level of triglyceride lipids per gram sample whereas the Utahan strain probably contains a greater proportion of phos-pholipids and/or sterol-type lipids.

The loss of the chorion, upon cyst hatching, resulted in a greater amount of lipid material per unit weight nauplii, except in the Italian strain. This would indicate that the chorion of the Italian strain contained more lipids than the shells of the other strains. The relative proportion of fatty acids of the cysts and nauplii remained the same before and after hatching and, when related to the increased lipid and FAME levels in the nauplii, indicate that the shell contains practically no fatty acids, with a possible exception of the Italian strain.

The nutrional quality of the five geographical strains used in this study has been recently determined for several species of marine larvae (Johns et al., 1978, 1980; Beck et al, 1980; klein-MacPhee et al., 1980). Johns et al. (1980), Working with the larval stages of the mud crab Rhithropanopeus harrisii and the rock crab Cancer irroratus found marked differences in the ability of the geographical strains of brine shrimp nauplii to promote high survival and growth. Crab larvae did not complete larval development when fed SP

1628 or the Utah strain but did so when fed the Brazilian, Australian or Italian strains. Klein-MacPhee et al. (1980) found similar results when winter flounder Pseudopleuronectes americanus larvae were reared using the five geographical strains as food sources. Survival through metamorphosis was high in fish larvae fed Australian, Brazilian or Italian strains while it was markedly lower for larvae fed SP 1628 or Utah brine shrimp.

Beck et al. (1980), also working with the larvae of a marine fish {Menidia mandia), found varying results. Survival and growth of the fish larvae were dependent on several factors including the previous dietary history of the fish larvae. Differences in survival between the fish fed the various geographical strains were less prominent when all larvae were fed the Brazilian strain rather than the SP 1628 strain prior to the start of the experiment. In an earlier study, Johns et al. (1978) had found that both SF 313 and SF 321 promoted good survival throughout larval development of the mud crab. Beck et al. (1980) also showed that these two strains promoted higher (but not significant) survival in Menidia than menidia the SP 1628 strain.

The relatively good survival and growth of the marine organisms fed the Italian strain indicates that the lipid levels and energy content are probably adequate. The total energy values of the five strains were in close agreement with those of Paffenhofer (1967) who found a level of 5 953 calories (2.49 x 104 J) per gram organic substance for an unidentified brine shrimp strain. Because the lipid levels and total energy values for the SP 1628 and Utah strains were greater, all strains were considered sufficient in energy and are probably not a significant contributor to the poorer growth and survival of the SP 1628 and Utah fed organisms.

The fatty acid composition of the nauplii appeared to be much more closely related to the biological effects of the five Artemia strains. The fatty acid composition of the strains evaluated here may have been different enough to affect their potential nutritional value. Strains that are higher in 20 : 5ω3 (Italy, SF 321, Brazil, and Australia) would presumably be better diets for marine organisms (Owen et al, 1972; Yone and Fujii, 1975b; Guary et al., 1976; Watanabe et al, 1978). Furthermore, the high level of 18 : 3ω3 (> 30% of the total FAME ) and the low level of 20 : 5ω3 in the Utah and SP 1628 nauplii may have induced a nutritional stress in marine consumer organisms.

Watanabe et al. (1978) stated «that the class of EFA (essential fatty acids) contained in Artemia (principally 20 : 5ω3 and 22 : 6ω3) is the principle factor in the food value of Artemia to fish. This was determined by modifying the fatty acid composition of a San Francisco Bay Brand Artemia (1976), deficient in 20 : 5ω3 and 22 : 6ω3, by feeding them either a marine chlorella or a yeast-supplemented squid liver oil, both of which are rich in these long chain 0)3 PUFA. The enriched Artemia induced better growth and survival of their test organism, the red sea bream Chrysophys major,

Artemia have generally been shown to contain either a fatty acid predominance of 18 : 3ω3 or 20 : 5ω3 (Enzler et al, 1974; Benijts et al, 1976; Gallagher and Brown, 1975; Claus et al, 1977, 1979; Watanable et al., 1978; Fujita et al., 1980). Similar results were obtained in this study. Also, a variation was found in the fatty acid composition of cysts collected within the same year at a similar location (Table III, SF 313 versus SF 321) (personal communication, A. Schmidt, 1978).

One other important factor which relates to the lipid character of a food organism is the interaction of lipids with contaminants. This is important because a number of cyst strains are collected from salinas in locations near commercial and agricultural regions (e.g. San Francisco Bay, San Pablo Bay, and Great Salt Lake). An analysis of the five

geographical strains for chlorinated hydrocarbons revealed that all are contaminated to some extent; the Italian strain contained more of the DDT family of pesticides, the Utah and SP 1628 contained higher levels of dieldrin and the SP 1628 contained substantially higher levels of dieldrin and much higher chlordane levels than the other strains (Onley et al., 1980).

The lipid content of a diet affect the accumulation of pesticides which can thereby alter lipid metabolism. For example, Phillips and Buhler (1979) found that rainbow trout fed dieldrin-contaminated tubificid worms (15% lipid) experienced a decrease in lipid accumulation while fish fed an artificial diet (10% lipid) containing dieldrin exhibited norma rates of lipid accumulation. These authors suggested that the increased dietary lipid level created a greater reservoir for pesticide accumulation which, in turn, may have altered lipid matabolism. Furthermore, Durham (1967) has shown that dieldrin affects the metabolism of unsa-turated fatty acids and accentuates the symptoms of a deficiency of essential fatty acids.

Considering the biological results of the mud crabs (Johns et al., 1980) and winter flounder (Klein-MacPhee et al., 1980) that were fed SP 1628 and Utahan strains of Artemia, it is possible that the excess 18 : 3ω3 and/or minimal quantities of 20 :5ω3 could result in a nutritional stress which, in the presence of a dietary contaminant, could be manifested in a synergistic fashion. Both a nutrional stress and/or a pesticide contaminant could potentially exert a physiological response which may be elevated to a critical point at the time of metamorphosis. This may explain why the results of the five strains as diet on survival of Atlantic silversides (Beck et al., 1980), which has a much less distinct metamorphosis, was not as dramatic as in mud crab and winter flounder. However, specific investigations will be required to evaluate these conditions.

ACKNOWLEDGEMENTS This publication is a result of research sponsored by NOAA Office of Sea Grant, Department of Commerce, under Grant no. NA 79AA-D-00096, and the University of Rhode Island Agricultural Experiment Station, contribution no. 1921

The authors thank Mr Allan Beck and the marine culture team at the EPA-Environmental Research Laboratory - Narragansett for supplying the newly hatched nauplii.

LITTERATURE CITED ABREU-GROBOIS F. A. and J. A. BEARDMORE.1980. International Study on Artemia, II. Genetic characterization of Artemia populations - an electrophoretic approach. P. 133-146. In : The brine shrimp Artemia. Vol. 1. Morphology, Genetics, Radiobiology, Toxicology. Persoone G.P. Sorgeloos, O. Roels, and E.Jaspers (Eds). Universa Press, Wetteren, Belgium. 345 p.

BECK A. D., D. A. BENGTSON, and W.H. HOWELL. 1980. International Study on Artemia. V. Nutrional value of five geogrphical strains of Artemia : effects on survival and growth of larval Atlantic silverside Mendia mendia. p. 249-259. In : The brine shrimp Artemia. Vol. 3.Ecology, Culturing, Use in Aquaculture. Persoone G.,P. Sorgeloos, O. Roels, and E. Jaspers (Eds). Universa Press, Wetteren, Belguim. 456 p.

BENIJTS F., E. VANVOORDEN, and P. SORGELOOS. 1976. Changes in the biochemical composition of the early larval stages of the brine shrimp, Artemia salina L.p. 1-9. In : Proc. 10th European Symp. Marine Biology, vol.1. Mariculture, Persoone G. and E. Jaspers (Eds). Universa Press, Wetteren, Belgium. 620 p.

BLIGH E. G. and W. J. DYER. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37 : 911-917.

BOWEN S. T,, J. P. DURKIN, G. STERLING, and L.S; CLARK. 1978. Artemia hemoglobins : genetic variation in parthenogenetic and zygogenetic populations. Biol. Bull. 155 : 273-287.

CASTELL. J.D. and J.F Covey. 1976. Dietary lipid requirements of adult lobsters (Homarus ameri-canus M.E.).J. Nutr. 106 : 1159-1165.

CASTELL J.D.J. LEE, and R.O.SINNHUBER. 1972a. Essential fatty acids in the diet of rainbow trout (Salmo gairdneri) : lipid metabolism and fatty acid composition. J. Nutr. 102 :93-99.

CASTELL J.D.,R.O. SINNHUBER, J.H. WALES, AND D.J. LEE. 1972b. Essential fatty acids in the diet of rainbow trout (Salmo gairdneri) : growth, feed conversion and some gross deficiency symptoms. J.Nutr. 102: 77-86.

CLARK L.S. and S.T. BOWEN. 1976. The genetics of Artemia salina. VII. Reproductive isolation. J. Hered. 67 : 385-388.

CLAUS C., F. BENIJTS, and P. SORGELOOS. 1977. Comparative study of different geographic strains of the brine shrimp Artemia salina. P. 91-105. In : Fundamental and applied research on the brine shrimp Artemia salina (L.) in Belgium. Persoone G. and E. Jaspers (Eds). European Mariculture Society Special Publication No.2. EMS. Bredene, Belgium. 110 p.

CLAUS C.F. BENIJTS, G. VANDEPUTTE, and W. GARDNER. 1979. The biochemical composition of the larvae of two strains of Artemia salina (L.) reared on two different algal foods. J. Exp. mar. Biol. Ecol; 36 : 171-183.

COWEY C.B., J. M. Owen, J.W. ADRON, and C. MIDDELETON. 1976. Studies on the nutrition of marine flatfish. The effect of different dietary acids on the growth and fatty acid composition of turbot (Scophthalmus maximus). Br. J. Nutr. 36 : 479-486.

DURHAM W. F. 1967. The interaction of pesticides with other factors. Residue Review 18 : 21-103.

ENZLER L.,V. SMITH. J.S .LINN, and H.S.OLCOTT. 1974. The lipids of Mono Lake brine shrimp (Artemia salina). J. Agr.Food Chem. 22 : 330-331.

FUJITA S.,T. WATANABE, and C. KITAJIMA. 1980. Nutrional quality of Artemia from different localities as a living feed for marine fish from the viewpoint of essential fatty acids. P. 277-290. In: The brine shrimp Artemia. Vol. 3.

INTERNATIONAL STUDY ON ARTEMIA*. XXVI. FOOD VALUE OF NAUPLII FROM REFERENCE ARTEMIA CYSTS AND FOUR GEOGRAPHICAL COLLECTIONS OF ARTEMIA FOR MUD CRAB LARVAE

C.R.SEIDEL, KM. JOHNS, P.S.SCHAUER and CE. OLNEY1

ABSTRACT. : Nauplii from 4 commercially available geographical collections of Artemia and nauplii hatched from the Reference Artemia Cysts were compared for their effects on survival and growth of Rhithropanopeus harrisii larvae. In addition, nauplii from these sources were analyzed for their fatty acid and chlorinated hydrocarbon contents. Despite differences in the amounts of a few important polyunsaturated fatty acids (18 : 3ω3, 20 : 5ω3), as well as in the chlorinated hydrocarbon content, there was little variation in the survival and development rates of R. harrisii fed these Artemia sources as food. However, growth of R. harrisit from hatching to megalopa was significantly higher on the strain from France, intermediate in the Reference, Brazil and Chinese strains, and poorest on the Chaplin Lake (Canada) strain. The Reference strain is shown to be one of the better sources of Artemia nauplii with regard to their use in crab culture and therefore represent a good standard for future research studies.

The brine shrimp Artemia is extensively used as food source in the culture of larval fish and crustaceans. Although relatively expensive, Artemia is convenient to use and supports better larval development and survival than other live artificial diets tested (Sulkin and Norman, 1976; Manzi and Maddox, 1980). There are, however, differences in Artemia composition that may alter the nutritional effectiveness of some commercially available sources of Artemia (Olney et al, 1980; Schauer et al, 1980).

The research reported here is a continuation of the effort initiated by the International Study on Artemia (Sorgeloos, 1980a) to characterize the biochemical composition and nutritional performance of commercially available sources of Artemia. Collections of Artemia tested were obtained from the following geographic areas: (1) Lavalduc, France, harvested 1979; (2) Tientsin, People's Republic of China, harvested 1979; (3) Chaplin Lake, Canada, harvested 1979; (4) Macau, Brazil, harvested 1978; (5) Reference Artemia Cysts (RAC), povided by the Artemia Reference Center, Ghent, Belgium. The RAC have been proposed as intercalibration material in studies using Artemia nauplii as food source (Simpson et al, 1980; Sorgeloos, 1980b).

Methods used for the detection of chlorinated hydrocarbons, as well as results of lipid analyses have been reported elsewhere (Olney et al., 1980; Schauer et al, 1980). For both of these analyses samples were run in triplicate.

Newly-hatched zoeae of the mud crab Rhithropanopeus harrisii were used as nutritional bioassay material. Methods for laboratory maintenance of gravid adults, procurement of newly-hatched zoeae and the experimental design used in this study have been described in detail by Johns et al. (1980).

The present study provides further information on variability in the biochemical compositions and food value between different geographical sources of Artemia. Differences were found in fatty acid composition, total lipid content and chlorinated hydrocarbon contamination levels. The most noteworthy differences between the 5 Artemia sources were the higher level of 18 : 3ω3 in the Canadian and French strains and the high level of 20 : 5ω3 in the Chinese strain (Table 1). All strains contained

substantial levels of 20 : 5ω3. (>8%). The total lipid levels of RAC, Brazilian and Chinese nauplii were higher (>200 mg g-1, dry wt sample) than the level found in the French and Canadian populations (<155 mg g-1, dry wt sample).

All values for chlorinated hydrocarbons were below 100 ppb, except for the total DDT's in the Chinese sample (172 ppb; Table 2). Lowest levels of CHC were found in RAC and Brazilian collections; French and Chinese Artemia were approximately 4 to 6 times more contaminated. Despite such chemical variation no significant differences were found in the ability of the various Artemia sources to support survival and developmental rate of R. harrisii larvae (Table 3).

Growth rates, however, were significantly higher in crab larvae fed nauplii from the French source; the poorest growth was found in crab larvae fed Canadian brine shrimp. Growth was fastest on the French strain which contained one of the lowest lipid levels, therefore it'appears that lipid levels were adequate in all Artemia strains. In addition, growth did not appear to be affected by the moderately high levels of contaminants found in the French strain. Although no significant differences were found in survival and developmental rates, the lowest mean values for survival and growth and the slowest development rate occurred with mud crab larvae fed Canadian brine shrimp nauplii. These trends suggest that the Canadian source of Artemia may be less effective in culturing larvae of R. harrisii.

The value of studies such as this would be of little interest if a single, reliable source of Artemia was commercially available. This has not been the case (Sorgeloos, 1980a). For example, Artemia from Macau, Brazil, which had been identified as one of the better food sources for a large variety of marine fish and crustacean larvae (Beck et al., 1980; Johns et al., 1980, 1981; Klein-MacPhee et al., 1980) are presently not commercially available (P. Sorgeloos, pers. comm.). The ISA series of studies, however, have highlighted major differences in the nutritional effectiveness of other commercially available sources of this indispensible source of food for marine and freshwater organisms (Sorgeloos et al., 1980a).

The use of Artemia of inferior quality could be an unexpected and confounding source of variation in experimental results. Although there is no ready solution to this problem, the use of RAC in expe-riments can give researchers a relative indication of the nutritional effectiveness of other Artemia sources they are using in the laboratory. Nauplii of the RAC have been tested for their nutritional performance in the culture of larval fish (Klein-MacPhee et al, in press) and crabs and have been found to be one of the better Artemia source. Thus far tested by the ISA. The use of RAC as an inter-calibration food source in experiments could reduce the number of incorrect inferences caused by the poor nutritive value of an uncharacterized laboratory diet.

Acknowledgement. We acknowlege the technial support of W. Berry for maintenance of crab larvae and Artemia.

Table 1. Artemia ssp. weight percent fatty acid composition of various geographical colections of newly-hatched nauplii. nd:

FAME RAC Brazil1 Canada China France14:0 1.79 1.57 0.83 1.80 1.7314:1 2.92 0.81 1.67 2.24 3.0316:0 12.70 15.42 9.99 11.40 11.9016:1ω7 16.78 10.79 9.03 19.06 11.3416:3ω4/17:1ω8 4.33 3.88 1.47 2.54 2.2018:0 4.07 2.79 5.12 3.99 4.2118:1ω9 30.37 35.89 28.24 26.81 24.7318:2ω6 9.62 9.59 7.95 4.68 6.-1418:3ω3 2.55 4.87 19.87 7.38 20.9018:4ω3 nd 0.96 1.60 1.26 2.0420:2ω6/20:3ω6 0.20 2.82 0.44 0.15 1.1320:3ω3/20:4ω6 5.82 nd 4.21 3.34 2.4520:5ω3 8.45 8.98 9.52 15.35 8.01Total % 99.60 98.37 99.94 100.00 99.81Total lipid mgg-1 dry wt 209.4±24.0 2020±8.0 142.9 ±34.0 201.7 ± 0.3 152 .1 ±29.01 Data from schauer et al. (1980); 0.67% 15:0 and 0.52% 20: 1 9 were also present

Table 2. Artemia ssp. chlorinated hydrocarbon content of various geographical collections of newly-hatched nauplii, results expressed as ng g-1 wet weight (ppb). nd: not detected.

RAC Brazil 1 Canada France China HCB 0.3 0.1 0.3 1.8 . 97.0PCB 1016 1.0 5.3 6.2 8.6 6.3PCB 1254/1260 0.2 1.6 5.6 32.0 43.0Σ PCB'S 1.2 6.9 12.0 41.0 49.0ppDDE 1.4 1.2 3.0 14.0 85.0ppDDD 0.4 0.4 0.4 3.8 22.0opDDT nd 0.4 nd nd 0.9ppDDT 0.3 1.9 nd 7.1 64.0Σ DDT's 2.1 4.3 3.4 25.0 172.0αBHC 0.2 1.1 1.6 0.3 23.0γBHC nd 0.8 nd 2.2 16.0t-Chlorodane 0.1 nd 0.1 0.3 0.4c-Chlorodane nd 0.1 nd 0.4 0.21 Data from Olney et al. (1980)

Table 3. Rithropanopeus harrissii. Summary of culture data for larvae fed various geografical collections of newly-hatched Artemia ssp. Data presented as mean ± one standard deviation. Means having the same grouping letter are not significantly different (P> 0.05) (n) sample size

Artemia source

Survival to

megalopa (%)

(n) Grouping Developpement time to

megalopa (d)

(n) Grouping Megalopa dry

weight fog)

(n) Grouping

French 89 ±13 60 A 11.1 ± 0.2 53 A 181 ± 10 53 A

Reference 89 ± 10 60 A 10.9 ± 0.2 50 A 166 ± 13 50. B

Brazilian 85 ± 10 60 A 10.7 ± 0.3 53 A 161± 13 53 B

Chinese 84 ± 17 60 A 10.9 ±0.3 51 A 168 ± 27 51 B

Canadian 72 ± 25 60 A 11.6 ± 0.6 43 A 144* 38 43 C

LITTERATURE CITED

BECK, A.D., BENGTSON, D.A.,HOWELL, W.H. (1980). International Study on Artemia V. Nutritional value of five geographic strains of Artemia: effects of survival and growth of larval Atlantic silversides, Menidia menidia. In: Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E. (eds.) The brine shrimp Artemia, Vol.3, Ecology, culturing, use in aquaculture. Universa Press, Wetteren, Belguim, pp. 249-259

JOHNS, D.M., BERRY, W.J., WALTON, W. (1981). International Study on Artemia XVI. Survival, growth and reproductive potential of the mysid, Mysidopsis bahia Molenock fed various geographical collections of the brine shrimp, Artemia. J.exp. mar. Biol. Ecol.53: 209-219

JOHNS, D. M., PETERS; M.E., BECK, A.D. (1980). International study on Artemia VI. Nutritional value of geographical and temporal strains of Artemia: effects on survival and growth of two species of brachyuran larvae. In: Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E. (eds.) The brine shrimp Artemia, Vol. 3, Ecology, culturing, use in aquaculture. Universa Presse, Wetteren, Belgium, pp. 291-304

KLEIN-MACPHEE, G., HOWELL, W.H., BECK, A.D. (1980). International Study on Artemia VII. Nutritional value of five geographical strains of Artemia to winter flounder (Pseudopleuronectes americanus). In: Persoone, G., Sorgeloos, P., Roels, O. Jaspers, E. (eds.) The brine shrimp Artemia, Vol. 3, Ecology, culturing, use in aquaculture. Universa Press, Wetteren, Belgium, pp. 305-312

KLEIN-MACPHEE, G., HOWELL, W. H., BECK, A.D. (In press). Comparison of a reference strain and four geographical strains of Artemia as food for winter flounder (Pseudopleuronectes america-nus) larvae. Aquaculture.

MANZI, J. J., MADDOX, M.B. (1980). Requirements of Artemia nauplii in Macrobrachium rosen-bergii (de Man) larviculture. In: Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E. (eds.) The brine shrimp Artemia, Vol. 3, Ecology, culturing, use in aquaculture. Universa Press, Wetteren, Belgium, pp. 313-330

OLNEY, C.E., SCHAUER, P.S.,MCLEAN, S., LU, Y., SIMPSON, K.L. (1980). International Study on Artemia VIII. Comparison of the chlorinated hydrocarbons and heavy metals in five different strains of newly hatched Artemia and a laboratory-reared

marine fish. In: Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E. (eds.) The brine shrimp Artemia, Vol. 3, Ecology, culturing, use in aquaculture. Universa Press, Wetteren, Belgium, pp. 343-352

SCHAUER, P.S., JOHNS, D.M., OLNEY, C.E., SIMPSON, K.L. (1980). International Study on Artemia IX. Lipid level, energy content and fatty acid composition of the cysts and newly hatched nauplii from five geographical strains of Artemia. In: Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E. (eds.) The brine shrimp Artemia, Vol. 3, Ecology, culturing, use in aquaculture. Universa Press, Wetteren, Belgium, pp. 365-373

SIMPSON, K.L., BECK, A.D.,SORGELOOS, P. (1980). Workshop I. Characterization of Artemia strains for application in

OVERVIEW GENETICS OF MEDITERRANEAN BISEXUAL ARTEMIA.

BY E.J.S. PILLA

* HOW DO WE DEFINE AN ARTEMIA BISEXUAL SPECIES IN GENETIC TERMS?

- FEW DIAGNOSTIC CHARACTERS, WHICH MAY BE NOT UNIVERSAL FOR THE SPECIES AS A WHOLE:

* CYTOLOGY

* REPRODUCTIVE ISOLATION

* MORPHOLOGICAL DIFFERENTIATION

* MORPHOMETRIC DIFFERENTIATION

* GENETIC DIFFERENTIATION:

- NUCLEAR DNA (ALLOZYMES, REPETITIVE SEQUENCES)

- MITOCHONDRIAL DNA (RFLP, GENE ORGANISATION)

* LIFE HISTORY TRAITS (NO. OFFSPRING, % CYSTS, CYST SIZE, etc)

* BIOCHEMICAL FEATURES (e.g. H.U.F.A; CONTENTS)

* PHYSIOLOGICAL FEATURES (e.g. ADAPTATION TO SPECIFIC WATER COMPOSITION)

* MOST PRUDENT IS TO USE A RANGE OF FACTORS

* OLD WORLD (EURASIA AND AFRICA): ESTIMATED CIRCA 70% OF POPULATIONS ARE PARTHENOGENETIC, REMAINING (INCLUDING A. TUNISIAN!} ARE BISEXUAL.

* ARTEMIA TUNISIANA - ANCESTRAL SPECIES TO OLD WORLD BISEXUAL (e.g. A. URMIANA AND A. SINICA WHICH GAVE RISE TO PARTHENOGENETIC FORMS)

* ONLY BISEXUAL SPECIES, TO DAT, WITH DOCUMENTED CONSPICUOUS NATURAL COEXISTENCE WITH OTHER (PARTHENOGENETIC) SPECIES

* MEDITERRANEAN BASIN: NORTH AFRICA (TUNISIA, ALGERIA, EGYPT, LIBYA?, MOROCCO?), CYPRUS, SPAIN (INCLUDING BALEARIC ISLES) ITALY (INCLUDING SARDINIA AND SICILY), SOUTHERN AFRICA?

* ENDURANCE OF A. TUNISIANA INTERESTING PHENOMENON

* NO WIDE COMMERCIAL USE. HOWEVER,

* BIOLOGICAL PROPERTIES OF POTENTIAL USE IN AQUACULTURE.

* POPULATIONS STUDIED TO DATE DISPLAY SOME USEFUL FEATURES FOR PRODUCTION AND USE IN AQUACULTURE:

- WELL ADAPTED TO LOW TEMPERATURES (e.g. 15° C)

- HIGH FEMALE ENCYSTMENT RATES (85-100%)

* DRAWBACKS TO BE ADDRESSED:

− LOW REPRODUCTIVE OUTPUT

− SHORT REPRODUCTIVE CYCLE (BUT ONLY SIGNIFICANTLY. SO WHEN COMPARED WITH, IN THE OLD WORLD, POLYPLOID PARTHENOGENS)

CYTOLOGY* MAJORITY OF POPULATIONS ANALYSED (HOWEVER, ONLY ITALIAN AND

SPANISH POPULATIONS LOOKED AT) FOR NAUPLII MITOSES 2N=42, BUT CONSPICUOUS 2N=44 AND 40,44,46,48 AND 50.

* HETEROPLOIDY/ANEUPLOIDY NOT CONFIRMED IN ADULT POPULATION. PHENOMENON LIMITED TO LARVAL STAGE?

* CHROMOSOME NUMBERS DO SEEM TO INFLUENCE INTRA OF INTRPOPULATION FERTILITY

* LACK OF CHROMOCENTRES (IN COMPARISON WITH A. FRANCISCANA)

− USEFUL DISCRIMINATORY TOOL TO ASSESS EXTENT OF DISSEMINATION OF INTRODUCED A. FRANCISCANA, ESPECIALLY IN NORTH AFRICA

Summary of the genetic variability in bisexual populations of Artemia.

Population Mean sample size

(s.e.)

Mean n° alleles per locus (s.e.)

% polymorphic

loci*

H (s.e)

H (s.e.)

A. franciscana (SFB)

65.7 (1.3)

1.4 (0.1)

30.0 0.049 (0.027)

0.058 (0.031)

A. tunisiana (TUN)

64.7 (2.6)

1.6 (0.2)

35.0 0.057 (0.028)

0.075 (0.039)

Artemia sp. (RUS)

61.7 (1.4)

2.0 (0.2)

60.0 0.096 (0.033)

0.108 (0.037)

Artemia sinica (SIN)

66.3 (2.0)

2.0 (0.3)

55.0 0.097 (0.033)

0.097 (0.033)

Artemia umiana (URM)

78.9 (1.8)

2.3 (0.3)

65.0 0.080 (0.024)

0.096 (0.028)

Artemia sp. (YIM)

49.0 (0.4)

1.9 (0.2)

55.0 0.084 (0.035)

0.095 (0.037)

* 0.99 criterion..

** = unbiased estimate (see Nei, 1978).

ALLOZYME DATA GENETIC DIFFERENCTIATION * MEAN CONSPECIFIC GENETIC DISTANCESALLOZYME FREQUENCIES ARE CONVERTED INTO A MEASURE OF DIVERSITY BETWEEN POPULATIONS AND SPECIES: A. TUNISIANA (7 populations): MEAN D= 0.091 (+0.061)

* Other bisexual spp.: A. FRANCISCANA (21 pop.): MEAN D= 0.126 (+0.067) A. SINICA (6 POPULATIONS): MEAN D=0.014 (+0.001) * MEAN CONGENERIC GENETIC DISTANCES

OLD WORLD Vs. NEW WORLD

A. TUNISIANA VS. A. FRANCISCANA D= 1.501 (+0.355)

NEW WORLD Vs. NEW WORLD

A. FRANCISCANA Vs NEW WORLD

A. FRANCISCANA Vs. A. MONICA D = 0.098 (+0.050)

A. FRANCISCANA Vs A. PERSIMILIS D = 1.073 (+ 0.299)

OLD WORLD Vs. OLD WORLD

A. TUNISIANA Vs. A. URMIANA D = 0.664 (+ 0.154)

A. TUNISIANA Vs. A. SINICA D = 0.868 (+ 0.206)

A. SINICA Vs. A. URMIANA D = 0.355 (+0.045)

REPROCUCTIVE ISOLATION * NO COMPREHENSIVE STUDY INTO FERTILITY OF A. TUNISIANA PERFORMED

TO DATE

* SOME SIGNS OF MINOR AND MAJOR GENETIC INCOMPATIBILI-TY (e.g. BETWEEN ITALIAN AND TUNISIAN BRINE SHRIMP: SAN BARTOLOMEO + TRAPANI Vs. CHOTT ARIANA + SFAX)

* INFERTILITY COULD BE THE RESULT OF «RING SPECIES» PAT-TERN OF DIFFERENTIATION

MORPHOLOGY AND MORPHOMETRICS

* MORPHOLOGY: - MORE USEFUL IN CONGENERIC (i.e. BETWEEN SPECIES ) TERMS,

ABSENCE OF LOWER THORACIC PROTUBERANCE IN A. TUNISIANA

FEMALES, AND PRESENCE IN FEMALES OF OTHER SPECIES

MORPHOLOGY OF MALE CLASPER KNOB REQUIRES DETAILED (S.E.M.) ANALYSIS

FURCA SHAPE AND NUMBER OF SETAE SUBJECT TO SALINITY CHANGES

* MORPHOMETRICS: - MORE USEFUL FOR STUDYING VARIATION BETWEEN POPULATIONS OF THE

SAME SPECIES (CORRELATING WITH OTHER DATA)

GENETICS * ALLOZYMZ DATA

GENETICS VARIABILITY

A. TUNISIAN A SITE Alleles/ locus Polymorphic loci (%) Heterozygosity (%)

Barbarena (Spain)

1.41 (± 0.18)

36.4 12.6 (± 4.9)

Salin di Poetto (italy)

1.46 ( ± .24)

27.3 9.9 (± 4.3)

Chott Ariana (Tunisia)

1.32 (± 0.14)

31.8 9.3 (± 3.7)

Larnaca (Cyprus)

1.17 (± 0.17)

44.4 6.3 (± 3.9)

San Felix (Spain)

1.36 (± 0.17)

27.3 8.5 (± 3.6)

San Pablo (Spain)

1.43 (± 0.23)

38.1 12.5 (± 5.4)

Santa Pola (Spain)

1.18 (± 0.10)

22.7 7.4 (± 3.6)

Quartu (Italy)

1.33 (± 0.18)

33.0 9.0 (± 4.0)

AVERAGE 1.60 (± .20)

35.0 (± 3.7)

7.5 (± 3.9)

* POPULATION SUBSTRUCTURING

FROM ALLOZYME SURVEY (FREQUENCIES), IT IS POSSIBLE TO ESTIMATE AMOUNT OF GENETIC VARIATION ATTRIBUTABLE TO POPULATION SUBDIVISION (i.e.,RELATIVE DIFFERENCES BETWEEN POPULATIONS OF A SPECIES) BY USING A STATISTIC KNOWN AS Fst, AND TO TEST WHETHER IT IS SIGNIFICANTLY DIFFERENT FROM ZERO.

A. TUNISANA (7 POPULATIONS) Fst = 0.117 (+ 0.118)

In other words, 12% of total genetic variation is due to population substructuring (Not significantly different from 0)

* Other bisexual spp.:

AFRANCISCANA (21 POPULATIONS): Fst = 0.240 (+0.045)

24% of total genetic variation is due to differences between populations (P<0.05)

A. SINICA (6 POPULATIONS): Fst = 0.064 (+0.010)

Differences between populations account only for 6% of total variation (not significantly different from 0)

ALTHOUGH ARTEMIA POPULATIONS CLEARLY DO NOT FORM CONTINUOUS DEMES, IT IS IMPORTANT TO ESTIMATE HOW MUCH MIXING MAY BE OCCURING BY BIRDS, MAN, WIND, ETC

* GENE FLOW/MIGRATION

Fst FIGURES FOR EACH SPECIES MAY BE CONVERTED INTO MEASURES OF GENE FLOW BETWEEN POPULATIONS (MAKING SOME ASSUMPTIONS ON PATTERNS OF GENE FLOW). Nm = NUMBER OF MIGRANTS PER GENERATIONS.

* TO PREVENT POPULATIONS FROM DIVERGING COMPLETELY, Nm NEEDS TO BE > 1.

A. TUNISIANA Fst = 0.117

Nm = 1.88 (i.e. MIGRATION OF THE ORDER OF 2 INDIVIDUALS PER GENERATION)

* Other bisexual spp.:

A. FRANCISCANA Fst = 0.24

Nm = 0.8 (i.e. MIGRATION OF LESS THAN 1 INDIVIDUAL PER GENERATION)

(HENCE INCIPIENT SPECIATION)

A. SINICA Fst = 0.064

Nm = 3.65 (i.e. CIRCA 4 INDIVIDUALS PER GENARATION)

UPGMA DENAOGRAM OF NER'S GENETIC DISTANCE FOR BISEXUAL AND PARTNENOGENETIC SPECIES OF ARTEMIA

THE NUTRITIONAL VALUE OF ARTEMIA

P. LEGER, D. A BENGTSON, P. SORGELOOS,K. L SIMPSON and A.D. SEEK

INTRODUCTION

Successful rearing of larval stages of aquatic organisms is aa challenger for aquarium hobbysts, a tool for aquatic ecologists and ecotoxicologists/and necessity for the aquculturists. All these people agree the primery problem in any type of larval rearing is that of food. Ideally, one would prefer to feed larvea their natural diet, which is usually characterized bay a wide diversity of nutritious live organisms.

Although not a «natural» food, Artemia have been successfully that such food could be attained with a food from such unusual (i.e., hypersaline) environment. Some recent experiences suggest that the use of Artemia does not absolutely guarantee success (see reviews by Sorgeloos, 1980, and Simpson et al, 1983). Explantions for and remedies to this variable success will be covered in this review through an analysis of the larval organism's requirements for food (see Fig. 1). A more complete review of the nutritional value of Artemia is presented by leger et al.

PRATICAL REQUIREMENTS FOR THE CULTURIST

A food organism must first meet the nutrional needs of the predator. In addition, other practical requirement have to be met to satisfy the culturist. The consistent availability of food organisms is of utmost importance for continuous cultures. In this respect, Artemia is superior to all other live foods since it is available as an off-the-shelf food in the form of dormant cysts. From those cysts, nauplii are obtained through simple hatching procedures (Sorgeloos et al., 1983). Ideally, a food organism should also be hardy and easily cultured. Artemia nauplii fulfill this requirement quite well, since they are very tolerant to various culture environments, resistant to even rough handling and may be disinfected resulting in a biologically uncontaminated live food (Sorgeloos et all, 1983).

The wide size range of Artemia and their different physical forms (Fig.2) make them very versatile in use. Since they are easily cultured, Artemia nauplii and later stages may be fed according'to the changing needs of the predator during its development. Also a smaller food particle may be used in the form of decapsulated cysts, which are some 50% smaller than freshly hatched nauplii and have several other advantages: a) they are disinfected and separated from the cyst shells during the decapsulation process (Sorgeloos et al., 1977, b) hatchability of the embryos is improved (Bruggemann et al, 1980), so that otherwise nonhatchable cysts increase in value, c) the energy content is higher (Vanhaecke et al., 1983), leading to a higher naupliar biomass production per gram of cysts and a smaller, more energy-rich food particle for the larval organism.

A last example of the versatility of Artemia as a food is the possibility of using Artemia (nauplii or adults) as a carrier for components which are otherwise difficult to administer to fish and crustacean larvae. Indeed, essential nutrients, pigments,, prophylactics and therapeutics may be bioencapsulated in Artemia and introduced to the consumer organism (see Fig. 3; Leger et al., 1985).

PHYSICAL REQUIREMENTS Physically, a food organism has to be clean, free from alien organisms and materials and especially free from contagious diseases. Artemia may be disinfected and fed as a clean food, although cysts are often heavily loaded with microorganisms. Austin and

Allen (1981) did not find an intimate microbial contamination of the nauplii and they demonstrated that bacteria surrounding the nauplii may be easily removed by simple washing procedures or even better by disinfecting the cysts prior to hatching incubation by a dipping procedure. No direct evidence exists for Artemia-borne infections in larvae.

A second physical requirement is that a food organism has to be accepted by the predator. Acceptablity of food is determined by several factors. The bright color of Artemia nauplii and their continuous movement make them easily perceptible. Perceptibility may be enhanced further by staining techniques, as demonstrated for sole larvae (Dendrinos et al., 1984). Artemia nauplii are easily caught because they lack an effective escape response. Palatability is apparently adequate, since Artemia is often used as a gustatory attractant in artificial diets (Barahona-Fernandes et al., 1977; Gatesoupe and Luquet, 1981/1982). Ingestibility of a palatable food is governed primarily by its size; Size of Artemia nauplii is therefore the first important consideration. Indeed, most first-feeding marine fish species and some decapods, such as penaeids, can not ingest (or handle) Artemia nauplii. Even species which may accept some Artemia nauplii as a first food sometimes face ingestion and prey-handling problems. Vanhaecke and Sorgeloos (1980) have demonstrated considerable variation in naupliar size (422 - 517 m) and volume (7.638 - 13.604 • 106 m3) among different geographical strains. The effect of size in feeding nauplii tofish to fish has been described by Beck and Bengtson (1982). They fed freshly nauplii from eight different Artemia strains to Atlantic siverside (Menidia menidia) larvae. The correlation between size of nauplii and mortality of the fish larvae indicated that at least 20% mortality could be expected when nauplii larger than 480 um were fed as a first food.

NUTRITIONAL REQUIREMENTS

In addition to physical requirements, a food organism also has to meet certain nutritional requisites, including digestibility. Watanabe et al. (1978a) found high digestibility rates for Artemia fed to carp and rainbow trout and reported high values for net protein utilization and protein efficiency ratio. Enzymes such as amylase and trypsin that are found in Artemia (Samain et al., 1980) may also play an important role in enzymatic autolysis during the transit of the nauplii through the larval gut, ant thus contribute to digestion.

Even when easily digested, a food organism still may not meet the nutrient requirements of the predator. The main problem in evaluating Artemia in this respect is our lack of knowledge of the nutrient requirements of most predators to which Artemia are fed. A proximate analysis of Artemia (Table I) reveals an equilibrated high protein diet indicating that macronutrient requirements probably are satisfied for most predators. However, several investigators report considerable variation in larval culture success (review in Leger et al., 1986).

Variation in larval growth rate has been attributed to significant differences in individual energy content (0.0366 - 0.0725 J) and dry weight (1.61 - 3.33 ug) of Artemia nauplii from different geographical origin (Vanhaecke et al, 1983). Selection of high energy strains is therefore recommended; Varying growth rates may also result from the use of older unfed instar stages which contain up to 39% less energy and 34% less dry weight than freshly hatched nauplii (Fig. 3) (Vanhaecke et al., 1983). This energy and dry weight loss can be avoided by storing instar I nauplii at lower temperatures, since they survive well for 24 hr at 2-4°C without significant losses in dry weight (Leger et al., 1983). This cold storage technique further allows a complete automation of feeding, permitting a 24 hr feeding of energy-rich instar I. Starved nauplii not only contain less

energy and dry weight, which may be less able to meet the requirements of the predator, but they are also less visible, larger and faster swimming, so their acceptability is reduced. Starved nauplii have a lower free amino acid content (Dabrowski and Rusiecki, 1983), which may reduce their digesitibility. All these negative factors may be reflected in poor growth of the larval predator.

Decapsulated cysts, on the other hand, constitute the highest energy

Artemia form and are more favorable for use except when a predator feeds only on moving prey.

Besides variable growth rates, other problems have been attributed to the use of Artemia. A relationship has been found between the use of particular geographical strains of Artemia and the appearance of various symptoms in fish and crustacean larvae, such as lethargy, lack of coordination, abnormal development, prblems at metamorphosis, abnormal pigmentation, and even mortality (Wickins, 1972; Campillo, 1975; Beck et al., 1980; Johns et al., 1980; Klein-MacPhee et al., 1980, 1982). Several authors have tried to explain these observations and they have formulated diverging and sometimes contradictory explanations. For example, Bookhout and Costlow (1970) suspected that high levels of DDT caused the problems, but Wickins (1972) research suggested that a nutritional deficiency was involved.

INTERNATIONAL STUDY ON ARTEMIA

The elucidation of the nutritional variability of Artemia was one of the major concerns of the participants in the International Study on Artemia (ISA) (Sorgeloos, 1980). Nutritional bioassays with several larval fish and crustacean species fed various Artemia strains confirmed previous reports of varibility in food value among different strains of Artemia (Table II). However, this variability was not a factor in culture tests with freshwater fish larvae. Major problems seemed to occur only in tests with marine species. Certain Artemia strains guaranteed good culture success for all marine spcies tested (Brazil, San Francisco Bay and Reference Artemia). The 1978 batch San Pablo Bay (SPB) Artemia nauplii, on the other hand, were consistently poor for all spcies. Utah Artemia in some cases gave poor culture results, whereas intemediate success was obtained with the Canadian strain. Generally good results were obtained with the Australian, Chinese, French and Italian strains, except for Atlantic silverside larvae, which, as mentioned before, were adversely affected by the large nau-pliar size. Major problems seemed to occur primarily when SPB and Utah naupliar size. Major problems seemed to occur primarily when SPB and Utah nauplii were fed to marine organisms, especially to those spcies, such as crab and flatfish, whose larvae undergo a pronounced metamorphosis. In those species almost all mortality was suffered at the onset of matamorphosie (Figs. 4 and 5). This information coincides with numerous reports from authors culturing decapod and flatfish larvae, such as crab, turbot and plaice (review in Leger et al., 1986),

The ISA has developed various correlations to try to explain strain differences in nutritional value, based on results of the bioassays and the chemical and biochemical analyses of the strains. Abnormalities, effects on growth, and mortality may be an expression either of nutrient deficiency of toxicity. For this reason a detailed analysis of chlorinated hydrocarbons (CHCs) was carried out on the Artemia strains used in the bioassays (Olney et al., 1980; Seidel et al., 1982). The most heavily contaminated strains, in terms of total CHCs, (the Italian and Chinese strains) however gave excellent results in the bioassay. On the other hand, Utah ranked among the cleanest strains. The only similarity between SPB and Utah samples was their higher level of dieldrin. Furthermore, SPB 1628 had the highest level of chlordane and high molecular PCBs.

Follow- up studies by Johns et al. (1981) and McLean et al. (1985) have demonstrated that Brazilian nauplii purposely contaminated with the suspected CHCs did not cause mortality in crab larvae or post-metamorphic flounder, although growth was reduced in flounder fed on Artemia with moderate levels of CHCs. CHCs therefore were probably not a principal factor controlling the dietary value of the Artemia strains tested. Heavy metals were also probably not significant factor since no metal common to SPB and Utah was present in dramatically higher levels (Olney et al., 1980). The prescence of toxic materials in Artemia is still a concern, however, because CHC and heavy metal contamination is anthropogenic and thus subject to variations. For example, copper levels have been shown to vary considerably in Utah Artemia (Blust, personal communication) and very high pesticide levels have been reported in a batch of Philippine Artemia (Simpson et al., 1983). Shelbourne (1968) hypothesized that Utah Artemia may have accumulated toxins produced in dinoflagellate blooms, but measurable amounts of paralytic shellfish poison have not been detected in SPB 1628 (Olney et al., 1980). Apparently, differences in nutritional value between the Artemia sources tested are not related to the presence of toxic contaminants.

The hypothesis that nutrient deficiency mayt explain nutritional variability is borne out by the difference in results of feeding Artemia strains to freshwater and marine species, which indeed have different dietary requirements. In-depth biochemical profiles of the different strains tested showed that differences in amino acid profile coud not explain differences in culture results (Seidel et al. 1980). All strains were found to meet the essential amino acid requirements for chinook salmon, though nethionine appeared to be the first limiting amino acid. Similarly, the varying culture success could not be explained by differences in carotenoid (Soejima et al., 1980), mineral (Watanabe et al., 1978a), caloric or lipid content (Schauer et al., 1980). However, pronounced differences were found in fatty acid profiles. As compared to other strains, Utah espcially SPB 1628 Artemia contained high levels of 18 :.3ω3 and particularly low levels of 20 : 5ω3 (Schauer et al, 1980; Seidel et al, 1982). The relative lack of 20 ; 5ω3 may explain the poor results in feeding the Utah and SPB strains to marine organisms. The highly unsaturated fatty acid (HUFA) 20 : 5ω3 known to be essential for marine fish and crustacean larvae (Teshima, 1978; Yone, 1978; Kanazawa et al, 1979). Canadian nauplii, in spite of hight levels of 20.-: 5ω3, provided intermediate results in the bioassays, which indicates that for this strain other factors, possibly energetic ones, may be involved.

IMPORTANCE OF ESSENTIAL FATTY ACIDS

Leger et al, (1985c) further studied the relationship between 20 : 5ω3 level and nutritional value of Artemia by evaluting different batches of the San Francisco Bay strain together with Reference Artemia and SPB 1628 as, respectively, positive and negative controls. Levels of 20 : 5ω3 varied considerably among different batches within the same strain. Batches with high 20 : 5 ω3 levels yielded high biomass production in culture tests with mysids, while batches with low 20 : 5ω3 levels consistenly yielded less biomass (Fig. 6). CHC analyses were also performed on these different batches and again considerable differences were found, but could not be correlated with the biomass figures (Fig.7).from these studies we may conclude that the content of the essential fatty acid 20 : 5 oo3 seems to be most important factor determining the nutritional value of Artemia nauplii to marine organisms. This is supported by the observation that Utah and San Pablo Bay nauplii provided good survival of freshwater fish which do not require highly unsaturated fatty acids such as 20 : 5ω3 in their diet.

Watanabe et al. (1978) classified Artemia strains into marine type Artemia, which contain high levels of 20 : 5co3, and freshwater type Artemia, which contain low levels of 20 : 5ω3. They obtained good survival of red seabrean larvae fed with the marine type nauplii and poor survival of those fed the freshwater type. However, when the freshwateer type nauplii were fed on 20 : 5ω3-rich diets, such as marine Chlorella or oo-yeast, before they were fed to the fish, superior survival rates of the seabream larvae were achieved. Watanabe et al. (1982) have demonstrated that 20 : 5ω3 could be incorporated into Artemia by feeding them for 24 h on 20: 5ω3-rich diets. Nauplii prefed oo-yeast also contained significant levels of 22 :6ω3 and red seabream larvae did best on these nauplii. Similarly, Leger et al. (1985c) have shown that feeding a HUFA-enrichment diet to San Pablo Bay 1628 Artemia increased its levels of 20 : 5ω3 and 22 : 6ω3 and markedly enhanced its nutritional value for penaeid shrimp larvae. The nutritional improvement of fattyacid- enriched Utah Artemia has also been demonstrated for mysids, two penaeid species and seabass larvae (Van Ballear et al., 1985; Amat et al., 1985; Leger et al., 1985b).

If the abundance of certain essential fatty acids governs the nutritional value of Artemia nauplii, what exactly determines their respective levels in Artemia? Schauer and Simpson (1985) have demonstrated that Artemia have a limited need to produce their own 20 : 5ω3 and Millamena et al (1985) reported that Artemia HUFA levels strongly resembled those of the algal diets on which they were fed. Lavens et al. (in preparation) grew nauplii containing about 5% 20: 5ω3 on two different diets, one containing 6.7% and the other only 0.7% 20:5ω3. The resulting adult populations were induced to produce cysts in a controlled cyst production unit (Lavens and Sorgeloos, 1984). The cysts produced by adults grown on the 20 : 5ω3-rich diet contained high levels of 20:5ω3 while the others contained very low levels of this fatty acid. This experiment clearly demonstrated that Artemia cysts reflects 20 : 5ω3 levels of the diet available for the parental population.

If these results can be extrapolated to wild populations, different food conditions in Artemia ponds and lakes probably explain differences in fatty acid profile between Artemia strains- and even within the same strain. Compilation of data from literature and our own analyses (Table III) (see review in leger et al, 1986) shows that 20 : 5ω3 levels may indeed vary considerably among and within strains. Variability is particularly great in strains produced in solar salt works, e.g., SFB, Brazilian and Chinese Artemia. Variability is small in Candian and especially Utah Artemia, both of which are produced in inland salt lakes characterized by a more stable environment. Food composition in solar salt ponds is more diverse and often completely different from one pond to another. The nutritional quality of Artemia cysts produced in solar salt ponds is therefore subject to uncontrolled variability and is difficult to predict.

ARTEMIA ENRICHMENT

The technique of feeding Artemia nauplii on HUFA enrichment diets markedly increases the nutritional value of inferior strains and batches and hence reduces strain and batch differences. The application of Artemia enrichment with algae was pioneered by Forster and wickins (1967) and wickins (1972) and further developed by Japanese, French, and Belgian researchers using prepared diets (see review in Leger et al., 1986). Since those methods have been covered elsewhere in this Symposium (Leger et al., 1985b; Robin et al, 1985; Watanabe et al., 1985), we will not review the techniques here, but summarize the advantages of Artemia enrichment. Enriched nauplii have an improved nutritional composition, i.e., they have a higher energy content and contain all essential fatty acids including 22 :6 3, which is generally absent in nauplii from all strains. Through

enrichment techniques other nutrients, prophylactics and therapeutics may be passed to the predator via Artemia nauplii. The application of enriched Artemia is reflected in improved performances in larval culture, in terms of both survival and growth, and consequently improved performances are obtained in later stages (Leger, unpublished data; Chamorro, personal communication). Larvae fed on enriched Artemia are indeed healthier and more resistant to stressful conditions, such as inflections, weaning of fish , or transfer of shrimp from hatchery tanks to nursery ponds. The only disadvantage of enriched Artemia is their larger size, which may be a problem for early larval stages. For those cases where size is a problem, freshly hatched, high quality instar I nauplii may be used as food for the first few days, followed by a gradual switch to enriched metanauplii as soon as the predator's size permits ingestion of larger particles. Optimized enrichment procedures may also reduce the disadvantage of size by obtaining similar enrichment levels in less time (Leger e shrimp larvae. imilar enrichment techniques may also be applied to juvenile and adult Artemia which may be used as a carrier for essential nutrients and other components to be administered to postlarval shrimp, juvenile fish and lobster larvae.

CONCLUSIONS AND RECOMMENDATIONS

To summarize, Artemia is an excellent food for a wide variety of cultured marine and freshwater organisms. The major constraint of Artemia as a food organism for marine predators is its variable nutritional quality. However, we recommend some measures to remedy this problem:

− for the problem of size and variable energetic content between strains and instar stages, one should:

a) select suitable strains

b) use freshly hatched first instar nauplii (i.e., through application of optimized hatching procedures, cold storage of nauplii and optimized feeding strategies

c) when possible, use decapsulated cysts

− for the problem of variable nutritional composition, one should:

a) apply enrichment techniques

b) select high quality lots for early larval stages and as a reference material in ecological studies. Especially for those studies where reproducibility of culture results is of utmost importance, an urgent need exists for the fully controlled production of standard high quality Artemia cysts.

Finally, realizing that nutritional quality of Artemia may be so poor that complete mortality of certain predators may result, we wonder how mariculture would have developed if Seale (1933) and Rollefsen (1939) had used a poor quality Artemia sources when they pioneered the use of Artemia for larval fish culture back in the 1930's. Despite variability among geographical strains in size, caloric content, nutritional compostion and contaminants, Artemia has proven to be the most widely used and successful diet for aquaculture activities.

ACKNOWLEDGEMENTS

Our research contributions for this review have been sponsored by the Belgian National Science Foundation (NFWO) grant FKFO 30.0012.82, the Belgian Institute for the Promotion of Industry and Agriculture (IWONL), the Belgian Administration for Development Cooperation, the Belgian Company NV Artemia Systems, the United

States Environmental Protection Agency, and the United States Sea Grant Program. PS is senior scientist with the Belgian National science foundation.

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SCHAUER PS. and K.L. SIMPSON. 1985. Bioaccumulation and bioconversion of dietary labeled fatty acids in Artemia and winter flounder (pseudopleuronectes americanus). Can. J. Fish. Aquat. Sci. 42: 1430-1438.

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SEIDEL C.R., J. KRYZNOWEK, and K.L. SIMPSON. 1980. International Study on Artemia. XI. Amino acid composition and electrophoretic protein patterns of Artemia from five geographical locations. P. 375-382. In: The brine shrimp Artemia. Vol. 3. Ecology, Culturing, Use in Aquaculture. Persoone G., P. Sorgeloos, O. Roels and E. Jaspers (Eds.). Universa Press, Wetteren, Belgium. 456 p.

SHELBOURNE J.E. 1968. The culture of marine fish larvae with special reference to the plaice (plenronectes platessa) and the sole (Solea solea). Thesis, London University, 143 p.

SIMPSON K.L., G. KLEIN-MCPHEE, and A.D. BECK. 1983. Zooplankton as a food source, p. 180-201. In: Proceedings of the Second International conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition. Pruder G.D., C. Langdon and D. Conklin (Eds.). Division of Continuing Education, Louisiana State University, Baton Rouge, Louisiana, USA.

SOEJIMA T., T. KATAYAMA, and K.L. SIMPSON. 1980. International Study on Artemia. XII. The carotenoid composition of eight geographical strains of Artemia and the effect of

diet on the carote-noid composition of Artemia. P. 613-622. In: The brine shrimp Artemia. Vol. 2. Physiology, Biochemistry, Molecular Biology. Persoone, G., P. Sorgeloos, O. Roels and E. Jaspers (Eds.). Universa Press, Wetteren, Belgium. 664 p.

SORGELOOS P. 1980. The use of the brine shrimp Artemia in aquaculture. p. 25-46. In: The brine shrimp Artemia. Vol. 3. Ecology, Culturing, Use in Aquaculture. Persoone G.,P. Sorgeloos, 0. Roels and E. Jaspers (Eds.). Universa Press, Wetteen, Belgium. 456 p.

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.

Table I : Average proximate composition of Artemia nauplii and adults as calculated from data presented in 26 and 15 references, respectively.

Nauplii Adults Protein 52.2 ± 8.8% 56.4 ± 5.6% Lipid 18.9 ± 4.511.8 ± 5.0 Carbohydrate 14.8 ± 4.8 12.1 ± 4.4 Ash 9.7 ± 4.617.4 ± 6.3

TABLE II: Summary of nutritional bioassay results for several categories of aquatic organisms fed 10 geographical strains of Artemia

Artemia geografical strain

AU

STR

ALI

A

BR

AZI

L

CA

NA

DA

CH

INA

FRA

NC

E

ITAL

Y

UTA

H

SA

N P

AB

LO B

AY

SA

N F

RA

NC

ISC

O B

AY

- R.A

.C.

+ + ± + + + - - + +

+ + ± + + + + - + +

+ + + + + + - - +

± + ± ± ± - ± - + +

+ + + + + + + + +

Table III: Intra-strain variability in levels of the essential fatty acid 20: 5w3. Data are given as percentage of total fatty acid methyl esters and represent analyses of samples taken over serveral seasons or years.

Artemia Geographical Strain 20: 5w3 content (area %)

San Francisco Bay 0.3- 13.3

Brazil 3.5 - 10.6

China 1.3-15.4 .

Canada 5.2 - 9.5

Utah - Southern Arm 2.7-3.6

- Northern Arm 0.3 - 0.4

Fig. 1 Summary of the requirements a food must meet to be a practical diet for larval organisms. Fig. 2 The size ranges of various Artemia life stages.

Fig. 3 Change in energy content and dry weight of different forms of Artemia. Instar I nauplii (newly hatched) are considered to have 100% values for those variables. The percent decrease or increase from 100% is shown for, successively, instar II-III nauplii, cold-stored instar I nauplii, and decapsulated cysts.

Fig. 4 Percent survival of winter flounder larvae fed on five geographical strains of Artemia (after Klein-MCPhee et al., 1980).

Fig. 5 Percent survival of mud crab larvae fed on five geographical strains of Artemia (after Johns et al., 1980).

Fig. 6 Linear relationship between the essential fatty acid (20:5w3) content of several Artemia collections from San Francisco Bay and the biomass of mysids to which the Artemia were fed (data from Leger et al, 1985c).

Fig. 7 Linear relationship between the chlorinated hydrocarbon content of several Artemia collections from San Francisco Bay and the biomass of mysids to which the Artemia were fed (data from Leger et al., 1985c).

Fig. 1: Food requirements for larval organisms

For the culturist—

— consistent availability

— simple production procedures

— euryplasticity and versatility

— salinity / temperature tolerance

— handling

— disinfection

— different sizes and forms

— use as a carrier

For the predator—

— physical requirements

— clean

— no alien materials

— no diseases

— acceptable

— perceptible

— cacenable

— palatable

— ingestable

— nutritional requirements

— digestible

— nutrient requirements

Figure 2

Figure 3

Figure 4

Figure 5

IMPROVED LARVICULTURE OUTPUTS OF MARINE FISH, SHRIMP AND PRAWN

P.SORGELOOS and PH. LÉGERBELGIUM

Much progress was made in the late 1980s in the commercial and experimental larviculture of different species with aquaculture interest, e.g., different species of penaeid shrimp, Macrobrachium prawn, European and Asian bass and bream species, Japanese flounder, Atlantic turbot and halibut, Pacific mahimahi. Improved nutrition, especially through the application of enrichment diets for enhancing the nutritional value of the live feed organisms Brachionus plicatilis and Artemia spp., as vell as the use of live feed supplements and substitutes, and the application of improved zootechni-cal methods, all have contributed to a significant increase in larval survival and quality. Better and more predictable hatchery outputs at reduced production costs offer higher guarantees for successful commercial larviculture. More competitive industrial larviculture, especially with the marine fish species will only be possible by further improvements in larval nutrition, zootechnical aspects and disease diagnosis as well as control.

Dependable availability of fry, fingerlings or postlarvae is one of the critical factors in the commercial success of the industrial production of marine fish or crustaceans (FAO 1989). Only for a few species with unique ecological and ethological characteristics can wild sources of seed be used for the stocking of growout ponds or cages. For example, in Ecuador penaied shrimp postlarvae are being collected from coastal areas by the billions per year. This cheap source of readily available seed has been at the origin of the multimillion US dollar shrimp farming industry in Ecuador (Lee 1989). Only wild fry are used for annual production of several hundred thousand tons each of the milkfish Chanos chanos in the Philippines and Indonesia, and the yellowtail Seriola quinqueradia-ta in Japan. However, when wild penaeid seed provisions suddenly decreased a number of years ago in Ecuador, extended pond areas could not be kept in operation (Spurrier 1988). Milk-fish and penaeid shrimp farming in the Philippines are often restricted because of seasonal fry shortages. The Japanese government resorted to restrictions on yellowtail cage farming to prevent overfishing of wild fry resources.

When applying intensive growout techniques, shrimp farms need to operate at maximum capacities in order to show a good profit and therefore require guaranteed provisions of shrimp postlarvae on a year-round basis. In recent years serveral species of marine fish (e.g., several bass and bream species, mahimahi, turbot, halibut, etc.) have been identified as new aquaculture candidates. Their growout in cages or land based systems have proven to be commercially attractive provided a regular supply of fry can be guaranteed. For most fish and crustaceans with aquaculture potential this can only be realized by the domestication of the species, i.e., the development of appropriate techniques for controlled reproduction and larviculture.

Larviculture nutrition, particularly first feeding by the early larval stages, appears to be the major bottle-neck for the industrial upscaling of the aquaculture of fish and shellfish. With a few selected species such as salmon, minimal problems had to be overcome. Within a few years billions of hatchery produced fry were used in the commercial production of over 100,000 tons of marketable salmon per year. The early development of salmon does not involve feeding problems as the larvae at hatching carry a large yolk sac with enough food reserves for the first three weeks of their development. Once the yolk is consumed and exogenous feeding begins, larvae already

have a large mouth and can thrive on formulated feeds. With most other marine fish, egg production might not pose the major problem, but larval size at hatching and first feeding is at the origin of forming difficulties (Table 1). Most marine fish with aquaculture potential have very limited yolk reserves at hatching, mostly lasting for not more than one or two days. At first feeding they still have small mouths, often with an opening of less than 0.1 mm (Glamuzina et al. 1989 ; Kohmo et al. 1988). In shrimp larvae feed size is not the only problem ; larvae pass through different larval stages changing from a herbivorous filter into a carnivore.

Table 1. Size of eggs and larval length at hatching in different species of fish (Jones and Houde 1981)

Species Egg diameter

(mm) Length of larvae

(mm)

Salmon (Salmo salar) 5.0-6.0 15.0-25.0

Trout (Salmo gairdneri) 4.0 12.0-20.0

Carp (Cyprinus carpio) 0.9-1.6 4.8-6.2

Bass (Dicentrarchus labrax) 1.2-1.4 7.0-8.0

Bream (Sparus aurata) 0.9-1.1 3.5-4.0

Turbot (Scophthalmus maximus) 0.9-1.2 2.7-3.0

Sole (Solea solea) 1.0-1.4 3.2-3.7

Milkfish (Chanos chanos) 1.1-1.25 3.2-3.4

Grey mullet (Mugil cephalus) 0.9-1.0 2.2-3.5

Grouper (Epinephelus tauvina) 0.77-0.90 1.4-2.4

Bream (Acanthopagrus cuvier) 0.78-0.84 1.8-2.0

The natural diet of most aquaculture fish and crustacean species consists of a wide diversity of phy-toplankton species (diatoms, flagellates, etc.) and zooplankton organisms (copepods, cladocerans, decapod larvae, etc.) found in great abundance in the natural environment. Relying on the collection of wild plankton as a larval food source in intensive aquaculture has proven neither to be a relable nor a commercially feasible strategy. Over the past two to three decades trial and error approaches have resulted in the adoption of selected larviculture diets. This science has been very empirical and can a posteriori elegantly be split up in selection criteria that best suit either the larvae's requirements or the farmer's restrictions (fig.l).

Today three groups of live diets are widely applied in industrial larviculture of marine fish and crustaceans : 1) different species of 2-20 µm large micro algae; 2) the 50-200 µm rotifer Brachionus pli-catilis ; 3) the 200-500 µm brine shrimp Artemia spp. In recent years different formulations of supplementation and substitution products have been added to this list.

MICROALGAE

Diatoms and green algae are the two dominant groups of cultured microalgae (De Pauw and Pruder 1986). Food species have been selected on the basis of their mass culture potential, cell size, digestibility, and overall food value, much more by trial and error than any other scientific selection process.

The most suitable species still pose many problems for large scale culture, not least with regard to contamination problems. As a result most farms still apply labor intensive and expensive batch production systems. Even when production targets can be maintained with regard to cell numbers produced, shrimp farmers for example have experienced temporal variations in algal food values resulting in inconsistent hatchery outputs (R. Chamorro, personal communication).

Figure 1 : Selection criteria for larval foodsources from the viewpoint of the culturist and the cultured larva (Méger et al 1987a).

Olsen (1989) reviewed the literature and illustrated that the content of the (n-3) highly unsaturated fatty acids (HUFA's) 20:5 (n-3) and 22:6 (n-3) can greatly vary among algal species but even from culture to culture within a given species (Fig. 2). Using penaeus stylirostris as a test organism, Léger et al. (1985a) demonstrated that the content of 20:5(n-3) and 22:6(n-3) in the zoea diet had a major impact on survival and growth in later stages, when animals had already been switched to another diet (fig. 3). This provided the rationale to look for alternatives or supplements to live microalgae. Today different approaches and formulations are already being applied at the commercial level, and many new developments in more cost-effective products are to be expected. Freeze-dried algae (Taylor 1990), manipulated yeasts (Léger et al 1987b; Léger and Sorgeloos, in press; Coutteau et al. 1989), micro-encapsulated feeds (Jones et al. 1979), and different kinds of microparticulate diets (Langdon and Waldock 1981 ; Kanazawa et al. 1982; Chamberlain 1988) are gradually reducing the need for and might eventually totally replace the microalgae in commercial larviculture in the near future.

THE ROTIFER BRACHIONUS PLICATILIS

Rotifers are mostly used as a starter diet in marine fish larviculture (Fukusho 1989). Their culture appears to be simple, with microalgae (often Chlorella spp.) supplemented with bakers' yeast as their feed. However, many fish hatcheries have reported that they experience considerable problems in maintaining large culture and producing on a predictable basis the massive numbers of rotifers that are needed to feed the hundreds of housands to millions of baby fish they have in culture.

Besides zootechnical aspects (e.g., water management) food appears to be one of the key elements in the successful mass production of rotifers. For convenience, fresh

bakers' yeast is mostly used as the main diet ingredient. However, its freshness, a criterion which is difficult to evaluate by the farmer, can greatly influcence the dietary value of the yeast for the rotifers, and as a consequence determine rotifer culture success. Many farmers supplement the baker's yeast with microalgae, a procedure which at the same time ensures an increase of the level of (n-3) essential fatty acids in the rotifers. This (n-3) HUFA enrichment is critical in enhancing the food value of the rotifers for marine fish larvae (Fukusho 1989).

Different microparticulate (Rimmer and Reed 1989) and emulsified formulations (Watanabe et al. 1983; Léger et al. 1987c, 1989) are used as a booster-of essential fatty acids and other components. The treatment is performed for 4 to 24 hours prior to feeding the rotifers to the fish larvae. A new tendency is to simplify procedures by using feed-products for the combined culture and enrichment of Brachionus (Komis et al. 1989).

Figure 2 : Content of the essential fatty acids 20 : 5(n-3) in marine diatoms, green algae and dino-flagelattes (Olsen 1989)..

Figure 3 : Effect of (n-3) HUFA content in the larval diet on postarval survival in Penaeaus styli-rostris (modified after Léger et al. 1985a).

THE BRINE SHRIMP ARTEMIA SPP. Of the live diets used in larviculture, brine shrimp Artemia nauplii constitute the most widely used species. Although its production and use appear to be most simple, considerable progress has been made in the past decade in improving and increasing its value as a larval diet. It appeared indeed that many small details in hatching procedures, which in the past have often been overlooked, e.g., light and pH, could significantly affect cyst hatching outputs (Sorgeloos 1980; Sorgeloos et al. 1986). The optimization in Artemia cyst use was also realized by the commercial provision of high quality cysts products. Although recent harvests of cysts have been plentiful, particularly at Great Salt Lake (Utah, USA), increased competition in the market has contributed to the development of improved methods for cyst cleaning and processing, resulting in the adoption of a more rigorous quality control (Bengtson et al. 1991).

Better knowledge of the biology of Artemia was at the orgin of the development of methods for cyst disinfection and decapsulation (Sorgeloos et al. 1986). These procedures are being applied at several large fish and shrimp hatcheries to sterilize the cysts and remove the shells as to reduce the problems at naupliar harvest.

For a long time farmers have overlooked the fact that Artemia nauplii in their first stage of development cannot take up food and thus consume their own energy reserves. At the high water temperatures which are applied for cyst incubation, the freshly-hatched Artemia nauplii develop into the second larval stage within a matter of hours, eventually losing up to 30% of their energy reserves and food value (Benijts et al. 1976). This is not only a significant financial loss for the farmer, as he has to consume up to 30% more cyst product to produce the same quantity of food, he is furthermore feeding his fish or shrimp with a less suitable prey as the older Artemia have grown bigger, swim faster, are

less visible, and have a reduced food value. Therefore, rigorous standardization of hatching procedures is a must. The cyst should be incubated at constant water temperatures.and the nauplii harvested when they are still in their most nutritious stage. Several hatcheries have switched to a practice of daily multiple hatching and separation procedures. An easier and already common practice is to apply cold storage of the freshly-hached nauplii in concentrations of several million nauplii per liter at temperatures of 5-10 C (Léger et al. 1983). Aeration needs to be provided in order to prevent suffocation of the Artemia which barely move at these cold temperatures and eventually sink to the bottom of the container. Applied for periods of 24 h or even longer the cold-stored Artemia remain viable without consuming their energy reserves. This allows the farmer not only to ensure the availability of a better quality product but at the same time to consider more frequent food distribution from one single hatching. This appeared to be benefical for fish and shrimp larvae as food retention times in the larviculture tanks can be reduced and hence growth of the Artemia in the culture tank can be minimized. For example, applying one or even two feedings per day, shrimp farmers often experienced growth of the Artemia in their larviculture tanks so that the Artemia and shrimp were competing for algae.

Easy hatching and disinfection procedures, however, appeared not to be the sole parameters in ensuring the success of using Artemia as a larval food source. Several other Artemia characteristics may influence the suitability of a particular brine shrimp product for one or another type of larviculture. One of these is nauplius size which can greatly vary from one geographical source of Artemia to another (Vanhaecke and Sorgeloos 1980). This is particularly critical for several species of marine fish that have a very small mouth size and swallow their prey in one bite. For example, using the marine silverside Menidia menidia as a test-organism, Beck and Bengtson (1982) were able to illustrate a high correlation between Artemia nauplius size and larval fish mortality during early development; with the largest strains of Artemia, up to 50% of the fish could not ingest their prey and starved to death. Another important dietary characteristic was identified in the late 1970s to early 1980s when many fish and shrimp farmers reported unexpected problems when switching from one geographical source of Artemia to another (Sorgeloos 1980). Japanese, American and European researchers studied these problems and soon confirmed variations in nutritional value of different geographical sources of Artemia for fish and shrimp species (Watanable et al. 1983; Léger et al. 1986). The situation became more critical when significant differences in production yields were obtained with distinct batches ot the same geographical origin of Artemia Multidisciplinary studies in Japan (Watanabe et al. 1983) and by the International Study on Artemia (Léger et al. 1985b, 1987a) revealed that the concentration of the essential fatty acid eicosapentaenoic acid (EPA) 20:5(n-3) in the Artemia nauplii determines the nutritional value of this particular batch of Artemia for the larvae of various marine fish and crustacean species. Fig. 4 illustrates the results obtained with different batches of the same geographical Artemia source, containing different amounts of EPA and yielding proportional results in growth and survival of Mysidopsis bahia fed these Artemia.

Figure 4. Linear relationship between the 20:5(n-3) content of several Artemia collections from San Francisco Bay orgin and the biomass of Mysidopsis bahia to which the freshly hatched Artemia were fed (Léger et al 1987a)

Table 2 : Intra-strain variability of 20:5 (n-3) content in Artemia. Data represent the range (area percent) and coefficient of variation of data as compiled by Léger et al. 1986 and 1987a.

Artemia geographical strain 20:5(3-3) range (area %) Coefficient of variation %

USA-California: San Francisco Bay 0.3-13.3 78.6

USA-Utah Great Salt Lake (S arm) 2.7-3.6 11.8

USA-Utah Great Salt Lake (N arm) 0.3-0.4 21.2

Canada-Chaplin Lake 5.2-9.5 18.3

Brazil-Macau 3.5-10.6 43.2

PR China-Bohai Bay 1.3-15.4 50.5

As can be seen in Table 2, EPA levels in Artemia can greatly vary, even from one batch to another within the same strain. Cyst products from inland sources appear to be more constant in composition, however, at low levels of EPA. As a result concentrations of the (n-3)HUFA EPA need to be taken into consideration when selecting the most appropriate batch of Artemia cysts. In this respect the introduction of quality certificates to characterize commercial batches of Artemia cysts is highly recommended.

Commercial provisions of small-sized Artemia cysts containing high EPA-levels are limited. Their use, therefore, should be limited to the feeding period when size of the prey is most critical. Indeed even the best natural Artemia products do not meet all the nutritional requirements of the predator larvae, most particulary with regard to the other essential fatty acid for marine organisms, docosa-hexaenoic acid (DHA) 22:6(n-3), which is never available in significant amounts in Artemia cysts (Léger et al. 1986; Bengtson et al. 1991).

It is fortunate that Artemia, because of its primitive feeding characteristics, allows a very convenient way to manipulate its biochemical composition. Since Artemia, once it has molted into the second larval stage (i.e., about eight hours following hatching), is non-selective in taking up particulate matter, simple methods have been developed to incorporate any kind of product into the Artemia prior to offering it as a prey to the predator larva. This method of «bio-encapsulation», also called Artemia enrichment or boosting, is widely applied at marine fish and crustacean hatcheries all over the world for enhancing the nutritional value of Artemia with essential fatty acids. British, Japanese and Belgian researchers developed enrichment products and procedures using selected microalgae and/or micro-encapsulated products, yeast and/or emulsified preparations, using self-emulsifying concentrates and/or micro-particulate products respectively (Léger et al. 1986; Bengtson et al. 1991). The highest enrichment levels in Artemia as well as in the rotifer Brachionus are obtained when using emulsified concentrates (Table 3) (Léger et al. 1987a; Bengtson et al. 1991).

PROGRESS IN LARVICULTURE NUTRITION

The use of (n-3) HUFA - enriched Artemia as a more adequate food source has without any doubt been at the origin of a real break-through in the larviculture of many marine fish species (Watanabe et al. 1983; Sorgeloos et al. 1987, 1988). For example, for the European bass Dicentrarchus labrax and seabream Sparus aurata, the adoption of this «bio-encapsulation» methodology has allowed the transition from pilot to commercial larviculture of these species (Frentzos and Sweetman 1989). The effects of feeding (n-3) HUFA-enriched Artemia and Brachionus indeed are significant. Dicentrachus larvae died off before day 35 when fed (n-3) HUFA-deficient, freshly-hatched Artemia nauplii from Great Salt Lake cysts (Van Ballaer et al. 1985). Enriched Artemia of the same batch fed to the larvae resulted not only in increased survival but also in the production of bigger larvae which better resisted stress conditions (Franicevie et al. 1987).

Similar observations of increased survival and growth when feeding (n-3)HUFA-enriched diets have been confirmed for several species of penaeid shrimp where sometimes effects of diet composition only came to expression in later stages (Léger et al. 1987b; Tackaert et al. 1989; Léger and Sorgeloos, in press). A good illustration of this is the resistance to salinity stress in PL-10 stages of a batch of Penaeus monodon fed on three different larval diets that varied in (n-3)HUFA levels. Differences in survival among the three treatments were not significant at PL-10 before the stress test. However, differences in PL quality, expressed as their ability to survive the salinity stress applied, were very pronounced (Fig. 5). resistance to salinity shocks, which can easily be applied at the hatchery level, is now being used as a quality criterion for determining the appropriate time for PL transfer from the hatchery to the ponds (Sorgeloos 1989).

With the freshwater prawn Macrobrachium rosenbergii (n-3)HUFA-requirements of the larvae were anticipated not to be very ciritical in view of the fact that they spend most of their life in freshwater. These assumptions, however, were largely contradicted by the results of Devresse et al. (1990) and Romdhane et al. (1990) who used Artemia enriched with different (n-3)-HUFA emulsions. Besides the improved growth rate, a distinct difference having an important impact for the commercial farmer was the more precocious and more synchronous metamporphosis as well as the higher stress resistance of the Macrobrachium postlarvae that had received (n-3)HUFA-enriched Artemia in the larval stages.

Table 3. (n-3) HUFA content in rotifers (Brachionus plicatilis) and Artemia enriched with different products (Léger et al. 1989.

(n-3) HUFA Content 20:5(n-3) 22:6(n-3)_ £(n-3)HUFA

Treatment Area % mg/ga Area % mg/g Area % mg/g

Brachionus plicatilis grown on algae (Tetraselmis sp. and marine Chlorella) plus baker's yeast = C1 3.0 2.0 1.0 0.6 4.8 3.1

C l + 8 h enrichment with Isochrysis galbana 3.8 1.8 2.8 1.3 8.7 4.0

C l + 8 h enrichment with Nannochloropsis spp. 6.2 3.5 1.3 0.7 9.0 5.1

C l + 24 h enrichment with Chlorella japonica 10.9 9.3 0.6 0.5 12.8 10.9

C l + 24 h enrichment with w-yeast 6.6 3.9 5.5 3.1 13.8 8.0

C l + 6 h enrichment with SELCOb 11.3 22.8 5.6 11.4 20.1 40.4

C l + 6 h enrichment with SUPER SELCOb 19.5 32.8 21.3 36.0 44.9 77.0

Artemia

Freshly hatched Great Salt Lake (UT-USA) Artemia nauplii = C2 3.5 4.3 4.0 4.9

C2 + 24 h enrichment with PROTEIN SELECOc 9.4 12.6 3/7 4.9 14.2 19.0

C2 + 24 h enrichment with SELCO 9.9 21.3 5.9 12.7 17.8 37.4

C2 + 24 H enrichment with SUPER SELCO 15.4 28.4 12.4 22.9 30.2 55.7a mg fatty acid methyl ester per gram dry weight rotifer or Artemia. b self emulsifying (n-3)HUFA enrichment concentrates (Artemia Systems, S.A., Ghent, Belgium). c protein based (n-3) HUFA enrichment product (Artemia Systems, S.A., Ghent, Belgium).

Figure 5. Survival of Penaeus monodon PL-10 cultured on larval diet combinations that contained low, medium and high levels of (n-3)HUFA's after 60 min transfer from 35 ppt to 7 ppt seawater (Tackaert et al 1989).

Where as for a number of species larviculture outputs have been improved through developments in nutritional manipulation of the live feeds (various papers presented at the Larviculture Special Session of Aquaculture '90, Halifax, Canada), with other species such as mahimahi, turbot, halibut and others, research is still in progress to better define quantitative (n-3)HUFA requirements; it appears that for many species of marine fish optimal dietary levels have not been reached yet in Brachionus and Artemia. Furthermore, although (n-3)HUFA's might have proven most critical so far, other nutrients, e.g. other lipid classes, particular peptides, free amino acids, pigments, sterols and vitamins might appear equally important and in some species may be more critical. In view of the better results obtained with the use of natural plankton, consisting mostly of marine copepods, in cul-turing turbot and mahimahi (S. Kraul, personal communcation), the challenge remains to identify the vital components in copepods in order to have these incorporated in the convenient dietary system consisting of enriched Brachionus and Artemia.

Figure 6. Artemia concentrator-rinsor as manufactured by Artemia Systems SA, Ghent, Belgium (Léger and Sorgeloos, in press).

ZOOTECHNICAL ASPECTS

In the past five years the number of capacities of marine fish and crustacean hatcheries have escalated all over the world. The intensification of hatchery activities, however, brough about several new problems not seen at an experimental scale. For example, skeletal deformities and absence of swim bladder inflation, long blamed to be genetic disorders, appeared to be caused by imperfect zootech-nical procedures (Foscarini 1988). An invisible oil-film at the tank's surface hampered the larval fishes' ability to gulp air and fill their swim bladders. As a result, these fish needed to spend more swimming efforts to stay in the water column, which eventually caused spinal deformities. These problems are largely overcome today by application of more rigorous washing procedures of the live food, especially after enrichment with the high lipid containing products. New equipment and materials were introduced in the hatcheries. For example, welded wedge filters (Fig. 6) are not only very efficient in washing and cleaning; at the

same time they guarantee that the Artemia are not physically damaged during this process. Oil skimmers (Fig.7) installed in the fish culture tank ensure final prevention of the build-up of an oil film, which might also be enhanced by algae metabolites when applying the green water technique.

SORGELOOS AND LEGER

Figure 7. Shematic drawings of four types of surface cleaners used in Europe in the larviculture of seabass Didentrarchus labrax and seabream Sparus aurata:A) model fixed to the side (BB) of the culture tank: B) C) and D) are floating models; the arrow shows the distribution of compressed air (Dewavrin and Chauveau 1990).

DISEASE PROBLEMS

Bacterial and viral outbreaks have caused enormous interferences in the successful industrialization of fish and crustacean larviculture. lack of basic hygienic precautions has been and still is at the origin of most problems (Brown 1989); however, all kinds of chemotherapeutics are applied both for desease prevention and treatment. Products have been used following trial and error procedures, not based at all upon expert advice. A wide range of broad spectrum antibiotics became routine application in shrimp and marine fish operations. Products such as chloramphenicol, tetracycline, and furazolidone are often applied at daily doses up to 50 ppm in the case of marine fish. Short-term benefits soon turned into disasters. Worst affected has been the shrimp culture industry in which at some times and places all larviculture activity had to be suspended (Chamberlain 1988).

Although there is still much room for improvement, not the least by a better identification and documentation of the disease problems, better preventive measures are being implemented. For example, routine disinfection procedures of culture tanks and live food culture facilities, as well as regular dry-outs between two to three consecutive larviculture runs, are widely adopted now and have resulted in more predictable hatchery outputs.

In marine fish larviculture it appears that microbial problems are less critical in those hatcheries that are operating recirculation systems using biological filters. In this respect there is a great need to document the microbial environment in fish and crustacean hatcheries not only at critical moments but also when outputs are optimal.

As was recently demonstrated, the bioencapsulation methodology with Artemia and Brachionus can also be considered for more effective transfer of therepeutics through oral administration of antibiotics (Léger et al. 1990).

FUTURE PROSPECTS

In the past decade marine fish and crustacean hatcheries have evolved from hit and miss ventures into profitable ventures. With many fish species the larviculture industry has not yet reached the state of competitive maturity. In Europe, for example, the

situation is artificial because fry outputs cannot meet present demands. As a result hatcheries can sell their fingerlings at prices that range from approximately 1 US dollar per individual fingerling of bass and bream up to 4 US dollars per turbot fry. Profit margins are high; however, one should not forget that many of these hatcheries have lost money for years when they were pioneering marine fish larviculture. The number of marine fish hatcheries is increasing very fast in Europe and the ones already in operation are steadily increasing their capacities (Artemia Systems NV/AS, personal communication). The situation may soon turn, similarly to what has been experienced in recent years with the prices of salmon smolts and penaied shrimp postlarvae. In Taiwan, Thailand, and Ecuador, market prices of Penaeus monodon and P. van-namei crashed from a year-long top price of 20 US dollars per 1,000 PL to extremely competitive prices of a few dollars only. Big hatcheries with high investment and over-head costs may only remain viable if they can operate on a year-round basis and at maximal efficiency. Some of the big operations in Ecuador for example manage to yield survival rates exceeding 80% at harvest of the PL's.

Backyard hatcheries (e.g., in Thailand and Indonesia) are a peculiar phonomenon as these are mostly hit and miss operations (Yap 1990). They are managed with limited expertise, mostly under poor water quality and hygienic standards. Nonetheless they have become more and more important and mushroomed into the thousands during the last couple of years in some SE Asian countries. As these family activities involve minimal operational costs they can afford low success rates and still remain profitable at PL prices which are beyond the limits for profitable production by the large hatcheries. However, they impose considerable risks for total collapses of local aquaculture activities as was the case with the once very successful shrimp farming industry in Taiwan (Liao 1989). In order to become a stabilized industrial activity that meets the requirements of local growout capabilities the larviculture of marine fish and crustaceans needs to further improve its outputs. With survival rates in marine fish larviculture rarely reaching 20-30%, it is clear that there is still much room for improvement and incrased cost-effectiveness. In the short term the following fields need more exploration: 1) how can egg quality be better defined and controlled?; 2) present knowledge about qualitative and quantitative nutritional requirements is still very limited for most species; 3) too limited attention has been paid to zootechnical aspects in relation to upscaling, automation, recirculation systems, intensive versus extensive systems, etc.; and 4) better understanding of the microbial environment in the hatchery as well as the larval immune system will allow better management with regard to disease prevention and control.

A final remark needs to be made regarding feeding strategies in larviculture, more particularly the future of live feeds versus formulated feeds. Off-the-shelf dry products are being developed and commercialized as a much more convenient application for the farmer. With some species, such as penaied shrimp, the moment is approaching for reducing live algae and Artemia feeding to virtual elimination from the hatchery operation. Other species, such as marine fish species, impose much more constraints than shrimp, not only in terms of nutritional requirements but also with regard to physical properties of the feed, e.g., water stability, buoyancy, paatability, etc. Appropriate process technologies will certainly be developed but their commercial applicability and price competitiveness might not be obvious. Feeding strategies in hatcheries of marine fish and crustaceans will probably never be standardized world-wide because of species differences and geographical discrepancies. The cost-effectiveness of live and formulated feeds will dictate their proportional use.

ACKNOWLEDGMENTS

This research has been sponsored by the Belgian National Science Foudation, the Belgian Administration for Development Cooperation, the Belgian Ministry for Science Policy, and the Belgian Company Artemia Systems S.A.

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THE USE OF BRINE SHRIMP ARTEMIA IN BIOLOGICAL MANAGEMENT OF SOLAR SALTWORKS

WIM TACKAERT1 AND PATRICK SORGELOOS2

1 SALT LAKE BRINE SHRIMP, INE, GRANTSVILLE, UTAH, USA2LABORATORY OF AQUACULTURE AND ARTEMIA REFERENCE CENTER,

UNVERSITY OF GHENT, BELGIUM

ABSTRACT In recent years, there has been a growing awareness of the hydrobiological aspects of the solar salt production process. Saltworks are man-managed artificial ecosystems that are highly vulnerable to biological disturbances, including uncontrolled proliferation of microalgae resulting in a reduced evaporation and contamination of the salt with gypsum and insoluble organic materials.

Optimal production of solar salt; both in terms of quality and quantity, requires a well-established balance between the primary and secondary producers, with brine shrimp Artemia grazing on phy~ toplankton constituting the major interaction. In this paper, we discuss the beneficial role of Artemia in balancing the hydrobiological activity of the salt pond system and highlight some of the critical aspects essential to proper management of Artemia, including selection and controlled introduction of the most suitable strain of Artemia.

Furthermore, the possibilities for establishing a vertically integrated aquaculture industry brough about by the opportunities for harvesting of Artemia cysts and biomas as valuable by-products pf the solar salt operation will be discussed. Results of experiences gained in different projects around the world will be presented.

THE NATURAL OCCURRENCE OF ARTEMIA

The brine shrimp Artemia is a small crustacean which is widely distributed on the five continents in hypersaline biotopes including salt lakes (coastal or inland waters riche in chloride, sulphate or carbonate) and especially in coastal salinas (man-made and/or managed solar saltworks). Detailed reviews can be found in Persoone and Sorgeloos (1980) and Sorgeloos et al. (1986). The very specific and large range of ecological characteristics of these Artemia habitats have resulted in the evolution of many geographical strains. At present over 350 different geographical strains are known (Vanhaecke et al, 1987)

In saltworks Artemia is found in the evaporation ponds only at intermediate salinity levels from about 100 ppt, the upper tolerance level of predators, to about 200-250 ppt, when food becomes limiting, and the Artemia need more energy for osmoregulation or when the water becomes more toxic in ionic composition as result of selective crystallisation of slats (see schematic outline in Fig. 1). At high salinities, depending in the local strain as well as the hydrobilogical conditions in the ponds (e.g. water retention time, water depth, pond productivity) cysts of Artemia (see Fig. 2) are produced seasonally or year-round. They float, tend to be driven by the wind, and often accumulate on the shores of the evaporation ponds.

HARVESTING OF NATURAL ARTEMIA HABITATS

Recent developments in aquaculture production of fish and shrimp have resulted in increased demands for Artemia as a valuable source of live feed. The present world market of Artemia cysts for use in aquaculture is estimated at over 700 t/year. Although

the large part of this cyst market is currently supplied by harvests from one single location, i.e. the Great Salt Lake, Utah, USA, there is a steadily-growing interest in the commercial harvesting of other Artemia biotopes which is brought about by local import restrictions and/or the increasing demand for Artemia products. The quality of the Artemia produced differs from strain to strain and from location to location as a result of genotypical and phenotypical variations (for reviews see Leger et al., 1986, 1987a). It largely reflects the food conditions of the local habitat; adults as well as cysts may be contaminated with high levels of heavy metals, and/or may be deficient in fatty acids essential for marine predators; furthermore, particular strains in specific habitats may produce cysts with unusually low content, e.g. the «sulphate strain» in Chaplin Lake, Canada (Vanhaecke et al., 1983). In this regard it is imperative to determine the nutritional quality of the adult Artemia and/or its cysts for specific aquaculture purposes prior to consider commercial use of natural Artemia biotopes.

Techniques for cyst/biomass harvesting and treatment are outlined by Sorgeloos et al. (1986). Maximum sustainable yields of cysts and biomass are influenced by the population dynamics of the local Artemia population. The recruitment rate of the population may be high in ponds where the dominant reproduction mode is ovoviviparity, and low in cyst-production ponds. In the low salinity ponds it may be influenced by the role cysts as inoculum, either after the winter or throughout the year. Furthermore, production by water birds needs also to be taken into consideration. The determination of maximal havesting rates is complicated by the heterogenous distribution of the Artemia, which makes accurate sampling and consequently precise population estimates very difficult (for more details, see Wear and Haslett, 1987 a,b). Natural recuitment can eventually be increased by introduction of a more productive strain. Fertilization of the Artemia ponds can also result in increased production potentials.

In most Artemia habitats population densities are very low as a result of food limitation due to low nutrient contents of the intake waters. Some solar saltworks, especially those located in highly eutro-phicated areas have, however, a very high productivity, e.g. Leslie saltworks in the San Francisco Bay, California, USA, and the solar saltworks along the Bohai Bay in P.R. China. In the latter area harvesting of cysts and especially biomass, used in local hatcheries and grow-out of white shrimps, has become a considerable industry employing several hundreds of people (Tackaert and Sorgeloos, 1992).

BENEFICIAL ROLE OF ARTEMIA IN SOLAR SALTWORKS

Since early times, man has developed systems to concentrate seawater and to harvest sodium chloride'as a basic need for his nutrition and health. Over the centuries hundreds and thousands of hectares of salt pans have been constructed, all over the world, in tropical and sub-tropical belts, for so-called solar salt making. The annual production presently amounts to about 200 Mt/year. Less than 10% is used for human consumption, the bulk being consumed by chemical industries (e.g. the chlorine-alkali industries). Seawater contains salts of almost every chemical element including gold in at least trace amounts. Solar salt is normally produced by pumping seawater from one evaporation pond into another, allowing carbonates and gypsum to precipitate, and finally draining NaCl-saturated brine or «pickle» (just before the so-called «salting point» is reached) into crystallizer ponds where sodium chloride precipitates. Before all the NaCl has crystallized out, the mother liquor, now called bittern, has to be drained off to reduce contamination of the sodium chloride with bromides and other salts that begin to precipitate at these elevated salinities. The technique of solar salt production thus

involves fractional crystallization of the salts in different ponds to obtain sodium chloride in the purest form possible, e.g. up to 99,7% on a dry-weight basis.

Fig. 1. Schematic diagram of solar salt operation with natural occurrence of Artemia (from Sorgeloos et al., 1986).

Fig. 2 Schematic diagram of Artemia life cycle (from Sorgeloos et al, 1986)

The hydrobilogical activity in a solar-salt operation largely determines the quality and quantity of salt produced (Davis, 1978, 1980; Sorgeloos, 1983). In many sites the natural conditions ensure a maximal salt production (e.g. in France, Brazil and South Africa); in other locations, however, proper biological management is needed (e.g. in P.R. China, India, Italy, Autralia, Bahamas and Venezuela). Algal blooms, induced by natural availability of organic and inorganic nutrients, are generally beneficial since they ensure increased solar heat absorption resulting in faster evaporation and increased yields of salt. However, if they are not metabolized in time algal excretion and decomposition products, such as dissolved carbohydrates, act as chemical traps and consequently

prevent early precipitation of gypsum which will contaminatate the sodium chloride in the crystaliizers and reduces salt quality. Furthermore, such organic impurities as algal agglomerations, which turn black on oxidation, may contaminate the salt and reduce the size of the crystals and hence the salt quality. In the worst situations, high water viscosities may completely inhibit salt crystal formation and pre-cipation. The presence of the brine shrimp Artemia in sufficient numbers is essential not only for controlling algal blooms (Davis, 1980), but also for providing essential nutrients from Artemia metabolites and/or decaying animals as suitable substractes for the development of Halobacterium in the crystallisation ponds (Jones et al., 1981). High concentrations of red halophilic bacteria promote heat absorption, thereby accelerating evaporation, and reduce concentrations of dissolved organics. Lower viscosity levels promote the formation of larger salt crystals, and thereby improve salt quality (Sorgeloos, 1983; Haxby and Tackaert, 1987). In many salt operations natural recruitment of Artemia from cysts dispersed by wind and water birds assures the presence and development of sufficient numbers of brine shrimp for optimal salt operation. In some situations, however, the salt producer should not rely on this opportunistic dispersion of Artemia. In saltworks with short water-retention times in their evaporation ponds, a rapid dilution may wash away the Artemia population; a hurricane or season of exceptionally heavy rainfall may eliminate or so reduce the lcoal population that it cannot effectively cope with the algae blooms. Some saltworks may be completely isolated from natural sources of Artemia dispersion. In such cases salt producers should optimize the hydrobiological activity in the evaporation ponds through a controlled introduction of brine shrimp. Situations have also been observed where the local Artemia population has a poor productivity and remains too small to control the algae and ensure an optimal hydrobiological activity for the salt production. The introduction of a foreign strain, better adapted to the prevailing conditions or with better production characteristics, may improve conditions for production of high quality salt. It is not possible to formulate a general strategy with regard to Artemia introductions in solar-salt operations. Each situation needs to be analyzed for specific requirements, with regard to selection of a suitable Artemia strain. The quality and quantity of Artemia to be introduced must be determined in consideration of the water retention times in evaporation reservoirs, food concentrations, water temperatures, etc. (Sorgeloos et al., 1986).

Proper Artemia management should lead not only to improved salt production outputs but also provide opportunities for the harvesting of the valuable by-product Artemia, as cysts and biomass.

INTRODUCTION OF ARTEMIA

Although Artemia is clearly cosmopolitan, a closer look at the regional level reveals that its distribution is discontinuous. Artemia does not occur in every existing body of seawater. Brine shrimps cannot migrate from one saline biotope to another via the sea, because they lack anatomical defences against predation by such carnivorous aquatic organisms as larger crustaceans and fish. The Artemia found in several saltworks have probably been accidentally introduced by man. Following and old custom, some salt farmers seeded new saltpans with salt, often containing Artemia cysts, from and operational saltwork. All Artemia populations in Australia were probably originally introduced by man and now compete, at least in low salinity ponds, with the endemic brine shrimp Par-artemia spp. (Geddes and Williams, 1987). The absence of a migration route of water birds probably explains why along the northeast coast of Brazil the very large salinas (several 10,000 ha in total area) contained no brine shrimp until Artemia franciscana was introduced in 1977 by man in just one saltern in Macau. A few years

later it had already been dispersed by local water fowl from Macau to most of the saltworkds of north-east Brazil, over a distance of more than 1,000 km (Camara and De Castro, 1983; Canara and De Medeiros Rocha, 1987).

Controlled introduction of Artemia by man into suitable biotopes not only provides interesting opportunities for aquaculture production but is also an interesting tool to balance the hydrobiological activity of those salt farms where Artemia is absent or too few to effectively cope with algal blooms. However, much caution is needed if one is to preserve the genetic diversity of indigenous brine shrimp populations, especially on the Australian continent, where several endemic species might be endangered by the presence of Artemia (see Geddes, 1980, 1981 ; Geddes and Williams, 1987). On other contients, detailed ecological analyses as well as collection and storage of viable cysts should precede any such new introductions.

Commercial considerations might eventually justify new Artemia introductions in solar salt operations where the salt production, quality and quantity, may be impaired by the absence or poor performance of local strains of Artemia (e.g. in Inida, Italy, Venezuela, Bahamas). Various so-called natural or indigenous Artemia populations may be illadapted to their environment because their local habitat has been modified by man in order to accommodate or improve salt production, resulting in new (sub-optimal) ecological conditions, e.g. in the deep Lago Salpi near Margherita di Savoia in Italy, which was converted into shallow evaporation ponds in which water temperatures in the summer rise above 30°C, lethal temperatures for the local A. parthenogenetica strain (Bargozzi and Trotta, pers. commun., 1980; Vanhaecke et al., 1984). Other examples are N. African Artemia populations of local commercial solar saltworks, which used to inhabit highly seasonal biotopes that filled up during winter precipitation periods and dried up during the summer. Originally adapted to maximize population growth at relatively low temperatures, they do not readily tolerate the high summer temperatures to which they are now exposed in the salinas. A critical aspect regarding Artemia introduction is the selection of the strain to be inoculated. An accidental introduction of A. parthenoge-netica from P.R. China into the solar salt operation on Great Inagua, Bahamas, resulted in significant reductions in salt quality and output (Morton Salt Cie, pers. commun., 1983). However, production returned to normal after the introduction of A. franciscana, which had previously been shown to control algal blooms under the local climatic conditions (E. Haxby, pers. commun., 1984). Serious problems resulting in sub-optimal conditions for solar salt production may also arise when natural re-colonization of the salt ponds after the winter season is retarded due to particular cimato-logical conditions. This is the case in the solar saltworks of the Bohai Bay (P.R. China). These solar saltworks are fed by highly eutrophicated waters causing an excessive accumulation of organic matter detrimental to salt production (Davis, 1991). Despite the abundant availability of food under the form of unicellular algae, Artemia densities in these salt ponds remain very limited especially during spring and are unable to remove sufficient amounts of organic matter. Overwintering cysts to repopulate the biotope during spring only hatch in the low salinity ponds due to absence of rain during this period. As a result, rapid colonization of the entire saltwork is prevented, because the high salinity ponds become populated with Artemia only when brine together with animals flow from the low salinity ponds to the downstream ponds. Repopulation of the biotope is furthermore exacerbated by the limited productivity of the local parthenogenetic strain at the lower temperature regimes prevailing in the ponds during spring as well as the poor resistance of this strain to high salinity (Tackaert and Sorgeloos, 1991). A recent inoculation trial, using Artemia franciscana from San Francisco Bay, USA, a strain selected for its high productivity at relatively low

temperatures and good resistance to high salinities (Vanhaecke and Sorgeloos, 1988), in a local experimental salt-works largely out-competed the parthenogenetic strain confirming its better suitability for the Chinese saltworks. Although not yet scientifically verified, better salt quality and higher yields of Artemia biomass were also reported in the Artemia franciscana inoculated salt ponds.

A new Artemia should be introduced only when one can be reasonably sure of its success, and certainly not before enough viable cyst material of the locally occuring strain has been collected to safeguard the conservation of this Artemia genepool. In accordance with a resolution adopted at the Second International Artemia Symposium, «...all possible measures (should) be taken to ensure that the genetic resources of natural Artemia populations are conserved». Such measures include the establishment of gene banks (cysts), close monitoring of inoculation policies and, where possible, the use of indigenous Artemia for inoculating Artemia-free ponds (Beardmore, 1987). Selection of the inoculated strain should be based on the available data on temperature and salinity tolerances (Vanhaecke et al., 1984); Vanhaecke and Sorgeloos, 1988), growth and production, reproductive characteristics, etc. Whenever possible, culture tests with various Artemia strains should be performed in simulated conditions, using the untreated waters of the habitat as culture medium. Competition between parthenogenetic and bisexual strains might favour the first when dealing with European bisexuals (A. tunisiana), although co-existence has been reported (Amat, 1983) with dominance of the parthenogenetic strain in the summer months. On the other hand we can confirm that A. franciscana strains always outcompete any other Artemia strain (Browne, 1980). Strain selection might also be restricted by the intended application of the produced Artemia in local aquaculture, e.g. and Artemia strain producing small cysts might be selected for use in sea-bass farming. Such introductions generally result in the permanent establishment of an Artemia population; introduction of an unsuitable strain cannot be readily rectified. Furthermore, adaptation of a newly inoculated strain may result in phenotypical and genotypical variations in the pre-existing stocks, eventually yielding a new Artemia genotype (Vanhaecke and Sorgeloos, 1988).

CONCLUSION

Now that it has been shown that salt making and Artemia production go hand in hand, one can envisage attractive joint ventures for shrimp and fish aquaculture operations to integrate with solar saltworks in some of the many thousands of hectares of salinas in the tropical-subtropical areas, often in climates that favour crustacean or fish farming. Furthermore, it can lead to an extra source of income for families in many developing countries (Sahavacharin, 1981).

ACKNOWLEDGEMENTS

This study has been supported by the Belgian National Science Foundation (NFWO-FKFO), the Belgian Administration for Development Cooperation and the Belgian Ministry of Science Policy.

REFERENCES AMAT, T., 1983. Zygogenetic and parthenogenetic Artemia in Cadiz sea-side salterns. Mar. Ecol. Prog. Ser., 13(2-3): 291-293.

BEARDMORE, J.A., 1987. Concluding remarks. In: P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors), Artemia Research and its applications. Vol. 1. Universal Press, Watteren, Belgium, pp. 345-346.

BROWNE, R.A., 1980. Competition experiments between parthenogenetic and sexual strains of the brine shrimp, Artemia salina. Ecology, 61(3): 471-774.

CAMARA, M.R. and DE CASTRO, E.V., 1983. Artemia salina L. (Anostraca): Uma opeao para aquicultura do Nordeste do Brasil. Revta Bras. Zool., S. Paulo, 1(3): 145-147.

CAMARA, M.R. and DE MEDEIROS ROCHA, R., 1987. Artemia culture in Brasil : an overview. In: P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors), Artemia Research and its Applications. Vol. 3. Universa Press, Wetteren, Belgium, pp. 195-199.

DAVIS, J.S., 1978. Biological communities of a nutrient enriched salina. Aquatic Botany, 4:23-42.

DAVIS, J.S., 1980. Experiences with Artemia at solar salt-works. In: Persoone, P. Sorgeloos, O. Roels and E: Jaspers (Editors), The Brine Shrimp Artemia. Vol. 3. Ecology, Culturing, Use in Aquaculture. Universa Press, Watteren, Belgium, pp. 51-55.

DAVIS, J.S., 1991. Biological management for the nutrient-rich Chinese solar saltworks. In : L. Cheng (Editor), Proceedings of International Symposium on Biotechnology of Salt-ponds, Salt Research Institute. Tanggu, Tianjin, PR China, 128-132 pp.

GEDDES, M.C, 1980. The brine shrimp Artemia and Parartemia in Australia. In : G. Persoone, P. Sorgeloos, O. Roels and E. Jaspers (Editors). The Brine Shrimp Artemia. Vol. 3. Ecology, Culturing, Use in Aquaculture. Universa Press, Wetteren, Belgium, pp. 57-65.

GEDDES. M.C, 1981. The brine shrimps Artemia and Parartemia. Comparative physiology and distribution in Australia. Hydrobiologia, 81: 169-222.

GEDDES, M.C. and WILLIAMS, W.D., 1987. Comments on Artemia introductions and the need for conservation. In: P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors). Artemia Research and Its Applications. Vol. 3. Universa Press, Wetteren, Belgium, pp. 19-26.

HAXBY, R.E. and TACKAERT W., 1987. Workshop report: Role of Artemia in solar salt operations. In: P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors). Artemia Research and Its Applications. Vol. 3. Universa Press, Wetteren, Belgium, pp. 291-293.

JONES, A.G., EWING, CM. and MELVIN, MY, 1981. Biotechnology of solar saltfields. Hydrobiologia, 82: 391-406.

LÉGER, P.H., BENGTSON, D.A., SIMPSON, K.L. and SORGELOOS, P., 1986. The use and nutritional value of Artemia as a food source. Oceanogr. Mar. Biol. Ann. Rev., 24:521-623.

LÉGER, PH., BENGTSON, D.A., SORGELOOS, P., SIMPSON, K.L. and BECK, A.D., 1987a. The nutritional value of Artemia, a review. In: P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors). Artemia Research and Its Applications. Vol. 3. Universa Press, Wetteren, Belgium, pp. 357-372.

PERSOONE, G. and SORGELOOS, P., 1980. General aspects of the ecology and biogeography of Artemia. In: G. Persoone, P. Sorgeloos, O. Roels, and E. Jaspers (Editors). The Brine Shrimp Artemia. Vol. 3. Ecology, culturing, use in aquaculture. Universa Press, Wetteren, Belgium, pp. 3-24.

SAHAVACHARIN, S., 1981. Ways to convert salt farm to Artemia farm (in Thai, with English abstract). Thai Fisheries Gazette, 34(5): 467-480.

SORGELOOS, P., 1983. Brine Shrimp Artemia in coastal salt-works: inexpensive source of food for vertically integrated aquaculture. Aquaculture Magazine, 9:25-27.

SORGELOOS, P., LAVENS, P., LÉGER, PH., TACKAERT, W. and VERSICHELE, D., 1986. Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture. Artemia Reference Center, State University of Ghent, Belgium, 319pp.

TACKAERT, W. and SORGELOOS, P., 1991. Biological management to improve Artemia and salt productions at Tank Gu Salt-works in the PR. China In : L. Cheng (Editor), Proceedings of International Symposium on Biotechnology of Salt-ponds, Salt Research Institute, Tanggu, Tianjin, PR China, pp. 78-83.

TACKAERT, W. and SORGELOOS, P., 1992. Salt, Artemia and shrimp. Integrated production in the Peoples' Republic of China: The Tang Gu Saltworks. World Aquaculture, in press.

VANHAECKE, P., LAVENS, P. and SORGELOOS, P., 1983. International Study on Artemia. XVII Energy consumption in cysts and early larval stages of various geographical srains of Artemia. Annls. Soc. r. zool. Belg., 113(2): 155-164.

VANHAECKE, P., SIDDAL, S.E. and SORGELOOS, P., 1984. International Study on Artemia. XXXII. combined effects of temperature and salinity on the survival of Artemia of various geographical origin. J.Exp. Mar. Bio. Ecol, 80(3): 259-275.

VANHAECKE, P. and SORGELOOS, P., 1988. International Study on Artemia. The effect of temperature on cyst hatching, larval survival and biomass production of different geographical strains of Artemia. Submitted for publication.

VANHAECKE, P., TACKAERT, W. and SORGELOOS, P., 1987. The biography of Artemia: an updated review. In: P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors), Artemia Research and Its Applications. Vol. 1. Universa press, Wetteren, Belgium, pp. 129-159.

WEAR, R.G. and HASLETT, S.J., 1987a. Studies on the biology and ecology of Artemia from Lake Grasmere, New Zealand. In : P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors), Artemia Research and Its Applications. Vol. 3. Universa Press, Wetteren, Belgium, pp. 101-126.

WEAR, R.G. AND HASLETT, S.J., 1987B. A minimal strategy for assessing Artemia biomass har-vestable from production salinas. In : P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Editors), Artemia Research and Its Applications. Vol. 3. Universa Press, Wetteren, Belgium, pp. 217-220.

SEMI-INTENSIVE CULTURING IN FERTILIZED PONDS

WIM TACKAERTand PATRICK SORGELOOS

INTRODUCTION

Artemia biomass, and cysts can be produced in intensive as well as semi-intensive and extensive conditions. Intensive production is performed in indoor tank systems under completely controlled conditions (see Chapter 12). Semi-intensive and extensive production refer to the culture of Artemia in outdoor conditions. The former is performed in small and managed salt pond systems (mostly seasonal solar saltponds); e.g. with some degree of control over salinity, water retention time, and feed availability. The latter (extensive production) consists of the harvesting of mostly natural Artemia populations from large biotopes with year-round high salinity conditions such as large solar salt operations or salt lakes.

Since Artemia is highly susceptible to predation, a major prerequisite to semi-intensive and extensive production is the availability of brine of sufficiently high salinity that is free from fish and invertebrates. In most saltpond locations/natural biotopes, this situation is reached at salinities from > 80 to 100 g/1, although situations have been reported where fish and insects were still present at salinities of > 100 to 130 pp.

Natural populations of Artemia are widely distributed over five continents in a variety of isolated biotopes such as inland salt lakes, coastal lagoons and especially coastal salterns associated with commercial solar salt production.2.3 A recent list of natural Artemia sites compiled by Vanhaecke et al.3 extends to over 350 localities.

In most natural Artemia populations, densities are low, mainly as a result of food limitation due to a low nutrient content of the water. The few exceptions which have higher productivity are those biotopes (mostly large solar saltworks) which are located in highly europhic areas (e.g., near population centers, estuaries, or mangrove areas) such as the Leslie Saltworks in the San Francisco Bay, and the salinas of the Bohai Bay in China.4 Production estimates for a few natural Artemia biotopes are given in Table 1. As a result of their generally low productivity, most of the natural biotopes offer opportunities for extensive harvesting of Artemia biomass only. Production of cysts in these biotopes, especially when the local ecological conditions are fairly stable, occurs only occasionally or is erratic. Moreover, since the quality of Artemia cysts differs from strain to strain and even from harvest to harvest5.6 it is imperative to determine its nutritional value for specific application in aquaculture, prior to commercial development.

A schematic outline of a typical solar saltwork is given in Figure 1. sea water flows over a series of successive ponds in which salinity gradually increases as sea water evaporates. During this process, salts with low solubility precipiate as carbonates and later as gypsum (see Figure 2). Finally, when the sea water has evaporated to approximately one tenth of its original volume, mother brine is transferred to the crystallizers where pure sodium chloride is deposited. Artemia is only found in the evaporation ponds of intermediate salinity, i.e., from approximately 90 g/1 (= upper tolerance level of predators) to approximately 200 to 250 ppt. At elevated salinities Artemia die as a result of either starvation because of increased energy associated with hyperosmoregulatory physiology7 and/or increased toxicity of the brine due to drastic changes in ionic composition caused by gradual precipitation and enrichment of different salts.

Very extended solar salt industries (i.e., highly mechanized operations of hundreds to thousands of hectares each with individual evaporation ponds of up to hundreds of hectares) are localized in climatic zones with high evaporation rates and restricted rainfall (e.g., various regions in Australia, South America, Mexico, United States, China, southern France, and southeast Italy) and are usually in production (though not necessarily harvesting) on a year-round basis. In contrast, numerous small cottage-scale units for artisanal solar salt production are in production in the tropical-subtropical belt only during a restricted period of the year; i.e., cycles of 3 to 6 months during the dry season when conditions are favorable for making salt.

Table 1 : Production Estimates for Natural Artemia Populations

Site Maximum Country

production Period Ref.

Lake Rezaiyeh Iran 1.2 aduslts/l 68 Sivash Salt Lakes U.S.S.R 400/1 69 Slagbaai Bonaire,

Netherlands Antilles

200-360/1 Oct. —June 70

4 adults/1 12 nauplii/1 June—Sept. 71

Mono Lake California

400/1 Aug.—Sept. 72 10/1 73 Great Salt Lake Utah 100—200g dw/m2 per year 74 10-100/1 March—Oct. 75 0.02-0.2 g/l ww

Salin de Giraud Camargue, France

16,000/m2 76 Long Island Salina Bahamas 25-100/1 May—Sept. 51 Alviso Salt Ponds California 13 g/m3 dw summer 77 San Francisco Bay saltponds

California (harvest)

5 kg/ha ww per week 78

250 kg/ha October 79 Crimea Salt Lakes U.S.S.R. 3000 kg/ha June 2.75 g/1 adults ww 0.93 g/1 juveniles June—Sept. 80

Burgas-Pomorije saltworks Bulgaria

0.5 g/1 nauplii 4.2 g dw/m3 Nov.—May 81,82 Lake Grassmere New Zealand 4.9 g dw/m3 Nov.—April 81,82

some data compiled from Persoone and Sorgeloos.83

The large operations can only be managed with regard to Artemia presence, especially to control the opportunistic dispersion of Artemia3 when better hydrobiological conditions are required for solar salt production. 4.8.10 As a result, the big-pond systems can be tapped for extensive harvesting only. The small units are much more versatile and provide realistic possibilities for human-managed production of Artemia biomass and cysts through inoculation with selected strains, through salinity control, pond retention times, food availability, predator presence, etc. This chapter will cover the basic

principales and strategies for semi-intensive production of Artemia in seasonal saltponds.

II. SEASONAL PRODUCTION OF ARTEMIA IN SMALL SOLAR SALTFARMS A. Typical characteristics of seasonal saltfarms

Seasonal saltfarms generally have an area of less than a few hectares, while individual ponds can be as small as a few hundred m2. The ponds are usually shallow with a water depth of < 10 cm. Sea water of mangrove or estuarine origin is usually supplied by tidal inflow, although some farms use pumps, windmills, and/or manually operated waterscoopers to permit better manipulation of water levels.

During winter (e.g., in China, southern Spain, and Sicily, and Sicily) or monsoon season (e.g., in Central America and Southeast Asia) salt production is abandoned. In monsoon climates, salt evaporation ponds are enventually converted into paddy fields or shrimp/fish ponds (e.g., Southeast Asia). In some farms deep brine reservoirs (pickle ponds) are available for storage of the brine which remains available at the end of the dry season (e.g., Philippines and Indonesia); at the onset of the dry season, brine is pumped back into the evaporation ponds and allows a faster start-up of salt production.

Figure 1. Schematic diagram of a solar salt operation with nautral occurence of Artemia. (From Sorgeloos, P., Léger, P., havens, P., andTackaert, W, Aquacult Dév. Cahiers Ethologie Appliquée, 7, 43, 1987. With permission.)

B. Pond modifications

Successful production of Artemia in seasonal solar-salt fields involves minor pond modifications to increase the water depths in the future Artemia ponds (in the salinity range of 100 to 180 g/l).to a maximum level of about 40 to 50 cm (preferably 70 to 100 cm). High water depths are essentiel not only to prevent lethal high temperature

conditions for Artemia, but also promote the development of phytoplankton which through its shading effect inhibits the development of phytobenthos. In contrast to phytoplankton, the latter is undesirable because it is too large to be ingested by Artemia, and as it starts to float, it may further reduce evaporation and eventually contaminate the harvests of Artemia biomass and cysts as well as the salt. Since Artemia is a planktonic organism, deep ponds can also sustain a larger production per surface area than shallow ponds. High water depths and associated phytoplankton result in a dark coloration of the brine which is also benficial for salt production since it enhances the evaporation efficiency through increased absorption of solar heat.9 Higher water depths can easily be achieved by digging and inner perimeter ditch and using the soil from the ditches for heightening the dikes (Figure 3). However, the water levels in the Artemia ponds will then be higher than in the nonmodified upstream evaporation ponds, which implies that the brine has to be relifted (by pump or windmill) into the first Artemia pond from where it further gravitates into the following downstream ponds.

Figure 2. Deposition of salts during concentration of sea water (from Bradley, personal communication).87

C. Preparation of ponds Prior to Artemia production, it is recommended that ponds be completely emptied to expose bottom soil for a period of one to two weeks, followed by raking the upper layer of the soil to enhance mineralization of accumulated organic matter, Fish left in remaining mud holes may be killed by the use of rotenone or tea-seed cake or by application of lime in combination with ammonium sulphate.11-12 Coastal saltfields may be located in mangrove areas associated with acid sulphate soils containing pyrite. Upon exposure to air, pyrite is oxidized to iron oxides and sulphuric acid, especially in newly excavated ponds. The release of the acid entails very low pH values, resulting in aluminium and iron leaching from the soil. The latter conditions are unfavorable for most aquatic organisms including Artemia; phytoplankton production is also inhibited through stripping of phosphorus from the water.13 In a number of cases, soil acidity may be

visually observed; i.e., air-exposed soils turn yellow to brownish-red. Addition of lime neurtralizes the acid soil conditions, allowing for a-better bio-availability of nutrients, which in turn enhances phytoplankton growth and production of Artemia. Lime, furthermore, aids in decomposition of pond muds, long term buffering of pH (which is essential in ponds where heavy organic fertilization is applied), and in the killing of undesirable fish eggs and pests through the toxic action of caustic components. There are several forms of lime : (1) calcium oxide, CaO, or quicklime has a neutralizing efficiency of 173% CaCO3, and is used for fast action in ponds with very low pH, in the range of 3.5 to 5.0; (2) calcium hydroxide, Ca(OH2), or hydrated lime has a neutralizing efficiency of 135% CoCO3 and also acts quickly to increase the soil pH; (3) calcium carbonate, CaCO3

, or agricultural lime (ground limestone) acts relatively slow and therefore may be used for long-term acidity control. CaCo and Ca(OH)2 may be used in newly excavated ponds while CaCO3 is used in older, more stabilized ponds. To raise the pH by 0.1 unit, about 500 kg of CaCO3 is applied per hectare. 13 Lime is applied to dry pond bottoms, although CaCO3 at low rates not exceeding 400 kg/ha, may also be used in ponds filled with water. Best results are obtained by spreading lime over the entire bottom or surface. Dikes should also be limed to prevent acidic runoff in case of rainfall.

Figure 3. Longitudinal section through a modified Artemia pond. (Modified from Tackaert, W., Léger, P., Lavens. P., and Sorgeloos, P., El cultivo del Camaron, Langostino y Congrejo en el Mundo : Bases y Technologias (The Aquaculture of Shrimp, Prawn, and Crawfish in the World: Basics and Technologies), Chavez Justo, C. and Sosa Nishizaki, O., EDS., McGraw-Hill, Mexico, 1989. With permission.)

D. Intake of sea water and increase of required salinities and water depths Most artisanal saltfarms are designed to permit intake of sea water by tide. Some are provided with sluices in order to obtain the maximum water level in the reservoir as close to the high-tide level as possible. Even then, and especially in the case where modified Artemia ponds are operated at higher water depths, it is that the brine can travel through the entire system by gravity alone. Pumps or windmills (see Figure 4) are required to supplement or take the place of the tidal gates. While water dephts in the reservoir should always be as high as possible (to maximize supply of the brine to the downstream ponds), evaporators and Artemia ponds should initially only be filled to a level of 10 to 15 cm, in order to ensure maximum evaporation and to create high water temperatures harmful to predators. Predators should furthermore be avoided by screening the intake water upon filling the ponds. If the pH of the water in the modified Artemia ponds is still lower than 7.0, it is advisable to wash out the remaining acidity by repeated flushing of the pond with sea water. If the pH is higher than 7.0, low water

levels in the Artemia ponds are allowed to evaporate until a salinity of about 100 g/1 is reached : i.e., high salinity in combination with high temperatures obtained in thin waterlayers will eliminate copepods and other predators. At this point, gradual intake of sea water is restarted at a rate to maintain the salinity in the Artemia ponds around 100 g/1 and be continued until the desired levels in the Artemia ponds (50 cm or more) are reached. While some predators (e.g., Cyprinodon variegatus and Aphanius fasciatus) are able to adpt to gradullay incrasing salinities, they will not resist the severe salinity shock created by the above practice of water intake. As soon as the Artemia ponds are filled to their maximum level, the rate of sea water intake is adjusted to maintain these depths. Consequently a density gradient typical for a normal salt operation will be established in the successive Artemia ponds and evaporators. Between the Atemia ponds, the brine is perferentially bottom-drawn not only to prevent temperature and salinity stratification but also to enhance disribution and release of organic matter which accumulates on the bottom.

Figure 4. Windmill used in seasonal solar salt farming in Thailand. (From Sorgeloos, P., lavens, P., Léger, P., Tackaret, W., and Versichele. D., Manual for the culture and Use of Brine Shrimp Artemia in Aquaculture. Artemia Eeference Center, State University of Ghent, Belgium, 1986. With permission.)

E. Fertilization

1. General Requirements

Before introducing Artemia in the ponds, enough particulate food should be present in the water to guarantee high population productivity. Water with a green-brown color and a transparency of less than 20 cm mostly contains high concentrations of organic detritus particules and/or algae that can be used as food by the Artemia. This is

generally the case in saltfarms associated with mangrove areas or eutrophic estuaries. The availability of the nutrients from mangrove of estuaries can be maximized by pumping at low tide.14 In this situation of good food availability, fertilization is not required, at least not before the introduction of the Artemia nauplii. Water with only a slight coloration and a high transparency (>30 cm) is not productive enough and requires fertilization to. increase the availability of natural food, 3 to 7 days prior to inoculation of Artemia.

Since the goal is to stimulate phytoplankton and not phytobenthos, it is essential to apply the fertilizer only to ponds already filled to maximum water levels. In flow-through systems, it is best to fertilize the low salinity ponds as we have often experienced difficulties in initiating a phytoplankton bloom when fertilizing high salinity ponds; i.e., chemical interactions limit the nutrient availability for a restricted number of algal species. In the latter systems, phytoplankton-rich water is ultimately drained into the high salinity ponds. Two kinds of fertilizers or a combination of both can be used: (1) organic fertilizers, such as dried chicken manure, and (2) inorganic fertilizers (commercial products used in local agriculture) with a high nitrogen and phosphorus content. Generally inorganic fertilizers stimulate phyoplankton growth more rapidly, while organic fertilizers act more slowly but provide a long-range effect since they first have to be degraded by bacterial action to release plant nutrients. In addition, some organic fertilizers such as dried ground chicken manure will easily disperse into the water column. Since they still contain up to 20% protein, they may also act as a direct food source for Artemia. Organic products are cheaper than inorganic fertilizers but much more bulky, and therefore involve more labor in their use. Moreover, organic fertilizers, especially when not properly distributed, may accumulate and decay at the pond bottom and create anaerobic zones resulting in oxygen deficiency, acidity, and toxicity through production of hydrogen sulphides.

Optimal rates of application are difficult to predict since they will vary from location to location due to climatic differences and quantitative/qualitative fertility of the local soil and water. Morales 86 reported that minimum concentrations of nitrogen and phosphorus in the water in order to obtain blooming of phytoplankton should be 1 to 2 mg/1 and 0.1 mg/l, respectively. Different fertilization programs also favor different types of food in the ponds; e. g., plankton is favored by a high ratio of nitrogen to phosphorus whereas organic manures, which are usually high in phosphorus, enhance the growth of undesirable filamentous algae. The fertilization program in Artemia ponds to be adjusted to optimize the availability of phytoplankton. The following doses can be recommended as a guideline. These concentrations have proven to be effective but other application rates and combinations of both organic and inorganic fertilizers are not excluded.

2. Organic Fertilizers

Best results to date have been obtained with chicken manure. Although cow and goat dung have been successfully used in some cases, chicken manure, in contrast to some other manures, such as from cattle (low nitrogen to phosphorus ratio : 1.5 ; high contents of undigested insoluble material) is more effective in inducing a phytoplankton bloom since it has a relatively high N/P ratio (3.5) and good dispersiblity, proving a larger surface for bacterial breakdown. In addition , it does not accumulate on the pond bottom since it is highly soluble. Van der Zanden15 reported a significant increase in the development of phytobenthos or «lab-lab» whenever cow dung was used.

Chicken manure needs to be dried and sieved for removal of debris, bran, feathers, etc., and it is preferable to grind it to incrase its availability as a driect food source for Artemia.

Nonetherless, good results have been reported by Jumalon et al.11 when using chicken manure suspension (1:1 ratio of manure to sea water).

Dry chicken manure is applied at rates of 0.5 to 1.25 ton/ha at the start with dressings of 100 to 200 kg every 2 to 3 days.

3. Inorganic Fertilizers

• Combination of 100 kg/ha mono-ammonium-phosphate (N:P:K ratio expressed in weight percentage of 16:20:0) and 50 kg/ha ammonium-nitrate (33:0:0); weekly dressings of 50 and 25 kg ha, respectively.

• Combination of 50 kg/ha di-ammonium-phosphate (18:46:0) and 50 kg/ha urea (44:0:0); weekly dressings of 25 and 20 kg/ha, respectively.

• 100 kg/ha urea (44:0:0); weekly dressings of 40 kg/ha (e.g., in ponds where natural levels of phosphorus are high).

Recently a by-product from the industrial production of monosodium glutamate derived from cassava or sugar cane molasses has been succesfully used as a cheap fertilizer for Artemia ponds. Application rates of up to 2500 1/ha have been found very effective in inducing dense phyoplankton blooms. In view of its acidity the effluents of the monosodium glutamate fermentation should be applied in small quantities but on a frequent basis.

Both organic and inorganic fertilizers should be very evenly spread over the pond surface. Slow dissolving pelleted fertilizers (e.g., 16:20:0) are first made into concentrated solutions (prepared over-nigh) or are placed on a platform in areas of active water flow (e.g., near the brine intake), 15 to 20 cm below the water surface in order to ensure a more even release and mixing and prevent any trapping of nutrients into the soil.

Although the fertilizer is generally applied directly to the Artemia ponds, several farmes are now working with separate «food production ponds» from which they feed the Artemia ponds; these may be low salinity ponds, 50 to 80 g/l, which are not necessarily integrated in the brine circuit. Availability/production of phytoplankton in these ponds is maximized through intake of «green water» from fish/shrimp ponds or through supplemental fertilization with fecal droppings from a vertically integrated poultry farm.11.12

F. ARTEMIA STRAIN SELECTION

In view of the high degree of genetic variation16 associated with the diversity of biometrical, and physiological characteristics5.17 found among strains of Artemia the selection of the strain best adapted to the particular ecological conditions (espacially temperature regimes) of the saltfarm and/or most suitable to its later application in aquaculture farms, is very important. Strain selection can be based on the available data of growth and production performance,18.19 reproductive characteristics,20 anion concentration tolerance,21 and especially temperature/salinity tolerance.22 In addition, whenever possible, a comparative bioassay culture test should be performed in closely simulaed conditions using the untreated brines of the habitat as culture medium. Strain selection might also be restricted by the intended application of the produced Artemia in local aquaculture; e.g., if small nauplii are needed for the production of the early larval stages of fish and shrimps, a strain producing small cysts and nauplii is to be preferred.23 On the other hand, if local aquaculture is primarily interested in Artemia biomass as a nursery/weaning or shrimp maturation diet, then a strain showing good growth and

survival, as well as dominant ovoviviparous reproduction characteristics will be the most interesting.

G. INOCULATION PROCEDURES

Inoculation of Artemia should be performed as early as possible in the brine circuit where no predators are found (i.e., usually at salinity levels of around 100 g/l). In flow-through systems with short pond-retention times, downstream ponds at higher salinity need not be inoculated since they will be gradually stocked with Artemia drained from the inoculated pond. Although Jumalon and Robles24 reported an optimum Artemia production at an inoculation density of 50 anuplii per 1, Vu Quynh and Nguyen Ngoc Lam25 reported faster growth and maturation as well as higher fecundity at densities <20 nauplii per 1. From our observations it is clear that small inocula (10 to 20 nauplii per 1) are generally as effective (at least under normal temperature conditions) and more economical than large inocula since they exhibit a faster population increase when compared to the latter. The quantity of cysts needed to obtain the number of nauplii needed for inoculation (and taking into account a 30% mortality at stocking) is calculated from the pond volume and the hatching efficiency of the selected cyst batch.26 Cysts are preferably hatched close to the ponds. Optimal hatching conditions26 are often difficult to achieve under field situations; nonetheless, the following aspects should be taken into consideration :

• Shaded or roof-covered area should be used to prevent excessive heating of the hatching containers by direct sunlight.

• Transparent, preferably funnel-shaped hatching containers, should be used. If flat-bottomed containers have to be used, they should be equipped with several aeration lines on their bottom so as to provide good mixing and aeration of the water.

• Hatching containers should be illuminated during the night to provide the essential light stimulus27 especially when cysts are incubated in late afternoon or evening.

• Air should be supplied from a blower, compressor, or several aquarium pumps powered by the net current, a generator, or batteries.

• Clean 20 to 35 ppt sea water should be used, filtered through a fine filter cloth (100 µm).

• Solium bicarbonate should be added at a rate of 1 g/lhatching medium so as to buffer the sea water.

• Cyst densities should not exceed > 1 g/1, espacially under suboptimal hatching conditions.

It is essential to harvest the nauplii in the first instar stage. This is determined from subsamples taken at regular intervals or from hatching rate and synchrony data for the given strain or batch.28 Older instar stages will not survive the salinity shock when transferred from natural sea water into 100 g/1 of salt water. After hatching, the nauplii should be screened over a 125 µm filter, thoroughly washed and transferred to clean sea water or pond water or pond water at half their hatching density. They are now ready for inoculation into the pond. If the pond is not within walking distance from the hatching site, aeration should be provided during transport (battery-operated pump or oxygen tank) to prevent mortality. If transport takes several hours, it is best to cool the nauplii container to 0—5°C using cooled sea water or adding iced bags. At these low temperatures the naupliar metabolism and motoric activity are strongly reduced without

affecting their viability.29 Moderate aeration, e.g., with a battery-operated aquarium pump, has to be provided to keep the motionless animals in suspension.

In this way about 100 million nauplii can be successfully transported for a period of several hours in a 20 liter plastic bag packed in a cooled styrofoam box. The best time of the day to inoculate a pond is during late evening when the water temperature is low and will continue to drop until early morning. When the nauplii have been transported at low temperature it is essential to allow the temperature in the containers to rise so that the animals can resume their motoric activity before they are introduced in the pond. Under heavy wind conditions it is important to siphon the nauplii on the leeward side of the pond to avoid having them driven on shore by heavy wave action.

H. FACTORS AFFECTING POPULATIONS GROWTH

During the first days after inoculation it is very difficult to see nauplii since thet have lost their dis-tinc orange color and tend to concentrate in the deeper marts of the pond. It is only when the nauplii have grown into adults that one may evaluate if the inoculation has been successful. In fertilized ponds, operated under optimal conditions (i.e., temperatures within the tolerance range for the selected strain; pH between 7.5 and 8.5; intermediate salinity levels of 100 to 150 g/1; and presence of sufficient quantities of particulate feed, especially phytoplankton) sexual maturity may already be attained 7 to 10 days after inoculation.25.30 Under these conditions the parental and the first generations will generally reproduce by ovoviviparity, resulting in a fast increase of the population. The size to which the population will grow is determined by the carrying capacity of the pond. The principal factors which affect this carrging capacity are ponddepth, food availability (determined by the concentration of nutrients in the water), and the frequency of water intake which will improve the water quality and result in an extra nutrient influx and better mixing of the nutrients which accumulate on the pond bottom. Aside from quantity, quality of the planktonic algal population may also affect the population growth of Artemia. Green algae (e.g., Tetraselmis and Dunaliella) and diatoms (e.g., Chaetoceros, Navicula, and Pleurosigma) are a much better food for Artemia than planktonic filamentous blue-green algae (e.g., Lyngbya and Oscillatoria). The latter are too big for Artemia to ge ingested and clog their thoracopods resulting in starvation ot the Artemia. Filamentous blue-green algae might predominate in stagnant waters (since green algae and diatoms sttle) and in conditions of high concentrations of organic matter, high pH, and low CO2 levels.31.33 In this regard, Jumalon and Ogburn33 found a high correlation between the arrest of water intake rich in CO2, the collapse of green algal populations, and the development of planktonic filamentous cyanophytes. This implies that regular water intake is important in pond management; i.e., aside from affecting the amount of food, continuous flow also stimulates blooming of particular phytoplankton species more suitable for Artemia development.

I. FACTORS CONTROLLING CYST PRODUCTION

Although the factors controlling the mode of reproduction are not fully understood, oviparity in Artemia is generally considered to be induced by environmental stress.34,35 In pond systems, cyst production is often observed when the population is exposed to high salinities (e.g., when the Artemia reach the high salinity ponds). Salinity shocks have also been found effective in switching the population toward cyst production36.37 i.e., abrupt lowering or raising of the salinity through rapid intake of brine of a very different density or at heavy rainfall. In addition, low oxygen concentrations or considerable fluctuations in dissolved oxygen levels reportedly induce oviparous reproduction in Artemia.34.38 This condition stimulates the synthesis of hemoglobin and the consecutive

excretion by the brown shell gland of its metabolic end product, hematin, which is the main constituent of the cyst shell of Artemia.39.41 Oxygen stress in pond conditions may be accomplished by raising the salinity and/or by increasing the rate of fertilization to induce blooms of phytoplankton, creating extensive diurnal fluctuations in dissolved oxygen.

Several authors postulated that the frequency of oviparity in Artemia is not only correlated with environmental stress, but is also influenced by the geographical strain of Artemia which has been used for inoculation.20.42.43 In this regard, Gajardo and Beardmore44 suggested that encystment in A. franciscana is under genetic control and is associated at least in part with the levels of heterozygosity found in the females. In view of the considerable interstrain differences in the distribution of heterozygosity16 care must be taken in selecting a strain showing high heterozygosity levels; e.g., a strain inhabiting a variable and stressful environment, if cyst production is preferred. Even then, pond management should be directed toward creation of stress conditions to retain the genetic variability in the population and consequently prevent a decline in the cyst production.

It has been observed on several occasions in tropical habitats that newly introduced populations initially exhibit a high rate of oviparous reproduction, followed, however, by a drastic decrease in cyst production as soon as the population has become fully established (adapted to the new environment?) and/or the biotope has become completely stablizied, e.g., in Brazil,45 Thailand,12 and Vietnam.46 This phenomenon was recently been observed for the second time in Macau, Brazil : reappearance of cyst production at the end of 1987 and beginning of 1988 was associated with transformation of the local shrimp farm into evaporation ponds for salt production presenting a «new bio-tope» for Artemia. Barthélémy-Okazak and Hedgecok47 presumed that this decline in cyst production may be due to harvesting of the cysts leading to a removal of the genotypes predisposed towards oviparity form the population. They suggested that cyst production could possibly be revived by rei-noculation with a highly oviparous strain.

J. MONITORING OF ENVIRONMENTAL CONDITIONS AND FOOD PRODUCTION IN PONDS

A basic prerequisite for correct pond management implies regular evaluation of the environmental conditions of the ponds. The physico-chemical parameters to be monitored include(l) dissolved oxygen, readily measured with an oxygen electrode or by Winkler titration;48 (2) brine densities at the water surface and the pond bottom measured with a refractometer or with Baumé scale hydrometers (see conversion tables for brine density and degrees Beaumé, as well as corrections for temperature in Tables 2 and 3) pH-values using a pH meter; (4) air and water temperature at water surface and pond bottom with a minimum-maximum thermometer; and (5) water depth, read from a depth gauge.

Since maintenance of a healthy phytoplankton population is considered to be one of the most important keys for a successful Artemia production, regular monitoring of nutrient levels and associated standing crops of phytoplankton are important for proper pond management, i.e., rate of water intake, fertilization dressings, and biomass harvesting. The concentration of the major nutrients should be regularly controlled to ensure that no deficiency causing inhibition of algal growth is developing and/or that the ratio of N/P is not becoming too low. This involves analysis of reactive inorganic phosphate (the major form of phosphorus required for algal cells), reactive nitrate, and ammonia, preferably by standard colorimetric procedures.48.49 The phytoplankton population should be analyzed

at least once a week. Phytoplankton densities may be determined from a representative sample by direct microscopic counting using a counting chamber. Whenever possible, species composition and cell size of the algal population should be determined, as the first may directly affect the nutritional value of the Artemia produced,5 and the latter determines whether the algal cells (especially when forming chains or colonies) are small enough (< 50 µm) for ingestion by Artemia.50 Records of phytoplankton species commonly found in nutrient enriched salinas were published by Davis51 and Wongrat.52 Other parameters to follow in respect to phytoplankton densities are water turbidity may be measured using a colorimeter or a Secchi disk. Procedures for measuring dry weight, chlo-rophyl, and primary productivity are decribed in Vonshack.53 Strickland and Parsons,48 and Boyd.54In situations where the water contains little or no silt, a good correlation is found between the latter parameters and the water turbidity, the measure of which still is the easiest and most rapid way to determine changes in phytoplankton densities.

Table 2 : Conversion Table for Various Units of Salinity

Density (g/ml)

Degree Beaumé

(°Be) Salinity

(g/l) Density Degree BeauméSalinityDensity

DegreeBeauméSalinityDensity

Degree BeauméSalinity

1.020 2.8 28.6 1.061 8.4 1.102 13.4 1.141 17.8 1.021 3.0 1.062 8.5 1.103 13.5 1.142 17.9 1.022 3.1 1.063 8.7 1.104 13.6 1.143 18.0 1.023 3.3 1.064 8.8 1.105 13.7 1.144 18.1 1.024 3.4 1.065 8.9 1.106 13.8 1.145 18.2 1.025 3.6 1.066 9.0 1.107 . 14.0 1.146 18.3 1.026 3.7 1.067 9.2 1.108 14.2 1.147 18.5 1.027 3.8 1.068 9.3 1.109 14.3 1.148 18.6 1.028 4.0 1.069 9.4 1.110 14.4 159.5 1.149 18.7 1.029 4.1 1.070 9.5 99.4 1.111 14.5 1.150 18.8 222.11.030 4.2 42.4 1.071 9.6 1.112 14.6 1.151 19.0 1.031 4.4 1.072 9.7 1.113 14.7 1.152 19.1 1.032 4.5 1.073 9.9 1.114 14.9 1.153 19.2 1.033 4.7 1.074 10.0 1.115 15.0 1.154 19.3 1.034 4.8 1.075 10.1 1.116 15.1 1.155 19.4 1.036 4.9 1.076 10.2 1.117 15.2 1.156 19.5 1.037 5.0 1.077 10.3 1.118 15.3 1.157 19.6 1.038 5.1 1.078 10.5 1.119 15.4 1.158 19.7 1.039 5.3 1.079 10.6 1.120 15.5 175.1 1.159 19.8 1.040 5.4 56.4 1.080 10.7 114.1 1.121 15.6 1.160 19.9 237.81.041 5.5 1.081 10.8 1.122 15.7 1.161 20.0 1.042 5.7 1.082 11.0 1.123 15.8 1.162 20.2 1.043 5.8 1.083 11.1 1.124 15.9 1.163 20.3 1.044 6.0 1.084 11.2 1.125 16.0 1.164 20.4 1.045 6.1 1.085 11.3 1.126 16.2 1.165 20.5 1.046 6.2 1.086 11.5 1.127 16.3 1.166 20.6 1.047 6.4 1.087 11.6 1.128 16.4 1.167 20.7 1.048 6.5 1.088 11.7 1.129 16.5 1.168 20.8 1.049 6.6 1.089 11.8 1.130 16.6 190.6 1.169 20.9 1.050 67 70.6 1.090 11.9 128.6 1.131 16.7 1.170 21.0 253.71.051 6.8 1.091 12.0 1.132 16.8 1.171 21.1 1.052 7.0 1.092 12.1 1.133 16.9 1.172 21.2 1.053 7.2 1.093 12.3 1.134 17.0 1.173 21.3 1.054 7.3 1.094 12.4 1.135 17.1 1.174 21.4 1.055 7.5 1.095 12.5 1.136 17.3 1.175 21.5 1.056 7.6 1.096 12.6 1.137 17.4 1.176 21.6 1.057. 7.7 1.097 12.7 1.138 17.5 1.177 21.7

7.9 1.098 12.8 1.139 17.6 1.178 21.8

Table 3 : Temperature Corrections (to 20°C) For Density Readings of Concentrated Sea Water

Density range (g/ml at 20°C) Tem (°C)

From 1.00 to 1.05

1.05 1.10

1.10 1.15

1.15 1.20.

1.20 1.25

1.25 1.30

10 Subtract 0.002 0.002 0.003 0.003 0.003 0.003 11 correction 0.002 0.002 0.003 0.003 0.003 0.003 12 from 0.001 0.002 0.002 0.003 0.003 0.002 13 measured 0.001 0.002 0.002 0.003 0.002 0.002 14 density 0.001 0.001 0.002 0.002 0.002 0.002 15 0.001 0.002 0.002 0.003 0.003 0.002 16 0.001 0.001 0.001 0.002 0.002 0.001 17 0.001 0.001 0.001 0.001 0.001 0.001 18 _ 0.001 0.001 0.001 0.001 0.001 19 _ _ 0.001 0.001 _ _ 20 _ _ _ _ _ _ 21 _ _ 0.001 0.001 0.001 _ 22 Add 0.001 0.001 0.001 0.001 0.001 0.001 23 correction 0.001 0.001 0.001 0.002 0.002 0.001 24 to measured 0.001 0.002 0.002 0.002 0.002 0.002 25 density 0.002 0.002 0.003 0.003 0.003 0.002 26 0.002 0.002 0.003 0.003 0.003 0.003 27 0.003 0.003 0.004 0.004 0.004 0.004 28 0.003 0.003 0.004 0.005 0.005 0.004 29 0.004 0.004 0.005 0.005 0.005 0.005 30 0.004 0.004 0.006 0.006 0.006 0.006 31 0.004 0.005 0.006 0.006 0.006 0.006 32 0.005 0.006 0.006 0.007 0.007 0.007 33 0.005 0.007 0.007 0.007 0.007 0.007 34 0.006 0.007 0.007 0.008 0.008 0.008 35 0.006 0.007 0.008 0.008 0.008 0.008

K. MONITORING OF ARTEMIA PRODUCTION PERFORMANCE

Precise estimates of Artemia densities are difficult to make because of the heterogenous distribution in ponds which is influenced by wind, water temperature, light, pond depth, etc.5.56.57 Nevertheless, rough estimates of Artemia densities, among other field data, may provide a valuable tool to assess the rate of biomass harvesting. Water samples should be taken at weekly intervals from fixed stations scattered throughout the pond, in the ditch as well as in the central part or along fixed transects. Sampling is preferentially done as early as possible in the morning when Artemia are more uniformly distributed.24.25 Samples may be taken with a specific sampler24 or with a variety of containers such as beakers, buckets, etc. When using the latter sampling procedure, it is advisable to thoroughly mix the water column so to stir up bottom-dwelling Artemia.58 The number of samples to be taken from a pond depends on the distribution of the Artemia as well as on the volume of the sample and the abundance of Artemia. This may be estimated by calculating the coefficient of variance (CV, variance/mean density), i.e., the lower the CV value, the more precise the sampling.

Table 4 : Examples of population Composition in Artemia Pond at Various Time (Athrough H)

Sampling time Nauplii Juveniles Preadults Adults Cysts A ++ - - - - B - - + ++ - C ++ - - + - D + + + + - E + - - ++ - F - - - ++ - G - - - ++ + H + + + + +

Of essential importance in pond management (e.g., rate of biomass harvesting, fertilizer addition, pond retention time, etc.) is the population composition, which provides valuable information on the population dynamics of Artemia in the ponds. The population composition should be analyzed from representative samples taken at weekly intervals from several places in the pond (e.g., combined with the density sampling). Samples containing large numbers ot animals may be subsampled until they contain 200 to 300 animals. The Artemia are categorized into five classes: cysts and/or nauplii (Instar I—IV), juveniles (Instar V—VII, larvae with developing thoracopods), preadults (adult size but not yet reproductively active), and adults. These classes may be distinguished under a dissection microscope or by pouring the Artemia over three successive filter screens with mesh sizes of about 500, and 125 µm which respectively retain adults and preadults; juveniles; and nauplii and cysts. The adults and preadults can easily be separated by eye. The (relative) presence of each Artemia class is expressed as percentage of the total number of Artemia counted in the plankton sample or is evaluated as follows : - absent; + present; + + dominant presence. Of further interest is the evaluation of the reproductive activity of the females; i.e., empty or full broodsacs and nauplii or cysts bearing. Table 4 shows a typical example of the population composition in an Artemia pond at various time intervals. The population changes over one-week intervals, from A through D, reveal a very healthy population; e.g., inoculation (A), growth up to preadult and adult stage (B), first generation of nauplii released (C), and continuous reproduction and good growth conditions (D). However, a population composition remaining for consecutive weeks (E) evenutally evolves into a situation which reveals food-limiting conditions (F); e.g., initial algal concentrations are still sufficient for the adults to ensure reproductive activity but too limited for the nauplii which have a lower feeding efficiency than adults; subsequently (F) food becomes too scarce even for the adults. When oviparity is the dominant mode of reproduction no population recruitment is observed (G); in heavily fertilized ponds a mixed reproductive activity is often observed (H).

L. STRATEGIES FOR CULTURE MAINTENANCE

The information collected from the monitoring programs is used to make appropriate decision about pond management. Optimal conditions for biomass production are at the lower salinity levels (100 to 150 g/1) and under conditions of very regular food availability. When transparency levels are high (>30 cm), and pond nutrient levels become undetectable or fall below levels found in the intake water, fertilizer dressing or intake of nutrient-rich water (e.g., from a feed production pond) should be considered. During temperature/salinity stratification (which causes lethal high temperatures for Artemia) or when planktonic blue-green algae become dominant, the bottom flow of

water from pond to pond should be maximized. Selection of desirable phytoplankton species and/or the prevention of the development of undesirable cyanophyes which have the ability to fix atmospheric nitrogen may also be aided by supplemental fertilization with specific nutrients: e.g., through application of nitrogen fertilizer during conditions where nitrogen is limiting but phosphorus is abundant, or through application of silicate in low salinity ponds to enhance the growth of diatoms, which are rich in the desired highly unsaturated fatty acids.

Sustained population growth also entails regular harvesting of the biomass so as not to exceed the carrying capacity of the pond. Insufficient harvesting may lead to a complete removal of the food (even in ponds with high nutrient levels) due to the high grazing pressure of increasing numbers of Artemia, eventually leading to a collapse of the population. Similarly, over-harvesting may reduce the grazing pressure on planktonic algal blooms which may deleteriously affect the salt production.49 Ideally, The rate of biomass harvesting should approach the maximum sustainable yields. Since a precise estimation of the latter is impossible, the rate of biomass harvesting should be assessed from estimation of density, population composition, fertility parameters, and phytoplanktonic standing crop (e.g., estimated by water turbidity levels). In situations where densities increase over time with a population composition showing all Artemia classes represented, combined with a dominant ovo-viparous reproduction, biomass is being renewed at a high rate. In this case and when transparency levels increase, indicating that the primary production rate cannot sustain the grazing pressure of the Artemia population. Frequent harvesting of biomass is recommended. If on the other hand, densities are decreasing and/or the population consists mostly of preadults and adults showing low fecundity and/or dominant oviparous reproduction, harvesting should cease.

In section ILL of this chapter we described the factors which induce cyst production. Production of cysts may naturally occur in the high salinity ponds. It may also be induced, even at low salinity levels, by increasing the rates of fertilization or by applying through salinity shocks of 10 to 20 g/1 rapid intake of low salinity brines. Oviparity, however, should only be induced when the population density is sufficiently high since during conditions of dominant cyst production, recruitment will be inhibited, eventually resulting in a gradual decrease of the population due to constant mortality.

M. HARVESTING AND QUALITY CONTROL

Produced cysts float at the surface and acumulate along the windward side of the pond. They can be easily collected from the water surface with a double-screen dip net (Figure 5). Cysts should be harvested as soon as possible after production (accumulation) in order to ensure maximum recovery and hatching quality because :

• Cysts washed ashore are difficult to harvest as well as to clean; in addition they will dry and may eventually become airborne.

• Cysts may be exposed to temperatures >40°C which are lethal or hydrated cysts.26

• Cysts may be exposed to repeated hydration/dehydration cycles (rainfall, high humidity) and lose part of their energy reserves59 resulting in a decreased hatchability.26

In order to prevent the cysts from being washed ashore and to facilitate harvesting, the windward pond corner or side should be steepened or lined with a cyst barrier, e.g., corrugated plastic. When winds develop heavy waves, foam is built up in which cysts are

trapped and then blowen away. In this case wave breakers should be installed in two or more rows parallel to the cyst barrier (Figure6).

Harvested cysts may undergo an on-site cleaning by washing the harvested product with saturated brine or water from the pond over screens with different mesh widths (e.g., 1000, 500, 125 µm) in order to remove debris larger and smaller than the cysts. This wet-dry product stored in brine or mixed with crude salt (for proper dehydration) may be an acceptable product for local use, provided it is consumed within a few weeks. For the production of high quality cysts with optimal hatchabi-lit, maximum purity, and storability, the following additional processing steps are however required : (1) density in brine to remove heavy debris in the same size range of the cysts; (2) rapid washing in fresh water to remove salt;.(3) density separation in fresh water to separate full cysts from empty cysts and other small light debris — this step should not take longer than 15 minutes in order to prevent elevated hydration levels which may initiate cysts metabolism; (4) removal of excess water by squeezing or centrifuging the cysts; and (5) drying in order to reduce the water level in the cysts below the critical level of 10% (preferably between 2 and 5%) in order to arrest the metabolic activity in the cysts. Optimal cyst quality in terms or hatching efficiency, hatching rate, and energy content is obtained by fast and homogeneous drying of all cysts at temperatures just below 40°C.84

Among the different drying techniques, optimal results are obtained when the cysts are kept in continuous movement in the drying air; i.e., each cyst is dried individually at the same time. This may be accomplished in a fluidized bed dryer (Figure 7) or a rotary dryer (Figure 8). If the latter equipment is not available, cysts may also be dried on drying racks on which the cysts are spread in thin layers of uniform thickness (few mm only). The drying racks are placed in the open air, protected from the sun to avoid temperatures higher than 40°C, or in a temperature-controlled room or oven at 35 to 38°C provided with good air exchange. Homogeneous drying is enhanced by granulating the cysts upon distribution on the drying racks through a 3 mm screen and by regular brushing (initially every h) of the cysts. Table 5 shows the effect of drying conditions on the hatching efficiency of cysts in Lavalduc, France. For more details with regard to cyst processing and drying, we refer to Sorgeloos et al.26

For long-term storage, cysts should be packed in air tight containers (cans) under vacuum or nitrogen.

Adult biomass may be harvested manually with a dip net (in small ponds) or with a conical net (see Figure 9) which can be towed over the entire pond. In highly eutrophic ponds optimal catches are made during the early morning after a calm night, when the dissolved oxygen concentration in the ponds is so low that the Artemia concentrate in very dense «blow-up» in the upper water layers where they perform so-called surface respiration.26 Biomass may also be harvested by static nets installed at the pond gates where active brine-flows drain large numbers of Artemia. The end part of the harvesting nets should have a small mesh size (<100 (µm) to prevent extrusion of the animals.

The nets must be harvested at intervals of less than 1 h since the Artemia accumulated at the end of the filter sac are exposed to anaerobic conditions which they cannot survive for more than 2 h. The harvesting efficiency of static nets can be improved by harvesting during the night light attraction to lake advantage of the positive phototactic behavior of the brine shrimp. Harvested biomass may be temporarily stocked (up to one week) in nylon screen cages (e.g., 1.5 x 2.0 x 0.5m) with a mesh width of 800 mm, which are suspended in the culture ponds (see Figure 10). For long distance live transport, the Artemia biomass may be packed at densities of 100 g/1 in plastic bags filled with one-

third cooled pond water (5 to 10°C) and two-third oxygen at atmospheric pressure. The bags are placed in a styrofoam box together with a few bags of ice (see Figure 11).

If not used directly, Artemia biomass should be frozen or dried after thorough washing with fresh water. Since Artemia is extremely prone to decomposition (due to proteolytic enzyme activity) it is essential to freeze the animals when still alive. In order to ensure optimal quality, the biomass should be frozen as quickly as possible in a blast or plate freezer (- 25°C or lower) in thin layers (maximum 1 cm thick) or small ice cube trays. A properly frozen product when thawed in water, yields only intac animals and does not pollute the water by leaching of body fluids (see Figure 12 for quality control).

Figure 5. Double screen dip net for cyst harvesting. (From Sorgeloss, P., Lavens, P., Léger, P., Tackaret, and Versichele, D., Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture. Artemia Reference Center, State University of Ghent, Belgium, 1986. With permission.)

Figure 6. Floating bamboo poles used as wave breaker for the harvesting of Artemia cysts. (From Sorgeloos. P., Levens. P., Léger, P., Tackaert W, and Versichele. D., Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture. Artemia Reference Center. State University of Ghent. Belgium. 1986. With permission.)

N. SPECIFIC MODIFICATIONS FOR FURTHER OPTIMIZATION OF ARTEMIA PRODUCTION

Since the production of Artemia requires the availability of high salinities to exclude predators, the production season in monsoon climates is basically limited to the dry season only. Nevertheless, a significant extension of the production season may be realized by the installation of overflow devices (e.g., PVC turndown pipes and level controlled gates) in order to allow decanting of stratifying layers of rain water, combined by rigorous control of predators (e.g., through screening of intake water by means of a bag screen or semi-cicular screen mounted to or surrounding the gate/pump). Year round production of biomass has been succesfully applied in both the Philippines11 and Thailand12 at salinities of 60 to 80 and 70 to 90 g/1, respectively.

In farms having brine reservoirs, salinity control during the rainy season may further be facilitated by the recirculation of surplus brine from these reservoirs into the Artemia ponds. In addition, this practice allows for maximal water exchange (essential for good phytoplankton production) and salinity manipulation (e.g., salinity shocks for the induction of cyst production). Another specific modification beneficial for Artemia production under high temperature conditions involves the installation of shading platforms (e.g., made of coconut fronds). De los Santos et al.60 reported that Artemia tend to concentrate under this shade to escape lethally high temperatures occurring on sunny days.

1 Conical shape results in differences in air pressure and assures better mixing of the cysts 2 more pressure is needed at the start (heavy cysts containing much water before dehydration) than at the end (light cysts which low water content) to keep the cysts suspended in the drying chamber.

Figure 7. Schematic drawing of fluidized bed dryer for Artemia cysts. (From Sorgeloos, P. Léger. P., Tackert, W, and Versichele, D., Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture Artemia Reference Center, state University of Ghent, Belgium, 1986 with permission.)

Figure 8. Schematic drawing of rotary dryer for Artemia cysts. (From Sorgeloos, P., havens, P. Léger. P., Tackert, W., and Versichele, D., Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture Artemia Reference Center, state University of Ghent, Belgium, 1986 with permission.)

Table 5 - The Effects of During Conditions on the Hatching Efficiency of Cysts from Lavalduc, Francea

Drying conditions Hatching effeciency (nauplii/g cysts)

Method Tem (°C)

Thickness of cysts layer

(cm) Xb SDb

Oven dryer 30 1.5 69,120 9,760 30 0.5 149,600 10,240 (154,120) (7,600) 38 1.5 150,880 7,200 38 0.5 181,360 9,600 (179,200) (10,100) Fluidized-bed dryer 35 182,400 6,400 (181,960) (6,920) Control (unprocessed cysts) 178,640 (8,840) a Data compiled from Sorgeloos et al.26

b In parentheses, data for same cysts but after 1-month storage under vacuum.

III. PRODUCTION FIGURES OF ARTEMIA IN FERTILIZED PONDS

Table 6 shows production figures of Artemia cysts and biomass in different man-managed saltfarms. Although the most successful farms yield 10 to 20 kg dry weight (dw) cysts and/ or 375 kg wet weight (ww) biomass/ha month, there is considerable variation from farm to farm, mainly as a result of differences in farm management. A survey of salt cum Artemia farms in Thailand in 1983,61 revealed that poor production in the Samut sakorn and Phetburi area (see Table 6) were correlated with low pond water depths, inappropriate local conditions such as acidity of the soil, and insufficient fertilization. Poor farm management including lack of puming and application of cow dung instead of the previously used chicken manure resulted in the development of lab-lab and overall food-limiting conditions. These were also responsible for the sharp decrease in cyst productions in Vinh Chau, Vietnam (area not specified) in 1988 (29.1 kg ww) as compared to 1987 (120 kg ww).15 On the other hand, Vu Do Quynh and Nguyen Ngoc Lam25 found that the introduction of a flow-through type management in Cam Ranh Bay, Vietnam improved the cyst yield from 1.4 — 6.8 to 8.6 kg of dw cysts/ha/month.

Recently, biomass has been the product of preference (especially in Thailand) largely because it is easier to master than cyst production, which in most farms has remained inconsistent. Biomass production furthermore offers new local marketing opportunities; e.g., during the dry season in Thailand more than 3000 kg of locally produced biomass is being harvested and consumed on a daily basis as a starter feed in shrimp nursing.85

Figure 9. Conical harvesting net for Artemia biomass.

Figure 10. Storage net for Artemia biomass produced in seasonal salt ponds in S.E. Asia, (from Sorgeloos, P., havens, P, Léger, P., Tackaert, W., and Verishele, D., Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture, Artemia Reference Center, Salte University of Ghent, Belgium, 1986. With permission.)

IV. SOCIO-ECONOMIC ASPECTS AND BENEFITS OF SALT CUM AREMIA PRODUCTION

Seasonal solar-salt production as practiced in Southeast Asia, Central America, East Africa, etc. is a labor-intensive activity generating employment for thousands of families. Its profitability, however, is usually limited, largely because of the low yields and the poor quality of the salt produced, owing to the small scale and artisanal manufacturing practices of this type of salt operation. In Viet Nam, for exemple, the mean annual income/worker in 1987/1988 was equivalent to 30 kg of rice. 15 In Thailand, the revenue of solar salt production has drastically decreased due to competition from rock-salt mining.12 In fact, in many countries (e.g., Thailand. Panama, and Costa Rica) hundreds of those family-operated saltfarms are being abandoned for socio-economic reasons. The profitability of these seasonal saltfarms can be considerably improved by integrating Artemia production with solar-salt production, Based on a survey of five salt cum Artemia farms in Thailand, Vanhaecke61 estimated that the total cost required for pond modification and operation of one ha Artemia pond was about 2040 U.S. dollars. Assuming a production of 180 kg ww cysts and 500 kg ww biomas/ha (extrapolated from average production figures of farms that adopted proper pond modification and good biological management) at average market prices of $ 16 and $4.4/kg respectively, the average benefits from Artemia production amounted to $3040 and $3870 in the first year and $3870 in the following years of operation. This represents an additional income almost triple of that derived from salt.12

Figure 11. Live transport of Artemia — transport bag (A) and styrofoam box (B). (From Sorgeloos, P., Lavens, P, Léger, Ph., Tackaert, W. and Versichele, D., Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture, Artemia Reference Center, State University of Ghent, Belgium, 1986. With permission).

During recent years salt cum Artemia production has become a profitable business. The latest data for Thailand62 reveal annual revenues from Artemia biomass production of over $14,000 (for average production yields of 260 to 375 kg/ha/month on a year round basis and wholesale prices of about $ 4/kg). Integrated Artemia production is not only attractive from a socio-economic point of view, it also stimulates the development of local aquaculture (especially in those countries which do not have hard currency for importing Artemia cysts, e.g., Vietnam, Bangladesh, etc.) through the local availability of cheap Artemia products. .

Figure 12. Quality control of Artemia biomass. (From Sorgeloos, P., havens, P., Léger, P., Tackaert, W., and Versichele, D., Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture, Artemia Reference Center, State University of Ghent, Belgium, 1986. With permission).

Table 6 : Production of Artemia Cysts and Biomass in Man-Managed Saltfarms

Artemia production (kg) Country Location Cysts Biomass Ref.

Thailand Chonburi 23.1 (dw/ha/month) 52.5 (ww/ha/month) 61 Thailand Samut Sakorn 5.2 (dw/ha/month) 61.7 (ww/ha/month) 61 Thailand Phetburi 3.0 (dw/ha/month) 27.2 (ww/ha/month) 61 Thailand Cha-Choengsao 17.5 (dw/ha/month) 14.4 (ww/ha/month) 61 Thailand Samut Songkram 15.3 (dw/ha/month) 51.5 (ww/ha/month) 61 Thailand 25.0 (ww/ha/month) — 12 Thailand Tambon klong Tamru,

Chounburi — 260—375

(ww/ha/month) 62

Thailand Cha-Choengsao 5.0 (ww/ha/month) — 58 Philippines Barotac Nuevo 5.0—18.6 (dw/ha/month) 29.4 (ww/ha/month) 11 Viet nam Cam Ranh Bay 1.4-6-8.6 (dw/ha/month) — 25 Viet nam Vinh Chau 3.2-3.4 (dw/ha/month) — 15 Viet nam Vung Tau 5.0 (dw/ha/month) — 63 China Xuwen County 74.6 (dw/ha/year) — 64 Peru Virrilla 35.0 (ww/ha/month) 0.06 (standing

crop/m3) 65

Indonesia Madura Island 38.0 (dw/ha/month) — 66 Jamaica Portland Cottase 8.2 (dw/ha/month) — 67

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Documents

MEDRAP II MEDITERRANEAN ARTEMIA, TRAINING COURSE … · • 71Lipid composition of cysts of the brine shrimp Artemia sp. from Spanish populations. By J. C. Navarro, F. Amat and J