Development of an in vitro subculture system for the oka organ (Lymphoid tissue) of Penaeus monodon

Aqwulture ELSEVIER

Aquaculture 136 ( 1995) 43-55

Development of an in vitro subculture system for the oka organ (Lymphoid tissue) of Penaeus monodon Ya-Li Hsu aS*, Yin-Hsiu Yang a, Yu-Chin Chen a, MingChen Tung b, Jen-Leih Wu a, Mark H. Engelking ‘, Joann C. Leong ’ aInstitute of Zoology, Academia Sinica, Taipei, Taiwan, Republic of China of Veterinary Medicine, National Ping-Tung, Polytechnic Institute, Ping Tung, Taiwan, Republic of China ’ Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA

b Department

Accepted 23 March 1995

Abstract We tried to establish a subculture system for cells from the Oka organ (lymphoid tissue) of the grass prawn Penaeus mono&n. The basic culture medium was tested for osmolality, serum concentration, serum sources and pH. It was found that Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum, 5 g 1-l NaCl, pH 7.63-8.1, with final osmolality at 470-500 mmol kg- ’ allowed for enhanced cell attachment and growth; however, cells could not be maintained for more than 5 days. The supplementary nutrients were also tested for carbohydrates, amino acids, L-ascorbic acid, Buffalo rat liver( BRL)-condition medium and selenium. The basic culture medium + 1 g l- ’ glucose were found to enhance cell attachment and growth. Our collected lymphoid cells required 3 days of incubation to obtain 80% confluency; however, cells did not grow in subcultures. Several growth factors were tested for developing a subculture system of shrimp cells. Epidermal growth factor (EGF) or transforming growth factor/l (TGF p) did not foster cell growth. Cells treated with insulin or insulin-like growth factor I (IGF I) were capable of being subcultured, but resultant cells differed in terms of feeder layer, and were eventually discarded due to yeast contamination. Cells treated with basic fibroblast growth factor (bFGF) 20 ng ml- ’ (F-20) could be subcultured for more than 90 passages without a feeder layer. Some F-20-treated cells were capable of extending their extracellular matrices for cell attachment and piled up; some of them became suspended and lost their anchoragedependent and contact inhibition properties. Keywords:

Cell culture; grass prawn; Oka organ; Penaeus monodon

* Corresponding

author.

0044~8486/9.5/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOO44-8486(95)01048-3

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1. Introduction The penaeid shrimp is one of the world’s most commercially important cultured crustaceans. Rapid development and expansion of prawn farming has occurred throughout much of southeast Asia. Within the past two decades, seven penaeid shrimp viral diseases have been discovered at commercial culturing facilities, and the substantial losses of shrimp due to these viral diseases has attracted considerable attention from researchers (Couch, 1974a; Couch, 1974b; Lightner and Redman, 1981; Lightner et al., 1983; Lightner and Redman, 1985; Tsing and Bonami, 1984; Sano et al., 1981; Lu et al., 1991). So far, research on penaeid shrimp viruses has been limited to histopathological and transmission electron microscopic observations (Lightner, 1984; Lightner et al., 1987). A complete understanding of these viruses is dependent upon the development of laboratory techniques for the maintenance and culturing of these viruses and their host cells. The development of “in vitro” cell cultures of Crustacea tissue is still in the experimental stage, with some encouraging results recently being achieved with shrimp tissue (Chen et al., 1986; Chen and Kou, 1989; Hu, 1990; Luedeman and Lightner, 1992). This report describes the development of a subculture system for grass prawn (Penaeus monodon) Oka (lymphoid) organ tissue and the effects of various nutrients and growth factors on cultured cell growth. 2. Materials and methods 2.1. Experimental

animals

Thirty ( 15-20 g) Peneaus monodon 10-13 cm in length that were used in each experiment, were obtained from a commercial supplier. 2.2. Tissue culture materials Medium 199 was purchased from UBI, Leibovitz’s 1 X L-15, F12, and Dulbecco’s modified Eagle Medium were purchased from Gibco. Three batches of fetal calf Ferum (tested serum numbers 1,2 and 3) were purchased from Gibco, while tested serum number 4 was obtained from UBI. Defined fetal bovine serum (tested serum number 5), cosmic calf serum (number 6)) characterized fetal bovine serum (number 7)) fetalclone II (number 8)) and two batches of fetal clone bovine serum product (tested serum numbers 9 and 10) for hybridomas were all purchased from Hyclone (Logan, UT); Hepes (20 mM) was purchased from Gibco. Antibiotics, trehalose, proteinase K, arginine, methionine, L-ascorbic acid, were all purchased from Sigma Chemical Co. Buffalo rat liver (BRL) cells were purchased from the Riken cell bank (Tsukuba Science City, Japan). Selenium was purchased from Gibco. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor p (TGF p), and Insulin-like growth factor I (IGF I) were purchased from BM, and insulin was purchased from Collaborative Research Incorporated. 2.3. Primary cultures Live shrimp were anaesthetized in ice for 20 min and surface-sterilized in 0.02% iodine disinfectant for 5 min before tissue excision. Oka organs were excised and placed into cold

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phosphate buffer saline (PBS). Holding media consisted of 1 X Leibovitz’s L-15 with penicillin (10 000 unit ml-‘), streptomycin (10 000 ug ml-‘), amphotericin B (500 ng ml-‘), gentamycin ( 1 ug ml-‘) and 10% fetal calf serum (FCS). Tissues were washed several times in cold PBS after which they were minced and forced through a 23G X 1 l/4 gauge needle several times. The cell suspension was transferred to 35 X 10 mm petri dishes containing approximately 2 ml of one of the ten experimental culture media. Each culture procedure was repeated at least three times, and dishes were incubated with 5% CO* at 28 + 1°C in a CO, incubator. Cultured cells were observed daily with an Olympus CK inverted microscope, and the effects of various culture conditions, nutrients, and growth factors on cell attachment, survival, and growth were recorded. The Oka organs were excised and prepared as described above, a sample of the cell suspension was then added to each dish.

3. Results 3.1. Basic cell culture Cells from the gills, ovaries, hepatopancreata, hearts, Oka organs and muscle tissue of experimental grass prawns were cultured in 1 X L-15 with antibiotics and 10% FCS. Only primary cell cultures of Oka organ tissue showed consistently reliable results. Chen and Kou (1989) reported that monodon baculavirus can infect primary cell cultures of Oka organ tissue from Penaeus monodon, therefore, we chose the Oka organ as our target tissue. The cell culture, our measurement of the culture medium providing the best results, was based on both the percentage of surface area covered by attached cells and cell appearance. Optimal osmolality for Oka organ cell cultures was tested by adding 0 g l-‘, 5 g I-‘, 10 g l-‘, 15 g l-‘, and 20 g l- ’ NaCl to 1 X L-15 plus antibiotics. Osmolality levels were measured at 325,472, 573, 735 and 920 mmol kg- ‘, respectively (Fig. 1A). Cell cultures with 5 g 1-l NaCl (472 mmol kg-’ osmolality) had the best attachment percentage and appearance. In addition, the best results for cell morphology and attachment were observed for 10% and 15% fetal calf serum concentrations (Fig. 1 B). At 30% FCS, we observed only l/6 cell attachment. For testing the ten serums, cell suspensions were cultured in L- 15 plus 5 g 1- ’ NaCl and 10% testing serum (Fig. 1C); our results show that test serum number 2 (fetal bovine serum from Gibco had the best effect on cell culture, and so this serum was used for all subsequent experiments. Oka organ cell suspensions were cultured in L-15 media containing 5 g 1-l NaCl and 10% fetal bovine serum at various pH values. After 24 h incubation, cell morphology and percentage of attached cells appeared to be highest between pH 7.63 and 7.9 (Fig. 1D). Shrimp cells could not be subcultured with L- 15 as the basal medium. LHM + 199 (Hu, 1990) and LDF media (Collodi et al., 1992) were used to test cell growth. The osmolality of the different media was measured at approximately 500 mmol kg- I. After 24 h incubation, cells in LHM(3) +199(7) (30% LHM +70% 199) or LDF (50% L-15+35% DME + 15% F12) media showed a 90% attachment (Fig. 1E). In contrast, cells incubated with L- 15 showed a 50% attachment, and cells with LHM + 199 media produced 60% attachment

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325 472 573 735 920

Osmolality

( mmolikg

)

_ -

12345678910 Tested serum

7.3 number

7.63

7.9

8.1

PH

Medium

Fig. 1. Effects of basic culture conditions on primary cell cultures of the Oka organ of grass prawn. (A) Osmolality: 0 g I-‘, 5 g I-‘, 10 g l-‘, 15 g I-’ and 20 g I-’ of NaCl was added, respectively, to the L-15 medium, their osmolality expressed as 325,472,573,735, and 920 mmol kg-‘. (B) Serum concentration. (C) Serum sources, shown as tested serum number. (D) pH. (E) Media. Effects are expressed as percentage of attached primary culture cells in medium after 24 h incubation in A, B, D. and E, and after 3 days incubation at 28°C. (Fig. 1E). However, those cells incubated with L- 15 showed a much better fibroblast-like morphology (Fig. 2). Therefore, we used L-15 + 5 g 1- ’ NaCl + 10% FBS as our basic culture medium.

4. Supplementary

nutrients

Glucose, sucrose and trehalose can be used as energy sources for cultured shrimp (Alava and Pascual, 1987) .We added 1 g l- ’ of glucose, trehalose or sucrose to our basic culture medium. Only 80% of cultured cells attached themselves to the surface in medium with 1 g 1-l glucose, and those cells in medium with trehalose, sucrose, or without supplementary carbohydrate showed an attachment rate of 50% (Fig. 3A). After 24 h incubation, cells in medium with 1 g 1-i or 3 g 1-i glucose added showed the best attachment and growth rates. Deshimaru et al. ( 1985) reported that cultured shrimp need higher concentrations of arginine and methionine in their diet to enhance growth. However, after 24 h incubation, cells in basic culture medium plus 1 g 1-l glucose and 0.5 g 1-l arginine were unable to adhere to the surface of petri dishes (Fig. 3C). In addition, many vacuoles appeared in these cells in contrast to control cells, this was probably due to an increase in pH. Medium

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Fig. 2. Effects of several synthetic media on the morphology of the primary cell culture. Shown are photographs of the primary cell culture in the tested medium after 2 days incubation at 28°C. The tested medium contained synthetic medium plus 5 g I-’ NaCI, 10% FBS and antibiotics. (A) L-15; (B) LHM + 199; (C) 30% LHM + 70% 199; (D) LDF. Bar is 75 pm.

treated with methionine showed increases in attachment and cell growth (Fig. 3D), but these cells died within 10 days. Vitamin C is also important for shrimp cultures (Lightner et al., 1977; Magarelli and Colvin, 1978). With supplements of 0.1 mg 1-l vitamin C and 1 g 1-l glucose in basic medium, the cells attached to the surface and grew well, but could not be subcultured. As shown in Fig. 3E, cells in basic culture medium with higher concentration of vitamin C only show lack of attachment and consequently died within 2 days. Buffalo rat liver (BRL) cell-condition medium contains a growth factor which inhibits cell differentiation (Smith and Hooper, 1987). Shrimp cells in basic culture medium plus 1O-20% BRL-condition medium and 1 g l- ’ glucose attached themselves to the surface at a rate of approximately 50% compared to a rate of 25% for control cells (Fig. 3F). However, we observed a substantial increase in vacuoles in cells treated with BRL-condition medium (Fig. 4). From our nutrient supplement experiments, basic culture medium plus glucose appear to enhance attachment and growth rates of shrimp cells. Vitamin C and BRL supplements enhanced cell attachment but not cell growth. Therefore, L-15 +5 g 1-l NaCl + 1 g 1-l glucose + 10% fetal bovine serum (FBS) + antibiotics appears to be the best basic nutrient medium; however, while shrimp cells in this medium grew, they could not be subcultured.

5. Growth factors Epidermal growth factor was added to the basic nutrient medium. At levels of 0.5 ng ml-’ or 1 ng ml-’ EGF cells attached at much higher rates following 24 h incubation

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;

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‘E; 20 *

0 Co%. GIL.

A.

Carbohydrate

sic.

( g/l)

II

$

8o

‘hl Iso -s d 40 f B *

20 0 0

150 300 450 mg/l )

Methionine

3

60

;

40

$

20

*

0 00050105

Vitamin

I

3

(

5

C ( mg/l )

Fig. 3. Effects of supplementary nutrients on the percentage of attached primary culture cells after 24 h incubation. (A) Carbohydrate (glucose 1 g l-‘, trehalose 1 g I-‘, sucrose 1 g I-’ or control 0 g 1-l). (B) Glucose; (C) Arginine; (D) Methionine; (E) L-ascorbic acid; (F) BRL-condition medium.

(Fig. 5A). However, these cells contained a large number of vacuoles and eventually died in contrast to control cells. Transforming growth factor p (TGF p) had no particular effect on cell attachment. As shown in Fig. 5B, the percentage of attached cells decreased at higher concentrations of TGF /?.A11 TGF p treated cells died within 3 days. Czech ( 1985) reported that insulin may act as a growth factor for some cells. However, insulin supplement did not enhance cell attachment in our experiment, and higher concentrations of insulin were observed to be harmful to cell growth (Fig. 6A, B, and C). Cells treated with 3 pg ml - ’ insulin (I-3) aggregated and grew into a flower-like clump (5 pm15 pm) after 8 days of incubation (Fig. 6D). These cells could be subcultured, but they were eventually discarded due to yeast contamination. Shrimp cells treated with 10 pg ml- ’ insulin (I- 10) grew with a similar, flower-like morphology after 15 days incubation. After being subcultured three times, these I-10 p3 cells stopped growing and became suspended in the medium. To prevent this happening, a primary cell culture of shrimp Oka organ tissue was used as a feeder layer which the I-10 p3 cells could attach to and subsequently grow (Fig. 6E); the feeder layer cells eventually died. With the feeder layer,the I-10 p3 cells could grow and be subcultured to I- 10 p5, after which they became contaminated with yeast. Cells treated with 5 pg ml- ’ insulin (I-5) showed similar behavior to the I-3 cells, that is they were subcultured three times, then became contaminated with yeast.

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Fig. 4. Effects of BRL-condition medium on primary cell culture morphology. Shown are photographs of the primary cell culture (B) with or (A) without BRL-condition medium (25%) after 24 h incubation. Bar is 100 fim.

Another batch of primary shrimp Oka organ cell culture was treated with insulin-like growth factor I (IGF I). After 24 h incubation, these treated cells became suspended and developed large vacuoles (Fig. 7B) unlike the control cells (Fig. 7A). However, cells treated with 25 ng ml-’ IGF I showed fibroblasts and aggregated round cells following 4 days incubation (Fig. 7C). Later, the fibroblast-like cells disappeared, and the remaining round cells attached to the extracellular matrix (Fig. 7D). After 8 days incubation, IGF I25 cells grew into a flower-like aggregate similar to the above-mentioned I-3 cells (Fig. 7E). We observed that IGF I-25 p4 cells were different from I- 10 p3 cells in that they could not attach to the feeder layer (Fig. 7F). These cells also became contaminated with yeast.

Fig. 5. Effects of growth factors on the percentage (B) Transforming growth factor /3.

of attached primary culture cells. (A) Epidermal growth factor;

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Fig. 6. Effects of insulin on the morphology of the studies shrimp cell cultures. (A) Control, 24 h incubation. (B) I-3 (3 pg ml-’ insulin)-treated cells after 24 h incubation. (C) I-10 (10 pg ml-’ insulin)-treated cells after 24 h incubation. (D) I-3-treated cells after 8 days incubation. (E) I-lo-treated cells after three passages attached to feeder layer cells. Bar is 75 pm.

Basic fibroblast growth factor (bFGF) is a mitogen for certain fibroblast cells (Ingber and Folkman, 1989; Presta et al., 1989). Shrimp cells in basic nutrient medium treated with bFGF could not attach to the petri dish (Fig. 8B), and subsequently suspended into an aggregated ball of a size ranging from 2.5 pm to 20 pm following 11 days incubation (Fig. 8C; controls Fig. 8A). After subculturing, these cells (F-20 means bFGF 20 ng ml-‘) were able to extend their extracellular matrix and attach to the petri dish, and attached to feeder layer cells( i.e. the primary cell culture of shrimp Oka organ tissue (Fig. 8D). After passage, these F-20 cells could grow and be subcultured without any feeder layer cells, and

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Fig. 7. Effects of insulin-like growth factor I (IGF I) on the morphology of the studied shrimp cell cultures. (A) Control after 24 h incubation. (B) 25 ng ml- ’ IGF l-treated cells after 24 h incubation. (C) IGF I-25-treated cells after 4 days incubation. (D) IGF I-25- treated cells after 5 days incubation. (E) IGF I-25-treated cells after 8 days incubation. (F) IGF.I-25treated cells after four passages with feeder layer cells. Bar is 75 pm. subsequently piled up and suspended. The F-20 cells could be subcultured for more than 90 passages, Fig. 8F shows the 60 passage of the F-20 cells. The morphology of these F20 cells changed from suspended cell (passage 39, Fig. 8E) to monolayers (Fig. 8F).

6. Discussion The yielded that of (Chen

L-15 medium with 10% FES, pH 7.63-8.1, and osmolality at 472 mm01 kg-‘, the best results. The pH range of this rich, basic culture medium is very similar to mammalian fibroblasts (Freshney, 1983) and to the primary cell culture of shrimps et al., 1986; Luedeman and Lightner, 1992)) but slightly higher than that found in

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Fig. 8. Effects of basic fibroblast growth factor (bFGF) on morphology of the studied shrimp cell cultures. (A) Control after 2 days incubation. (B) 20 ng ml-’ bFGF-treated cells after 2 days incubation. (C) bFGF (F-20)treated cells after 11 days incubation. (D) F-20-treated cells after three passages with the feeder layer cells. (E) F-20 treated cell after 39 passages. (F) F-20-treated cells after 60 passages. Bar is 75 pm.

insect cells (Grace, 1982). The osmolality (472 mm01 kg- ‘) of our basic culture medium was much lower than those reported for primary shrimp cell cultures (700-750 mm01 kg-‘) (Chen et al., 1986; Luedeman and Lightner, 1992). As Penueus monodon shrimps are euryhaline marine organisms, they are very strong osmoregulators (Cheng and Liao, 1986), and can survive over a wide range of temperatures. The shrimp used for our experiments were probably collected from brackish water which may explain why the osmolality of our grass shrimp was so low. As the primary shrimp cell cultures could not survive more than 5 days in this basic culture medium, these cells may need nutrient supplements. Previous studies have shown that glucose supplements are very important for insect cell cultures (Hink, 1976). Our results show that basic culture medium with 1 g l- ’ glucose can enhance the growth of

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primary culture cells. While grass shrimp cultures require higher methionine and arginine concentrations (Deshimaru et al., 1985)) it appears that L-15 medium contains adequate amounts of arginine (500 mg 1-l) and methionine (150 mg 1-l) (also similar to insect cell requirements, Hink, 1976). L-ascorbic acid can enhance the formation of collagen, and thus help in tissue repair and reconstitution (Jauncey et al., 1985). However, our results show that while L-ascorbic acid enhances the attachment of primary cell cultures, those cells cannot be subcultured. This may be due to our using an incorrect form of L-ascorbic acid. Finally, our results show that both selenium and BRL-condition medium appear to be harmful to grass shrimp cell cultures. Epidermal growth factor (EGF) is an acidic peptide of about 6100 daltons. It can either stimulate (Kawamoto et al., 1983) or inhibit cell proliferation (Barnes, 1982)) depending on the target cells. In human epidermoid carcinoma A43 1 cells, EGF binds to EGF receptors to induce the receptor-associated tyrosine kinase activity (Cohen et al., 1980; Ushiro and Cohen, 1980) ; this kinase activity can inhibit the proliferation of A43 1 cells. We found that higher concentrations of EGF (3 ng ml- ‘) inhibits the proliferation of grass shrimp Oka tissue cells, Transforming growth factor p can act as a growth factor on cell surfaces with receptors; however, it can also act as an inhibitor to cell growth (Moses et al., 1985). Our results show that TGF p does not enhance shrimp cell growth. Insulin, a peptide hormone, can enhance the transportation and utilization of sugar as well as the absorption of amino acids in cells, In addition, insulin can stimulate DNA synthesis in some cells and act as a growth factor (Czech, 1985). Insulin-like growth factor I( IGF I), which is very similar to insulin in structure and function, and is found in a variety of tissue cells (Cohick and Clemmons, 1993), and can stimulate cell proliferation after binding to cell receptors. We found that both insulin and IGF I stimulate the proliferation of shrimp Oka organ cells, and that those cells can extend their extracellular matrix for cell attachment and growth by growing into flower-like aggregates. However, while I- lo-treated cells adhered to feeder layer cells and subsequently grew, IGF-I-treated cells did not. Although it appears that both insulin and IGF-I can act as growth factors for shrimp cells and stimulate cell proliferation, however, both IGF-I and insulin-treated cells easily became contaminated. Basic fibroblast growth factor (bFGF), a 17 kd peptide, can bind to heparin to induce DNA synthesis (Presta et al., 1986) ; it is also a mitogen for fibroblast cells (Ingber and Folkman, 1989; Presta et al., 1989). Shrimp cells treated with bFGF developed an extracellular matrix for cell attachment and growth. Previous studies show that bFGF can bind to heparin-like molecules in the extracellular matrices of endothelial cells (Presta et al., 1989), and therefore stimulate cell growth and differentiation (Ingber and Folkman, 1989). Moscatelli and Quart0 ( 1989) found that bFGF-treated NIH 3T3 cells could be transformed. The bFGF receptor have down regulation to the high concentration of bFGF. This means that high bFGF concentrations can reduce the number of bFGF receptors and induce cell transformation. We found that bFGF (F-20) -treated shrimp cells could grow quickly, some of them became suspended, and some of them piled up. This might be a kind of transformed cell. At the present time, they cannot be subcultured for more than 90 passages and become monolayers.

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Acknowledgements The authors wish to thank Dr. David W. Barnes, Department of Biochemistry and Biophysics, Oregon State University for his technical advice. This study was supported by the cooperative research program in the Agricultural Sciences between the US Department of Agriculture (Project No. TW-AES-37, Grant No. FG-Ta- 134) and the Council of Agriculture, Republic of China (82- 1,5-Food-&I(5), 82- I,5secret-06(4) and 83-biotechnology 1.1 Food 61 (65)).

References Alava, V.R. and Pascual, F. P., 1987. Carbohydrate requirements of Penaeus monodon (Fabricius) juveniles. Aquaculture, 61: 211-217. Barnes, D.W., 1982. EpidermaJ growth factor inhibits growth of A431 human epidetmoid carcinoma in serumfree cell culture. J. Cell Biol., 93: 14. Chen, S.N. and Kou, G.H., 1989. Infection of cultured cells from the lymphoid organ of Penaeus monodon Fabricius by monodon-type baculovirus (MBV). J. Fish Dis., 12:73-76. Chen, S.N., Chi, SC., Kou, G.H. and Liao, I.C., 1986. Cell culture from tissues ofgrass prawn, Penaeusmonodon. Fish Pathol., 21:161-166. Cheng, J.H. and Liao, I.C., 1986. The effect of salinity on the osmotic and ionic concentrations in the hemolymph of Penaeus monodon and Penaeuspenicillatus. In: J. L. MacLean, L. B. Dizon and L. V. Hosillos (Editors), The first Asian Fisheries Forum. Asian Fisheries Society, Mania, Philippines, pp. 633636. Cohen, S., Carpenter, G. and Jr, L. K., 1980. Epidermal growth factor-receptor-protein kinase interaction. J. Biol. Chem., 255: 48344842. Cohick, W. S. and Clemmons, D. R., 1993. The insulin-like growth factors. Ann. Rev. Physiol., 55: 131-153. Collodi, P., Kamei, Y., Sharps, A., Weber, D. and Barnes, D. W., 1992. Fish embryo cell cultures for derivation of stem cells and transgenic chimeras. Mol. Mar. Biol. Biotech., 1: 257-265. Couch, J.A., 1974a. Free and occluded virus, similar to Baculovirus in hepatopancreas of pink shrimp. Nature (London), 247: 229-23 1. Couch, J.A., 1974b. An enzootic nuclearpolyhedrosis virus of pink shrimp: ultrastructure, prevalence and enhancement. J. Invertebrate Pathol., 24: 311-331. Czech, M.P., 1985. The nature and regulation of the insulin receptor: structure and function. Ann. Rev. Physiol., 47: 357-381. Deshimaru, O., Kuroki, K., Mazid, M.A. and Kitamura, S., 1985. Nutritional quality of compounded diets for prawn Penaeus monodon. Bull. Jpn. Sot. Sci. Fish., 51: 1037-1044. Freshney, RI. (Editor), 1983. The culture environment: II. media and supplements. Culture of animal cells, a manual of basic technique. Alan Liss, New York, pp. 67-78. Grace, T.D.C., 1982. Development of insect cell culture. In: K. Maramorosch (Editor), Invertebrate Tissue Culture. Academic Press, New York, pp. 1-8. Hink, W.F., 1976. A complication of invertebrate cell lines and culture media. In: K. Maramorosch (Editor), Invertebrate Tissue Culture, Research Applications. Academic Press, New York, pp. 319-369. Hu, Ke, 1990. Studies on a cell culture from the hepatopancreas of the oriental shrimp, Penaeus orientalis Kishinouge. Asian Fish. Sci., 3:299-307. Ingber, D.E. and Folkman, J., 1989. Mechanochemical switching between growth and differentiation during tibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J. Cell Biol., 109: 317330. Jauncey, K., Soliman, A. and Roberts, R.J., 1985. Ascorbic acid requirements in relation to wound healing in the cultured tilapia Oreochromis niloticus (Trewavas). Aquaculture Fish. Manage.,l6: 139-149. Kawamoto T., Sato, J. D., Le, A., Polikoff, J., Sat, G. H. and Mendelsohn, J., 1983. Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc. Natl. Acad. Sci. USA, 80: 1337-1341.

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