Laboratory culture of fairy shirmps using baker's yeast as ... · Note the dark cell wall of normal cells, and by contrast, the hyaline cell wall of ‘empty’ cells. more than 12

Hydmbiologia 298: 141-1.57. 199.5.

D. Belk, H. .I. Durnont & G. Muier (eds), Studies on Lurge Brunchiopod Biology and Ayuuculture II.

@ 1995 Khmer Acudenzic Publishers. Printed in Belgium. 141

Laboratory culture of fairy shrimps using baker’s yeast as basic food in a flow-through system

Alejandro M. Maeda-Martinez’>2, Hortencia Obregh-Barboza1a2 & Henri J. Dumont’ ’ Laboratory of Animal Ecology, University of Ghent, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium 2 Current address: Centro de Investigaciones Bioldgicas de1 Noroeste, SC., Division Biologia Marina, Apdo. Postal 128, La Paz, Baja California Sul; Mexico

Key words: Thamnocephalus, Branchinecta , growth, filter-feeders, feeding, clay, silicium

Abstract

We designed and standardized a culture method for freshwater anostracans using diets free of live algae. Thamno- cephalus platyurus and Branchinecta lindahli were used as test organisms.

We used baker’s yeast as basic food and added inert particles (clay or amorphic silicium dioxide) to improve the digestion of the yeast. A flow-through culture system was used, according to a fixed feeding schedule, to supply separately, culture medium (tap water), food, and inert particle suspensions. Three variants with baker’s yeast as basic food, were compared for survival, growth, and reproduction. A diet of solely baker’s yeast (diet 1) or baker’s yeast supplemented with vegetal oil containing ,&carotene (diet 2) was unsuitable for reproduction of i? platyurus. Cyst production was only achieved when diet 2 was supplemented with fish oil and Spirulina powder (diet 3). This suggests that not only a digestibility problem, but also nutritional deficiencies are present in baker’s yeast.

Introduction

Knowledge of growth and reproduction of freshwater phyllopods is fundamental to use them in aquaculture. Laboratory studies require standardized culture methods, usually based on live algae as food. The obtention of algae in the quantity and condition required is, however, laborious and costly, and rep- resents a major constraint for the culture of aquatic filter-feeders (Coutteau et al., 1992). Therefore, the objectives of this investigation were to design and stan- dardize a culture method for fairy shrimps using diets without live algae. We used Thamnocephalus platyurus Packard and Branchinecta lindahli Packard as test organisms.

Antecedents

Two general methods are used for the culture of fairy shrimps viz. static and flow-through systems. Although cultures have been in use since the last century (e.g. Gissler, 1883), Moore (1957) first described a laboratory static culture system for Streptocephalus sealii Ryder. The culture medium was a mixture of auto-

claved demineralized water and sterile soil extract, and the diet consisted of yeast. Moore pointed out that yeast was the best available food, and that monodiets of Chlamydomonas intermedia or Paramecium mul- timicronucleatum gave poor results. Prophet (1963a) utilized Moore’s method to study the cyst production of S. sealii, S. texanus Packard, Branchinecta packardi Pearse (cited as B. lindahli Packard: Belk, pers. comm.), and Eubranchipus serratus Forbes. Ander- son & Hsu (1990), Baqai (1963), Gaudin (1960), and Meade & Bulkowski-Cummings (1987) also used baker’s yeast to culture S. sealii. Sluzheuskaya (1975) cultured S. torvicornis (Waga) with brewer’s yeast, and Sluzheuskaya-Drobysheva (1982) reported a mixture of baker’s yeast and Chlorella as the best diet for the same species. Coutteau et al. (1992) reported that a monodiet of baker’s yeast gave poor results in culturing Artemia and Streptocephalus proboscideus (Frauen- feld). Dimentman etal. (1976) investigated the grazing efficiency of Branchipus shaefferi Fisher, Branchinec- ta ferox (Milne-Edwards), Streptocephalus torvicornis, Chirocephalus neumanni Hartland-Rowe, and C. bairdi (Brauer). They concluded that these species are effective in removing sewage-grown Scenedesmus obliquus (Turpin). For a study of the postembryon-

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ic development of Streptocephalus mackini Moore, Herrera-Colmenero (1986) used Ankistrodesmus con- volt&us. For the laboratory culture of S. mackini, Rodriguez-Garcia (1990) compared monodiets of A. convolutus, Chlorella sp, Scenedesmus sp, and dry baker’s yeast. Rodriguez-Garcia (1990) reported Chlorella sp as the best food for growth, biomass and maturation, and A. convolutus for survival.

De Walsche, Mertens & Dumont (1991) stressed the importance of economic factors in the aquacultural applications of S. proboscideus, and tested a series of low-cost diets using bacteria, cyanobacteria, and baker’s yeast as basic food components in static cultures. They found that the best diet for cyst production was composed of active silt of bacteria, Spirulina platen- sis, and baker’s yeast, while the best diet in survival tests consisted of Corynebacterium lilum, S. platen- sis, baker’s yeast and a suspension of clay particles. Mitchell (1991) studied the efficiency of S. macrourus to convert algal biomass into anostracan biomass in semicontinuously and continuously fed cultures. He reports S. macrourus to be tolerant of crowding, and to yield higher productions than rotifers and cladocerans (Mitchell, 1991). Maeda-Martinez (1991) described an argillotrophic method, which is in principle Banta’s classical soil-manure medium (Banta, 1921).

For culturing Streptocephalusproboscideus, Bren- donck et al. (1990) described a semiautomatic flow- through system, with a capacity of ca 336 animals per unit (a cage of ca 6 l), using Selenastrum capricornu- turn as food. Finally, using the system of Brendonck et al. (1990), Ali & Brendonck (199 ; this volume) tested micronized agro-industrial waste by-products as a diet for S. proboscideus. They report that in terms of survival, growth and fecundity, the wastes of pea and corn (YM20) gave the best results (Ali & Brendonck, 1995).

Material

Cysts of Thamnocephalusplatyurus were produced in a mass culture (Dr D. Weaver, California, USA) and supplied to our laboratory by Dr D. Belk. Dr Weaver’s mass culture was a flow- through system, with water quality and automatic feeders computer controhed, and a commercial artificial plankton supplied as food (Dr Belk, pers. comm.). Cysts of Branchinecta lindahli were obtained from Morrill, Nebraska, USA, collected by Dr M. Fugate.

Incubation method and use of larvae

All culture tests were carried out using larvae hatched under standard conditions. We considered the follow- ing factors (reviewed by Lavens & Sorgeloos, 1987): (1) salinity (Horne, 1967; Brown, 1969; Belk, 1972; Sam & Krishnaswamy, 1979; Scott & Grigarick, 1979), (2) pH (Scott & Grigarick, 1979), (3) temperature (Prophet, 1963b; Horne, 1967; Belk, 1977; Scott & Grigarick, 1979), (4) oxygen (Brown, 1969), and (5) light (Belk, 1972; Sorgeloos, 1973; Van der Linden et al., 1985). Our method consisted of:

1. Incubator. A screw-capped transparent polystyrol flask (67 mm height x 34 mm diameter) of 60 ml capacity. Cap and bottom of the flask consisting of a 100 pm gauze, glued with cyanoacrylate. Scott & Grigarick (1979) used glass vials (17.5 ml) covered by nylon to prevent cysts of Triops longicaudatus (LeConte) from floating and adhering to the walls of the incubation jar. They demonstrated that only 2.7% hatched if floating, while 82% hatched in the cyst containers (Scott & Grigarick, 1979).

2. Container for the incubator of 600 ml capacity. 3. Thermal bath. An aquarium of 74 x 36 x 34 cm,

controlled by a heater and a cooler (MGW Lauda MT).

4. Light. Constant light was provided by two fluorescent lamps of 30 Watt, installed 30 cm above the thermal bath.

5. Hatching medium. Distilled water, aerated for at least 2 hours before use.

6. Aeration. Constant aeration was supplied to the container of the incubator through Pasteur pipettes.

Cysts were placed in the incubator, which was sub- merged in the hatching medium. Air bubbles were removed with a pipette, such that all cysts were per- manently immersed in water. The aeration was strong enough to provoke a constant rotation of the incubator into the container. Belk (1977) reported that the optimal hatching temperatures for Thamnocephalus platyurus and Branchinecta lindahli from Arizona were 20 to 25 “C and 5 to 20 “C, respectively. On that basis, we adjusted the incubation temperatures to 25fl “C for T platyurus and to 12f 1 “C for B. lindahli.

The hatching percentages reached a maximum after the 72 and 120 h of incubation for both species. Muru- gan & Dumont (199 ; this volume) report the same timing of hatching in T platyurus. The larvae used in the test cultures were those which hatched during the maximum eclosion period, and differed in age by no

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Fig. 1. Normal (n) and ‘empty’ (e) yeast cells as seen under the light microscope (40x). Note the dark cell wall of normal cells, and by contrast, the hyaline cell wall of ‘empty’ cells.

more than 12 h for 7: platyurus, and 24 h for B. lindahli.

Fresh baker’s yeast as food

Two reasons made us decide to test baker’s yeast as food. First, its application in many culture methods from the literature. Second, a series of tests on the survival of 1: plutyurus larvae after three days of culture. We tested, using the flow-through system described below, six different products as monodiets in a concentration of 0.25 g I-‘, supplied at a rate of 1.25 ml min- ’ . The products were (1) dry baker’s yeast, (2) fresh baker’s yeast, (3) rice powder, (4) Spirulina powder, (5) soya powder, and (6) tetramin powder (commercial fish food). We found less than 5% of survival for all diets except for baker’s yeast, which gave between 25 and 55% (Fig. 8). We presume that these results were due to a food-availability problem, because the powders and dry yeast tended to form clumps or aggregates. Fresh baker’s yeast also formed aggregates, but less than the other products. Good buoyancy of baker’s yeast had already been reported by Coutteau & Lavens (1989). World-wide interest in yeast as food using molasse-grown strains of Succha- romyces cerevisiae (baker’s yeast) arose because of its high-quality concentrated protein, and the abundance

of B-complex vitamins (Peppler, 1969). Yeast food is used as an animal feed supplement (Bunker, 1963), and recently, it has been incorporated to aquacultural diets (Coutteau & Lavens, 1989).

In this study, we used an available commercial baker’s yeast (Koningsgist, Belgium). As a rule, it was utilized before the expiration date indicated by the producer.

Effect of suspended inert particles on the digestion of baker’s yeast

By adding clay to the cultures, a better growth and survival of the fairy shrimps was obtained. At the same time, we also noted that the number of ‘empty’ yeast cells (Fig. 1) present in faecal pellets, increased. Two experiments were performed to study a possible effect of the suspended inert particles on the number of ‘empty’ yeast cells in faecal pellets, as an indication of the digestion of baker’s yeast. We used polystyrol flasks of 60 ml volume as test chambers. The bottom of each test chamber consisted of a mesh of 2 mm. Below this, a flask with a mesh of 100 pm was used to collect the faecal pellets. All test chambers were placed into a thermal bath. The yeast-clay suspensions were prepared with commercial clay (montmorillonite). The size distribution of its particulate composition was determined

144

A

16

6

0 20 10 9 6 7 6 6 4 3 2 1 .Q 8 .i’ .8 .6 .4 .3 .2

partlcle size (urn)

100 B ul l-l l-l a’ 0

cl m Ill (u50- > K 4’ !

35

Ea -f i

** -70

** al

i .L

= .I7

s

0’ ’ I I 0.0 0.5 1 .o

clay (g 1-l) 1 .5

Fig. 2. A. Size frequency distribution (%) of particulates of clay type 1, determined by laser sizing. B. Percentage of ‘empty’ yeast cells in faecal pellets of Thamnocephalus plafyurus (five treatments; five replicates) at different concentrations of clay type I, and 0.2 g 1-l of baker’s yeast. Bars represent standard deviations. f* = significant at p<O.Ol (t-test), compared to treatment without clay.

by a Malvern Laser sizer (Jones, 1987)(Figs 2A & 1.25 ml min-‘. The culture medium was tap water, 3A). Yeast-clay suspensions were maintained by gen- previously aerated for a minimum of 24 h. The dura- tle aeration at the conical bottom of the container. The tion of the experiments was 6 h. Constant light (20 suspensions were distributed to the test containers by Watt fluorescent lamp) was supplied. The percent- a multi-channel peristaltic pump with a flow rate of age of ‘empty’ yeast cells was determined as follows.

145

A

,j~ 20 10 9 0 7 6 6 4 3

SI 1 .Q .0

partlcle ~I.29 (pm)

I 7 A .b .3 .s

IOOr

m l-4 l-l al 0

+J (0 m

;50 -

=

$

E .al =

G

B

i .44 **

OL I I 0.0

clay ";," 1-l)

Fig. 3. A. Size frequency distribution (%) of ptiiculates of clay type 11, determined by laser sizing. B. Percentage of ‘empty’ yeat cells in faecal pellets of Thutnnocephulus plcltyurus (five treatments; five replicates) at different concentration of clay type II, and 0.2 g 1-I of baker’s yeast. Bars represent standard deviations. * * * & * * = significant at p<O.OO I & p<O.Ol (t-test) when compared with treatment without clay,

Three hours before the experiments the animals were collectors. At the end of the experiment, the faecal fed only algae. Once transfered to the test chambers, pellets of each animal were transfered to a container it took them no more than 1 h 30 min to excrete the with 20 ml of clean water and kept at 2 “C. The count- algae in the form of faecal pellets. Thus, after 2 h of ing of the yeast cells was done within the next seven experiment, all faecal pellets were removed from the days. The faecal pellets were washed in tap water in

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Fig. 4. A-B. View of the flow-through culture system. cv. culture vessel (volume of the culture medium 0.5 I), fc. food container (10 I), fi. food filter (0.4 1), he. heater, ipc. container of the suspension of inert particles (10 1), pl & p2. multi-channel peristaltic pump 1 & 2.. t. 100 1 reservoir of the culture medium (tap water) & tb. thermal bath. Arrows in A indicate water flow from the 100 1 tank to the culture vessels through peristaltic pump 1.

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a petri dish, transfered to a glass slide with a drop of water and squashed with a cover slip. A sample was transfered to an improved Neubauer haemocytome- ter counting chamber. Normal and ‘empty’ cells were scored and their percentages calculated. Per sample not less than 400 cells were counted. Samples containing less cells were eliminated. Per animal, two counts were carried out, averaged, and the percentage of ‘empty’ cells in the faecal pellets of each animal calculated. The final percentage of ‘empty’ cells for each animal was obtained by subtracting the average from the backpolled ‘empty’ cells present in the yeast-clay suspensions of the treatments.

Experiment I

Five treatments with five one-animal replicates. Yeast- clay suspensions prepared with 0.2 g 1-l of baker’s yeast, and 0.0,0.17,0.35,0.70 & 1.4 g 1-l of clay (dry weight) type I (Fig. 2A), respectively. The test organisms were postlarvae of both sexes of Thamnocephalus plutyurus, with a mean total length of 17.3 mm (range 16.0-19.0 mm) (N= 10). Culture medium temperature 25.0-26.0 “C, pH 6.5-7.0, and conductivity 1030- 1090 PS cm-‘. Mean percentage of ‘empty’ cells in the yeast-clay suspension of the treatments 2.68%.

Experiment II

Five treatments with five one-animal replicates. Yeast- clay suspensions prepared with 0.2 g 1-l of yeast, and 0.0, 0.11, 0.22, 0.45 & 0.91 g 1-t of clay (dry weight) type II (Fig. 3A), respectively. The test animals were postlarvae of both sexes of T platyurus, with a mean total length of 14.6 mm (range 13.0- 18.0 mm) (N= 16). The culture medium temperature 25.0-26.0 “C, pH 6.5-7.5, and conductivity 1330- 1350 PS cm-‘. Mean percentage of empty cells in the yeast-clay suspensions of the treatments 0.68%.

The results demonstrate that the suspended inert particles affected the percentage of ‘empty’ yeast cells in faecal pellets (Figs 2B & 3B). In both experiments, the percentage of ‘empty’ yeast cells increased in all treatments containing clay. However, comparing with the treatment without clay, only those treatments with 0.35 & 0.70 g 1-l of clay type I, and with 0.11, 0.22 & 0.44 g 1-l of clay type II, were significant (t-test) (Figs 2 & 3). Fig. 5. A. Food filter. B. View of the connections of the peristaltic

pump 2 (~2) with the food filter (fi) and the circuit supplier of the inert~pakcle suspension (ci). tb. thermal bath & wp. water pump of the sub-system for the suspension of inert particles.

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Fig. 6. A. Culture vessel. B. View of the culture vessels (cv) (volume of the culture medium 0.5 1) installed in the thermal bath. 1-3. Distributing tubes of: 1. culture medium (tap water), 2. food suspension & 3. inert-particle suspension. Arrows indicate flow direction.

149

(o/o)60

1 r i I

20 10 Q 8 7 6 6 4 3 2 1 .Q .0 .7 .6 .6 -4 .3 .2

partlcle size (pm)

Fig. 7. Size frequency distribution (%) of particulate composition of commercial powder of amorphic silicium dioxide used as standard source of inert particles.

Flow-through system

The basic elements of the culture system are: I. A 100 1 tank (Fig. 4). This is the reservoir of the

culture medium. We used tap water as culture medium. Before use, the tap water was aerated for 48 h, and passed through an aquarium filter (3.8 1 min-’ flow rate) with synthetic cotton as filter element.

2. Peristaltic pump No. 1. This pump (multi-channel Watson Marlow 501 UII) was used to supply water from the reservoir to the culture vessels (Fig. 4A).

3. Peristaltic pump No. 2. This pump (multi-channel Watson Marlow 302 F) was used to supply food and inert particles separately (Fig. 4).

4. Sub-system for suspension of inert particles. This sub- system consists of a 12 1 plastic-container with a funnel at the bottom (Fig. 4B), and two water pumps. To maintain the particles in suspension, a constant water current in a closed circuit was produced by a pump. To supply the suspension of inert particles to the culture vessels, another circuit of constant water current was produced by a second pump. The tubes of the peristaltic pump No. 2 were connected to this circuit (Fig. 5B).

5. Food container and filter. The food container was an inverted 10 1 glass tank. To maintain the food in suspension, gentle aeration was supplied (Fig. 4). A

food filter, was installed between the food container and the peristaltic pump (Figs 4 & 5). Food-clumps were separated from the suspension by gravity. From this filter, the tubes of the peristaltic pump distributed food to the culture vessels.

6. Thermal bath. An aquarium of 115 x 40 x 25 cm, with a two cm-thick PVC plate as bottom (Figs 4 & 6). Water temperature was controlled by a heater and a cooler (MGW Lauda MT).

7. Culture vessels. Each vessel consisted of a transparent Plexiglas tube (19.0 cm high, 17.0 cm diameter & 0.3 cm thick), and a plastic funnel fixed at one of the open sides and used as an outflow. Between the Plexiglas tube and the funnel, a gauze of specific mesh size (200, 500, 1000 or 2000 pm) was inserted (Fig. 6A). The culture vessels were placed in the thermal bath by fixing the tube of the funnel in the corresponding metal nipple at the bottom of the thermal bath. All culture vessels were covered with plastic petri dishes (Fig. 6B).

8. A fuorescent lamp of 20 Watt, installed at 40 cm above the culture vessels. Peristaltic pump No. 1, controlled by a timer, dis-

tributed water to the culture vessels at an intermittent flow rate of 20 ml min-’ , during 15 min, each 15 min. Peristaltic pump No. 2, distributed food to the culture vessels at a constant flow rate of I .25 ml min-‘. The

150

water level in the culture vessels was controlled by an outflow tube. The water flowing out from the vessels was discharged. A light regime of 16L/8D was maintained.

Basic feeding schedule

No literature data on specific yeast concentrations in a flow-through culture system are available. After a number of trials, we found that on culture day 10, a final concentration of cu 700-900 lo3 yeast cells ml-‘, was sufficient to obtain mature adults (ca 20.0 mm total length) at a density of ca 1 shrimp per 10 ml. These data served as our basis for determining a feeding schedule (Table 1). It was applied to all flow-through test cultures. After day 10, depending on the condition and size of the animals, the food supply was adjusted to avoid an excess by daily inspection on the yeast cell concentration, which was estimated using an improved Neubauer counting chamber. During the first culture periods (1 to 9 days), the food suspension was prepared every 24 h. From culture day 10, the food suspension was prepared every 12 h. To maintain the appropriate yeast concentration in the culture vessels, it was neces- sary to clean the food filter and distributor tubes, every two days.

A better utilization of the yeast food by the shrimps is obtained when inert particles are added. However, because the organic content of the clay was unknown, and because dissolved organic matter can be a source of particulate food for filter-feeders like Artemia (Bay- lor & Sutcliffe, 1963), we used a commercial amorphic silicium dioxide (powder containing 87% pure silici- urn acid, Flugge, Germany) as a standard source of inert particles. The size distribution of its particulate composition, revealed about 45% of 2 pm particles (Fig. 7). The concentrations of silicium in the feeding schedule, were determined from observations on the percentage of ‘empty’ yeast cells in faecal pellets of shrimps, during preliminary tests. We noted that at a concentration of 0.15-0.20 g 1-l of silicium during culture days 6-8 (flow rate 1.25 ml min-‘), the percentage of ‘empty’ cells was ca 50% or more. This concentration of silicium is similar to the concentrations of clay which gave high percentages of ‘empty’ cells in faecal pellets (Figs 1 & 2).

01,. ‘. 3 “. ‘. t. s. s 0 2 4 6 6 10 12 14

daym of cultura

Fig. 8. Survival of Thamnocephulus platyurus reared at 2.5.5f 1 .O ‘C in the flow-through system using diet I. The test culture started with new-born larvae. A. Survival ufter 1-3, 3-6, 6-9 and 9-15 (days) culture periods. Bars represent standard deviations. B. Cumulative survival.

General procedure of the test cultures

All test culturing was conducted using the basic feeding schedule (Table 1). Survival and growth were determined at the end of subsequent culture periods, which corresponded to days 3, 6 & 9 for Thumnocephulus plutyurus and days 5, 7, 9 & 11 for Brunchinecta lin- duhli. To start each culture period with an equal number of shrimps in all replicates, their density was adjusted by randomly selecting a fixed number of surviving shrimps. Their growth was estimated by measuring standard length (from tip of head to posterior margin of telson). Measurements were made with an eye-piece micrometer on a stereo microscope Wild 3 to the near- est 0.18 mm. In the first tests, we used culture vessels with a mesh of 200 pm. At every inspection of survival and growth, the culture vessels were changed for oth- ers with a bigger mesh size (500, 1000 & 2000 pm). After the first culture period, the cumulative survival was determined by subtracting the proportional mortality of the actual culture period from the cumulative survival of the foregoing period. The proportional

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Table 2. Basic feeding schedule. Supply of food (baker’s yeast g I-‘) and inert particles (amorphic silicium dioxide g 1-t) at a flow rate of 1.25 ml mitt-‘. Minimum and maximum represent the expected range of suspended yeast cells (lo3 cells ml-‘) in culture vessels.

Culture day 1 2 3 4 5 6 I 8 9 10

yeast 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 minimum 70 140 210 280 350 420 490 560 630 700

maximum 90 180 270 360 450 540 630 720 810 900 silicium 0.025 0.05 0.075 0.1 0.12 0.15 0.17 0.2 0.22 0.25

mortality is the corresponding value of the mortality percentage when taking the cumulative survival of the foregoing period as 100%. The test were carried out at 25.5fl.O “C, pH of 6.7-7.2 and conductivity 580-630 pS cm-‘.

Survival and growth of Thumnocephalus platyurus

Diet I

We initially ran a number of test cultures using solely baker’s yeast as food. We faced two major prob- lems, i.e. a high mortality in the first larval stages, and absence of cyst production (Fig. 8). Always, a mortality of more than 55% occurred during the first culture days (days O-3). After this, mortality decreased (Fig. 8). This high initial mortality may mean that the first larval stages are not yet able to feed on baker’s yeast, or that the yeast concentration (ca 70-270 lo3 cell ml-‘) was too low. Therefore, we investigated alternatives to obtain a better larval survival.

Standard culture method for the first larval stages

In a static culture and adding treated liquid soya as food, a larval survival of more than 80% was obtained. The soya food was prepared as follows: 10 ml of commercial liquid soya (soya drink, without sugar or salt) was diluted in 390 ml of clean 24 h-aerated tap water. The solution was heated for 2.5 minutes in a microwave oven (the solution’s temperature reached ca 75 “C). Then, the solution was stirred at 8500 rpm for 30 sec-

onds (Ultra Turrax homogenizer). After this, the foam was separated with a 50 pm gauze.

To find a good ration of soya solution, four treatments were tested in a48-h experiment. The treatments were different quantities of soya food added to 400 ml of culturemedium: (1) 0 ml of soya, (2) 6 ml, (3) 12 ml & (4) 18 ml and (4) 0.0 ml (soya (ml) v/v= 0, 0.015, 0.03 & 0.045). We used three replicates per treatment with 40 newborn larvae each. At the end of 24 h, the larvae were counted and placed in a new container with fresh solution. The final count was made after 48 h. A gentle aeration was supplied to each test vessel. The best ration found was 6.0 ml (0.015 ml soya per each ml of culture medium), giving a mean survival of 96.4%, while the rations of 12.0, 18.0 & 0.0 ml gave 88.1, 84.8 & 1.2%, respectively (Fig. 9). This procedure was adopted as a standard method to obtain 24 h-old larvae for the flow-through test cultures.

In subsequent cultures using Diet 1, larval survival at day 3 was about 70%. However, no improvement was obtained at day 9, with only about 30% of cumulative survival (Figs 10 & 11). The observed growth rate was 1 .O mm d-l, with a slight decrease in males in the third culture period (Fig. 12). Observations in more advanced periods (days 12 to 18) showed that, although more than 50% of females were sexually mature, cyst production never occurred. At day 12, 18% of the animals suffered from black disease.

Diet 2

A second diet was prepared by adding to each 20 g of fresh baker’s yeast, 1.8 ml of a mixture of corn oil, vegetal triglycerides, ,&carotene, soya lecitin, and palm oil (Natrele, Belgium). This was manually mixed to obtain a homogeneous paste, and stored at 2.0 “C, for no more than 7 days.

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IOO- 1

;

80-

G

t-a :

60- -m-l 2 : 40-

0 1 2 3 days of culture

Fig. 9. Mean survival of Thamnocephalus pluryurus new-born larvae under four different doses of soya food at 24 and 48 h. I = 0.015,2 = 0.03, 3 = 0.045 & 4 = 0 ml of soya food per ml of culture medium.

A considerable improvement was observed in the three culture periods. The survival at day 3 was 94.7%, and at day 9, 63.1% (Figs 10 & 11). The observed growth rate was increased in the first two periods, but a considerable decline occurred in females (from 1.38 to 0.59 mm d-‘) (Fig. 12). Observations during more advanced culture periods (days 12 to 18) showed that more than 50% of the females were adult, but no cyst production occurred. At day 12, 11% of the shrimps suffered from black disease.

Diet 3

A third diet was prepared by supplementing Diet 2 with 1.8 ml of fish oil (Halibut liver oil, Ortis, Belgium) per 20 g of baker’s yeast, and by adding 0.3 g I-’ of Spiruli- na powder to the food suspension. Compared with the results of Diet 2, again an improvement was observed. A better survival was obtained in the first two culture periods, but the survival at the end of the third period decreased, giving a final cumulative survival of 56.4% (Figs 10 & 11). The growth rate increased in all culture periods, giving a mean standard length of ca 11.7 mm for both sexes at day 9, while diets 1 & 2 gave ca 9.1& 10.0 mm, respectively (Fig. 12). However, the major improvement with Diet 3 was that now cyst production

was achieved at day 13. On day 12,5% of the shrimps suffered from black disease.

Survival and growth of Branchinecta lindahli

Cysts of B. lindahli were incubated at 12 “C. The new- born larvae were first reared for three days in static cultures with soya food (see above). On days 1 & 2, temperature was increased to 15 & 18 “C, respectively. On day 3, the larvae were transferred to the flow- through system and cultured at 20.0-2 1.5 ‘C, pH 6.7- 7.2, and conductivity 580-630 PLS cm-‘. Diet 3 was used as the standard food. From day 3 onwards, we used four culture periods of two days each. A survival of more than 75.0% was obtained in all culture periods, giving a final cumulative survival of 59.9% at day 11 (Fig. 13). In the second culture period, a remarkable growth rate of 2.93 mm d-’ in males, and 2.57 mm d-’ in females was observed (Fig. 14). More than 50% of the females reached sexual maturity on day 7. Cyst production started at day 8. On day 11, 2% of the animals showed black disease.

153

‘“i 0 2 4 6 e 10

Fig. 10. Survival of Thammcephalus platyurus reared at 25.5fl.O “C in the flow-through system using diets 1 (A), 2 (B) & 3 (C). Test cultures (A-C) started with 24 h-old larvae. Bars represent standard deviations.

Discussion

The present culture system fulfills the key require- ments of the cultivation of invertebrates (Persoone & Sorgeloos, 1975), namely: (1) to oxygenate the medium without disturbing the animals, (2) to maintain food in suspension to insure its availability, (3) to auto- mate the procedure, while (4) avoiding complex systems, (5) to use, whenever possible, inert sources of food, and (6) avoiding laboratory systems impossible to adapt to mass culturing. Regarding rule 5, although

fresh baker’s yeast is alive, its use does not represent any problem given its availability and low cost. Similar flow-through culture systems have been described by Sorgeloos & Persoone (1972, Fig. 2) for growth experiments with Artemia larvae, and by Lampert (1976, Fig. 2) for long-term experiments with Duphnia.

Our first results showed that a mortality of more than 50% occurred during the first culture period (day O-3). This was explained by assuming that the first larval stages are not able to take up baker’s yeast, or that the yeast concentration was too low. The first assumption is supported by Coutteau et al. (1990a), who found baker’s yeast an inadequate food for the brine shrimp Artemia. However, after this critical period, 7: platyurus and B. lindahli can be reared to reproduction on baker’s yeast (Diet 3).

The addition of inert particles to improve the utilization of the yeast, has also been studied in bivalve molluscs. Coutteau et al. (1990b) found that the addition of kaolin to the diet, significantly improved the growth of both Tapes semidecussata and Mercenaria mercenariu. This improvement was related to an increment of filtration rate and to the delivery of soluble nutrients via adsorption to the particles (see Coutteau & Lavens, 1989). Murken (1976) reported that suspended silt plays an important role in digestion and utilization of animal debris by the mussel Mytilus edulis L.

Coutteau & Lavens (1989) suggested that the inef- fectiveness of baker’s yeast is mainly a digestibility problem. Coutteau et&. (1990a) proposed that Artemia is unable to grow on baker’s yeast because its digestive enzymes cannot penetrate the outer mannoprotein layer of the yeast cell wall. The improved digestion of baker’s yeast seen in the increased number of ‘empty’ cells in faecal pellets (resulting in better survival and growth) suggests that the inert particles cause a retar- dation of the rate of the intestinal transit, exposing the yeast cells longer to the digestive enzymes and better extracting the cell content. Yet, the cell wall is not digested. Coutteau (1992) found that the faecal material of Artemia fed mutant yeast strains contained a large fraction of ‘empty’ cells, while Artemia fed untreated yeast, also contained ‘empty’ cells but in much lower numbers. He suggested that the apparently intact cell wall of digested yeast cells may indicate that digestive enzymes are capable of penetrating the cell wall at weak sites, but are unable to degrade its skeletal structure consisting of glucan fibrils (Coutteau, 1992: 125). A diet consisting of only baker’s yeast (Diet 1) or supplemented with vegetal oil containing p-carotene

154

-3 - BO- rt 2 .r( =

60- B

z c

:

: 40- rl a A z

20-

-0 2 4 6 days of culture

8 10

Fig. 11. Cumulative survival of Thamnocephalus platyurus reared at 25.5fl.O OC in the flow-through system using diets 1 (A), 2 (B) & 3 (C). Test cultures started with 24 h-old larvae.

(Diet 2) was unsuitable for reproduction in Tharnno- cephalus platyurus.

Cyst production was only achieved when Diet 2 was supplemented with fish oil and Spirulina powder (Diet 3). These results strongly suggest that probably not only a digestibility problem exists, but that there are also nutritional deficiencies in baker’s yeast. The same was indicated by Coutteau & Lavens (1989) and Coutteau et al. (1990a) in Artemia, fed baker’s yeast. Studies on the composition of Saccharomyces cerevisiae showed that although all essential amino acids are present, the contents of methionine, cystine and tryp- tophan are low (Peppler, 1969; Watanabe et al., 1983). Also, baker’s yeast contains little or no carotenes, ascorbic acid (vit. C), and cyanocobalamine (vit. B- 12) (Bunker, 1963). Watanabe et al. (1983) reported S. cerevisiae to contain a fairly high amount (52-82%) of monoethylenic fatty acids, 16: 1 and 18: 1, but no w3 highly unsaturated fatty acids (w3-HUFA). In order to improve the nutritional value of baker’s yeast for rotifers and Artemia, these authors supplemented the yeast with an emulsion of fish oil (rich in w3-HUFA) and a small amount of raw egg yolk (Watanabe et al., 1983). Similarly, an improvement of the nutritional value of baker’s yeast can be expected by our method of preparing a paste of yeast with vegetal and fish oils. Also, the protein and vitamin content of Diet 3

was increased by the addition of Spirulina powder. Spirulina has a crude protein content of 56-65% (see Kharatyan, 1978; Johnson, 1980; Soeder, 1980).

Thamnocephalus platyurus and Branchinecta lindahli are species which occur in North America (Belk, 1975). They may live together, but T platyurus is a thermophilic species, which occurs during the warm months of the year (Belk, 1977; Eng et al., 1990; Horne, 1971; Prophet, 1963c), while B. lindahli is less thermophilic (Eng et al., 1990), and restricted to habitats with temperatures of less of 36 “C (Belk, 1977). These fairy shrimps also exhibit different life cycles. Our observations on growth rate and maturation, show that individuals of T. platyurus had a mean size of 0.88 mm at day 1, and 11.7 mm (11.71 mm males, 11.76 mm females) at day 9. Their mean size increment of 10.8 mm in 8 days (1.3 mm d-i) rep- resents a specific growth rate of 153.6% (length) d-’ at 25.5f 1 .O “C. 7: platyurus females reached maturity on day 12, at a standard length of 18.0-20.0 mm, and started to produce cysts on day 13. Comparable data were obtained by Mackay et al. (1990) from a wild population of a May flood playa in New Mexico. They reported that 10 days after flooding no i7 platyurus females were gravid, but after day 13, all females were carrying eggs, and had a total length of 20-22 mm (Mackay et al., 1990).

155

t gronth rate (mm day-')

15

growth rate (mm day')

days Of culture

Fig. 12. Growth of Thamnocephalus platyurus reared at 25.5fl.O “C in the flow through system using diets 1 (A), 2 (B) & 3 (C). Test cultures (A-C) started with 24 h-old larvae. Bars represent standard deviations.

Cultured individuals of Branchinecta lindahli had a mean size (standard length) of 1.13 mm at day 3, and 14.79 mm (males) and 12.48 mm (females) at day 11. They had a mean size increment of 13.66 mm (males) and 11.35 mm (females) in 8 days (1.70 mm d-’ in males, 1.41 mm dd’ in females), which represent a specific growth rate of 15 1.1% (length) d-’ males and 125.5% (length) d-’ females at 20.0-21.5 “C. B. lindahli females reached maturity at day 7, at a standard

0 2 4 6 10 12 days of culture

Fig. 13. Survival of Branchinecfu lindahli reared at 20.0- 21.5 “C in the flow through system using diet 3. Test culture started with 3 days-old larvae. A. Survival after 3-5, 5-7, 7-9 & 9-11 (days) culture periods. Bars represent standard deviations. B. Cumulative survival.

length of 8.0-10.0 mm, and started to produce cysts on day 8. Early development and maturity are an advan- tage in small and temporary waters, and this explains the occurrence of this species in smallest, extremely temporary and erratic pools in California, USA (see Eng etal., 1990).

Other authors have also reported high growth rates for anostracans in static cultures. Prophet (1963a) reported on Branchinecta packardi (cited as B. lindahli: Belk, pers. comm.) a rapid preadult development and sexually maturity within nine days when cultured at 20 “C. He observed copulation at 10 mm length (Prophet, 1963a). Moore (1957) mentioned that S. se&ii fed yeast and reared at 25 “C, reached maturity within 8 days at a length of ca 12 mm. This repre- sents a size increment of ca 1.4 mm d-’ , and a specific growth rate of ca 237% (length) d-’ . Similar data were recorded by Sluzheuskaya-Drobysheva (1982) in cultures of Streptocephalus torvicornis using yeast and

156

18

15 - growth rate ,(mm day- 1)

12-

6-

3-

2 4 6 8 10 12 days of culture

Fig. 14. Growth of Brunchinecta lindahli reared at 20.0-21.5 ‘C in the flow-through system using diet 3. Test culture started with 3 days-old larvae. Bars represent standard deviations.

Chlorella as food at 28 “C. Lake (1969) reported a lower growth rate in Chirocephalus diaphanus Pre- vost, which reached sexual maturity on day 12 at a standard length of lo-12 mm, when cultured at 25 “C and fed Scenedesmus.

Acknowledgments

We thank Dr Denton Belk, and Dr Michael Fugate for the cysts of T platyurus, and B. lindahli. Also, we thank Dr S. S. S. Sarma for useful comments. The first author has a doctoral fellowship with Consejo National de Ciencia y Tecnologia (CONACYT), Mexico.

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Laboratory culture of fairy shirmps using baker's yeast as ... · Note the dark cell wall of normal cells, and by contrast, the hyaline cell wall of ‘empty’ cells. more than 12