ARTEMIA BIOENCAPSULATION I. EFFECT OF PARTICLE SIZES ON ...

JOURNAL OF CRUSTACEAN BIOLOGY, 21(2): 435–442, 2001

ARTEMIA BIOENCAPSULATION I. EFFECT OF PARTICLE SIZES ON THE FILTERING BEHAVIOR OF ARTEMIA FRANCISCANA Rolando Gelabert Fernández Centro de Investigaciones Marinas, Universidad de La Habana, Cuba. Present address: Laboratorio de Biología Marina Experimental, UNAM, Calle 26 No. 1 Playa Norte, Ciudad del Carmen, Campeche, C.P. 24140, Apdo. Postal No. 69. México (e-mail: [email protected], [email protected]) A B S T R A C T Artemia is not believed to be a selective filter-feeding organism; however, evidence of an influence of the size of the nutrient particles on the filtration process of this crustacean is presented. To evaluate the influence of the size of food on the filtration process, assays with latex particles and with starch granules were made. A relation between the frequency distribution of size of the particles found in the digestive tract of the animals and the frequency distribution of size of the particles found in the medium is shown. The results indicate that Artemia has a preference for food of specific size. The behavior of different size-classes of animals analyzed is shown, but I conclude the size of food for Artemia must range between 6.8 and 27.5 µm, with the optimum about 16.0 µm.

“Bioencapsulation involves the entrapment of biologically active materials in gel microspheres or membrane-bound microcapsules. The resulting microspheres may contain enzymes, DNA, vaccines, viable cells, or other biologically or pharmacologically active materials.” (Poncelet, 1996). Bioencapsulation in aquaculture can be defined as a process where a live organism incorporates a certain product either orally or by phagocytosis, pinocytosis, or endocytosis and modifies its original composition. This organism becomes a live capsule. The nature of the product given can vary depending on the desire of the aquaculturist. The process is generally directed to the incorporation of some essential element for the diet. Modification of the nutritional value of Artemia for aquaculture purposes has been done for years. Different papers show nutritional differences among Artemia strains (Lovell, 1990; Chen, 1997) and indicate the necessity of improving them to guarantee adequate nourishment of their predators (Dhert et al., 1990; Devresse et al., 1994). Various materials have been bioencapsulated; microalgae (Watanabe et al., 1978, 1980, 1982, 1983; Olsen et al., 1997), yeasts enriched with different oils (Gatesoupe, 1991), liposomes (Hontoria et al., 1993, 1994; Ozkizilcik and Chu, 1994; McEvoy et al., 1996), emulsions (Leger et al., 1987a; Kontara, 1991; Clawson and Lovell, 1992),

and bacteria (Gorospe et al., 1996; GomezGil et al., 1998). Different nutritional levels in Artemia are shown, depending on the type of food and concentrations of polyunsaturated fatty acids (McEvoy et al., 1997; Navarro et al., 1997). Today there are many papers about bioencapsulation. Many of them deal with different commercial or noncommercial products for Artemia and rotifer bioencapsulation, but the way that the products must be used is not always clear. Not all of the papers offer scientifically tested information about the conditions needed to bioencapsulate. Many of those papers do not agree about the time, concentration, kind of food employed, and density of animals used for bioencapsulation. This paper tries to provide information on one of the aspects involved in this procedure because references about it are scarce. Dobbeleir et al. (1979) proposed Artemia should be fed with a food smaller than 50 µm, but Gelabert and Solis (1994) showed the size of the food should be between 7 and 29 µm. The present results show the importance of particle size in the filtration process by Artemia franciscana and the relevance of particle size to efficient bioencapsulation. MATERIALS AND METHODS To determine the selectivity of food by Artemia, assays comparing particle-size frequency distribution in the gut of animals and in the media were made.

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Fig. 1A. Experiment I. Comparison between latex particles in the media and in the gut of animals. (Particles in the media n = 400, particles in gut n = 750, animals sampled n = 50.) B. Experiment II. Particle size frequency distribution in starch particles suspension. C. Selectivity for size class 1. (Particles in gut n = 1,386, animals sampled n = 29.)

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Table 1. Sample size (n), the correlation coefficient (R2), F value, and probability (P) from each regression line. Size-class

n

R2

F

P

1 2 3 4 5 6 7 8 All size classes

13 20 17 25 27 24 27 24 27

0.86 0.68 0.61 0.62 0.47 0.43 0.36 0.79 0.61

18.73 11.48 6.78 11.29 6.99 5.05 4.37 25.56 12.00

3.28–4 2.89–4 5.00–3 1.27–4 1.64–3 9.10–3 1.41–2 4.78–7 6.21–5

For the first experiment, a suspension of 5- and 15µm diameter latex particles was used as food. The concentration in the medium of 5-µm particles was 159,000 particles/ml (68%) and for 15-µm particles was 74,000 particles/ml (32%) (Fig. 1). About 50 animals were put into the suspension for 1 h. In this experiment about 6 or 7 animals were studied for each size class. In a second experiment, a suspension of starch particles between 1 and 69 µm was prepared. A mix of 440 mg soluble starch (BDH, ANALR analytical reagent) and 60 mg wheat starch put through a 71-µm sieve was used. Both quantities were put in a flask with filtered and boiled 35‰ sea water and vigorously shaken. A suspension of the particles (25 ml) was taken and put in a 500-ml flask with enough sea water to get a concentration of 50 mg/l. About 50 individual Artemia of different size classes were put in the flask, which was inverted every 60 seconds to avoid precipitation and selection of small particles. After ten minutes, the animals were collected on a 400µm sieve and rinsed with boiled and filtered sea water to remove uningested particles. The Artemia were killed with lugol and were put into an iodo-glycerin preparation to avoid water evaporation and to color the starch. Artemia individuals were measured between the beginning of the cephalothorax and the furca, not including spines. A stereomicroscope (MBC–9) with an ocular micrometer and 16× resolution was used to measure 211 organisms included in 8 size classes, with the first size class from 1 to 1.99 mm, the second from 2 to 2.99 mm, and the last from 8 to 8.99 mm. Animals were dissected to recover the particles ingested, which were measured to obtain their frequency distribution. About the same quantity of particles for each size class of animals was measured to avoid bias. For both experiments, distribution of particle size in the culture media was made to compare with the distribution in the gut. For the first experiment, a Chi-square test was made to compare the proportions of the different particles inside and outside of the gut. Comparison of the particle proportion present in the gut and in the suspension was done with the Ivlev selectivity formula (Salazar and González, 1986) S = G – E/G + E where S = selectivity index, G = percentage of particles in the gut, and E = percentage of particles in suspension. If the frequency of a given particle size in the gut is greater than the frequency found in the suspension, the animal selects those particles positively. If the reverse, the selection is negative. The value of S is between –1

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and +1. Selectivity index < 0 indicates negative selectivity of little acceptability, whereas an index > 0 indicates preference or acceptability for a particle size. An additional processing to smooth the individual variations of the results was made. A third-order polynomial regression of the selectivity data with respect to the sizes of the particles ingested by the individuals from different size classes was calculated. It shows the ingestion limits for each of the size-classes of the animals used. The F-value and resulting P-value were used as an overall F test of the relation between the dependent variable and the independent variables.

RESULTS Experiment I: Latex Particles As shown in Fig. 1A, concentration of 5-µm particles in the media was 68% and for 15-µm was 32% (n = 400). For the animals studied (n = 50), concentrations of these particles in the gut show an inverse behavior. Those of 5-µm represent 37% and 15-µm represent 63% (n = 750). The Chi-square comparison of the distributions of particles was significant α = 0.05 (P = 0.0000). Selectivity index for 5-µm particles was negative (–0.295), whereas for the 15-µm particles it was positive (0.326). Artemia prefer 15-µm particles rather than 5-µm particles. Experiment II. Starch The size-frequency distribution of starch granules found in the suspension is shown in Fig. 1B. Granules greater than 36 µm appeared at very low frequency. Table 1 shows the sample size (number of selectivity index used for each regression line (n), correlation coefficient (R2), F-value, and probability (P) from each regression line obtained by plotting size of particles against selectivity index. All of the regression models are significant (P < 0.05). The first size class, 1–1.99 mm (Fig. 1C), has an ingestion limit between 4.1 and 17.7 µm, with 10 µm the size of greatest frequency in the gut. Animals between 2 and 2.99 mm (sizeclass 2, Fig. 1D) incorporated particles greater than 4.3 but smaller than 22.5 µm. The peak was at 13 µm. For the individuals of size class 3 (3–3.99 mm, Fig. 1E), the polynomial regression model does not show a pronounced peak, and it appears the ingestion range is from about 7 µm to 29 µm. Particles with the greatest frequency in the gut, therefore those of greatest acceptability, were 21 µm. Individuals of size class 4 (4–4.99 mm, Fig.

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Fig 1D. Selectivity for size class 2. (Particles in gut n = 650, animals sampled n = 24.) E. Selectivity for size class 3. (Particles in gut n = 719, animals sampled n = 25.) F. Selectivity for size class 4. (Particles in gut n = 743, animals sampled n = 26.)

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Fig. 1G. Selectivity for size class 5. (Particles in gut n = 842, animals sampled n = 27.) H. Selectivity for size class 6. (Particles in gut n = 733, animals sampled n = 27.) I. Selectivity for size class 7. (Particles in gut n = 703, animals sampled n = 27.)

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Fig. 1J. Selectivity for size class 8. (Particles in gut n = 729, animals sampled n = 26.) K. classes grouped.

1F) mainly ingested particles between 7.4 µm and 27.8 µm, with 16.4 µm those of greater acceptance. In Fig. 1G (5–5.99 mm), positive selectivity is between 8.8 and 32.0 µm, with a peak at 18.7 µm. The particles between 7.7 and 30.4 µm were those accepted best by individuals between 6 and 6.9 mm length, with 18.0 µm the size most frequently taken into their digestive tract (Fig. 1H). For the individuals of size class 7 (Fig. 1I, 7–7.99 mm), ingestion limits were between 6.7 and 26.4 µm, with a peak at 15.7 µm. For the largest size class (8–8.9 mm) (Fig. 1J) the ingestion limits were between 8.0 and 28.0 µm, with those 17.0 µm the most frequent.

Selectivity for all size

Figure 1K shows the behavior of the selectivity index of all the animals from all the size-classes. This analysis shows that Artemia has a selectivity range for ingestion between 6.8 and 27.5 µm. For all sizeclasses, particles of 16.0 µm were the mostoften digested. DISCUSSION That Artemia is a selective filter-feeding organism is now not in question because results indicate that there is a preferential ingestion related to the size of the particles found in the medium. The importance of particle size in the filtration process of Artemia franciscana

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is corroborated. Not all particles are ingested in the same way. In a medium where both 5 µm and 15 µm latex particles are present, Artemia incorporates a greater percentage of 15-µm particles. These results are the same for all size classes tested with the starch particles. Analyzing the information in the literature and the results obtained in these assays, we see that Artemia ingests a wide range of foods with sizes over a wide range less than 50 µm (Dobbeleir et al., 1979). In my samples, individuals were observed with food particles as large as 69.6 µm in the digestive tract; however, large particles were not abundant. Hontoria et al. (1994) obtained maximum ingestion and liposome accumulation from Artemia placed into a labeled liposome suspension for 30 hours. These liposomes had sizes ranging between 268 nm and 1,314 nm. It would appear such sizes of food are difficult for Artemia to ingest, so an extended time was required to achieve those results. In general, enrichment trials done with Selco as an enrichment agent extend for more than 12 hours, but less than 30 hours (Cappellaro et al., 1993). This diet is an autodispersed lipid emulsion that produces fine globules of approximately 2-µm diameter (Leger et al., 1987b). With respect to time, Selco enrichment protocols are better than those with liposomes, presumably because of the sizes of the micelles found in the enrichment media. Southgate and Lou (1995), using gelatinacacia microcapsules containing cod liver oil or squid oil for enrichment of Artemia nauplii, noted these microcapsules were readily ingested by Artemia nauplii and were visible in the gut a few minutes after the start of enrichment. Ingestion of microcapsules containing cod liver oil or squid oil resulted in a significant increase in the HUFA content of nauplii after only one hour. The microcapsules had a mean diameter of 4.85 µm (± 2.84, n = 100) or 5.07 µm (±3.15, n = 100) depending on the oil used in their preparation. From the result of these assays, I presume for Artemia bioencapsulation or feeding, it is better to use particle sizes allowing the digestive tract to fill easily. It can reduce energetic waste in feeding and reduce the time to fill the digestive tract (Gelabert, unpublished data), and those results are obtained with particle sizes ranging between 6.8 and 27.5 µm.

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Results shown here reveal the necessity to look for more efficient bioencapsulation processes. Determining the time, concentration of food, and density of animals is necessary to find out how to make the bioencapsulation more efficient. Defining the range of sizes ingested will have repercussions related to biomass production, and could be used as a model for feeding other Artemia strains. ACKNOWLEDGEMENTS Thanks to the Polymer Laboratories for the gift of the latex particles used in the trials. Thanks to Dr. Ellis Glazier for editing the English-language text.

LITERATURE CITED Cappellaro, H., L. Gennari, L. Achene, and G. Brambilla. 1993. Artemia salina as medicated feed for marine fry.—Bollettino Societa Italiana di Patologia Ittica 5: 29. Clawson, J. A., and R. T. Lovell. 1992. Improvement of nutritional value of Artemia for hybrid striped bass/white bass (Morone saxatilis × M. chrysops) larvae by n-3 HUFA enrichment of nauplii with menhaden oil.—Aquaculture 108: 1, 2. Chen, L. 1997. Application of multivariate analysis in nutritional evaluation of Artemia.—Marine Science Bulletin. Haiyang Tongbao 16: 66–75. Devresse, B., P. Leger, P. Sorgeloos, O. Murata, T. Nasu, S. Ikeda, J. R. Rainuzzo, K. I. Reitan, E. Kjorsvik, and Y. Olsen. 1994. Improvement of flat fish pigmentation through the use of DHA-enriched rotifers and Artemia.—Aquaculture 124: 1–4. Dhert, P., P. Lavens, M. Duray, and P. Sorgeloos. 1990. Improved larval survival at metamorphosis of Asian seabass (Lates calcarifer) using omega 3-HUFA-enriched live food.—Aquaculture 90: 63–74. Dobbeleir, J., N. Adam, E. Bossuyt, E. Bruggeman, and P. Sorgeloos. 1979. New aspects of the use of inert diets for high density culturing of brine shrimp. The Brine Shrimp Artemia 3: Ecology, Culturing, Use in Aquaculture. P. 456 in G. Persoonne, P. Sorgeloos, O. Roels, and E. Jaspers, eds. Universa Press, Bélgica. Evjemo, J. O., P. Coutteau, Y. Olsen, and P. Sorgeloos. 1997. The stability of docosahexaenoic acid in two Artemia species following enrichment and subsequent starvation.—Aquaculture 155: 1–4. Gatesoupe, F. J. 1991. Managing the dietary value of Artemia for larval turbot, Scophthalmus maximus: the effect of enrichment and distribution techniques.— Aquacultural Engineering 10: 111–119. Gelabert, R., and L. Solis. 1994. The selection of food particle size by Artemia from Guantanamo, Cuba.— Revista de Investigaciones Marinas 15: 141–145. Gomez-Gil, B., M. A. Herrera-Vega, F. A. Abreu-Grobis, and A. Roque. 1998. Bioencapsulation of two different Vibrio species in nauplii of the brine shrimp (Artemia franciscana).—Applied and Environmental Microbiology 64: 2318–2322. Gorospe, J. N., K. Nakamura, M. Abe, and S. Higashi. 1996. Nutritional contribution of Pseudomonas sp. in Artemia culture.—Fisheries Science 62: 914–918.

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Hontoria, F., J. H. Crowe, L. M. Crowe, and F. Amat. 1993. Bioencapsulation of liposomes in Artemia nauplii. Potential use as delivery system in larviculture.— Actas Del IV Congreso Nacional De Acuicultura. Cervion, A: 497–502. ———, ———, ———, and ———. 1994. Potential use of liposomes in larviculture as a delivery system through Artemia nauplii.—Aquaculture 127: 2, 3. Kontara, E. K. 1991. Growth and survival of Penaeus monodon postlarvae fed with Artemia nauplii enriched with (n-3) highly unsaturated fatty acids. Pp. 74–75 in P. Lavens, P. Sorgeloos, E. Jaspers, and F. Oliverr, eds. Symposium on Fish and Crustacean Larviculture Larvi 91. Leger, P., D. Bengtson, P. Sorgeloos, K. L. Simpson, and A. D. Beck. 1987a. Nutritional value of Artemia: a review. Pp. 359–370 in P. Sorgeloos, D. A. Bengtson, W. Decleir, and E. Jasper, eds. Artemia Research and Its Application. Vol. 3. Ecology, Culturing, Use in Aquaculture. Universa Press, Wetteren, Belgium. ———, E. Naessens-Foucquaert, and P. Sorgeloos. 1987b. Techniques to manipulate the fatty acid profile in Artemia nauplii and the effect on its nutritional effectiveness for the marine crustacea Mysidopsis bahia M. Pp. 411–424 in P. Sorgeloos, D. A. Bengtson, W. Decleir, and E. Jasper, eds. Artemia Research and Its Application. Vol. 3. Ecology, Culturing, Use in Aquaculture. Universa Press, Wetteren, Belgium. Lovell, T. 1990. Variation in quality of Artemia for feeding larval fish.—Aquaculture Magazine 16: 77, 78. McEvoy, L. A., J. C. Navarro, F. Amat, and J. R. Sargent. 1997. Application of soya phosphatidylcholine in tuna orbital oil enrichment emulsions for Artemia.— Aquaculture International 5: 517–526. ———, ———, F. Hontoria, F. Amat, and J. R. Sargent. 1996. Two novel Artemia enrichment diets containing polar lipid.—Aquaculture 144: 339–352. Navarro, J. C., L. A. McEvoy, M. V. Bell, F. Amat, F. Hontoria, and J. R. Sargent. 1997. Effect of different dietary levels of docosahexaenoic acid (DHA, 22:6 omega-3) on the DHA composition of lipid classes in sea bass larvae eyes.—Aquaculture International 5: 509–516.

Olsen, A. I., A. Jensen, J. O. Evjemo, and Y. Olsen. 1997. Effect of algal addition on stability of fatty acids in enriched Artemia franciscana.—Hydrobiologia 358: 1–3. Ozkizilcik, S., and F.-L.-E. Chu. 1994. Uptake and metabolism of liposomes by Artemia nauplii.—Aquaculture 128: 1, 2. Poncelet, D. 1996. New: BRG-Forum–Bioencapsulation Research Group Forum.—BRG-Forum on [email protected]. Salazar, O., and M. A. González. 1986. Intensidad de la alimentación diaria en postlarvas de Ictiobus cyprinellus.—Boletín Técnico, Ministerio de la Industria Pesquera, Empresa Nacional de Acuicultura, Cuba 31: 1–4. Southgate, P. C., and D.-C. Lou. 1995. Improving the n-3 HUFA composition of Artemia using microcapsules containing marine oils.—Aquaculture 134: 1, 2. Watanabe, T., M. Ohta, C. Kitajima, and S. Fujita. 1982. Improvement of dietary value of brine shrimp Artemia salina for fish larvae by feeding them on omega 3 highly unsaturated fatty acids.—Bulletin of the Japanese Society Scientific Fisheries 48: 1775–1782. ———, F. Oowa, C. Kitajima, and S. Fujita. 1978. Nutritional quality of brine shrimp, Artemia salina, as a living feed from the viewpoint of essential fatty acids for fish.—Bulletin of the Japanese Society Scientific Fisheries 44: 1115–1121. ———, ———, ———, and ———. 1980. Relationship between dietary value of brine shrimp Artemia salina and their content of omega 3 highly unsaturated fatty acids.—Bulletin of the Japanese Society Scientific Fisheries 46: 35–41. ———, T. Tamiya, A. Oka, M. Hirata, C. Kitajima, and S. Fujita. 1983. Improvement of dietary value of live foods for fish larvae by feeding them on omega 3 highly unsaturated fatty acids and fat-soluble vitamins.—Bulletin of the Japanese Society Scientific Fisheries 49: 471–479. RECEIVED: 23 November 1999. ACCEPTED: 31 July 2000.