Relationship of ovarian and marsupial development to the female molt cycle in Acanthomysis robusta (Crustacea: Mysida)

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1 Blackwell Science, LtdOxford, UK FISFisheries Science Blackwell Science Asia Pty Ltd 695October Reproductive cycle in A. robusta T Dkumura /j x Original Article BEES SGML FISHERIES SCIENCE 2003; 69: Relationship of ovarian and marsupial development to the female molt cycle in Acanthomysis robusta (Crustacea: Mysida) Takuji OKUMURA* National Research Institute of Aquaculture, Nansei, Mie , Japan ABSTRACT: In the present study, the relationship of ovarian development and marsupial development to the female molt cycle was examined in Acanthomysis robusta (Crustacea: Mysida). Yolk accumulation in ovaries started at stage C and advanced during stages D 0 D 2 of the female molt cycle. As females at stages A B exhibited newly spawned eggs in their marsupia and non-developed ovaries, oviposition likely occurred at stage A of the molt cycle. Larvae in the marsupia were at the egg-shaped embryonic stage during the female molt stages A C. Larval hatching occurred at stage C, and the larvae were at the nauplioid stage during stages C D 1 of the female molt cycle. Larvae at the postnauplioid phase appeared at stage D 1, indicating that naupliar molting occurred in stages D 0 D 1 of the female molt cycle. At stage D 2, empty marsupia were observed, and any remaining brood were at the postnauplioid phase, suggesting that young are released at stage D 2 of the female molt cycle. The brood size (8 53 embryos and larvae) was significantly correlated to female carapace length. These results indicate that ovarian development and marsupial development are closely related to the female molt cycle of A. robusta. KEY WORDS: reproduction. Acanthomysis robusta, Crustacea, development, molt cycle, mysid, ovary, INTRODUCTION Various species of mysids are widely distributed throughout the coastal regions of the world and are potential prey for coastal fish. Mysids occupy an important position in coastal marine ecosystems and, hence, it is necessary to study the reproduction and growth of mysids. Mysids grow by molting and continue to molt after reaching sexual maturity. Females extrude eggs into the marsupium and incubate the larvae within the marsupium after hatching; following a period of development, the larvae are released from the marsupium. It is known that both molting and reproduction are related in females: release of the young precedes ecdysis, and copulation and oviposition take place after ecdysis. 1 Under this sequence of physiological events, females do not lose their eggs by inappropriate molting during the incubation period. Details of the relationship between the female reproductive cycle and molt cycle have been reported in the mysid Siriella armata; 2 however, in other species, such information remains limited. *Corresponding author: Tel: Fax: takuji@affrc.go.jp Received 9 September Accepted 8 May Acanthomysis robusta, a common mysid species in sandy beach areas in the northern Japan Sea, 3 serves as the major food of juvenile Japanese flounder Paralichthys olivaceus, one of the most important fish in commercial fisheries and aquaculture in Japan. 4 The abundance of A. robusta in Niigata (Japan) sharply increases in June July when juvenile flounders settle in shallow beach areas. 4 As population increases may affect the survival of juvenile flounder, elucidation of this phenomenon is important from the point of view of resource enhancement of flounder. The rapid increase in mysid abundance requires high reproductive activity and, therefore, the reproductive cycle of A. robusta must be better understood. The aim of the present study was to investigate the female reproductive cycle in A. robusta and to examine the relationship between female reproduction and the molt cycle in detail. MATERIALS AND METHODS The mysid Acanthomysis robusta was sampled at 4 m depth off Igarashi Beach, Niigata, Japan (N , E , Fig. 1) in June June

2 996 FISHERIES SCIENCE T Okumura is the month when mysid abundance starts to increase during the annual cycle. 4 Sampling was done using a bottom plankton sampling net (width, 60 cm; height, 40 cm; mesh size, 0.76 mm). The sampling net was equipped with a cod-end to reduce sampling damage to the mysids. Adult female A. robusta with developed marsupia (n = 76) were sorted for use in this study. Live sampled female mysids were fixed individually in 10% formalin in seawater. Carapace length (from the base of the eyestalk to the posterior mid-dorsal margin of the carapace) and body length (from the base of the eyestalk to the end of the telson) were measured under a microscope. Molt stages were determined by observing the setogenesis of the uropods according to the criteria of Cuzin-Roudy and Saleuddin 5 with suitable modification. Molt stages A and B were not divided in the present study due to difficulty in making clear determinations. Molt stages were as follows: A B, thin cuticle after ecdysis; C, completed cuticle with maximal thickness; D 0, progressive retraction of epidermis; D 1, splitting of matrix for new setae; D 2, deposition of new cuticle and appearance of barbules on new setae. Ecdysis occurs between D 2 and A. Samples at ecdysis were not collected in the present study, because ecdysis occurs at night and is completed within a short duration. 1 Embryos and larvae were removed from the marsupia of incubating females, and the number of embryos and larvae was counted. Furthermore, embryo length (long diameter of egg) and larval length (from the top of the head to the end of the tail) were measured, and the marsupial developmental stages (Table 1) were determined according to the criteria of Cuzin-Roudy and Tchernigvtzeff. 2 As the larvae in the marsupium develop almost synchronously, 1 one marsupial stage represented all larvae of each incubating female. The thoraxes of mysids fixed as above were embedded in paraffin, sectioned at 4 mm, and stained with hematoxylin-eosin for histological observations of the ovarian development. The data were analyzed using the Tukey Kramer test for multiple comparison, linear correlation analysis, linear regression analysis, and the Spearman rank correlation test. RESULTS Ovarian development and the molt cycle Fig. 1 Sampling of Acanthomysis robusta. Arrow indicates the sampling station. During stages A B of the female molt cycle, the ovaries of female mysids were occupied by only non-vitellogenic oocytes (60 80 mm in diameter) and oogonia (Fig. 2a). At stage C, oocytes with eosin-stained yolk globules in the cytoplasm appeared in some females, indicating the start of Table 1 Stage Marsupial developmental stages of Acanthomysis robusta Remarks 1 Embryo. Egg-like and enveloped by an egg membrane that is shed at the end of this stage. 2 Early nauplioid. Larvae have developing thoracic appendages, and segmentation of the thorax and abdomen progresses. 3 Late nauplioid. Segmentation of the thorax and abdomen is completed, and the eyes are pigmented. Larval molting occurs at the end of this stage. 4 Early postnauplioid. Molted larvae have eyes on completed stalks. Eyestalks and thoracic appendages are mobile. 5 Late postnauplioid. Thoracic appendages of the larvae are elongated and highly developed, and yolk is resorbed into the digestive gland.larvae molt at the end of this stage, and are released from the marsupium.

3 Reproductive cycle in A. robusta FISHERIES SCIENCE 997 Fig. 2 Ovarian development in Acanthomysis robusta. (a) Previtellogenic stage at molt stages A B; (b) start of yolk accumulation at molt stage C; (c) early vitellogenic stage at molt stage C; (d) early vitellogenic stage at molt stage D 0 ; (e) late vitellogenic stage at molt stage D 2. FC, follicle cell; OC, oocyte; OG, oogonia; Y, yolk globule. Scale bar = 100 mm. yolk accumulation (Fig. 2b), and some females showed more developed ovaries (Fig. 2c). The vitellogenic oocytes reached mm in diameter, and were enveloped by follicle cells. At stage D 0 of the female molt cycle, yolk accumulation advanced, the cytoplasm of the oocytes was filled with large numbers of yolk globules, and the diameter of the vitellogenic oocytes increased to mm (Fig. 2d). However, non-vitellogenic oocytes still existed in the ovaries (not shown). At stage D 2, yolk globules nearly filled the cytoplasm of the vitellogenic oocytes, and oocyte diameter reached 350 mm (Fig. 2e). These developed oocytes were capable of being spawned and successfully fertilized. Marsupial development and the molt cycle Fig. 3 Correlation between frequency in marsupial developmental stages and female molting in Acanthomysis robusta. At the female molt stages A B just after ecdysis, marsupial development was at stage 1 (Fig. 3). At molt stage C, marsupial stages ranged between 1 and 2, indicating that hatching occurred at this molt stage. At molt stage D 0, marsupial stage was at stages 2 3. The developmental stage 4 appeared at molt stage D 1, showing that naupliar ecdysis occurred in stage D 1. At molt stage D 2, empty marsupia were observed, and any remaining brood was all at stages 4 5. The empty marsupia were also observed at molt stages A B. These findings suggest that young were fully developed and released at molt stage D 2 before female ecdysis. The female molt stage and marsupial developmental stage were significantly correlated (P < 0.001, r s = 0.903, Spearman rank correlation test). Embryo and larval length increased during marsupial development (Fig. 4). Length at each marsupial stage differed significantly (P < 0.001, Tukey Kramer test for multiple comparison), and both were significantly correlated (P < 0.001, r s = 0.945, Spearman rank correlation test). Critical increases

4 998 FISHERIES SCIENCE T Okumura Fig. 6 Correlation between body length and carapace length in female Acanthomysis robusta. Fig. 4 Correlation between embryo and larval length and marsupial development in Acanthomysis robusta. BL = CL (n = 76, P < 0.001, r = 0.901, linear regression analysis). DISCUSSION Fig. 5 Correlation between brood size and female carapace length in Acanthomysis robusta. occurred at the hatching of embryos (stages 1 2) and during the postnauplioid stage (stages 4 5). Brood size Brood size ranged between eight and 53 embryos and larvae (Fig. 5). The brood size and carapace length were significantly correlated (n = 71, P < 0.001, r = 0.629, linear correlation analysis). Body length and carapace length The relationship between body length (BL) and carapace length (CL) of females is shown in Fig. 6: The present study has revealed a correlation between ovarian development and the molt cycle in A. robusta. Yolk accumulation started at molt stage C and advanced during molt stages D 0 D 2. At molt stages A B, developed oocytes were not observed, and newly spawned eggs (stage 1) and empty marsupia were observed. These results suggest that oviposition occurs between molt stages A B. This timing deduced in A. robusta parallels previous reports in mysid species. 1 The above relationship is similar to that in Siriella armata; 2 however, ovarian development was not examined histologically in S. armata. The correlation between ovarian development and molt cycle is known to exhibit various patterns among crustacean species. 6 8 The nature of the relationship in A. robusta is of the type seen in amphipods and carideans (e.g. ovarian development is completed in one molt cycle with copulation and oviposition following ecdysis) 6 8 and, in particular, is very similar to the pattern reported in two Macrobrachium species. 9,10 Marsupial development within the marsupium was also related to the female molt cycle in A. robusta. Embryos hatched at molt stage C, naupliar ecdysis occurred at molt stages D 0 D 1, and late postnauplioid larvae were released at molt stage D 2. The timing of hatching is similar to that in S. armata; however, the timing of the naupliar ecdysis differs (stages C D 0 in S. armata 2 ). The release of young is known to occur just before ecdysis of the female in several mysid species. 1,11

5 Reproductive cycle in A. robusta FISHERIES SCIENCE 999 This larval release before ecdysis enables female mysids to avoid the loss of larvae in the marsupium due to ecdysis. The timing of release at molt stage D 2 in A. robusta is likely similar to that of other mysid species, because stage D 2 occurs just prior to ecdysis. As the A. robusta females that had released their young exhibited empty marsupia and had developed ovaries, females most likely repeated spawning and brood development in each molt. The number of spawning occasions of each female was not determined in the present study, as laboratory studies are necessary to determine the spawning times and spawning intervals. In the mysid Neomysis intermedia the spawning cycle is 8 9 days at 21 C, 7 days at 25 C, and 6 days at 30 C, and there are two to six total spawning times during a lifetime. 12 Brood size was eight to 53 embryos and larvae. This brood size is comparatively large among mysid species, 1 indicative of the high fecundity of this species. Brood size was positively related to female body size, as has been shown in many other species of mysid. 1 This relationship indicates that fecundity increases with the growth of the females due to molting after reaching sexual maturation. Although a seasonal variation in the brood size is known in some species, 1 the seasonality in this species was not examined in the present study. The relationship between body length and carapace length of females is shown in Fig. 6. Both parameters are indices of growth, and increase in a stepwise manner following molting. This highly correlated relationship indicates that the conformational proportion of body shape does not change with growth in adult female A. robusta as in other reported species. 1 Under the synchronous processes of ovarian development, marsupial development and molting, the coordination of female growth and reproduction becomes possible. Parental growth increases the number of spawned eggs, and the repetition of spawning increases the total number of eggs during the lifetime of females, indicating the potentially high productivity of this species. These reproductive features may explain the drastic increase in mysid abundance during early summer in Niigata, whereas mysid abundance in Niigata rapidly decreases in late summer. 4 Effects of environmental factors, such as temperature and trophic requirements, are possible factors contributing to rapid decreases. Further studies are necessary to reveal the productivity of this species. Mysids are often used in toxicological tests because of their high sensitivity to pollutants. 13 Recently, the phenomenon of endocrine disruption, which brings about sex change, reproductive failure, and abnormalities of the reproductive organs, has become a major issue in environmental protection. Endocrine disruption is a cause of concern in regard to its potential effects on marine invertebrates, including mysids as well as vertebrate species. 14,15 As the coordination of reproduction and molting is governed by endocrine regulatory mechanisms, examination of these processes in mysids may lead to the development of a suitable biomarker for use in endocrine disruption studies. ACKNOWLEDGMENTS This work was supported in part by the Integrated Research Program for Effects of Endocrine Disrupters on Agriculture, Forestry, and Fisheries and Their Action Mechanisms on Domestic Animals and Fishes (ED-II-304). REFERENCES 1. Mauchline J. The biology of mysids and euphausiids. 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