Evaluation of microalgae diets for Litopenaeusvannamei larvae using a simple protocol

MAXIMIANO NUÑEZ1, CÉSAR LODEIROS1,*, MARCOS DE DONATO2

and CÉSAR GRAZIANI2

1Instituto Oceanográfico de Venezuela, Universidad de Oriente, Núcleo de Sucre, Cumaná, 6101, Ven-ezuela; 2Dpto. Biología, Escuela de Ciencias, Universidad de Oriente, Núcleo de Sucre, Cumaná, 6101,Venezuela; *Author for correspondence (e-mail: clodeiro@sucre.udo.edu.ve)

Received 8 October 2001; accepted in revised form 5 February 2002

Key words: Food, Mieroalgae, Penaeus, Shrimp, Venezuela, Zoea

Abstract. We evaluated native marine microalgae isolated from Northeastern Venezuela, as food forLitopenaeus vannamei larvae, compared to six microalgae commonly used in aquaculture. We evaluatedthe microalgal ingestability calculating the biomass consumed by larvae, later we evaluated threemonoalgal diets on protozoea stages of L. vannamei over 72 hr based on the larval biomass, RNA/DNAindex and the proportion of stage retarded larvae. The presence of every microalgae species in the pro-tozoea’s digestive tract indicated their consumption. Chaetoceros spp. showed high ingestability andTetraselmis spp. and Skeletonema sp. Ch1 showed low ingestability. The native microalgae Chaetocerossp. A1 and Chaetoceros sp. G1 were evaluated as monoalgal diets compared to Chaetoceros gracilis.Ch. gracilis demonstrated the best results when the evaluation was performed with the RNA/DNA in-dex. However, the survival rate, larval biomass and percentage of stage retarded larvae fed with Cha-etoceros sp. G1 were not statistically different from those fed with Ch. gracilis. Taking into account thatnative microalgae produced higher biomass in shorter times when they were cultured in field conditions,the results suggest that native microalgae are adequate for large-scale culture conditions, thus reducingcosts. The determination of microalgal size and the larval consumption, dry and organic larvae biomassand specially the RNA/DNA index, were valuable features of this evaluation. The protocol developed inthis study could be used to evaluate microalgae quality under different environmental conditions andproduction regimes.

Introduction

In aquaculture, the production of larvae and juveniles in good nutritional and healthconditions is one of the most important factors to assure success. Larvae generallyfeed from different species of microalgae, and the amount and quality of food aresome of the most critical factors affecting larval development. Thus, the biochemi-cal composition of the food used for feeding the larvae provides the basic elementfor their present and future metabolism, growth and reproduction.

The nutritional value of a microalgal species depends on the main molecularcomponents such as proteins, carbohydrates and lipids (specially fatty acids), quali-fying them as good or bad quality food (Napolitano et al. 1990). At the same time,the biochemical content of the microalgae depends on several factors, such as the

Aquaculture International 10: 177–187, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

strain, type and amount of nutrients used in the culture medium, temperature andlight conditions, stage of the growth curve at harvest and water quality (Abalde etal. 1994; Duerr et al. 1998).

In general, the nutritional elements of a microalgae required for high survival ofpenaeid larvae are a protein content higher than 25% of its dry weight, 8–30% car-bohydrate content, and around 10% lipid content, specially including certain typesof lipids such as the fatty acids C20:5w3 and C22:6w3 (Tobias-Quinito and Ville-gas 1982). Several species of Chaetoceros have been shown to meet these require-ments, and serve as an excellent food for penaeid larvae (Simon 1978; Tobias-Qui-nito and Villegas 1982; D’Souza and Loneragan 1999).

Several studies have found that indigenous species of microalgae perform betterthan some of the exotic species commonly used for culture. For example, Sanchez(1986) found that larvae of Litopenaeus vannamei cultured in Florida and fed witha native microalgae Isochrysis sp. exhibited the highest survival from larval stagesof protozoea I to postlarvae I, compared to those fed with other non-indigenousspecies. Renaud et al. (1994) evaluated the biochemical composition of 12 microal-gae native to Australia and found that these species contained a higher proportionof essential fatty acids and other biochemical components, compared to the non-indigenous species used for culture.

Marín et al. (1994) compared the growth rate, dry biomass and bacteriologicalquality of 18 strains of exotic microalgae and 7 native species isolated from North-eastern Venezuela, under large scale culture conditions. They found that, from theexotic strains, only Tetraselmis chuii showed satisfactory results for mass cultureunder “tropical” conditions. They also found that among the native species, Tetra-selmis sp. G1, Chaetoceros sp. G1, Chaetoceros sp. A1 and Skeletonema sp. Ch1,showed good growth and high dry biomass. They suggested that these native spe-cies could be cultured as food for larvae of marine organisms such as fish, mol-lusks and crustaceans.

Since shrimp culture in the tropics is one of the most important and largest in-vestment activities in aquaculture, the improvement of any aspect of shrimp larvi-culture would have a positive effect on this industry. In this study we evaluated thein vivo use of the above-mentioned species of microalgae as food for larvae of L.vannamei using a simple protocol, comparing the non-native to native species.

Methods

Microalgal ingestability study

We determined the microalgal ingestability using protozoea of the Pacific whiteshrimp Litopenaeus vannamei. For this, we tested the following native species ofmicroalgae isolated by Marín and Lodeiros (1990) in the northeast of Venezuela: a)Chaetoceros sp. A1 isolated from the intake channels of the Araya Saltwork, b)Skeletonema sp. Ch1 isolated from the Chacopata marine lagoon, c) Chaetoceros

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sp. G1 and Tetraselmis sp. G1 isolated from the shallow waters of the Golfo deCariaco. In addition, we tested 6 non-indigenous species widely used in aquacul-ture, from the collection of the Laboratorio de Acuicultura of the Instituto Ocean-ográfico de Venezuela, Universidad de Oriente: Skeletonema costatum, Ch. calci-trans, Ch. gracilis, T. chuii, Isochrysis galbana and Thalasiossira pseudonanna.

Cellular biomass of each microalgae was determined in order to quantify theamount of biomass supplied to the shrimp protozoea. For this, all species were cul-tivated intensively in the medium F/2 (Guillard 1974), continuously increasing thevolume of the cultures, starting from a 5 ml, 250 ml and 2 l culture, with 3 replicasper species. During the exponential growth phase, 300 ml of culture of each spe-cies were harvested by mechanical filtration through a fiberglass Whatman GFF fil-ter (with a particle retention of 0.7 �m), using vacuum Millipore equipment. Thefilters containing the samples were dried for 12 hr at 60 °C and weighed to deter-mine the microalgal biomass. The cellular concentration was determined using theNeubauer chamber (hematocytometer) and the weight was used to determine thecellular biomass of each algae.

The L. vannamei larvae used in this study were obtained from the Aquamarinade la Costa hatchery, Santa Fe, Estado Sucre, Venezuela. They were transported, atthe nauplius stage, to the laboratory. The transport of the larvae at this stage per-mitted assays to begin 24 hours after arrival at the lab, when they reach the proto-zoea I stage. They were carried to the lab. in 10 -l plastic bags with 1/3 volumefilled with water and 2/3 with oxygen, at a temperature of 26–27 °C and a concen-tration of 1,000 larvae/l. The bags were placed inside 50 l tanks for acclimation tothe experimental temperature of 29–30 °C, which is the optimal temperature forlarval development in this species (Wyban and Sweeney 1991). After 2 hrs, the lar-vae were placed in the same tanks with moderate aeration.

Larval density was determined by extrapolation of the concentration of larvameasured from aliquots in a Bogorov chamber. The experimental treatments startedwith 40 larvae in a sterile flask containing 200 ml of sterilized seawater. Each treat-ment consisted of a monoalgal diet with three flasks per treatment. The feedingwith the microalgae was done by supplying a biomass of each algae equivalent tothe biomass of Ch. gracilis at a concentration of approximately 150.000 cells·ml−1,which is the standard feeding concentration for the larval development of L. van-namei in many hatcheries (Simon 1978; Wyban and Sweeney 1991), includingAquamarina de la Costa.

After 2 hours of feeding, we evaluated the ingestability by the presence of thealgae inside the digestive tract of the larva. Observations were done by harvestingthe larvae with a mesh of 80–100 �m, fixing the larvae with a 10% formaldehydesolution and inspecting them with a stereoscopic microscope. In addition, we cal-culated the ingested microalgal biomass, which was the difference between the ini-tial and final biomass measured in the larvae flasks following mechanical stirringto mix the contents.

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Evaluation of monoalgal diets

The nutritional value of the monoalgal diets was evaluated using the developmentand moulting of the protozoea I stages of L. vannamei larvae over a 72 hour pe-riod. The algae were cultured as described above, and 600 larvae were placed in 4l glass containers with 3 l of filtered and UV irradiated water. The larvae were fedafter the period of acclimation (1–2 hrs post arrival) and every 6 hr. One ml watersamples were taken from each treatment to determine the microalgae concentra-tions. In order to maintain the same algal abundance throughout the experiment puremicroalgal culture was added periodically. The replacement amounts of pure cul-ture were calculated by the following formula:

Va � Vt�Da/Dt

where:Va = Volume of pure culture to be addedVt = Volume of water in the larval cultureDa = Cell concentration needed for the larval cultureDt = Cell concentration in the pure cultureAt the end of the experiment, the larvae in each container were harvested using

a mesh. We determined the survival, dry and organic biomass, RNA/DNA index,and the proportion of stage retarded larvae from the proportion of protozoea atstages II and III. Dry biomass was determined with about 200 larvae, in triplicateper treatment, after drying the samples for 18 hrs at 60 °C. The organic biomasswas determined from the difference between the dry mass and weight after bakingat 450 °C for 2 hours.

Total nucleic acids were extracted from samples of about 20 mg of tissue (10larvae) in each replicate and the RNA/DNA index was determined by a fluorimet-ric method according to Canino and Calderone (1995). The tissue was homogenizedin vials, digested in 100 ml of sarcosine (0.1%), mixed with 900 �l of TE buffer(50 mM of Tris-HCl, 0.5 nM of EDTA, pH 7.5) and centrifuged at 2500 rpm for 15min. Two 1 -�l aliquots of the supernatant were mixed in 500 �l of water contain-ing, one 4 �g.l−1 ethidium bromide and another 5 �g.l−1 Hoechst 33258, and theabsorbance was determined in a fluorometer (Sequoia-turner model 450) at 450 and585 nm, respectively. The RNA/DNA index was determined from the RNA + DNA(Ethidium bromide staining) and DNA (Hoechst staining) quantities in the sample(calculated from calibration curves from their respective absorbances).

Statistical analysis

The ingestability of the microalgae was analyzed through the consumption of mi-croalgal biomass using a one-way analysis of variance (ANOVA I), taking each mi-croalgae as a treatment. Similarly, in the evaluation of monoalgal diets for the lar-val development, the RNA/DNA index, larval dry biomass and proportion of stageretarded larvae, were analyzed using the same statistical test. In all cases we used

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the average measure of samples from each replicate treatment as the value for thereplicate used in the analysis. In those cases where the ANOVA I test showed sig-nificant differences (p < 0.05), a Scheffè test (p < 0.05) was used to determine thegrouping of the treatments. The RNA/DNA index and the proportion of stage re-tarded larvae were analyzed using an arcsine transformation, in order to normalizethe data, according to the recommendations of Zar (1984).

Results

Microalgal ingestability

Microalgal dry biomass in this study was similar to that reported in the studiescompiled by Yúfera and Lubián (1990) for exotic algae. The cellular masses weresignificantly different, and the Scheffè test revealed two distinctive groups: a highbiomass group, containing the microalgae from the genus Tetraselmis, and a lowerbiomass group, containing all the other species (Figure 1). Tetraselmis sp. G1showed the highest biomass (272 pg/cell) and Isochrysis galbana showed the low-est (24 pg/cell) and the biomass of the rest varyed from 60 to 95 pg/cell.

The presence of all species used in the protozoea’s digestive tract and the de-crease in microalgal concentrations from the cultures indicated that they were con-sumed by the larvae. However, the amount consumed by the larvae varied signifi-cantly and was dependant on the species. Groups of high ingestability (Chaetocerosgracilis and Ch. calcitrans), medium ingestability (Chaetoceros. sp. G1, Thalassio-sira pseudonanna, Skeletonema costatum, Isochrysis galbana and Chaetoceros sp.A1), and low ingestability (Tetraselmis chuii, Tetraselmis sp. G1 and Skeletonemasp. Ch1) were found (Figure 2).

Dietary evaluation

Only the species Chaetoceros sp. G1 and Chaetoceros sp. A1 were used in this partof the study because of their high ranking of ingestability among the native mi-croalgal species. Ch. gracilis was used for comparison because of its high ingesta-bility and its worldwide use in the culture of Litopenaeus vannamei (Wyban andSweeney 1991).

At the end of the assays, the protozoea fed with Ch. gracilis and Chaetocerossp. G1 showed slightly higher survival (76 and 74%, respectively), than Chaetoc-eros sp. A1 (66%; Figure 3); but the differences were not statistically significant.

The final dry and organic biomass of the larvae fed with the monoalgal dietstested, showed significant differences. The larvae fed with the exotic species Ch.gracilis developed the highest biomass both dry and organic (Figure 3), with 61and 28 ng/larvae, respectively. These weights were not significantly different fromthe ones shown by the larvae fed with Chaetoceros sp. G1, which achieved dry andorganic biomass of 44 and 13 ng/larvae, respectively, but were significantly differ-

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ent from the weights of the larvae fed with Chaetoceros sp. A1, which showed dryand organic biomass of 29 and 8 ng/larvae, respectively.

The RNA/DNA index of the larvae exhibited the same pattern as the weights(Figure 3). However, larvae fed with Ch. gracilis exhibited a significantly higherRNA/DNA index (6.25) than the ones fed with Chaetoceros sp. G1 and A1 (4.7and 3.5, respectively).

Even though all the larvae were the same age at the beginning of the assay, atthe end not all the larvae had molted to the protozoea III stage. The percentage ofstage retarded larvae (at protozoea II stage) was 6, 9 and 13% of the larvae fedwith Ch. gracilis, Chaetoceros sp. G1 and A1, respectively. These proportions werenot statistically different.

Figure 1. Cellular dry biomass of native and exotic microalgae species used for the microalgal inge-stability study. The vertical lines represent the standard error and the horizontal lines represent speciesshowing no significant differences in biomass, according to the Scheffè test (P < 0.05).

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Discussion

The biomass consumed by Litopenaeus vannamei larvae differed depending on thealgae used for feeding, even though all the species tested were ingested by the lar-vae. Of all the microalgae used in this experiment, the diatoms produced the high-est consumable biomass. This demonstrates the advantage of using diatom speciesas food for larval development of shrimp and other marine organisms (Simon 1978).

The variation in the biomass of microalgae consumed by L. vannamei could bedue to several factors, including the size of the algae. According to Mc Vey andFox (1983), the adequate cellular diameter for feeding shrimp protozoea rangesfrom 3 to 30 �m. However, detailed studies on the effect of the size of microalgae

Figure 2. Average percentage of consumed biomass of the native and exotic microalgae species usedfor feeding Litopenaeus vannamei larvae. The vertical lines represent the standard error and the hori-zontal lines represent the species with consumed biomass showing no significant differences, accordingto the Scheffè test (P < 0.05).

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on feeding (Tobias-Quinito and Villegas 1982; Okauchi and Hirano 1986; Clark etal. 1988; Jones and Canavate 1988) have indicated that protozoea prefer diameterssmaller than 10 �m. These reports agree with our results, where the smallest mi-croalgae (Chaetoceros gracilis and Ch. calcitrans) were consumed at a higher ratecompared to larger microalgae (Tetraselmis chuii and Tetraselmis sp. G1). An ex-ception to this was Skeletonema sp. Ch1 which had a low consumed biomass inrelation to its small size (7–8 �m). This result could be related to the small setaesize (15–17 �m), compared to the setae sizes in Chaetoceros and Skeletonema cos-tatum (25–30 �m), making it more difficult for the protozoeae to capture the mi-croalgae. This suggests that cellular morphology as well as volume are factors

Figure 3. Percentage of survival, dry (solid) and organic (stripped) biomass, RNA/DNA index and av-erage of stage-retarded of Litopenaeus vannamei larvae fed with three microalgae species. The verticallines represent the standard error and the horizontal lines represent species showing no significant dif-ferences, according to the Scheffè test (P < 0.05).

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which affect microalgae consumption. Thus, it is important to take into account theappendages of diatoms when selecting a microalgal diet.

In this study, Ch. gracilis showed the highest consumed biomass, which agreeswith the general use of this microalgae by the major shrimp producers in Venezuela(Aquamarina de la Costa, Siembramar and Aquacam) and other countries (Labora-tory of Larviculture of Mariculture S.A, in Costa Rica, and The Oceanic Instituteof Honolulu, in Hawaii, USA) (Simon 1978; Wyban and Sweeney 1991).

Larvae fed with Ch. gracilis and Chaetoceros sp. G1 had a higher survival rate,although not significantly different from those fed with Chaeroceros sp. A1. The66–76% survival of L. vannamei larvae obtained in this study is high, consideringthe conditions of culture, high density (200 larvae/l) and no water exchange. Thismay be compared to a survival rate between 70–90% under normal culture condi-tions of commercial hatchery operations, with densities of 100–150 larvae/l andwater exchange (Simon 1978; Arellano 1990).

Microalgal diet affected the dry and organic biomass as well as the RNA/DNAindices of shrimps larvae. The larvae fed with Ch. gracilis showed a higher dry andorganic biomass than those fed with Chaetoeros sp. A1. Larvae fed with Chaeto-eros sp. G1 did not show statistical differences in performance with either of the 2groups. This fact suggests the Chaetoeros sp. G1 is a suitable microalgae for shrimplarviculture. The RNA/DNA index, however, was significantly higher for the larvaefed with Ch. gracilis. This results demonstrate the greater sensitivity of the RNA/DNA index as an indicator of the physiological condition of shrimp larvae, makingit a good tool not only to quickly assess the effect of diet on organisms, but also ofother factors that affect growth, such as environmental conditions and presence ofxenobiotic compounds. This index has been used to establish a relationship betweenthe amount of RNA and the RNA/DNA index with different diet conditions within24 h (Moss 1994). In this study, the average RNA/DNA index, also known as in-stantaneous growth index, was slightly higher for larvae fed with Ch. gracilis (6.25)than that reported by Moss (1994) in juveniles of L. vannamei fed with a commer-cial microencapsulated food (6.15) and with microalgae of the Chaetoceros genus(6.01).

In our study we found that 83–94% of the larvae reached protozoea III stage,independently of the microalgal diet. This is comparable with Sanchez (1986) whofound that 90% of the L. vannamei larvae passed to protozoea II stage when fedwith Isochrysis sp. which is of similar size to the Chaetoeros species used in thisstudy.

Because the biochemical composition of each microalgae species varies, the useof monoalgal diets could produce a shortage of essential nutrients needed for theadequate development of penaeid shrimp (Gopalakrishnan 1976; Yúfera and Lu-bián 1990). Even though the use of a combination of several microalgae as a dietfor shrimp larvae is recommended, monoalgal diets have been widely used forcommercial culture due to reduced contamination risks and costs, as well as easymanipulation and feeding regimes.

The species belonging to the genus Chaetoceros have an adequate biochemicalcomposition of essential polyunsaturated fatty acids, mainly omega 3 highly unsat-

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urated fatty acids (Akiyama et al. 1992; D’Souza and Loneragan 1999), requiredfor the growth of several aquatic organisms. The species Ch. gracilis and Chaeto-ceros sp. G1 have high levels of lipids (> 12%), mainly from polyunsaturated fattyacids (Lopez 2000).

Although Ch. gracilis demonstrated the best results as a diet for L. vannameilarvae in this study, Chaetoceros sp. G1 produced a survival rate which was notstatistically different. Previous comparisons between native and exotic microalgae(including Ch. gracilis) have shown that native microalgae produce a higher bio-mass in a shorter time than the exotic ones, when cultured in a large scale and un-der field conditions (Marín and Lodeiros 1990; Marín et al. 1994). This suggeststhat native microalgae are adequate for large scale culture conditions, saving costsand producing a higher shrimp biomass. Additionally, Chaetoceros sp. A1 showeda high division rate, and maintained a high microalgal concentration throughout theassay.

The protocol developed here helped to assess the evaluation of the tested mi-croalgae as diets for shrimp larvae. The determination of microalgal size, dry andorganic biomass and larval consumption, dry and organic larval biomass and espe-cially the RNA/DNA index, were valuable features of this evaluation. This proto-col could be used to evaluate microalgae quality and nutritional value under differ-ent environmental conditions and production regimes.

Acknowledgements

This study was possible due to the facilities provided by the Aquamarina de la CostaCompany. We are particularly indebted to C. Howell, R. Ramirez, R. Verginelli, J.Belmonte and R. Manrrique for the larvae supply and useful comments of themanuscript. We also want to acknowledge the corrections made by Frances Osbornon the English style. The research was supported partially by grants of Fondo Na-cional de Investigaciones Científicas y Tecnológicas of Venezuela to C.L.

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