Original Article. Takuji OKUMURA*,a AND Katsumi AIDA

Transcription

1 FISHERIES SCIENCE 2000; 66: Original Article Hemolymph vitellogenin levels and ovarian development during the reproductive and non-reproductive molt cycles in the giant freshwater prawn Macrobrachium rosenbergii Takuji OKUMURA*,a AND Katsumi AIDA Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo , Japan SUMMARY: In this study, ovarian development and vitellogenin levels were determined during the reproductive and non-reproductive molt cycles in the giant freshwater prawn Macrobrachium rosenbergii in order to examine the relationship between vitellogenesis and molting. During the reproductive molt cycle, the ovary developed synchronously with the advancement of molting stage. At molting stages B C 0, the ovaries contained oogonia and oocytes at the previtellogenic and endogenous vitellogenic stages, and hemolymph vitellogenin levels were relatively low ( mg/ml). At stage C 1, yolk accumulation commenced in oocytes at the endogenous vitellogenic stage, after which oocytes entered the exogenous vitellogenic stage. Vitellogenin levels increased (4.9 mg/ml) at this stage. During premolt stages D 0 D 3, vitellogenin titers were high ( mg/ml), and gonadosomatic index (GSI) increased rapidly due to the yolk accumulation in the oocytes. Following ecdysis, yolk accumulation was completed at stage A 0, and oviposition occurred. Entering stage A, vitellogenin levels became low ( mg/ml). In contrast, throughout the non-reproductive molt cycle, GSI remained low, and oocytes did not develop beyond the previtellogenic or endogenous vitellogenic stage. Some atretic oocytes at the exogenous vitellogenic stage were observed in a few females. During the nonreproductive molt cycle, vitellogenin levels did not show distinct changes in relation to the molt cycle. KEY WORDS: giant freshwater prawn, Macrobrachium rosenbergii, molt cycle, vitellogenesis, vitellogenin. INTRODUCTION *Corresponding author: Tel: Fax: takuji@affrc.go.jp a Present address: Japan Sea National Fisheries Research Institute, Niigata , Japan. Received 8 November Accepted 25 February In crustaceans, the ovary develops rapidly during the process of vitellogenesis (see reviews 1,2 ). Vitellogenesis is characterized by the appearance in the hemolymph of vitellogenin, the precursor of the major yolk protein, vitellin, and its uptake into the oocytes. Within the oocytes, vitellogenin is processed into vitellin. The accumulation of yolk protein causes a rapid increase in oocyte diameter and size. Yolk protein is subsequently utilized as a nutritional source during the process of embryogenesis. In most crustacean species, the female continues to molt after reaching sexual maturity. 1,2 In such species, the process of vitellogenesis must be coordinated to molting, because females of most species mate and spawn immediately after ecdysis; the occurrence of ecdysis during the brooding of embryos may cause their loss. While there are obviously regulatory mechanisms which ensure the coordination of molting and reproduction, these mechanisms have not been fully clarified. Detailed study concerning the relationship between reproduction and molting is expected to provide useful and basic information which will enable further understanding of this physiological functioning. In this investigation, the giant freshwater prawn Macrobrachium rosenbergii was used to examine the nature of the relationship between vitellogenesis and the molt cycle. This prawn is commercially available from many aquaculture farms, and females exhibit an interesting feature in their reproductive and molting physiology. Females undergo two types of ecdysis: reproductive ecdysis or pre-spawning ecdysis accompanied by ovarian development and followed by oviposition; and nonreproductive ecdysis or somatic ecdysis accompanied by growth only. Both molting types are repeated in a random fashion. 3 This feature allows comparative studies

2 Vitellogenesis in Macrobrachium rosenbergii FISHERIES SCIENCE 679 between reproduction and molting. Thus far, information concerning ovarian development and hemolymph vitellogenin in this species has been reported by several authors. 2,4 9 However, these investigations did not examine in detail the dynamics of reproduction in the context of advancing ovarian maturity and changing hemolymph vitellogenin levels during the molt cycle. In this investigation, we followed changes in ovarian development and hemolymph vitellogenin levels during the molt cycle, and compared them between the reproductive and non-reproductive molt cycles. MATERIALS AND METHODS Animals Male and female M. rosenbergii were obtained from local aquaculture farmers in Osaka and Kumamoto Prefectures. Individual prawns were kept in divided chambers maintained at 28 C under a photoperiod of 15L:9D. Prawns were fed commercial pellets and minced clam meat daily, and acclimated in these chambers at constant environmental conditions for at least 2 weeks prior to use in three experiments: (i) examination of ovarian development during the molt cycle; (ii) examination of female hemolymph vitellogenin levels during the molt cycle; and (iii) western immunoblotting analysis of male and female hemolymph and mature ovary. For female prawns, times of ecdysis, occurrence of oviposition and appearance of ovigerous setae on the first four pairs of pleopods were recorded. Reproductive ecdysis was determined based on the occurrence of oviposition and the appearance of ovigerous setae following ecdysis. Under these conditions, the reproductive ecdysis and the non-reproductive ecdysis (common ecdysis) are observed to occur in a random fashion. 3 Molt staging Molting stages were determined based on observations of setogenesis occurring in the setae of the pleopods according to previously described criteria. 10,11 The distal portion of the fourth or fifth pleopod was taken, and the state of setogenesis was observed using a differential phase contrast microscope. The molt cycle was divided into four overall stages: postmolt, intermolt, premolt and ecdysis. In addition, each stage was further divided into substages: postmolt (A and B), intermolt (C 0 and C 1 ), premolt (D 0, D 1, D 2 and D 3 ) and ecdysis (E). Ovarian development For microscopic observation of ovarian development during the molt cycle, female prawns (body weight ranging from 12.9 to 30.0 g) were killed at differing molt stages. Body weight was determined, and ovaries were quickly excised and weighed. Gonadosomatic index (GSI) was calculated as gonad weight (g) 100/body weight (g). Ovaries were fixed in Bouin s solution, embedded in paraffin, and sectioned at 3 5 mm. Sections were stained with Carazzi s hematoxylin-eosin or Carazzi s hematoxylin PAS (periodic acid Schiff) for purposes of histological observation. For immunohistochemistry, the sections were stained with rabbit antiserum raised against purified vitellin from the freshwater prawn, Macrobrachium nipponense 12 and the avidin biotin complex (ABC) method according to a previous report. 4 To examine the presence of lipid in the oocytes, ovaries were fixed with 10% formalin, and were postfixed with 1% osmium tetraoxide in 0.1 M cacodylate buffer, ph 7.5. The fixed samples were embedded in paraffin, and sectioned at 4 mm. Hemolymph sampling Hemolymph sampling ( ml) was conducted from female prawns (body weight ranging from g) at intervals of 10 days or longer in order to avoid the influence of stress to the experimental animals. Individual female prawns were followed throughout the course of sampling in order to determine molting type based on observation of ovarian development and occurrence of oviposition. Hemolymph was withdrawn from the pericardial cavity via a syringe with needle, and stored at 70 C until analysis. Vitellogenin enzyme-immunoassay Hemolymph vitellogenin levels were measured by enzyme-immunoassay (EIA) according to procedures described previously. 11 Vitellin purified from matured ovaries of M. rosenbergii using gel filtration and ionexchange chromatography according to a method described by Han et al. 12 was used as a standard. Antibody used in this assay was raised against purified vitellin from M. nipponense. In this system, the standard curve and absorbance curve for the serially diluted hemolymph of mature female were seen to be parallel. Sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotting Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4 20% acrylamide gradient slab gels which were purchased from TEFCO (Tokyo, Japan). Hemolymph from male and female prawns and crude extract of ovaries in 20 mm Tris-HCl buffer (ph 8.7) containing 0.15 M NaCl were used in this

3 680 FISHERIES SCIENCE T Okumura and K Aida analysis. Sample preparation was done by mixing sample with dilution buffer (20 mm Tris-HCl ph 6.8, containing 4% SDS, 40% glycerol, 2% mercaptoethanol, and 0.005% bromophenol blue). Samples were heat-treated at 100 C for 5 min. For purpose of comparison, vitellin purified from M. rosenbergii ovary was also run concurrently. For molecular weight determination, high molecular weight markers (Bio-Rad molecular weight marker kit; Bio-Rad Laboratories, Hercules, USA) were employed. For immunochemical determination, immunoblotting was carried out following SDS-PAGE according to methods previously reported. 4 Proteins were transferred electrophoretically from gels to PVDF membranes (Millipore, Bedford, USA), and the blotted membrane was immuno-stained with biotin-labeled IgG raised against vitellin of M. nipponense using the Vectastain ABC kit (PK-6100; Vector Lab. Inc., Burlingame, USA). Statistics Duncan s multiple range test and the Student s t-test were used for statistical analysis after log-transformation of the data. RESULTS Ovarian development during the molt cycle Changes in GSI and the state of ovarian development during the molt cycle are shown in Figs 1 and 2. At molting stages B C 0, GSI was low (<0.49%, mean), and the ovary contained oogonia and oocytes without yolk at the previtellogenic and endogenous vitellogenic stages (Fig. 2a). During these molting stages, it could not be distinguished whether females were in the reproductive molt cycle or in the non-reproductive molt cycle based on GSI and microscopic observation of the ovaries. Thus, GSI data for individuals at these stages could not be divided into two molt groups as shown in Fig. 1. Oocytes in the previtellogenic stage exhibited hematoxylin-stained cytoplasm, and those in the endogenous vitellogenic stage possessed oil globules (Fig. 2f) and PAS-positive but eosin-negative vesicles (Fig. 2a) in the cytoplasm. The central area of the ovary itself consisted of oogonia, and the peripheral area of the ovary was filled with oocytes at the previtellogenic and endogenous vitellogenic stages described above. At molting stage C 1, four of 18 females showed relatively high GSI (0.79 ± 0.10%, mean ± SE) and accumulation of yolk globules was seen to have commenced in those oocytes which were in the exogenous vitellogenic stage (Fig. 2b). These females were determined as being in the reproductive molt cycle. On the other hand, 14 of 18 females showed relatively low GSI (0.51 ± 0.03%), and had not commenced accumulation of yolk globules Fig. 1 Changes in gonadosomatic index (GSI) during the molt cycle. The GSI values in females during the ( ) reproductive and ( ) non-reproductive molt cycles. ( ) GSI of females for which molt type could not be determined (either reproductive or non-reproductive molt cycle). Symbols and vertical bars represent the mean ± SE. Numbers beside symbols indicate the number of animals employed in a single determination. (*) Stage A and B after reproductive ecdysis or nonreproductive ecdysis; stage A following reproductive ecdysis is divided into stage A 0 before oviposition and stage A 1 after oviposition. (**) Data of stage A 0 are not included in those of stage A due to high GSI at stage A 0 before oviposition. in the oocytes. These females comprised those individuals which were in the non-reproductive molt cycle as well as those in the reproductive molt cycle which had not yet begun yolk accumulation. During molting stages D 0 D 3, females in the reproductive molt cycle could be distinguished based on high GSI and extensive yolk accumulation in oocytes. During the reproductive molt cycle in these females, oocytes accumulated yolk globules with a rapid increase in size (Fig. 2c), and GSI increased especially during stages D 0 D 3. Ovaries were almost completely filled with oocytes at the exogenous vitellogenic stage. Oogonia and oocytes at the previtellogenic and endogenous vitellogenic stages were observed only in the central area of the ovary. Using immunohistochemistry, it was seen that oocytes at the exogenous vitellogenic stage showed a distinct positive reaction against anti-vitellin serum; in contrast, oocytes at the previtellogenic and endogenous vitellogenic stages did not show any positive reactions at molting stage D 0 (Fig. 2g). After ecdysis, GSI reached 9.16 ± 0.29% at stage A 0 prior to oviposition. Following the occurrence of oviposition between molting stages A 0 and A 1, mature oocytes

4 Vitellogenesis in Macrobrachium rosenbergii FISHERIES SCIENCE 681 Fig. 2 Ovarian development during the molt cycle. Each photo shows a cross-section of ovarian tissue. (a) Periodic acid Schiff (PAS) staining at stage B; (b) HE staining at stage C 1 of the reproductive molt cycle; (c) HE staining at stage D 2 of the reproductive molt cycle; (d) PAS staining at stage A 1 following oviposition; (e) PAS staining at stage D 2 of the non-reproductive molt cycle; (f) osmium tetraoxide postfixation at stage C 0 ; (g) immunohistochemistry at stage D 0 of the reproductive molt cycle. O, oogonium; PO, previtellogenic oocyte; EN, endogenous vitellogenic oocyte; EX, exogenous vitellogenic oocyte; EF, empty follicle after ovulation; AO, regressing atretic oocyte; OG, oil globule. Scale bar = 100 mm. nearly disappeared in the ovary (Fig. 2d) and GSI became low (1.1%). However, in the ovaries of some females at molting stage A 1, unspawned mature oocytes were observed to have remained (Fig. 2d). In contrast, throughout the non-reproductive molt cycle, ovaries were filled with oogonia and oocytes at the previtellogenic and endogenous vitellogenic stages (Fig. 2e). The GSI was constantly low (<0.48%, mean)

5 682 FISHERIES SCIENCE T Okumura and K Aida during stages D 0 D 3. However, throughout the molt cycle, some females possessed atretic mature oocytes within the ovaries, and showed relatively high GSI ( %). Hemolymph vitellogenin levels during the molt cycle Figure 3 shows hemolymph vitellogenin levels during the reproductive and non-reproductive molt cycles. In the reproductive molt cycle, vitellogenin levels were low during molting stages A C 0 ( mg/ml). Subsequently, levels increased at molting stage C 1 concomitant with the start of yolk accumulation, reaching significantly high titers during molting stages C 1 D 3 ( mg/ml, P < 0.01). When yolk accumulation was completed at molting stage A 0, vitellogenin levels decreased (1.3 mg/ml). During the non-reproductive molt cycle, changes in vitellogenin levels were not statistically significant (P > 0.05). Levels at each molt stage showed a wide range of variation (non-detectable levels up to >4 mg/ml). Some animals even showed titers higher than 9 mg/ml. Some of such animals showing high vitellogenin levels were sampled in order to observe the ovaries histologically. These individuals possessed atretic mature oocytes within the ovaries (Fig. 4). From a comparison of both molt cycles, it was seen that differences in vitellogenin levels were not significant at molting stages A C 0 (P > 0.05). However, during molting stages C 1 D 2, levels in the reproductive molt cycle became significantly higher than those in the nonreproductive molt cycle (P < 0.01). At molting stage D 3, levels did not differ significantly between both molt cycles. However, levels at stages A 0 B after reproductive ecdysis became significantly lower than did those at stages A B after the non-reproductive ecdysis (P < 0.01). SDS-PAGE and immunoblotting Vitellogenin and vitellin were analyzed using SDS- PAGE and western immunoblotting (Fig. 5). Vitellin purified from M. rosenbergii ovary and crude ovarian extract showed two distinct bands of 102 and 90 kda. Hemolymph in females with mature ovaries showed two major bands of 102 and 90 kda and three weak bands of 199, 64 and 60 kda. Hemolymph in non-vitellogenic females in the non-reproductive molt cycle showed no positive bands. However, in females in the nonreproductive molt cycle which had atretic oocytes, hemolymph showed the positive bands which were similar to those of mature females. Hemolymph obtained from male and juvenile females did not show any positive bands. DISCUSSION In this study, the relationship between vitellogenesis and molting was examined in detail in M. rosenbergii, and the process of vitellogenesis was seen to be closely related to that of molting during the reproductive molt cycle. Fig. 3 Hemolymph vitellogenin levels during the reproductive and non-reproductive molt cycles. Vitellogenin levels in females during the ( ) reproductive and ( ) non-reproductive molt cycles. Symbols and vertical bars represent the mean ± SE. Numbers beside symbols indicate the number of animals employed in a single determination. (*) Stage A and B after reproductive ecdysis or non-reproductive ecdysis; stage A after reproductive ecdysis is divided into stage A 0 before oviposition and stage A 1 after oviposition. Fig. 4 Cross-section of the ovary showing regressing atretic oocytes at stage D 2 of the non-reproductive molt cycle (HE staining). O, oogonium; PO, previtellogenic oocyte; AO, regressing atretic oocytes. Scale bar = 100 mm.

6 Vitellogenesis in Macrobrachium rosenbergii FISHERIES SCIENCE K 116K 97K 66K 45K Fig. 5 Examination of hemolymph vitellogenin by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS- PAGE) and western blotting. Lane 1, adult male; lane 2, juvenile female; lane 3, immature adult female; lane 4, crude ovarian extract; lane 5, purified vitellin; lane 6, mature adult female; lanes 7 and 8, immature adult females having regressing atretic oocytes in the ovaries. Numbers on the left side of the figure indicate the molecular weights of the molecular weight markers. At intermolt stage C 1, hemolymph vitellogenin levels became elevated, and GSI increased greatly due to the accumulation of yolk globules within the oocytes. This indicates that the synthesis and uptake of vitellogenin become highly active at molting stage C 1. During premolt stages D 0 D 3, hemolymph vitellogenin levels remained high and the ovaries developed even more rapidly. Based on this, it may be considered that vitellogenin synthetic activity was maintained in the later molting stages. When oviposition occurred at postmolt stages A 0 A 1, vitellogenin levels became low, indicating that vitellogenin synthesis had ceased and accumulation of yolk globules was completed by stage A 0 after the reproductive ecdysis. Our results are consistent with previously reported profiles of vitellogenin titers 2,9 and ovarian development in M. rosenbergii. 6,7 However, the present study demonstrated in more detail the relationship between vitellogenesis and molting. We had also obtained similar results previously in M. nipponense. 11 The close relationship between ovarian maturation and molting seems to be a common feature in many of the Macrobrachium species. In general, the process of vitellogenesis is understood to comprise the synthesis of vitellogenin and the uptake of vitellogenin from the hemolymph into the oocytes accompanied by the accumulation of yolk globules. 1,2 In this study, the immunoblotting analysis indicated the existence of similar immunoreactive substances in both the ovary (vitellin) and hemolymph (vitellogenin) of mature females, and immunohistochemistry has shown the existence of positive immunoreactivity in oocytes at the exogenous vitellogenic stage, as reported in previous studies. 4,13 Lee et al. 13 reported the existence of three subunits (170, 100 and 89 kda) in purified hemolymph vitellogenin and the biochemical characteristics of vitellogenin and vitellin in M. rosenbergii. They suggested that the 100 and 89 kda vitellogenin subunits give rise to the dominant 100 and 89 kda subunits of vitellin, respectively, and that the 170 kda vitellogenin subunit is transformed into the 100 kda vitellin subunit via processing mechanisms. These results support the abovementioned scheme of vitellogenesis in M. rosenbergii. The sites of vitellogenin synthesis are known to differ according to species. In prawns of the Macrobrachium genus including M. rosenbergii 14 and M. nipponense, 12 the existence of an extraovarian source of vitellogenin has been suggested. During the non-reproductive molt cycle, the ovary did not develop and the oocytes remained at the previtellogenic and endogenous vitellogenic stages, whereas the molting process advanced as in the reproductive molt cycle, indicating that molting is independent of ovarian development. The absence of ovarian development during the non-reproductive molt cycle is consistent with a previous report in M. rosenbergii. 6 However, in this study, some females showed very high vitellogenin titers in spite of the absence of ovarian development, while most females showed low or even undetectable titers. Such females with high titers of vitellogenin possessed atretic oocytes containing yolk. Since some females exhibited unspawned oocytes in the ovaries after oviposition, the atretic oocytes are possibly derived from unspawned oocytes. Consequently, the reabsorption of unspawned oocytes and the release of the absorbed vitellin to the hemolymph seem to be a possible cause of this phenomenon. However, in immunoblotting analysis, hemolymph from females with atretic oocytes showed three weak bands in addition to two dominant bands. The existence of the 199 kda band suggested that the immunoreactive substances in these females were identical to hemolymph vitellogenin in mature females but different from ovarian vitellin, indicating that the immunoreactive substances in these females contain vitellogenin-specific components. However, it is possible that the 199 kda band reappears through the processes of vitellin absorption and transfer to the hemolymph, and it is also possible that both vitellogenin synthesis and transfer of vitellin to the hemolymph occur simultaneously. Further study is needed to clarify the origin of immunoreactive substances in hemolymph observed in some nonreproductive females. In contrast, throughout the non-reproductive molt cycle in M. nipponense, ovary does not develop and vitellogenin titers remain at undetectable levels. 10,11 This is likely to be due to the distinct seasonal reproductive physiology of M. nipponense. 10 During the reproductive

7 684 FISHERIES SCIENCE T Okumura and K Aida season, females repeatedly undergo reproductive molting. However, during the non-reproductive season, the females undergo only non-reproductive molting. During this season, females remain immature and do not exhibit unspawned mature oocytes in the ovary, and thus, vitellogenin does not appear in the hemolymph during this time. This relationship between ovarian maturation and molting during the reproductive and non-reproductive molt cycles is under the regulation of an endocrine system. Current understanding of this system in Crustacea is as follows. The X-organ-sinus gland complex located in the eyestalk secretes crustacean hyperglycemic hormone (CHH) family, 15,16 which includes moltinhibiting hormone (MIH), 17,18 vitellogenesis-inhibiting hormone (VIH) 19 and mandibular organ-inhibiting hormone (MOIH). 20 The MIH inhibits ecdysteroid synthesis in the Y-organ. 21 The VIH possibly inhibits vitellogenin synthesis directly and/or indirectly. 1,2,22 The MOIH inhibits the synthesis of methyl farnesoate (MF), which is also thought to be involved in reproduction and molting. 23,24 Based on knowledge of this endocrine system, the following can be postulated concerning mechanisms of interaction between ovarian maturation and molting during the reproductive and non-reproductive molt cycles in M. rosenbergii. In the reproductive molt cycle, oocytes initiate yolk accumulation at molting stage C 1, which is completed at molting stage A 0, while ecdysteroid levels increase during premolt stages D 0 D 3 and decline sharply at late D 3 stage. 25,26 These results suggest that hemolymph levels of both VIH and MIH decrease synchronously in the intermolt stages and increase at the late D 3 stage, although there is no direct evidence for this due to the lack of a suitable assay system. On the other hand, during the non-reproductive molt cycle, ovarian development does not proceed, although changes in ecdysteroid levels are similar to those during the reproductive molt cycle. 26 These results suggest that VIH does not decrease, but that MIH may decrease as in the reproductive molt cycle. Because MF shows similar fluctuations during both the reproductive and non-reproductive molt cycles, MF may not play a key role in this interaction. 27 The synchronous regulation of VIH and MIH secretion is probably an important factor in linking ovarian maturation and molting. It is possible that a decrease in VIH secretion could be the determining factor of molt type, e.g. reproductive ecdysis or nonreproductive ecdysis through the initiation of vitellogenesis in the intermolt stages. In order to elucidate the above hypothesis, we are conducting further studies. ACKNOWLEDGMENT We thank Dr M. N. Wilder, Japan International Research Center for Agricultural Sciences, Ministry of Agriculture, Forestry and Fisheries, for critical reading of the manuscript. REFERENCES 1. Charniaux-Cotton H, Payen G. Crustacean reproduction. In: Laufer H, Downer RGH (eds). Endocrinology of Selected Invertebrate Types. Alan R. Liss, New York, 1988; Meusy JJ, Payen G. 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