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Biochemical characterization of gonadal development in the shrimp Penaeus vannamei
Comp. Biochem. Physiol. Vol. 105B,Nos 3/4, pp. 579-584, 1993
0305-0491/93$6.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
BIOCHEMICAL CHARACTERIZATION OF GONADAL DEVELOPMENT IN THE SHRIMP PENAEUS VANNAMEI MARIOSA. CARIOLOU* and CONSTANTINN. FLYTZANIS Baylor College of Medicine, Department of Cell Biology, One Baylor Plaza, Houston, TX 77030, U.S.A. (Fax 713 790-1275) (Received 30 November 1992; accepted 15 January 1993)
Abstract--1. Mini-two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze polypeptides extracted from eggs and gonadal tissues from different developmental stages of male and female P. vannamei shrimp. 2. Gel patterns of polypeptides from immature and mature ovaries showed notable differences. 3. Several biochemical differences between ovarian and egg samples indicated that the majority of egg polypeptides may be synthesized towards the end of oogenesis. 4. Five polypeptides present in the eggs, immature and mature ovaries may be synthesized early in oogenesis. 5. Qualitative polypeptide differences between testes from adult and juvenile shrimp suggest differential expression of genes in a developmentally controlled program.
INTRODUCTION The white shrimp P. vannamei is considered one of the most important penaeid species for commercial production (Lawrence et al., 1985; Sandifer et al., 1987, 1988). Successful and profitable cultivation of this shrimp is heavily dependent on several parameters, including the constant and reliable availability of seedstock supplies, and hence, the yearround existence of reproductively mature shrimp. Although P. vannamei has been studied extensively at research centers and commercial hatcheries, its reproduction in captivity remains difficult (Aquacop, 1979; Lawrence et al., 1985; Wyban et al., 1987). The analysis of the biological complexity of polypeptides in shrimp gonadal tissues is an essential first step towards the understanding of the role they play in reproduction. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) has been used in several laboratories in order to identify the major shrimp-egg yolk proteins (Tom et al., 1987; Yano and Chinzei, 1987; Quackenbush, 1989; Rankin et al., 1989). We demonstrate herein that 2D-PAGE can be used successfully to resolve the complex mixture of polypeptides from immature and mature gonads. Our studies revealed many notable differences between mature and immature ovarian preparations in polypeptides localized within the molecular mass range of 22--66 kDa. Although we observed many differences in the proteins resolved by this procedure, at least five polypeptides, with molecular masses of 24, 38, 47, 48 and 54 kDa were shown to be present in both ovarian and *Present address: The Cyprus Institute of Neurology and Genetics, P.O. Box 3462, Nicosia, Cyprus.
egg samples. In addition, we present results from 2D-PAGE analysis of testes extracts which strongly suggest that male gonadal maturation is also characterized by differential testicular polypeptide expression which increases in complexity with the size, and hence the age, of the male shrimp.
MATERIALS AND METHODS
Recirculating system
Our artificial seawater system consists of seven rearing tanks (1.2 x 0.55 x 0.30 m), one biofilter (0.90 x 0.65 x 0.60 m), and a coarse filtration unit (0.48 x 0.56 m). The tanks were filled with artificial seawater (salinity 29.0 + 1.0 ppt) made with Hawaiian marine mix-salt (Hawaiian Marine Imports, Houston, TX). The total volume of water in each rearing tank, and the biofilter, was 35 and 80 gallons, respectively. One ½HP self-prime pump (3600 gph) was used to circulate water through the tank system, and there was a vertical filtration system consisting sequentially of mechanical (30 mm), chemical (activated carbon), and UV sterilization units (Rainbow Lifeguard, El Monte, CA). A 300 W and two 150 W Visi-therm heaters (Aquarium Systems, Mentor, OH) were submerged in the biofilter tank to maintain water temperature in the rearing tanks at 28.0 _+ 1.0°C. Water quality parameters such as pH, nitrite, and dissolved oxygen were measured with Hach (Hach Co., Loveland, CO) and Taylor (Taylor Technologies, Inc., Sparks, MD) test kits. Ammonia was measured by the salicylate-hypochlorite method (Bower and Holm-Hansen, 1980). Salinity and temperature were measured daily. All other parameters were measured weekly. 579
580
MARIOS A. CARIOLOU and COr~STAN~N N. FLYTZANIS
Ovarian maturation
Female and male P. vannamei shrimp weighing more than 45 g, were purchased from Granada Corporation (Agromarina d6 Panama), Houston, Texas, and Amorient Aquafarm, Inc., Kahuku, Hawaii. Animals were also raised to adulthood in our facility from nauplii (stages N3/N4) which were provided by Granada Corporation. All shrimp were acclimated in our system for at least three weeks prior to any ablation attempt. To induce ovarian maturation, at the end of the acclimation period and four days following ecdysis, female shrimp were unilaterally ablated by cutting with sharp scissors the eyestalk close to the carapace and immediately cauterizing the wound. An equal number of unablated female shrimp was used as controls. No males were ablated. Maturation feed (Rangen, Inc., Buhl, ID) was provided five times daily as food source (8% Biomass/day). Ovarian development was monitored daily and classified as described by Yano et al. (1988). Shrimp in advanced stages of ovarian development were transferred to 10-gallon tanks for spawning and egg collection. M i n i - 2 D - P A G E and sample preparation
Mini-2D-PAGE analysis of ovarian, egg, and testicular polypeptides from P. vannamei was performed essentially as described by O'Farrell (1975). Equilibrium pH gradient electrophoresis was used in the first dimension and S D S - P A G E in the second dimension. Carrier ampholytes (Bio-Rad Laboratories, Richmond, CA) were used at concentrations of 1.6% (pH range 5-7) and 0.4% (pH range 3-10). Maturation and synapsis stage ovaries (Yano et al., 1988) were obtained from ablated mature and immature shrimp (body weight > 43 g), respectively. Eggs were collected immediately after the spawning of shrimp. Male animals with individual weights of 11, 25 and 43 g were sacrificed and their testes dissected. The samples were processed by homogenization in extraction buffer (0.05 M Tris pH 7.0, 8M urea, 1% SDS, 1%0 b-mercaptoethanol, 360 mg/ml TPCK (N-tosylL-phenylalanine chloromethyl ketone), 180 mg/ml TLCK (N-tosyl-L-lysine chloromethyl ketone), 3 mg/ml pepstatin, 240 mg/mi A - P M S F (4-amidinophenylmethanesulfonyl fluoride) and 3 mg/ml leupeptin) and centrifugation at 14,000 g and 10°C, for 15 min in an Eppendorf centrifuge. Total soluble protein in each sample was determined using the BCA protein assay (Pierce, Rockford, IL). Prior to electrophoresis, samples were diluted 1:1 with dilution buffer (9.5M urea, 8% Nonidet P-40, 4% ampholytes, in the same ratio as the gel, and 5% b-mercaptoethanol) to give a Nonidet P-40/SDS ratio of 8:1 and final ampholyte concentration of 2%. Approximately 20 #g of protein from each sample were loaded on the IEF tube gels (inside diameter 1.5 x 73mm). Samples were electrophoresed with 350 V for 8 hr at room temperature. Electrophoresis
in the first dimension was carried out, for all samples, on the same day. S D S - P A G E was performed at constant current (15mA/gel) using 1.5mm thick polyacrylamide gels (85 x 65 mm) and was terminated when the tracking dye (Bromphenol Blue) eluted from the gel. Following electrophoresis in the second dimension, gels were silver-stained using the procedure of Wray et al. (1981). Molecular weight markers for S D S - P A G E were phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5kDa) and lysozyme (14.4 kDa) (Bio-Rad). RESULTS
The water condition in our recirculating system is presented in Table 1. The overall survival of the animals in this system was excellent. Fewer than 5% of the animals died during the course of the investigation and subsequent histological examination did not reveal pathological conditions (Table 2). Ovarian maturation was observed only in ablated female shrimp. The majority of unilaterally ablated shrimp matured and spawned 15 + / - 6 days after ablation (Table 3). Male shrimp matured without ablation. Figure 1 illustrates the 2D-PAGE separation of polypeptides from immature ovaries (1A), mature ovaries (IB), and eggs (1C). Approximately 125 and 250 polypeptides stained with silver were readily detected in the gels from immature (Fig. 1A) and mature ovaries (Fig. 1B), respectively. Although the electrophoretic pattern was similar both qualitative and quantitative differences were detected between the immature and mature ovary. The most notable qualitative differences occurred in two polypeptides with similar molecular mass of 63 kDa, but different isoelectric points, and three other polypeptides with molecular masses of 24, 29.5, and 57 kDa. These five proteins, which are marked in Fig. 1 with arrowTable 1. Mean + standard deviation, maximum and minimum values for six water quality parameters measured during ablation trials Parameter
Mean
Dissolvedoxygen(rag/I) Biofilter 8.5 _+0.6 Rearing tanks 7.3 _+1.4 Ammonia nitrogen (mg/l) Biofilter 0.040 _+0.007 Rearing tanks 0.089± 0.014 pH Biofilter 8.1 -t-0.2 Rearing tanks 7.6 + 0.4 Temperature (°C) Biofilter 29.6 + 1.0 Rearing tanks 28.0 +_1.0 Nitrate (mg/1) Biofilter 0.16 + 0.08 Rearing tanks 0.19 ± 0.07 Salinity(ppt) Biofilter 29.0 __1.0 Rearing tanks 29.0 +_1.0
Maximum
Minimum
9.0 9.0
8.0 5.0
0.049 0.120
0.031 0.076
8.5 8.5
8.0 7.5
31.0 28.5
29.4 27.5
0.33 0.36 31 31
0.09 0.10 28 28
Shrimp gonadal development Table 2. Survival of shrimp in rearing tanks 60 days post-ablation Trial No.
Tank No.
1 I 1 2 2 3 3
Ablated (No.)
1 2 2C 3 3C 4 4C
2 2 0 1 0 12 0
60 days Beginning post-ablation 4/3* 2/3 2/3 1/2 1/2 12/0 12/0
2/3* 1/3 2/2 1/2 1/2 10/0 11/0
*Ratio of female to male shrimp in each tank. Tanks 2C, 3C and 4C contained control unablated shrimp.
heads, are present in mature (Fig. 1B), but are not detected in immature (Fig. 1A), ovarian extracts. Open brackets in Fig. 1 illustrate the most notable quantitative differences which were observed between ovary samples. The prevalence of a family of basic polypeptides (45~i6.2 kDa) and an acidic, 24 kDa protein was significantly increased from a low level in immature ovaries (Fig. 1A) to a relatively high concentration in mature ovaries (Fig. IB). As ovarian maturation proceeds, the prevalence of some proteins is diminished, most notably a 40 kDa protein which is present in immature ovaries (Fig. IA, triangle), is undetectable in mature ovaries (Fig. 1B). In contrast to the ovarian samples, fewer polypeptide species were resolved by 2D-PAGE in egg extracts (Fig. 1C). Despite the large dissimilarity of gel patterns from ovaries and eggs we were able to detect at least five polypeptides with molecular masses of 24, 38, 47, 48 and 54 kDa which were present in all three samples (Fig. 1, open circles). The streaking, which was observed with certain egg polypeptides localized at both the basic and acidic ends of the egg gels, may be due to secondary modifications, such as glycosylation, and/or the over-abundance of these polypeptides in the sample. Analysis of different dilutions of egg polypeptides extracted from the same sample or different egg samples, revealed identical gel patterns (results not shown). We conclude that alterations in the pH gradients caused by the over-abundance of certain egg proteins was highly unlikely and, therefore, we are confident that the interpretation of the gel patterns is correct. Our sample preparations did not include removal of nucleic acids which may also cause streaking of protein spots and an increase in the background staining of 2D-gels. Table 3. Spawning events and frequency of spawning for each ablated shrimp Trial No.
Tank No.
1 2 3
2 3 4
No. Ablated shrimp 1 1 2 2 I 1 1 1 1 1
Spawning events/shrimp 3 (9, 13, 26)* 1 (19) 0 I(II) 2(11,14) 1 (15) 2 (15, 25) 1 (19) 2 (27, 33) 1 (i 17)
*Number of days after ablation when spawning took place. Invariably, shrimp spawned between 2:00 and 3:00 a.m. CBP(B)I05-3/4--K
581
Figure 2 demonstrates that the complexity and heterogeneity of testes polypeptides increases as the shrimp grow older. Testes polypeptides were extracted from males of approximate weights of 11, 25 and 43 g. A large number of testes polypeptides from the 43 g shrimp were detected in 2D-gels (Fig. 2A). Electrophoretic analysis of testes extracts from 25 g shrimp also indicated the presence of several proteins (Fig. 2B). Although the overall pattern is very dissimilar, at least one 40 kDa polypeptide and two others, with different isoelectric points but similar molecular mass of 32 kDa, were readily detected in both testes preparations. These three polypeptides are marked with open circles in Fig. 2. We were able to detect only a small number of proteins by 2D-PAGE analysis of testes extracts from juvenile (11 g) shrimp (Fig. 2C). One of these polypeptides, illustrated with an open circle in Fig. 2C, with molecular mass of 32 kDa was shown to be present in all three preparations. Protein assays were carried out on all testes extracts in order to standardize samples and ensure the loading of an equal amount of protein on each gel. However, it is obvious from Fig. 2 that the total silver-stained testes polypeptides detected on gels from 11 g shrimp (Fig. 2C) are considerably fewer than those from 43 g shrimp (Fig. 2A). The high degree of reproducibility and consistency of both the protein assay and the 2D-PAGE analysis of testes extracts lets us speculate the presence of an unknown factor in testes from juvenile shrimp that interferes with the protein assay we have employed, causing overestimation of total protein in this particular sample. As the shrimp grow older this factor may be diluted out. It is also possible that proteins extracted from testes of juvenile shrimp may not stain with silver as efficiently as the proteins of adult testes, or that immature testes contain a high concentration of proteases, which cleave the proteins to sizes that are not resolved in our gels. DISCUSSION
In order to begin to understand the molecular events that underlie the gonadal development of P. vannamei, it is essential to learn more about the biological complexity of proteins present in shrimp gonadal tissues. We present herein our first observations on the polypeptide species present in the immature and mature ovaries, and testes from P. vannamei. Using high-resolution mini-2D-PAGE we were able to resolve and identify several qualitative and quantitative differences in polypeptides extracted from these tissues. Immature and mature ovarian samples displayed many qualitative differences, although the overall migration pattern of the polypeptides appeared quite similar. Except for one protein that is clearly absent from the mature ovary, the major differences between the two samples result from additional polypeptides present in the mature ovary. These proteins may be exclusively found in the
582
MARIOS A. CARIOLOU and CONSTANTINN. FLYTZANIS
IEF
+
A. 97.4~
66.2-"45--"31---
21.5--,.14.4-,.-
B. 97.4"--=-
66.2--.¢n 0 03
45-,-
31~
21.5~ 14.4~
97
66
21 14
Fig. 1. 2D-PAGE patterns of silver stained polypeptides extracted from shrimp immature ovary (A), mature ovary (B), and eggs (C). Procedures for the 2D-PAGE and sample preparations are as described in Materials and Methods. Open circles indicate common polypeptides. Arrowheads point to positions where major polypeptides are present in mature but absent in immature ovaries. The position where a major polypeptide is present only in immature ovary is illustrated with a triangle. Open brackets illustrate polypeptides with significant quantitative and qualitative differences. The protein standards in the second dimension are indicated in kDa. The amount of protein loaded in the first dimension was 20/~g.
583
Shrimp gonadal development IEF
4-|
97.
66. u') 4 o~ 3
21. 14.
C. 97.4~ 66.2~ 45---
31---
21.5-14.4---
Fig. 2.2D-PAGE patterns of silver-stainedpolypeptides extracted from the testes of male shrimp weighing 43 g (A), 25 g (B), and 11 g (C). Procedures for the 2D-PAGE and sample preparations are described in Materials and Methods. Open circles indicate common polypeptides. The protein standards indicated in the second dimension are in kDa. The apparent amount of protein loaded in the first dimension was 20/~g. ovarian tissue and not the mature oocytes since we could not identify them in the spawned eggs. Very few similarities were observed between the ovarian and the egg sample. Thus, the majority of the polypep-
tides deposited in the egg seem to be synthesized towards the end of oogenesis. The migration pattern, and the abundance of the major egg polypeptides, does not indicate that they were simply hidden within
584
MARIOSA. CARIOLOUand CONSTANTINN. FLYTZANIS
the more complex ovarian population. The few (at least five) proteins that were found to be common in both the spawned eggs and the immature ovary, suggest that at least some egg-specific polypeptides may be synthesized very early during ovarian development. These qualitative and quantitative differences in the proteins extracted from immature and mature ovaries, and the spawned eggs, suggest that the differential synthesis and accumulation of several polypeptides during ovarian maturation may not only be involved in oogenesis per se (i.e. synthesis of proteins and maturation of the oocyte) but may also play an important role in the processes that govern overall ovarian development. As male P. vannamei grow older and larger in size, testes development is characterized by an increased complexity in the number of different polypeptides resolved by the 2D-PAGE analysis. This strongly suggests that male gonadal maturation is characterized by differential synthesis of testes proteins in a developmentally controlled program. Although not easily detected, the immature testes polypeptides may all be present in the mature sample and thus may not comprise an immature specific set of proteins. It is interesting that the number of polypeptides detected by the 2D-PAGE increased dramatically during spermatogenesis but not oogenesis. One explanation for this may be the unequal contribution of ovarian and testicular tissue to the analyzed samples at the different developmental stages. During ovarian development we were able to distinguish polypeptides which may belong exclusively to the eggs and polypeptides contributed by the ovarian tissue. Since we did not analyze sperm-only samples, we cannot make the same distinction for the testes. After their capture from the wild, mature shrimp normally undergo gonadal regression. Due to this complex biological process we did not wish to compare polypeptides extracted from laboratory-reared and wild-caught animals. Therefore, some proteins found in the animals reared in our facility may differ from those found in wild shrimp. Also, we did not attempt to identify specific proteins in our samples during the course of this investigation. This can be accomplished by the use of specific antibodies raised against proteins from other animals or by the purification of the most prevalent of the analyzed proteins. Our emphasis in the future will be given to the study of those proteins, which are either common between developmental stages, or stage-specific. Such future studies
may begin to elucidate the role of specific proteins during gonadal development. Acknowledgement--This research was supported by a grant
from Granada BioSciences, Inc. to CNF. REFERENCES
Aquacop (1979) Penaeid reared broodstock: closing the cycle of Penaeus monodon, Penaeus stylirostris, and Penaeus vannamei. Proc. Worm Maricult. Soc. 10, 445-452. Bower C. E. and Holm-Hansen T. (1980) A salicylatehypochlorite method for determining ammonia in seawater. Can. J. Fish. Aquat. Sci. 27, 794-798. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680--682. Lawrence A. L., McVey J. P. and Huner J. W. (1985) Penaeid Shrimp Culture. In Crustacean and Mollusk Aquaculture in the United States (Edited by Huner J. V. and Brown E. E.), pp. 127-157. AVI Publishing, Westport, CT. O'Farrell P. H. (1975) High-resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250, 4007-4021. Panouse J. B. (1943) Influence de l'ablation due pendocule oculaire sur la croissance de l'ovaire chez la crevette Leander serratus. C. r. Acad. Sci., Paris 217, 553-555. Quackenbush L. S. (1989) Vitellogenesis in the shrimp, Penaeus vannamei: in vitro studies of the isolated hepatopancreas and ovary. Comp. Biochem. Physiol. 94B, 253-261. Rankin S. M., Bradfield J. Y. and Keeley L. L. (1989) Ovarian protein synthesis in the South American white shrimp, Penaeus vannamei, during the reproductive cycle. Invert. Reprod. Dev. 15, 27-33. Sandifer P. A., Hopkins J. W. and Stokes A. D. (1987) Intensive culture potential of Penaeus vannamei. J. Worm Aquacult. Soc. 15, 94-105. Sandifer P. A., Hopkins J. S. and Stokes A. D. (1988). Intensification of shrimp culture in earthen ponds in South Carolina: progress and prospects. J. World Aquacult. Soc. 19, 218-226. Tom M., Goren M. and Ovadia M. (1987) Purification and partial characterization of vitellin from the ovaries of Parapenaeus longirostris (Crustacea, Decapoda, Penaeidae). Comp. Biochem. Physiol. 87B, 17-23. Wray W., Boulikas T., Wray V. P. and Hancock R. (1981) Silver-stainingof proteins on polyacrylamidegels. Analyt. Biochem. 118, 197-203. Wyban J. A., Lee C. S., SweeneyJ. N. and Richards W. R. Jr (1987) Observations on development of a maturation system for Penaeus vannamei. J. Worm Aquacult. Soc. 18, 198-200. Yano I. and Chinzei Y. (1987) Ovary is the site of vitellogenin synthesis in Kuruma prawn, Penaeus japonicus. Comp. Biochem. Physiol. 86B, 213-218. Yano I., Tsukimura B., Sweeney J. N. and Wyban J. A. (1988) Induced ovarian maturation of Penaeus vannamei by implantation of lobster ganglion. J. World Aquacult. Soc. 19, 204-209.
0305-0491/93$6.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
BIOCHEMICAL CHARACTERIZATION OF GONADAL DEVELOPMENT IN THE SHRIMP PENAEUS VANNAMEI MARIOSA. CARIOLOU* and CONSTANTINN. FLYTZANIS Baylor College of Medicine, Department of Cell Biology, One Baylor Plaza, Houston, TX 77030, U.S.A. (Fax 713 790-1275) (Received 30 November 1992; accepted 15 January 1993)
Abstract--1. Mini-two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze polypeptides extracted from eggs and gonadal tissues from different developmental stages of male and female P. vannamei shrimp. 2. Gel patterns of polypeptides from immature and mature ovaries showed notable differences. 3. Several biochemical differences between ovarian and egg samples indicated that the majority of egg polypeptides may be synthesized towards the end of oogenesis. 4. Five polypeptides present in the eggs, immature and mature ovaries may be synthesized early in oogenesis. 5. Qualitative polypeptide differences between testes from adult and juvenile shrimp suggest differential expression of genes in a developmentally controlled program.
INTRODUCTION The white shrimp P. vannamei is considered one of the most important penaeid species for commercial production (Lawrence et al., 1985; Sandifer et al., 1987, 1988). Successful and profitable cultivation of this shrimp is heavily dependent on several parameters, including the constant and reliable availability of seedstock supplies, and hence, the yearround existence of reproductively mature shrimp. Although P. vannamei has been studied extensively at research centers and commercial hatcheries, its reproduction in captivity remains difficult (Aquacop, 1979; Lawrence et al., 1985; Wyban et al., 1987). The analysis of the biological complexity of polypeptides in shrimp gonadal tissues is an essential first step towards the understanding of the role they play in reproduction. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) has been used in several laboratories in order to identify the major shrimp-egg yolk proteins (Tom et al., 1987; Yano and Chinzei, 1987; Quackenbush, 1989; Rankin et al., 1989). We demonstrate herein that 2D-PAGE can be used successfully to resolve the complex mixture of polypeptides from immature and mature gonads. Our studies revealed many notable differences between mature and immature ovarian preparations in polypeptides localized within the molecular mass range of 22--66 kDa. Although we observed many differences in the proteins resolved by this procedure, at least five polypeptides, with molecular masses of 24, 38, 47, 48 and 54 kDa were shown to be present in both ovarian and *Present address: The Cyprus Institute of Neurology and Genetics, P.O. Box 3462, Nicosia, Cyprus.
egg samples. In addition, we present results from 2D-PAGE analysis of testes extracts which strongly suggest that male gonadal maturation is also characterized by differential testicular polypeptide expression which increases in complexity with the size, and hence the age, of the male shrimp.
MATERIALS AND METHODS
Recirculating system
Our artificial seawater system consists of seven rearing tanks (1.2 x 0.55 x 0.30 m), one biofilter (0.90 x 0.65 x 0.60 m), and a coarse filtration unit (0.48 x 0.56 m). The tanks were filled with artificial seawater (salinity 29.0 + 1.0 ppt) made with Hawaiian marine mix-salt (Hawaiian Marine Imports, Houston, TX). The total volume of water in each rearing tank, and the biofilter, was 35 and 80 gallons, respectively. One ½HP self-prime pump (3600 gph) was used to circulate water through the tank system, and there was a vertical filtration system consisting sequentially of mechanical (30 mm), chemical (activated carbon), and UV sterilization units (Rainbow Lifeguard, El Monte, CA). A 300 W and two 150 W Visi-therm heaters (Aquarium Systems, Mentor, OH) were submerged in the biofilter tank to maintain water temperature in the rearing tanks at 28.0 _+ 1.0°C. Water quality parameters such as pH, nitrite, and dissolved oxygen were measured with Hach (Hach Co., Loveland, CO) and Taylor (Taylor Technologies, Inc., Sparks, MD) test kits. Ammonia was measured by the salicylate-hypochlorite method (Bower and Holm-Hansen, 1980). Salinity and temperature were measured daily. All other parameters were measured weekly. 579
580
MARIOS A. CARIOLOU and COr~STAN~N N. FLYTZANIS
Ovarian maturation
Female and male P. vannamei shrimp weighing more than 45 g, were purchased from Granada Corporation (Agromarina d6 Panama), Houston, Texas, and Amorient Aquafarm, Inc., Kahuku, Hawaii. Animals were also raised to adulthood in our facility from nauplii (stages N3/N4) which were provided by Granada Corporation. All shrimp were acclimated in our system for at least three weeks prior to any ablation attempt. To induce ovarian maturation, at the end of the acclimation period and four days following ecdysis, female shrimp were unilaterally ablated by cutting with sharp scissors the eyestalk close to the carapace and immediately cauterizing the wound. An equal number of unablated female shrimp was used as controls. No males were ablated. Maturation feed (Rangen, Inc., Buhl, ID) was provided five times daily as food source (8% Biomass/day). Ovarian development was monitored daily and classified as described by Yano et al. (1988). Shrimp in advanced stages of ovarian development were transferred to 10-gallon tanks for spawning and egg collection. M i n i - 2 D - P A G E and sample preparation
Mini-2D-PAGE analysis of ovarian, egg, and testicular polypeptides from P. vannamei was performed essentially as described by O'Farrell (1975). Equilibrium pH gradient electrophoresis was used in the first dimension and S D S - P A G E in the second dimension. Carrier ampholytes (Bio-Rad Laboratories, Richmond, CA) were used at concentrations of 1.6% (pH range 5-7) and 0.4% (pH range 3-10). Maturation and synapsis stage ovaries (Yano et al., 1988) were obtained from ablated mature and immature shrimp (body weight > 43 g), respectively. Eggs were collected immediately after the spawning of shrimp. Male animals with individual weights of 11, 25 and 43 g were sacrificed and their testes dissected. The samples were processed by homogenization in extraction buffer (0.05 M Tris pH 7.0, 8M urea, 1% SDS, 1%0 b-mercaptoethanol, 360 mg/ml TPCK (N-tosylL-phenylalanine chloromethyl ketone), 180 mg/ml TLCK (N-tosyl-L-lysine chloromethyl ketone), 3 mg/ml pepstatin, 240 mg/mi A - P M S F (4-amidinophenylmethanesulfonyl fluoride) and 3 mg/ml leupeptin) and centrifugation at 14,000 g and 10°C, for 15 min in an Eppendorf centrifuge. Total soluble protein in each sample was determined using the BCA protein assay (Pierce, Rockford, IL). Prior to electrophoresis, samples were diluted 1:1 with dilution buffer (9.5M urea, 8% Nonidet P-40, 4% ampholytes, in the same ratio as the gel, and 5% b-mercaptoethanol) to give a Nonidet P-40/SDS ratio of 8:1 and final ampholyte concentration of 2%. Approximately 20 #g of protein from each sample were loaded on the IEF tube gels (inside diameter 1.5 x 73mm). Samples were electrophoresed with 350 V for 8 hr at room temperature. Electrophoresis
in the first dimension was carried out, for all samples, on the same day. S D S - P A G E was performed at constant current (15mA/gel) using 1.5mm thick polyacrylamide gels (85 x 65 mm) and was terminated when the tracking dye (Bromphenol Blue) eluted from the gel. Following electrophoresis in the second dimension, gels were silver-stained using the procedure of Wray et al. (1981). Molecular weight markers for S D S - P A G E were phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5kDa) and lysozyme (14.4 kDa) (Bio-Rad). RESULTS
The water condition in our recirculating system is presented in Table 1. The overall survival of the animals in this system was excellent. Fewer than 5% of the animals died during the course of the investigation and subsequent histological examination did not reveal pathological conditions (Table 2). Ovarian maturation was observed only in ablated female shrimp. The majority of unilaterally ablated shrimp matured and spawned 15 + / - 6 days after ablation (Table 3). Male shrimp matured without ablation. Figure 1 illustrates the 2D-PAGE separation of polypeptides from immature ovaries (1A), mature ovaries (IB), and eggs (1C). Approximately 125 and 250 polypeptides stained with silver were readily detected in the gels from immature (Fig. 1A) and mature ovaries (Fig. 1B), respectively. Although the electrophoretic pattern was similar both qualitative and quantitative differences were detected between the immature and mature ovary. The most notable qualitative differences occurred in two polypeptides with similar molecular mass of 63 kDa, but different isoelectric points, and three other polypeptides with molecular masses of 24, 29.5, and 57 kDa. These five proteins, which are marked in Fig. 1 with arrowTable 1. Mean + standard deviation, maximum and minimum values for six water quality parameters measured during ablation trials Parameter
Mean
Dissolvedoxygen(rag/I) Biofilter 8.5 _+0.6 Rearing tanks 7.3 _+1.4 Ammonia nitrogen (mg/l) Biofilter 0.040 _+0.007 Rearing tanks 0.089± 0.014 pH Biofilter 8.1 -t-0.2 Rearing tanks 7.6 + 0.4 Temperature (°C) Biofilter 29.6 + 1.0 Rearing tanks 28.0 +_1.0 Nitrate (mg/1) Biofilter 0.16 + 0.08 Rearing tanks 0.19 ± 0.07 Salinity(ppt) Biofilter 29.0 __1.0 Rearing tanks 29.0 +_1.0
Maximum
Minimum
9.0 9.0
8.0 5.0
0.049 0.120
0.031 0.076
8.5 8.5
8.0 7.5
31.0 28.5
29.4 27.5
0.33 0.36 31 31
0.09 0.10 28 28
Shrimp gonadal development Table 2. Survival of shrimp in rearing tanks 60 days post-ablation Trial No.
Tank No.
1 I 1 2 2 3 3
Ablated (No.)
1 2 2C 3 3C 4 4C
2 2 0 1 0 12 0
60 days Beginning post-ablation 4/3* 2/3 2/3 1/2 1/2 12/0 12/0
2/3* 1/3 2/2 1/2 1/2 10/0 11/0
*Ratio of female to male shrimp in each tank. Tanks 2C, 3C and 4C contained control unablated shrimp.
heads, are present in mature (Fig. 1B), but are not detected in immature (Fig. 1A), ovarian extracts. Open brackets in Fig. 1 illustrate the most notable quantitative differences which were observed between ovary samples. The prevalence of a family of basic polypeptides (45~i6.2 kDa) and an acidic, 24 kDa protein was significantly increased from a low level in immature ovaries (Fig. 1A) to a relatively high concentration in mature ovaries (Fig. IB). As ovarian maturation proceeds, the prevalence of some proteins is diminished, most notably a 40 kDa protein which is present in immature ovaries (Fig. IA, triangle), is undetectable in mature ovaries (Fig. 1B). In contrast to the ovarian samples, fewer polypeptide species were resolved by 2D-PAGE in egg extracts (Fig. 1C). Despite the large dissimilarity of gel patterns from ovaries and eggs we were able to detect at least five polypeptides with molecular masses of 24, 38, 47, 48 and 54 kDa which were present in all three samples (Fig. 1, open circles). The streaking, which was observed with certain egg polypeptides localized at both the basic and acidic ends of the egg gels, may be due to secondary modifications, such as glycosylation, and/or the over-abundance of these polypeptides in the sample. Analysis of different dilutions of egg polypeptides extracted from the same sample or different egg samples, revealed identical gel patterns (results not shown). We conclude that alterations in the pH gradients caused by the over-abundance of certain egg proteins was highly unlikely and, therefore, we are confident that the interpretation of the gel patterns is correct. Our sample preparations did not include removal of nucleic acids which may also cause streaking of protein spots and an increase in the background staining of 2D-gels. Table 3. Spawning events and frequency of spawning for each ablated shrimp Trial No.
Tank No.
1 2 3
2 3 4
No. Ablated shrimp 1 1 2 2 I 1 1 1 1 1
Spawning events/shrimp 3 (9, 13, 26)* 1 (19) 0 I(II) 2(11,14) 1 (15) 2 (15, 25) 1 (19) 2 (27, 33) 1 (i 17)
*Number of days after ablation when spawning took place. Invariably, shrimp spawned between 2:00 and 3:00 a.m. CBP(B)I05-3/4--K
581
Figure 2 demonstrates that the complexity and heterogeneity of testes polypeptides increases as the shrimp grow older. Testes polypeptides were extracted from males of approximate weights of 11, 25 and 43 g. A large number of testes polypeptides from the 43 g shrimp were detected in 2D-gels (Fig. 2A). Electrophoretic analysis of testes extracts from 25 g shrimp also indicated the presence of several proteins (Fig. 2B). Although the overall pattern is very dissimilar, at least one 40 kDa polypeptide and two others, with different isoelectric points but similar molecular mass of 32 kDa, were readily detected in both testes preparations. These three polypeptides are marked with open circles in Fig. 2. We were able to detect only a small number of proteins by 2D-PAGE analysis of testes extracts from juvenile (11 g) shrimp (Fig. 2C). One of these polypeptides, illustrated with an open circle in Fig. 2C, with molecular mass of 32 kDa was shown to be present in all three preparations. Protein assays were carried out on all testes extracts in order to standardize samples and ensure the loading of an equal amount of protein on each gel. However, it is obvious from Fig. 2 that the total silver-stained testes polypeptides detected on gels from 11 g shrimp (Fig. 2C) are considerably fewer than those from 43 g shrimp (Fig. 2A). The high degree of reproducibility and consistency of both the protein assay and the 2D-PAGE analysis of testes extracts lets us speculate the presence of an unknown factor in testes from juvenile shrimp that interferes with the protein assay we have employed, causing overestimation of total protein in this particular sample. As the shrimp grow older this factor may be diluted out. It is also possible that proteins extracted from testes of juvenile shrimp may not stain with silver as efficiently as the proteins of adult testes, or that immature testes contain a high concentration of proteases, which cleave the proteins to sizes that are not resolved in our gels. DISCUSSION
In order to begin to understand the molecular events that underlie the gonadal development of P. vannamei, it is essential to learn more about the biological complexity of proteins present in shrimp gonadal tissues. We present herein our first observations on the polypeptide species present in the immature and mature ovaries, and testes from P. vannamei. Using high-resolution mini-2D-PAGE we were able to resolve and identify several qualitative and quantitative differences in polypeptides extracted from these tissues. Immature and mature ovarian samples displayed many qualitative differences, although the overall migration pattern of the polypeptides appeared quite similar. Except for one protein that is clearly absent from the mature ovary, the major differences between the two samples result from additional polypeptides present in the mature ovary. These proteins may be exclusively found in the
582
MARIOS A. CARIOLOU and CONSTANTINN. FLYTZANIS
IEF
+
A. 97.4~
66.2-"45--"31---
21.5--,.14.4-,.-
B. 97.4"--=-
66.2--.¢n 0 03
45-,-
31~
21.5~ 14.4~
97
66
21 14
Fig. 1. 2D-PAGE patterns of silver stained polypeptides extracted from shrimp immature ovary (A), mature ovary (B), and eggs (C). Procedures for the 2D-PAGE and sample preparations are as described in Materials and Methods. Open circles indicate common polypeptides. Arrowheads point to positions where major polypeptides are present in mature but absent in immature ovaries. The position where a major polypeptide is present only in immature ovary is illustrated with a triangle. Open brackets illustrate polypeptides with significant quantitative and qualitative differences. The protein standards in the second dimension are indicated in kDa. The amount of protein loaded in the first dimension was 20/~g.
583
Shrimp gonadal development IEF
4-|
97.
66. u') 4 o~ 3
21. 14.
C. 97.4~ 66.2~ 45---
31---
21.5-14.4---
Fig. 2.2D-PAGE patterns of silver-stainedpolypeptides extracted from the testes of male shrimp weighing 43 g (A), 25 g (B), and 11 g (C). Procedures for the 2D-PAGE and sample preparations are described in Materials and Methods. Open circles indicate common polypeptides. The protein standards indicated in the second dimension are in kDa. The apparent amount of protein loaded in the first dimension was 20/~g. ovarian tissue and not the mature oocytes since we could not identify them in the spawned eggs. Very few similarities were observed between the ovarian and the egg sample. Thus, the majority of the polypep-
tides deposited in the egg seem to be synthesized towards the end of oogenesis. The migration pattern, and the abundance of the major egg polypeptides, does not indicate that they were simply hidden within
584
MARIOSA. CARIOLOUand CONSTANTINN. FLYTZANIS
the more complex ovarian population. The few (at least five) proteins that were found to be common in both the spawned eggs and the immature ovary, suggest that at least some egg-specific polypeptides may be synthesized very early during ovarian development. These qualitative and quantitative differences in the proteins extracted from immature and mature ovaries, and the spawned eggs, suggest that the differential synthesis and accumulation of several polypeptides during ovarian maturation may not only be involved in oogenesis per se (i.e. synthesis of proteins and maturation of the oocyte) but may also play an important role in the processes that govern overall ovarian development. As male P. vannamei grow older and larger in size, testes development is characterized by an increased complexity in the number of different polypeptides resolved by the 2D-PAGE analysis. This strongly suggests that male gonadal maturation is characterized by differential synthesis of testes proteins in a developmentally controlled program. Although not easily detected, the immature testes polypeptides may all be present in the mature sample and thus may not comprise an immature specific set of proteins. It is interesting that the number of polypeptides detected by the 2D-PAGE increased dramatically during spermatogenesis but not oogenesis. One explanation for this may be the unequal contribution of ovarian and testicular tissue to the analyzed samples at the different developmental stages. During ovarian development we were able to distinguish polypeptides which may belong exclusively to the eggs and polypeptides contributed by the ovarian tissue. Since we did not analyze sperm-only samples, we cannot make the same distinction for the testes. After their capture from the wild, mature shrimp normally undergo gonadal regression. Due to this complex biological process we did not wish to compare polypeptides extracted from laboratory-reared and wild-caught animals. Therefore, some proteins found in the animals reared in our facility may differ from those found in wild shrimp. Also, we did not attempt to identify specific proteins in our samples during the course of this investigation. This can be accomplished by the use of specific antibodies raised against proteins from other animals or by the purification of the most prevalent of the analyzed proteins. Our emphasis in the future will be given to the study of those proteins, which are either common between developmental stages, or stage-specific. Such future studies
may begin to elucidate the role of specific proteins during gonadal development. Acknowledgement--This research was supported by a grant
from Granada BioSciences, Inc. to CNF. REFERENCES
Aquacop (1979) Penaeid reared broodstock: closing the cycle of Penaeus monodon, Penaeus stylirostris, and Penaeus vannamei. Proc. Worm Maricult. Soc. 10, 445-452. Bower C. E. and Holm-Hansen T. (1980) A salicylatehypochlorite method for determining ammonia in seawater. Can. J. Fish. Aquat. Sci. 27, 794-798. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680--682. Lawrence A. L., McVey J. P. and Huner J. W. (1985) Penaeid Shrimp Culture. In Crustacean and Mollusk Aquaculture in the United States (Edited by Huner J. V. and Brown E. E.), pp. 127-157. AVI Publishing, Westport, CT. O'Farrell P. H. (1975) High-resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250, 4007-4021. Panouse J. B. (1943) Influence de l'ablation due pendocule oculaire sur la croissance de l'ovaire chez la crevette Leander serratus. C. r. Acad. Sci., Paris 217, 553-555. Quackenbush L. S. (1989) Vitellogenesis in the shrimp, Penaeus vannamei: in vitro studies of the isolated hepatopancreas and ovary. Comp. Biochem. Physiol. 94B, 253-261. Rankin S. M., Bradfield J. Y. and Keeley L. L. (1989) Ovarian protein synthesis in the South American white shrimp, Penaeus vannamei, during the reproductive cycle. Invert. Reprod. Dev. 15, 27-33. Sandifer P. A., Hopkins J. W. and Stokes A. D. (1987) Intensive culture potential of Penaeus vannamei. J. Worm Aquacult. Soc. 15, 94-105. Sandifer P. A., Hopkins J. S. and Stokes A. D. (1988). Intensification of shrimp culture in earthen ponds in South Carolina: progress and prospects. J. World Aquacult. Soc. 19, 218-226. Tom M., Goren M. and Ovadia M. (1987) Purification and partial characterization of vitellin from the ovaries of Parapenaeus longirostris (Crustacea, Decapoda, Penaeidae). Comp. Biochem. Physiol. 87B, 17-23. Wray W., Boulikas T., Wray V. P. and Hancock R. (1981) Silver-stainingof proteins on polyacrylamidegels. Analyt. Biochem. 118, 197-203. Wyban J. A., Lee C. S., SweeneyJ. N. and Richards W. R. Jr (1987) Observations on development of a maturation system for Penaeus vannamei. J. Worm Aquacult. Soc. 18, 198-200. Yano I. and Chinzei Y. (1987) Ovary is the site of vitellogenin synthesis in Kuruma prawn, Penaeus japonicus. Comp. Biochem. Physiol. 86B, 213-218. Yano I., Tsukimura B., Sweeney J. N. and Wyban J. A. (1988) Induced ovarian maturation of Penaeus vannamei by implantation of lobster ganglion. J. World Aquacult. Soc. 19, 204-209.