Effects of Different Diet Regimes on Development of Gill and Rostrum Spines of Pacific White Shrimp

Aquacultura Indonesiana (2013) 14 (XX): XX-XX ISSN 0216-0749

Hak cipta oleh Masyarakat Akuakultur Indonesia 2013

Effects of different diet regimes on development of Gill and Rostrum spines of

Pacific white shrimp Litopenaeus vannamei

Romi Novriadi

Aquaculture Hall

Directorate General of Aquaculture, Ministry of Marine Affairs and Fisheries

Jl. Raya Barelang, Jembatan III, PO BOX 60 subdistrict of Sekupang of Batam city of Riau Islands

Email : [email protected]

Telphone: (0778) 7027623 – 7027624, Faksimile: (0778) 3582557

A b s t r a c t

Romi Novriadi. XXXX. The effect of partial replacement of Artemia nauplii with artificial diets were

evaluated by analyzing the gill and rostrum spines development as well as the quality of white shrimp (Litopenaeus vannamei, Boone) post larvae (PL). The treatments were: (1) live food control Artemia nauplii, (2)

65% replacement of Artemia with artificial diet, and (3) 85% replacement of Artemia with artificial diet. All

treatments were also compared to the L. vannamei PL generated from commercial hatcheries. Instar II Artemia

nauplii were cultured as a starter live food and Frippak microencapsulated feeds were provided as the artificial

diet to the L. vannamei. A significantly better quality, gill and rostrum spines development was achieved by post

larvae (from PL1 to PL 5) fed with live Artemia or the 65% replacement of Artemia in comparison to 85%

replacement of Artemia. Interestingly, even had the lowest quality, PL fed with 85% replacement of Artemia

nauplii still recorded a superior quality compared to commercial hatchery products at the same PL stages,

suggesting that the nutrition standard performed at the commercial hatchery in the sampling area does not

support the optimal development of gill and rostrum spines as well as the quality of L. vannamei at the post

larva stages. Additionally, the findings are important in aquaculture where the appropriate concentrations of artificial diet are also able to demonstrate a better growth, osmoregulation and detoxification performance in L.

vannamei post larvae and this may contribute to the efficiencies towards a reduction of Artemia nauplii cost.

Keywords : Litopenaeus vannamei, Diet regime, Gill, Rostrum spines, Larva index quality

Introduction

Pacific white shrimp (Litopenaeus

vannamei, Boone), is an economically important

species that is widely cultured not only in their native regions at western pacific coast of Latin

America but also in the expansive Asia (Liao and

Chien, 2011). However, the success story of L. vannamei farming has always been crippled by

the lack of high-quality post larvae. One of the

major factors hampering the quality of hatchery-

reared post larvae is nutrition, especially during the “critical periode” where there is an extensive

morphogenesis in the digestive system occurs at

the first 10 days of post larvae (Lovett and Felder, 1989). The behavioural changes from herbivorous

(filter feeders) to carnivorous (hunters), which

occur due to the life cycle development of shrimps require appropriate nutrition and correct

prey size (Lavens and Sorgeloos, 1996).

Freshly hatched Artemia nauplii are the

most widely used as live feed for early stage crustacean larvae due to its high nutritional

quality and ease of use (Sorgeloos et al., 1986).

The suitability of Artemia nauplii as live feed in crustacean larviculture is also supported by the

presence of 20:5(n-3) fatty acid (eicosapentaenoic acid or EPA) (Leger et al.,

1986). Artemia nauplii is usually given in live

condition to the larvae to give an optimum

feeding regime especially on the mysis and early postlarval stages (Mc Fey and Fox, 1983). In

addition, several forms of Artemia have also been

used in most penaeid hatcheries to induce an optimal growth: i.e. heat killed nauplii, frozen

and blended Artemia (Wilkenfeld et al., 1984;

Wouters and Van Horenbeeck, 2003; Juarez et al., 2010).

Good post larval quality is a top priority

in penaeid hatchery. Several factors influence the

growth and quality performance of L. vannamei post larva. One of the factors is the development

of gill that play a key role in respiration,

osmoregulation and detoxification. In their real life, gills are in direct contact with the external

environment; therefore, their development are

crucial to enhance shrimp tolerance to external

biotic and abiotic factors (Clavero-salas et al., 2007). Other than gill, the development of

rostrum spines also play an important role,

particularly as an indicator for larval stage identification.



In penaeids hatchery, the increase of the

operational cost has been considered as a major contsraint in the use of live nauplii.

Consequently, novel approaches to substitute the

use of Artemia are needed. Recent research have

include the use of rotifers Brachionus plicatilis (Naessens et al., 1995), nematodes Panagrellus

redivivus (Biedenbach et al., 1989) and extending

the use of algal food for the late mysis and post larval stages as a potential alternatives for

substitute live feed Artemia (Gopalakrishnan,

1976). Moreover, micro-particulate and micro-encapsulated formulated diets can also be used to

reduce the amount of Artemia cysts (Galgani and

Aquacop, 1988; Wouters et al., 2009). Many

scientists have studied the effect of the supplement diets on survival, growth or total

length of penaeid shrimps (Biedenbach et al.,

1989; Hirata et al., 1985; Cobo, 2013; Brito et al., 2001 and Naessens et al., 1995). Moreover, Brito

et al. (2001) have stated that partial substitution

(50%) of Artemia by artificial diet and the use of algae beyond the first post-larval stage have a

benefit impact to the growth and nutritional state

of L. vannamei.

In present study, the use of Artemia nauplii as a live food and their combination with

Frippak© microencapsulated diet was evaluated in

L. vannamei post larvae. We examined variations in gill and rostrum spines development as well as

the quality of L. vannamei at the first five days of

post larval development as a response to the

different diet regimes included Artemia nauplii as live food control, 65% and 85% substitution of

Artemia with Frippak. We wished to determine if

the variations in quality, especially in gill and rostrum spines development that have an

important role to enhance the salinity tolerance of

L. vannamei, is related to any combination of food. Additionally, the result may eventually

contribute towards a reduction of Artemia cost to

support the sustainability of shrimp farming

production.

Material and methods

Experimental animal

Larvae of Pacific white shrimp (L.

vannamei, Boone) was used as an experimental

animal and obtained from a commercial hatchery in Chonburi – Thailand. They were reared until

Protozoea II in the concrete tank using a feeding

schedule proposed by SCRD Thailand. Even though the L.vannamei nauplii at this stage still

deplete internal reserves and do not feed on

algae, as much as 20 cells/mL of Chaetoceros

calcitrans was added to guarantee the feed supply once the animals reached the protozoea

stage. Feeds was provided 6 times per day and

the number of Chaetoceros calcitrans was counted by using Haemocytometer.

Experimental design

At stage protozoea III, larvae were transferred to spherical tank containing 175 L

saline water (30 mg/L). The spherical tank were

placed in hatchery containing water maintained

at 29±10 C using thermostatic heater. Each tank

were stocked with batches of 175 nauplii of L.

vannamei. The water quality parameters, the

photoperiod, temperature and the feeding regime were adjusted. Water was exchanged at a rate of

aproximately 50% per day after removing waste

and uneaten feed by siphoning. Several water

quality parameters, i.e., NH4-N, NO2-N, and NO3-N were measured every two days. Gentle

aeration was applied in all rearing tank to

maintain the dissolved oxygen concentration above 5 mg/L. The photoperiod was set at 12 h

light at an intensity of 900-1000lx with

fluorescent lamps at the water surface. Newly hatched Artemia (SepArt, New generation High 5

Artemia, INVE aquaculture) were used as live

food and different feeding regimes was applied

from Protozoea III until PL5 (Five days after metamorphosis) according to Table 1. The

Artemia dosage was calculated based on the

feeding level and the number of shrimp larvae in the tank and split over two feedings period at

07.00 h and 16.00 h.

Table 1. Type and amount of feed provided for each successive stage of larval development of Litopenaeus vannamei

Stage Live food (control) 65% substitution 85% substitution

Nauplii 5 C.calcitrans 20

cells/µL

C.calcitrans 20 cells/µl C.calcitrans 20 cells/µl

Protozoea

1-2

C.calcitrans 50

cells/µL

C.calcitrans 50 cells/µl C.calcitrans 50 cells/µl



Protozoea

3– Mysis 3

C.calcitrans 50

cells/µL

Artemia nauplii 2-

10/mL

C.calcitrans 50 cells/µl

65% substitution of Artemia

nauplii with Frippak

C.calcitrans 50 cells/µl



PL1 – PL5 Artemia nauplii 5-

10/mL





In our experiments, an open clear water system was used with a daily water exchange of 50 %.

During water exchange, the remaining (heat killed) Artemia and waste from the previous day

were removed by siphoning. This operation was

carried out with great care to avoid loss of larvae. Feeding was done after water exchange. Three

different treatments with four replicates each

were tested (Table 1), namely: life food as control

(Artemia nauplii only), 65 % substitution of Artemia nauplii with Frippak and 85 %

substitution of Artemia nauplii with Frippak,

respectively. The substitution was adjusted based on the daily growth (length, wet weight and dry

weight) and the estimation number of L.

vannamei in the experiment tank. The experiments was terminated after five days of

feeding at post larvae stage (PL-5) and quality,

length, gill and rostrum development were

determined.

Determination of gill and rostrum spines

development

Gill and rostrum spines development in each treatment was observed under light

microscope equipped with a Canon EOS digital

camera connected to a PC. The images were

compared with gill and rostrum development at the same post larvae stages from commercial

hatcheries in Chonburi-Thailand. The rearing

condition and feeding regime in commercial hatchery was also evaluated by direct interview to

obtain a relation to the gill and rostrum spines

development.

Post larva quality

Post larva (PL) quality was checked at

every stage of PL by analyzing the osmotic stress,

dry weight, wet weight, and average length among the treatments. Size of the PL was

measured according to Kitani (1986) from the

base of the antennal flagellum to the telson, and the mean of total length and coefficient of

variation were calculated for each treatment and

PL stages of sampling. To evaluate the PL to osmotic stress, a simple stress test was performed

according to Samocha et al. (1998) with some

modification. Sample for each treatment was left

for 60 minutes at different range salinity, 1, 2 and 3 ‰, respectively and then the number of

survivors PL was counted after the specified

time. Dry weight analysis were performed by drying 1 gram of PL in the oven for 24 hours at

60 0C and measured using analytical balance.

Wet weight obtained by measuring 1 gram and dividing by the number of existing PL. The post

larvae quality from the commercial hatchery was

also evaluated as a comparison to the post larvae

quality generated from the experiment.

Statistics

The results are presented as mean values

followed by the standard deviation and the percentage data were arcsine transformed for

statistical comparisons to satisfy normal

distribution and homoscedasticity requirements.

Data were then subjected to one way analysis of variances followed by Tukey’s multiple

comparison range using the statistical software

SPSS version 21.0 to determine significant differences among treatments. All significance

level of the statistical analysis was set at P<0.05.

Results

Gill and rostrum spines development

Microscopic analysis showed that the

development of gill and rostrum spines varies for

each treatment . At PL 1 stages, larvae fed only

live food (control) and fed Frippak 65 % have 3 dorsal rostral spines. Another growth indications

of spine was also observed between the first

spines (at the edge) and the second spines. Larvae fed frippak 85 % treatment have only 3

dorsal rostral spines without any indication of

further spines growth. At PL 2 stages, larvae fed only live food and fed frippak 65 % have 4 dorsal

rostral spines each, while the larvae fed frippak

85 % still have 3 dorsal rostral spines with new



indication of spines growth. At PL 3 stages,

larvae fed live food and fed frippak 65% have 4 dorsal spines each with the addition of setae and

the second spines. However, at larvae fed frippak

85 % there are variations in the number of spines

(3 and 4 dorsal spines) with or without setae. At PL 4 stages , larvae fed live food and fed frippak

65 % have 4 dorsal spines each with 2-3 setae at

the second dorsal spines, while the l;arvae fed

frippak 85 % also have 4 dorsal spines but only with 1-2 setae at the second dorsal spines. Finally

at PL 5, larvae fed live food have 5 dorsal rostral

spines, while larvae fed frippak 85 % have 4

dorsal rostral spines. The difference in rostrum spines became more and more pronounced

towards the end of the experiment (Fig.2).

Figure 1. Stage wise gill development of PL1 to PL5 of L.vannamei for each treatment

PL 1

PL 2

PL 3

PL 4

PL 5

Live food (control) 65 % frippak 85 % frippak



Figure 2. Stage wise rostrum spines development of PL1 to PL5 of L.vannamei for each treatment

The treatments with only Artemia nauplii (live food) and with Frippak 65 % at PL 2 indicate the

presence of 2-3 branched at their gill, while with

Frippak 85 % remain without branched. At PL 3,

treatment with only live food and with Frippak 65 % majority of shrimps have 5 branches in their

gill, while with Frippak 85 % varies from 2-5

branches. At PL 4, treatment with only live food and with Frippak 65 % indicated 7-9 branches,

while with Frippak 85 % varied from 5-7

branches. And finally at PL 5, treatment with only

live food and with Frippak 65 % showed 10-11

branches in each of the gill, while the branches in

Frippak 85 % varied between 3-9.

Post larva quality analysis

1. Average length and dry weight

Larvae fed with Artemia nauplii were

significantly longer (P < 0.05) in average length

at PL 5 (0.87 ± 0.011 cm) followed by 65 % frippak substitution treatment (0.79 ± 0.018 cm),

and this group was significantly different with

PL 2

PL 3

PL 4

PL 5

PL 1

Live food (control) 65 % Frippak 85 % Frippak



treatment of 85% frippak substitution of Artemia

nauplii resulted in the shortest average length ( 0.68±0.017 cm) at the end of experiment (PL5)

(Figure 3). Post larvae of L.vannamei fed with

Artemia nauplii (Live food) were significantly (P

< 0.05) heavier in terms of dry weight (0.218 ±

0.03 mg) than those other treatments. Larvae fed

with 65 % frippak substitution were significantly (P < 0.05) heavier in dry weight (0,191± 0.01

cm) than fed 85 % frippak substitution (0,175±

0.02 cm). Overall, the dry weight variation trends

were observed from PL3 stages.

Figure 3. Average length of L. vannamei from PL1 to PL 5, fed with Artemia naulii (LFC = Live Food), 65% S and 85% S (S= substitution of Artemia nauplii with Frippak INVE Commercial feed),

respectively. Values are average±SE, n=4. Different letters denote significant differences (P<0.05).

Different types of superscript denote different comparison.

Figure 4. Average dry weight of L. vannamei post larvae from PL1 to PL 5 fed with Artemia naulii

(LFC = Live Food), 65% S and 85% S (S= substitution of Artemia nauplii with Frippak INVE Commercial feed), respectively. Values are average±SD, n=4. Different letters denote significant

differences (P<0.05). Different types of superscript denote different comparison. 2. Salinity stress analysis

Post larvae of L.vannamei are maintained in saline water (30 ‰) at 30±0,5

0 C before

salinity shock. All the treatments (live food 65%

frippak substitution and 85% frippak substitution)

were exposed to three low salinity solution (1 ‰,

2 ‰ and 3 ‰) at ambient temperature and

survival determined after 1 h. Twenty post larvae

were used per trial at every post larvae stages



Figure 5. Mortality (%) of PL 1 to PL 5 L.vannamei post larvae fed Artemia nauplii with or without commercial feed. In the experiment, LFC : Live food (only fed with Artemia nauplii), 65 % S : 65 %

substitution of Artemia nauplii with frippak, and 85 % S : 85 % substitution of Artemia nauplii with

frippak. All the treatments exposed to 1 ‰ solution. Values are average±SD, n=3, Different letters

denote significant differences (P<0.05).

The results presented in Figure 5 showed that a significant low percentage mortality

(P<0.05) was observed in L. vannamei post

larvae fed with Artemia nauplii exposed to 1 ‰

salinity solution in comparison to 85% partial substitution of Artemia with frippak at every

stage of post larvae development. However, no

significant difference in mortality was observed between post larvae of L. vannamei fed with

Artemia in comparison to post larvae that receive

65% partial replacement of Artemia.

Similar trend was also noted at 2 ‰ and 3 ‰ treatments solution (Fig.6 and Fig.7). The

treatments with both live food and with 65 %

frippak substitution resulted in the lowest mortality percentage in comparison to 85%

partial replacement of Artemia nauplii (P<0.05)

Figure 6. Mortality (%) of PL1 to PL5 L.vannamei post larvae fed Artemia nauplii with or without commercial feed. in experiment, LFC : Live food (only fed with Artemia nauplii), 65% S : 65%

substitution of Artemia nauplii with commercial feeds, and 85% S : 85% substitution of Artemia

nauplii with commercial feeds. All the treatments exposed to 2 ‰ solution. Values are average±SD, n=3, Different letters denote significant differences (P<0.05).



wet weight Dry weight average of

( gr) ( gr ) length (cm) 1 g/L 2 g/L 3 g/L Total T (C) sal ( g/L)

Prom-Phong Hatchery Penaeus Vannamei Zoea : C. Calcitrans + artificial diet+ heat kil led Artemia PL 6 0,0013624 0,000326 0,71032 5 5 0 10 Brand "white bear" 50% 25 30

Mysis : C. Calcitrans + artificial diet+ heat kil led Artemia

PL 1-12 : Daphnia + Artemia + artificial diet

Pichitpon Farm Penaeus Vannamei Zoea : C. Calcitrans + artificial diet+ heat kil led Artemia PL 8 0,00223214 0,00047 0,91085 1 0 0 1 Brand "Galaxy" 60% 27 25 (rainy day)


PL 1-12 : Artemia + artificial diet+ Black flakes

Lab-Inter2 Farm Penaeus Vannamei Nauplii : C. Calcitrans PL 7 0,00174216 0,0004546 0,8326 0 0 0 0 Brand " Swan GSL" 50-60 % 30 30

Zoea : C. Calcitrans + artificial diet+ heat kil led Artemia


PL 1-12 : Artemia + artificial diet+ Black flakes

55 Farm Penaeus Vannamei Nauplii : C. Calcitrans PL 6 0,00110865 0,000276 0,7116 1 0 0 1 Brand "Eagle" Artemia 50% 30 30

Zoea : C. Calcitrans + artificial diet+ heat kil led Artemia


PL 1-12 : Artemia + Daphnia + artificial diet+ Black flakes

Phanh Tib Farm Penaeus Vannamei Nauplii : C. Calcitrans PL 12 0,00649351 0,0015633 0,9951 2 0 0 2 Brand " Aqua " AQP 50% 30 30

Zoea : C. Calcitrans + Spirulina + heat kil led Artemia


PL 1-12 : Artemia +artificial diet

Chaleam Farm 1 Penaeus Vannamei This hatchery is only as a transit step at PL 8-12 PL 8 0,0037594 0,00053867 0,9817 1 0 0 1 Brand "Galaxy" 50% 29 20

Artemia only applied when packaging to avoid the stress -

condition in shrimp

Chanchira Farm 2 Penaeus Monodon Mysis to PL 1 : heat kil led artemia (Instar-1) PL 2 0,0015008 0,00021067 0,704 20 20 20 60 Brand M.T.I > 80 % 30 30

Chanchira Farm 1 Penaeus Monodon Mysis to PL 1 : heat kil led artemia (Instar-1) PL 3 0,0013298 0,00019933 0,7548 20 20 20 60 Brand M.T.I > 80 % 30 30

Chanchira Farm Penaeus Vannamei Mysis to PL 1 : heat kil led artemia (Instar-1) PL 3 0,00065934 0,000096 0,4914 5 2 2 9 Brand M.T.I 60% 30 30

PL 1-12 : Artemia +artificial diet

Pasit Farm 1 Penaeus Monodon Nauplii : C. calcitrans PL 8 0,0117375 0,0002967 0,70165 20 20 20 60 Brand " Premium" > 80 % 30 30

Mysis to Zoea 1 : heat kil led Artemia (instar 1) Red and green package

Zoea 2 to PL 4 : Artemia

PL 5 onwards : Daphnia

Pasit Farm Penaeus Vannamei Nauplii : C. calcitrans PL 3 0,00105568 0,000099 0,5973 5 3 2 10 Brand " Premium" 60-70 % 30 30

Mysis to Zoea 1 : heat kil led Artemia (instar 1) Red and green package

Zoea 2 to PL 4 : Artemia

PL 5 onwards : Daphnia

Ma win Farm Penaeus Vannamei Zoea I-III : Spirulina powder + Chaetoceros PL 10 0,00325733 0,00043313 0,8103 2 0 0 2 Brand " M.C.C " 10-50% 30 30

zoea II they applied the heat kil led artemia

PL 1 onwards : Artemia + artificial diet

sometimes mix with Vitamin

Observation Parameter

SRwater quality (at sampling)Hatchery Farm Feeding schemeType of shrimp mortality with osmotic stress (1 H)

PL stagesType of Artemia

Figure 7. Mortality (%) of PL1 to PL5 L.vannamei post larvae fed Artemia nauplii with or without commercial feed. in experiment, LFC : Live food (only fed with Artemia nauplii), 65% S : 65%

substitution of Artemia nauplii with commercial feeds, and 85% S : 85% substitution of Artemia

nauplii with commercial feeds. All the treatments exposed to 3 ‰ solution. Values are average±SD, n=3, Different letters denote significant differences (P<0.05).

Comparison of post larva quality with

commercial hatcheries.

Twelve shrimp hatcheries was visited in

the frame of sampling campaign to obtain a comparative data between post larva quality

generated from trial work with post larva quality

from commercial hatchery. Sampling was carried out using local equipment and sample were kept

on plastic filled with oxygen during

transportation.

Table 2. Summary of sampling campaign in 12 commercial hatcheries



There were two commercial hatcheries

that could be compared due to the availability of PL stage based on the observed post larva stage,

namely: Chanchira Farm (PL 3 of L.vannamei)

and Pasit Farm (PL 3 of L.vannamei). Based on

the post larva quality and microscopic analysis, PL from commercial hatcheries appeared to grow

slightly lower in terms of dry weight and average

length even when compared with PL fed Frippak 85% substitution. Percentage mortality in 1 ‰

solution also slightly higher compared with

larvae fed with live food and with 65% frippak substitution treatments, but equal to the mortality

number of 85% frippak substitution treatment.

Similar trend was also seen in the 2 ‰ and 3 ‰

solution treatments, no replicate measurements are available for commercial hatcheries sample to

enable statistical evaluation. Gill and rostrum

development was also slower for both commercial hatcheries sample.

Figure 7. Rostrum spines and gill of L.vannamei at PL 3 in Pasit Farm (upper row : A and B) and

rostrum spines and gill of L.vannamei at PL 3 (lower row : C and D) images in Chanchira farm

Discussion

In this study, we investigated the effect of

different diet regimes on the pacific white shrimp

L.vannamei post larvae performance from PL1 to PL5 and on the rostrum spines and gill

development. During their life cycle, Shrimp will

be more herbivorous at zoea stages and therefore

require microalgae such as diatoms Chaetoceros spp., Thalassiosira spp. and Skeletonema spp

(Cook and Murphy, 1966; Emmerson, 1980;

Martins et al., 2006; Soares et al., 2006), whereas at the mysis and postlarva stage, penaeid shrimp

becomes more carnivorous and require

zooplankton (Lavens and Sorgeloos, 1996).). It is evident that the administration of

live Artemia nauplii from Protozoea III to Post

larvae 5 resulted in better gill and rostrum spines

development (Figure 1 and 2). Moreover, These

results showed no significant differences with

65% partial replacement of Artemia in respect to the gill and rostrum spines development along

with the quality of post larvae. In contrast, 85%

replacement of Artemia nauplii resulting in the lowest quality in comparison to other treatments.

In term of gill development, as an euryhaline

tropical shrimp, L. vannamei highly depend on gill, mainly for regulation of their

osmoregulatory capacity and tolerance of salinity

fluctuations in the water (Wickins and Lee.

2002). Salinity is one of the most important abiotic factors for marine and estuarine

organisms, which affect their growth and

survival (Saoud et al., 2003; Buranajitpirom et al., 2010; Péqueux, 1995; Fielder et al., 2001;

Atwood et al., 2003 and Kumlu et al., 1999 ).

Moreover, mass mortalities in grow out ponds of

penaeid shrimp is often related to the

A B

C D

http://www.sciencedirect.com/science/article/pii/S1744117X11000864#bb0375









fluctuations of salinity levels and less observed

in smaller sized organisms because they are better osmoregulators (Vargas-Albores and Ochoa,

1992).

Our results also indicate that the different

diets directly influenced the rostrum spines development (Fig. 2). Although there is no clear

relation, for post larvae it is likely that protein

quality in diet have an important role to induce the development of rostrum spines in L. vannamei

and this development are related to the quality

and exact stage of post larvae. Another important feature arising from

this trial is the impact of different feeding regime

to the growth and survival of L. vannamei post

larvae when tested with osmotic stress factor. Importantly, we found that control and 65%

substitution of Artemia nauplii, which had a better

development of gill and rostrum spines had essentially the same survival levels (Fig. 5) when

exposed to 1, 2 and 3 ‰ solution from 30 ‰ for 1

hour. Their survival was significantly higher in comparison to 85% substitution (P < 0.05).

Dietary manipulations have important roles to

improve the osmoregulatory abilities of culture L.

vannamei subjected to osmotically stressful conditions which in turn is expected to increase

the quality and productivity of aquaculture

production (Romano and Zeng, 2012). This mainly due to the osmoregulation process require

energy that are sourced from protein (Setiarto et

al., 2004, Rosas et al., 1999, and Silvia et al.,

2004) and/or lipids (Palacios et al., 2004; Luvizotto-Santos et al., 2003; Lemos et al.,

2001 and Sang and Fotedar. 2004). Therefore it

seems reasonable to assume that providing an easy digestible live food and readily available

energy supply with optimal substitution

concentration could effectively improve the survival of L. vannamei in extreme change of

environment salinity.

In addition to the importance of diet,

every stage of L. vannamei PL responded similarly to several feed and microencapsulated

diet (Cousin et al., 1993). If there is a difference

in digestibility, potential exist appear between larvae and postlarvae. Figure 3 showed that from

PL 1 to PL4, there is no significant difference (P

< 0.05) in average length between live food and 65% substitution treatment. However, both of the

treatments (life food and 65% frippak

substitution) have a significant differences (P <

0.05) compared to 85 % frippak substitution. Furthermore, at the end of observation (PL 5),

treatment live food had a larger average length

(P < 0.05) than 65% frippak substitution. From

PL 1 to PL 5, treatment of 85% frippak substitution resulted in poor growth in terms of

average length. The trials emphasized that native

protein seems to be better hydrolyzed than the

processed one (Zwilling et al., 1981). This is in agreement with studies of Cuzona et al (2004),

who reported that more digestible nutrients

absorbed promotes more growth to the cultured aquatic animals. The provision of feed by using

Artemia nauplii also appears to provide a better

growth, as the post larvae stage had a significantly heavier (P<0.05) dry weight

compared to 65% and 85% substitution of

Artemia nauplii. This difference is very evident

from PL 3 to PL 5 and 65% substitution still have a better performance than the 85% substitution.

In comparison of our PL stages with

those from several commercial hatcheries, the quality of L. vannamei post larvae was not as

expected. The gill and rostrum spines

development in both hatcheries: Pasit and Chanchira farm was slower in terms of gill

lamellae branch and rostrum spines. Microscopic

analysis show that the PL 3 stages from Pasit and

Chanchira farm is nearly equal to PL 3 of 85 % substitution and PL 2 of life food treatment,

respectively. In general, the quality performance

of PL from both hatcheries were still lower than PL fed with life food and with 65% substitution.

We note that these conditions were caused by

inadequate nutrition in their Artemia cyst due to

the diversity of cyst products in the market and improper Artemia hatching procedure and

feeding regime.

In the present study, appropriate concentrations of Frippak can also provide a

better growth and performance of L. vannamei

post larvae as well as gill and rostrum spines development in comparison to post larvae that

fed with Artemia nauplii. The results showed that

an appropriate concentrations of

microencapsulated diet in the diet will also make the feed more digestible and provide adequate

nutrition to induce gill and rostrum development

resulting in beneficial effects on L. vannamei post larvae performance.

Conclusion

Artemia nauplii had good dietary and promote the optimal development of gill and

rostrum spines for Pacific white shrimp

(Litopenaeus vannamei, Boone) at post larvae stages. The constant dietary availability on

http://www.sciencedirect.com/science/article/pii/S0044848611010179#bb0550












microencapsulated diet in Frippak show no

significant differences in gill and rostrum spines development compared to Artemia nauplii live

food only when 65% of Artemia was substituted

with this diet, while the differences become

significant when the live food substitution increase up to 85%. In this work, we found that

the development of gill increase the salinity

tolerance of L. vannamei PL mainly due to the higher survival percentage when exposed to lower

salinity solution.

In addition, from the sampling campaign activity, the quality of post larvae generated from

this study was better than the post larvae coming

from commercial hatchery. Even when we

compared to 85% Artemia substitution that have a lower quality of post larvae. The apparent

capacity of Artemia nauplii to promote better

quality of post larvae production offers an interesting reality that the substitution of Artemia

with microencapsulated diet in early development

of L. vannamei remains a challenge.

Acknowledgements

The author acknowledges the grant

support by De Vlaamse Interuniversitaire Raad (VLIR) Belgium for the Internship program and

technical assistance by Patipon Srianek.

References

Atwood, H.L., Young, S.P., Tomasso, J.R. and

Browdy, C.L. 2003. Survival and growth of

pacific white shrimp Litopenaeus vannamei

postlarvae in low-salinity and mixed-salt

environments. J. World Aquac. Soc., 34, pp.

518–523.

Biedenbach, J.M., Smith, L.L., Thomsen, T.K. and

Lawrence A.L. 1989. Use of the nematode

Panagrellus redivivus as an Artemia

replacement in a larval penaeid diet. Journal of the World Aquaculture Society (20): 61–

71.

Brito, R., Rosas, C., Chimal, M.E. and Gaxiola, G. 2001. Effect of different diets on growth and

digestive enzyme activity in Litopenaeus

vannamei (Boone, 1931) early post-larvae.

Aquaculture Research (32): 257-266

Buranajitpirom, D., Asuvapongpatana, S.,

Weerachatyanukul, W.,Wongprasert, K.,

Namwong, W., Poltana, P. and

Withyachumnarnkul, B. 2010. Adaptation of the black tiger shrimp, Penaeus monodon,

to different salinities through an excretory

function of the antennal gland. Cell Tissue

Res., 340, pp. 481–489.

Clavero-Salas, A., Sotelo-Mundo, R.R., Gollas-

Galva´n, T., Hernandez-Lopez, J.,

Peregrino-Uriarte, A.B., Muhlia-Almazan,

A. and Yepiz-Plascencia, G. 2007.

Transcriptome analysis of gills from the

white shrimp Litopenaeus vannamei infected with White Spot Syndrome Virus. Fish &

Shellfish Immunology (23): 459-472.

Cobo, M.L. 2013. Intensification of white shrimp

Litopenaeus vannamei (Boone) larviculture.

Ph.D. thesis, Ghent University, Belgium

Cook, H.L and Murphy, M.A. 1966. Rearing

penaeid shrimp from eggs to postlarvae. In:

Proceedings 19th Annual Conference

Southeast Association Game Fish

Community. pp. 283-288.

Cousin, M., Cuzon, G., Blanchet, E. and Ruelle, F. 1993. Protein requirements following an optimum dietary energy to protein ration for

Penaeus vannamei juveniles. In: Kaushik,

Luquet, P. (Eds.), 4th

Int. Symp. Fish

Nutrition and Feeding, Biarritz (France), 24–

27 Jun 1991 Colloq. INRA, vol. 6, pp. 599–

606.

Cuzona,G., Lawrence, A., Gaxiolac, G., Rosasc, C.

and Guillaumed, J. 2004. Nutrition of

Litopenaeus vannamei reared in tanks or in

ponds. Aquaculture (235): 513–551.

Emmerson, W.D. 1980. Ingestion, growth and development of Penaeus indicus larvae as a

function of Thalassiosira weissflogii cell

concentration. Marine Biology (58): 65-73.

Fielder, D.S., Bardsley, W.J. and Allan, G.L. 2001.

Survival and growth of Australian snapper,

Pagrus auratus, in saline groundwater from

inland New South Wales, Australia.

Aquaculture (201): 73–90

Focken, U., Schlechtriem, C., Von Wuthenau, M.,

García-Ortega, A., Puello-Cruz, A. and

Becker, K. 2006. Panagrellus redivivus mass

produced on solid media as live food for Litopenaeus vannamei larvae. Aquaculture

Research (37): 1429-1436.

Galgani, M.-L. and Aquacop, 1988. Replacement of

live unicellular algae by microparticulate diet

during larval rearing of zoeal stages of some

penaeid prawns. Aquaculture (65): 115-127.

García-Ortega, A., van Hoornyck, A., Segner, H.,

Coutteau,, P. And Verreth J. 1995. Effect

of heat treatment on the nutritional quality of

decapsulated Artemia cysts as food for

African catfish Clarias gariepinus larvae. In: Larviʼ 95- Fish & Shellfish Larviculture

Symposium (ed. by P. Lavens, E. Jaspers & I.

Roelants), pp. 281-284. European

Aquaculture Society, Special Publication

No.24, Gent, Belgium.

Gopalakrishnan, K., 1976. Larval rearing of the red

shrimp, Penaeus margindus (Crustacea).

Aquaculture (9): 145-154.



Hirata, H., Anastasios, M. And Yamasaki, S. 1985.

Evaluation of the use of Brachionus plicatilis

and Artemia for rearing prawn Penaeus

japonicus larvae on a laboratory scale.

Memoirs of the Faculty of Fisheries

Kagoshima University, Japan (34):27-36. Liao, I.C and Chien, Y.H. 2011. The Pacific White

Shrimp, Litopenaeus vannamei, in Asia: The

World’s Most Widely Cultured Alien

Crustacean, In the Wrong Place - Alien

Marine Crustaceans: Distribution, Biology

and Impacts, Volume 6, pp. 489-519.

Wickins, J.F. and Lee, D.O.C. 2002. Crustacean

Farming: Ranching and Culture. Second

edition. Blackwell Sciences, Ltd. pp. 145

Juarez, L.M., Moss, S.M. and Figueras ,E. 2010.

Maturation and larval rearing of the Pacific

white shrimp, Penaeus vannamei. In: The Shrimp Book (ed. by V. Alday-Sanz), pp. 920.

Nottingham University Press, UK.

Kitani, H. 1986. Larval development of the white

shrimp Penaeus vannamei Boone reared in

the laboratory and the statistical observation

of it’s naupliar stages. Bulletein Japanese

Society Science Fisheries (52): 1131-1139.

Kumlu, M., Eroldogan, O.T. and Aktas, M. 1999.

The effect of salinity on larval growth,

survival and development of Penaeus

semisulcatus (Decapoda: Penaeidae). Isr. J. Aquac.Bamid. (51): 114–121

Lavens, P. and Sorgeloos. P. 1996. Manual on the

production and use of live food for

aquaculture. FAO Fisheries Technical Paper

No. 361. FAO, Rome, Italy. pp. 2-5

Léger, P.H., D.A. Bengtson., K.L. Simpson. and P.

Sorgeloos. 1986. The use and nutritional value

of Artemia as a food source. Oceanogr. Mar.

Biol. Ann. Rev.(24): 521-623.

Lemos, D., Phan, V.N.and Alvarez, G. 2001.

Growth, oxygen consumption, ammonia-N

excretion, biochemical composition and energy content of Farfantepenaeus paulensis

Pérez-Farfante (Crustacea, Decapoda,

Penaeidae) early postlarve in different

salinities. Journal of Experimental Marine

Biology and Ecolog (261): 55–74.

Lovett, D.L. and Felder, D.L. 1989. Ontogeny of gut

morphology in the white shrimp Penaeus

setiferus (Decapoda, Penaeidae). Journal of

Morphology (201): 253-272.

Luvizotto-Santos, R., Lee, J.T., Branco, Z.P.,

Bianchini, A. and Nery, J.E.L. 2003. Lipids as an energy source during salinity

acclimation in the euryhaline crab

Chasmagnathus granulata Dana, 1851

(Crustacea-Grapsidae). The Journal of

Experimental Zoology (295A): 200–205.

Martins, T.G., Cavalli, R., Martion, R.C., Rezende,

E.M. and Wasielesky, W.Jr. 2006.

Larviculture output and stress tolerance of

Farfantepenaeus paulensis postlarvae fed

Artemia containing different fatty acids.

Aquaculture (252): 525-533.

McVey, J.P. and Fox, J.M. 1983. Hatchery

techniques for penaeid shrimp utilized by

Texas A&MNMFS Galveston laboratory Program. In: J.P. McVey (Editor), Handbook

of Mariculture. Vol. 1. Crustacean

Aquaculture. CRC Press, Boca Baton, pp.

129-153.

Naessens, E., Cobo, M.L., Van Hauwaert ,A., Van

Horenbeeck, M. and Sorgeloos, P. 1995. In:

Memorias del Segundo Congreso

Ecuatoriano de Acuicultura (ed. by J.

Calderón & P. Sorgeloos), pp. 101-108.

Advances of the Artemia Project,

Improvement of white shrimp Penaeus

vannamei larval production through feeding strategies based on live and formulated feed.

Escuela Superior Politécnica del Litoral,

Guayaquil-Ecuador.

Palacios, E., Bonilla, A., Pérez, A., Racotta, I. And

Civera, R. 2004. Influence of highly

unsaturated fatty acids on the responses of

white shrimp (Litopenaeus vannamei)

postlarvae to low salinity. Journal of

Experimental Marine Biology and Ecology

(299): 201–215

Péqueux, A. 1995. Osmotic regulation in crustaceans. J. Crust. Biol.(15): 1–60.

Romana, N. and Zeng C. 2012. Osmoregulation in

decapod crustaceans: implications to

aquaculture productivity, methods for

potential improvement and interactions with

elevated ammonia exposure. Aquaculture

(334): 12-23.

Rosas, C., Martinez, E., Gaxiola, G., Brito, R.,

Sánchez, A. and Soto, L.A. 1999. The

effect of dissolved oxygen and salinity on

oxygen consumption, ammonia excretion and

osmotic pressure of Penaeus setiferus (Linnaeus) juveniles. Journal of

Experimental Marine Biology and Ecology

(234): 41–57.

Sang, H.M. and Fotedar, R. 2004. Growth, survival,

haemolymph osmolality and organosomatic

indices of the western king prawn (Penaeus

latisculcatus Kishinouye, 1896) reared at

different salinities. Aquaculture (234): 601–

614.

Samocha, T.M., Burkott, B.J., Lawrence, A.L.,

Juan, Y.S., Jones, E.R. and McKee, D.A. 1998. Management strategies for production

of the Atlantic white shrimp Penaeus

setiferus as bait shrimp in outdoor ponds. J.

World Aquacult. Soc. (29): 211–220.

Saoud, I.P., Davis, D.A. and Rouse, D.B. 2003.

Suitability studies of inland well waters for

Litopenaeus vannamei culture. Aquaculture

(217): 373–383.



Setiarto, A., Strussmann, C.A., Takashima, F.,

Watanabe, S. and Yokota, M. 2004. Short-

term responses of adult kuruma shrimp

Marsupenaeus japonicus to environmental

salinity: osmotic regulation, oxygen

consumption and ammonia excretion. Aquaculture Research (35): 669–677.

Silvia, G.J., Antonio, U.R.A., Franscisco, V.O. and

Georginia, H.W. 2004. Ammonia efflux

rates and free amino acid levels in

Litopenaeus vannamei postlarvae during

sudden salinity changes. Aquaculture (233):

573–581.

Soares R., Peixoto S., Wasielesky W. and D Incao

F. 2006. Effect of different food items on the

survival and growth of Farfantepenaeus

paulensis (Pérez-Farfante 1967) postlarvae.

Aquaculture Research (37): 1413-1418.

Sorgeloos, P., Lavens, P., Leger, P., Tackaert, W.

and Versichele, D. 1986. Manual for the

culture and use of Brine shrimp Artemia in

aquaculture. FAO, Ghent, Belgium.

Vargas-Albores, F. and Ochoa, J.L., 1992. Variation

of pH, osmollality, sodium and potassium

concentrations in the haemolymph of sub-

adult blue shrimp (Penaeus stylirostris)

according to size. Comp. Biochem. Physiol.

A (102): 1-5.

Wilkenfeld, J.S., A.L. Lawrence. and F.D. Kuban. 1984. Survival, metamorphosis and growth

of penaeid shrimp larvae reared on a variety

of algal and animal foods. J. World Maricul.

Soc., (15): 31–49

Wouters, R and Van Horenbeeck,T. 2003. Larval Shrimp Feeds, Current Status. In:

Responsible Aquaculture for a Secure

Future, Proceedings of a Special Session on

Shrimp Farming (ed. by E. J. Darryl), pp. 90-

109.World Aquaculture 2003, Recife, Brazil.

The World Aquaculture Society, Baton

Rouge, Louisiana, USA.

Wouters, R., Cobo, M.L., Dhont, J., and Wille, M. 2009. Developments in feed formulations,

feeding practices and culture techniques for

marine shrimp larvae. In: The Rising Tide,

Proceedings of the Special Session on Sustainable Shrimp Farming, Aquaculture

2009 (ed. by C.L. Browdy & E. J. Darryl),

pp. 79-91. The World Aquaculture Society,

Baton Rouge, Louisiana, USA.

Zwilling, R., Dorsan, H., Torff, H.J. and Rodl, J.

1981. Low molecular mass proteases:

evidence for a new family of proteolytic

enzymes. FEBS Lett. 127 (1): 75– 78.

Documents

Effects of Different Diet Regimes on Development of Gill and Rostrum Spines of Pacific White Shrimp