Blackwell Science, LtdOxford, UKFISFisheries Science0919 92682004 Blackwell Science Asia Pty LtdDecember 2004706988995Original ArticleReproduction of female swimming crabK Hamasaki et al.

FISHERIES SCIENCE 2004; 70: 988–995

Ovarian development and induced oviposition of the overwintering swimming trituberculatus (Brachyura: ) reared in the laboratory

Katsuyuki HAMASAKI,1a* Hideyuki IMAI,2b Nobuhiko AKIYAMA3 AND Kyohei FUKUNAGA1c

1Tamano Station, National Center for Stock Enhancement, Fisheries Research Agency, Tamano, Okayama 706-0002 and 2Graduate school of Marine Science and Technology and 3Department of Fisheries, Faculty of Marine Science and Technology, Tokai University, Shimizu-orido, Shizuoka 426-8610, Japan

ABSTRACT: Females of the swimming crab Portunus trituberculatus were cultured under natural temperature and high temperature (21∞C) conditions to examine ovarian development and oviposition from autumn (October) to the beginning of breeding season in the following spring (May). Ovaries developed because of vitellogenesis of oocytes from October to December and their developmental state did not change during the overwintering period from December to March. In spring, from late March to mid May, some ovaries reached prematuration and maturation stages and ovipositions began from midÐlate April under natural temperature conditions. Females reared in tanks of high tem- perature regimes oviposited earlier than those reared in tanks of natural temperatures. The number of days to oviposition tended to decrease with advancement of the beginning times (early February to late April) of culture of females under high temperature regimes; induced ovipositions were achieved more easily with increasing photoperiod from c. 12 h in early February to c. 14 h in late April. There- fore, it is inferred that the temperature and photoperiod are important environmental factors controlling ovarian development and oviposition.

KEY WORDS: ovarian development, oviposition, photoperiod, Portunus trituberculatus, Swimming crab, temperature.

INTRODUCTION ovigerous females occur from April/May to August/September in the southern range of its dis- The swimming crab Portunus trituberculatus tribution in Japan.4–7 The females of P. tritubercula- (Miers) is distributed mainly on sandy and muddy tus are also known to oviposit several broods bottoms of bays and inlets from Hakodate, Japan, during a breeding season of the year.8 Larvae (first through to and .1 In Japan, this zoeas) used for seed production were obtained has been selected as a target species for from ovigerous females caught from the wild and stock enhancement programs through production from females oviposited in tanks.9 It has been sug- and release of juvenile .2 Recently, the annual gested that larvae from the first broods have the production of juvenile crabs at hatcheries has advantage of easy rearing because their body size is reached c. 58 million.3 larger than that of larvae from successive broods.9 Ovaries of P. trituberculatus develop from Therefore, it is considered that induced oviposition autumn (October) to the following spring4–6 and before the breeding season is useful in obtaining larvae from the first brood for a longer period. For several , studies have revealed *Corresponding author: Tel: 81-3-5463-0538. that temperature and photoperiod play an impor- Fax: 81-3-5463-0542. Email: [email protected] tant role in regulating ovarian development and aPresent address: Faculty of Marine Science, Tokyo University oviposition; for example, ovarian development is of Marine Science and Technology, Minato, Tokyo 108-8477, accelerated and oviposition is induced under long Japan. photoperiod and high temperature conditions.10–12 b Present address: Faculty of Science, University of the However, information on the reproductive biology Ryukyus, Nishihara, Okinawa 903-0213, Japan. cPresent address: Akkeshi Station, National Center for Stock of P. trituberculatus is very limited to reliably con- Enhancement, Fisheries Research Agency, Akkeshi, Hokkaido trol its ovarian development and oviposition for 088-1108, Japan. seed production; it is only reported briefly that ovi- Received 7 January 2004. Accepted 14 July 2004. positions were induced under high temperature Reproduction of female swimming SCIENCE 989

conditions from March–April before breeding were replaced in 70% ethanol solution after 24– season in a hatchery.13 72 h. Pieces of fixed ovary were dehydrated in The present study examined ovarian develop- ethanol, embedded in paraffin, sectioned 5-mm ment and oviposition of laboratory-reared P. tritu- thick, and stained with Mayer’s hematoxylin–eosin. berculatus from autumn (October) to the The ovarian developmental stage was expressed by beginning of breeding season in the following the most advanced stage of oocytes among 10 his- spring (May) under natural temperature condi- tological stages during oogenesis of P. tritubercula- tions. In addition, we analyzed the relationship tus reported by Imai et al.6 Five stages which were between the beginning times (early February to determined for the ovaries in this study were as fol- late April) of culture of females under high tem- lows: yolk granules are dyed with eosin in the yolk perature regimes and the number of days to granule stage; yolk granules fuse and become oviposition. larger peripherally to form yolk globules in the primary yolk globule stage; yolk globules increase in size and the nucleus begins to shrink and MATERIALS AND METHODS becomes basophilic in the secondary yolk globule stage; yolk globules partly fuse to one another and Ovarian development the nuclear membrane becomes ragged in the pre- maturation stage; and fusion of yolk globules Females which had copulated with males in the progresses and germinal vesicle breakdown occurs wild were obtained from natural habitats in the in the maturation stage. Maximum diameters of Seto Inland Sea of Japan: 43 from October–Decem- 200 oocytes, which were opaque as a result of vitel- ber 1991 and 10 in October 1992. Carapace width logenesis after yolk granule stage, from ovary lobes (CW) including lateral spines of females ranged fixed in formalin were measured using a Profile 148–235 mm (mean, 183 mm) and was larger than projector (V-12; Nikon Corp., Tokyo, Japan). the biological minimum (c. 120–130 mm in CW) of Although there were no histological differences this species.4,5 between oocytes from several parts of the ovary, Females were reared in our institute using 7-kL the collection site of subsamples for measure- (450 cm ¥ 183 cm ¥ 83 cm) tanks with bottoms ments of oocyte diameter were fixed at the top of covered with c. 10 cm of fine sand. Each female was the ovary. The relationship between modal values tagged with a numbered vinyl tape to facilitate in the frequency distributions of oocyte diameter individual identification. Tanks were located out- (x) and the GI values (y) was analyzed using a doors and covered with a blackout curtain (shade power function, y = axb. In the present study, the efficiency, 90%) that allowed natural light to enter. model to describe random error of y is exemplified Sand-filtered seawater was supplied using a flow- as a normal distribution Ny()ˆ,s22 yˆ , where the through system (15–24 L/min; salinity, 31–32 psu) standard deviation is proportional to the theoreti- and water temperatures were not regulated. cal value of yy()= ˆ by a certain factor s according to Females were fed with Manila clam Ruditapes Yamakawa and Matsuda.14 Parameters of the equa- philippinarum or frozen Euphausia superba tion were estimated by the maximum likelihood every day. method detailed in the previous paper.15,16 In this study, gonad index (GI), histology, and diameter of oocytes were used to identify the ova- rian developmental state. One to five females were Induced oviposition sacrificed once a month and the total of 28 and nine surviving females were dissected from Octo- From 1986 to 1992, a total of 541 females (141– ber 1991 to May 1992 and in October and Decem- 252 mm CW) were collected in the Seto Inland Sea ber 1992, respectively. Rearing periods of females and reared in two to three 7-kL tanks in each year to the time of sacrifice ranged 27–169 days except and a total of 380 surviving females were used for for females that were sampled after 1–6 days from culture trials of induced oviposition (Table 1). collection in October and November. The CW Induced ovipositions were attempted by rearing (mm) of the specimens were measured with a slide the females under high temperature regimes caliper, then their ovaries were removed. Their (=heating culture) for a certain period during the wet weights (g) were measured after paper-blotting overwintering period in each year from 1987 to and GI was calculated using the formula, 1993, as shown in Table 2. Five to 47 females were GI = (Gonad weight) ¥ 107/CW3. The ovary was H- used for each trial and reared in one to three 4-kL shaped. Right and left lobes of ovaries were fixed in (450 cm ¥ 150 cm ¥ 50 cm) tanks with bottoms Bouin’s solution and 10% neutral formalin solu- covered with fine sand. These tanks had flow- tion, respectively. Ovaries fixed in Bouin’s solution through systems (8–14 L/min; salinity, 31–32 psu) 990 FISHERIES SCIENCE K Hamasaki et al.

Table 1 Survival of females caught from the Seto Inland Sea until the beginning of culture trials for induced oviposition of Portunus trituberculatus in the laboratory Collection Females collected No. Date N CW† (mm) Survival (N/%) 19 Dec. 1986 31 213 (183–242) 19/61 2 11 Mar.-13 Apr. 1987 25 199 (155–238) 21/84 3 27 Nov.-30 Nov. 1987 74 209 (173–240) 43/58 4 20 Feb.-22 Apr. 1988 28 202 (158–233) 23/82 5 21 Nov.-24 Nov. 1988 53 218 (181–252) 43/81 6 24 Mar.-21 Apr. 1989 15 210 (182–235) 14/93 7 25 Nov.-13 Dec. 1989 94 203 (149–240) 29/31 8 11 Apr.-19 Apr. 1990 21 201 (175–224) 21/100 95 Dec.-7 Dec. 1990 65 200 (168–242) 57/88 10 29 Oct.-17 Dec. 1991 61 200 (171–235) 54/89 11 8 Apr.-10 Apr. 1992 11 163 (141–190) 11/100 12 25 Nov.-1 Dec. 1992 63 207 (167–241) 45/71 541 205 (141–252) 380/70

†Carapace width; mean (range).

and were located in an experimental room with Rearing water temperatures of ovigerous females windows that admitted natural light. Water tem- were controlled at designated temperatures peratures in these tanks were maintained at between 18 and 23∞C. 21 ± 0.5∞C with a heating system because the rear- ing temperatures of ovigerous females were regu- lated at around temperatures of 18–23∞C to control Temperature and photoperiod the hatching date in our hatchery.17 Adjustment to 21∞C from ambient temperature was done over 1–3 Natural temperatures in tanks were measured each days after the initiation of culture trials. The begin- morning (09.00–10.00 hours) from December 1986 ning times of heating culture varied from early to June 1993 and the mean temperatures for every February to late April. The temperature-controlled 10 days were calculated using these data. The period ranged between 5 and 95 days. In 1991– natural photoperiod was calculated from the hours 1993, 16–38 females were also reared in tanks of between sunrise and sunset at Tamano city natural temperatures (trials no. 2 in 1991, no. 2 in (34∞29¢N, 133∞53¢E) where our institute is located, 1992 and no. 3 in 1993). Females were fed daily with as reported by the Japan Weather Association, plus 30 min before sunrise and sunset (civil twilight) Manila clams. Dead and ovigerous females were 18 recorded daily during the culture period. Rearing according to the method of Aiken and Waddy. continued until all surviving females spawned except for trials no. 3 in 1988 and no. 1 in 1989 RESULTS (Table 2). The effects of beginning times of heating culture Temperature and photoperiod on ovipositions were evaluated by testing the Kendall’s rank correlation coefficient between cal- Natural temperature in tanks fluctuated season- endar days (January 1 = day 1) of beginning times ally, and minimum and maximum mean tempera- of heating culture and the number of days to des- tures were 8.6∞C in late February and 27.2∞C in late ignated cumulative percentage of ovipositions August (Fig. 1). The photoperiod also fluctuated after initiation of culture trials. Significance for seasonally; photoperiods of over 12 h were tests was designated as p < 0.05. observed from mid February to late October After oviposition, ovigerous females were trans- (Fig. 1). ferred to 0.5–2-kL tanks (3–10 females/tank) with running seawater at a flow rate of 1.7–2.5 L/min. A total of 1–2 days before hatching, each ovigerous Ovarian development female was transferred to a 0.7-kL cylindrical tank with still water and gentle aeration. Hatching was The mean GI value in mid October was 15; it verified in the early morning (06.00–07.00 hours). increased linearly to 57 in early December (Fig. 2). Reproduction of female swimming crab FISHERIES SCIENCE 991 ‡‡ 323/97 ( N /%) Hatching Hatching period Oviposition 24 Apr.– 12 May24 Apr.– 13/100 †† 13/100 334/98 ( N /%) Oviposition ¶ vival 9/90 9/100 Apr. 23 Apr.–29 8/89 24/838/100 24/10012/92 8/100 May 5 Apr.–18 12/100 May 17 Apr.–25 23/96 June 18 Apr.–5 8/100 12/100 13/936/100 13/1005/100 6/100 May 19 Apr.–12 5/100 May 13 Mar.–14 13/100 May 17 Apr.–5 6/100 5/100 36/97 33/92 May 5 Apr.–13 33/100 7/10010/91 7/100 10/100 Apr. 17 Apr.–19 May 29 Apr.–11 7/100 10/100 32/86 32/100 May 8 Apr.–19 30/94 15/75 15/100 May 17 Apr.–22 15/100 340/89 ( N /%) Sur § 7 8 8 2 3 4 3 4 4 6 9 9 10 25/93 25/100 June 10 Apr.–6 22/88 1212 14/93 13/93 14/100 13/100 May 15 Apr.–10 May 19 Apr.–15 14/100 13/100 12 13/81 1, 2 21/81 21/100 May 31 Mar.–12 17/81 5, 6 43/91 40/93 May 4 Apr.–16 40/100 no. 10, 11 34/89 34/100 June 16 Apr.–2 34/100 Collection (mm) ‡ 210 203 emales reared F (149–230) (175–200) (187–224) (155–238) (160–227) (198–226) (173–214) (173–227) (182–233) (158–228) (181–252) (182–235) (168–242) (180–227) (182–235) (192–230) (188–231) (141–235) (195–238) (141–252) CW N 37 206 47 217 20 200 38 186 16 eared in the laboratory eared 380 r – – – /(number of females oviposited). 2 period 10 controlled ¥ emperature- T 3 Apr.–12 Apr.3 Apr.–12 37 200 9 Feb.–14 May9 Feb.–14 6 207 8 Apr.–17 Apr.8 Apr.–17 27 204 25 Apr.–13 May 25 Apr.–13 21 Apr.–16 May 21 Apr.–16 tunus trituberculatus /(number of females survived). † 2 Por /(number of females reared). 2 10 ¥ 10 21 May 30 Mar.–13 29 195 2121 May 11 Apr.–13 May 16 Apr.–13 8 13 193 205 21 May 20 Mar.–12 26 214 2121 May 13 Apr.–12 21 14 May 29 Feb.–5 Apr., Mar.–19 30 194 5 198 21 Apr. 14 Apr.–19 7 208 2121 May 26 Apr.–11 Apr., Mar.–10 30 11 199 21 Apr. 21 Apr.–29 10 206 21 21 21 2121 Apr. 10 Apr.–25 Apr. 15 Apr.–26 15 14 206 207 NC NC NC ( ∞ C) Rearing temperature ial no. Tr C; NC, not controlled. ∞ Oviposition and hatching of 0.5 ± (number of females oviposited) (number of females oviposited) (number of females survived until hatching) (number of females survived) ¥ apace width; mean (range). = = = ee Table 1. ee Table % % % S 21, 21 Car † ‡ § ¶ †† ‡‡ able 2 30 Mar. 199030 Mar. 1 11 Apr. 199011 Apr. 199016 Apr. 2 19913 Apr. 3 1 T Date of beginning of trial culture 198720 Mar. 1 13 Apr. 198713 Apr. 19889 Feb. 2 198829 Feb. 1 198830 Mar. 2 3 14 Apr. 198814 Apr. 4 26 Apr. 198826 Apr. 198930 Mar. 5 1 21 Apr. 198921 Apr. 2 3 Apr. 19913 Apr. 2 8 Apr. 19928 Apr. 1 8 Apr. 19928 Apr. 2 10 Apr. 199310 Apr. 199315 Apr. 1 199315 Apr. 2 3 992 FISHERIES SCIENCE K Hamasaki et al.

Fig. 1 Seasonal changes of natural seawater tempera- ture in tanks and natural photoperiod at Tamano City where females of Portunus trituberculatus were cultured. Fig. 3 Relationship between the modal values in the Open circles show mean temperatures for every 10 days frequency distributions of oocyte diameter and the from December 1986 to June 1993. Data are shown from gonad index values of Portunus trituberculatus reared in October to September. The solid curve indicates photo- the laboratory. Symbols represent developmental stages period, calculated from the hours between sunrise and of oocyte as in Fig. 2. The solid curve indicates theoreti- sunset plus 30 min before sunrise and sunset. cal values calculated from the equation shown in the text.

Induced oviposition

A total of 334 and 340 females oviposited and sur- vived until the end of culture trials, respectively. Thereafter, 323 females survived to hatching (Table 2). Changes in the cumulative percentage of ovipositions ([cumulative number of females oviposited] ¥ 102/[total number of females ovipos- ited]) in each trial of respective years are shown in Fig. 4. The cumulative percentage of ovipositions tended to increase linearly to reach around 70%, Fig. 2 Changes of gonad index of Portunus tritubercu- then gradually increased to 100%. Ovipositions latus reared in the laboratory from October to May. were accelerated and were initiated from early to Gonad index = (Gonad weight) ¥ 107/CW3. Symbols rep- mid April in the tanks for which temperatures were resent histological developmental stages of oocytes and heated to 21∞C (trials no. 1 in 1991, no. 1 in 1992, mean GI values at each sampling date; YGS, yolk granule and no. 1 and no. 2 in 1993) in comparison with the stage; PYGS, primary yolk globule stage; SYGS, second- tanks of natural temperatures (trials no. 2 in 1991, ary yolk globule stage; PMS, prematuration stage; and no. 2 in 1992 and no. 3 in 1993) in which oviposi- MS, maturation stage. tions occurred from mid–late April to mid May/ early June. Induced ovipositions were also observed in trials conducted from 1987 to 1990 and Ovaries at the yolk granule stage and primary yolk the earliest oviposition occurred in mid March in a globule stage were observed until November; then tank of trial no. 1 in 1988, for which temperature all ovaries reached the secondary yolk globule was regulated at 21∞C from early February. stage. Mean GI values appeared almost constant, Figure 5 depicts the relationships between calen- around 60, during the overwintering period. From dar days of beginning times of heating culture and late March to mid May, GI increased and some ova- the number of days to initiation of oviposition and ries reached prematuration and maturation stages. the number of days when cumulative percentage of Figure 3 shows the relationship between modal ovipositions first exceeded 50% (50%£) after initi- values of oocyte diameter and the GI values. The ation of culture trials. The number of days to reach GI values increased exponentially with increasing 50%£ of the cumulative percentage of ovipositions modal values of oocyte diameter. The power func- was adopted because temperature-controlled tion between modal values of oocyte diameter periods lasted to those days in all trials. The (x) and GI values (y) were determined as y = number of days to oviposition tended to decrease 2.478 ¥ 10-5 x2.540, (N = 37, s = 0.1250, R = 0.9603). with increasing calendar days of beginning times Reproduction of female swimming crab FISHERIES SCIENCE 993

Fig. 4 Changes of cumulative percentage of ovipositions of Portunus trituberculatus reared in the laboratory. Cumulative percentage = (cumulative num- ber of females oviposited) ¥ 102/ (total number of females ovi- posited). See Table 2 for details of culture trials of females.

of heating culture. The Kendall’s rank correlation Almost identical patterns of ovarian development coefficients between these variables showed signi- examined by GI and histological method, as in this ficant negative values: -0.650 and -0.703 for initi- study, are reported for wild P. trituberculatus in the ation of oviposition and 50%£ of cumulative Seto Inland Sea,4 Tokyo Bay,5 and Suruga Bay.6 percentage of ovipositions, respectively. species generally have a well- defined breeding season in temperate zones; ovigerous females occur from May to August in the DISCUSSION red frog crab , off Hachijojima, Izu Islands19 and from April to September in Ovaries of laboratory-reared P. trituberculatus the Japanese spiny Panulirus japonicus, off developed because of vitellogenesis of oocytes Oshima Island, Tokyo, Japan.20 Moreover, it is from October to December. During the overwinter- reported that the spawning season extends from ing period from December to March, the GI values June to August for the lined shore crab Pachy- appeared almost constant and the histological grapsus crassipes collected along the coast of stage of ovary remained at the secondary yolk glo- Niigata, Japan.21 It is also known that yolk accumu- bule stage. In spring, from late March to mid May, lation in these species is initiated 1–3 months the GI values increased again and some ovaries before breeding season; that is, from February in reached prematuration and maturation stages, R. ranina19 and P. japonicus,20 from May in P. then, ovipositions began from mid–late April in crassipes.21 Subsequently, their ovaries grow rapidly tanks of natural temperatures in 1991–1993. for oviposition. In contrast, ovarian development 994 FISHERIES SCIENCE K Hamasaki et al.

wintering period under low temperatures below 12∞C (Figs 1 and 2). Therefore, it is suggested that temperature is the prominent environmental fac- tor controlling ovarian development of P. trituber- culatus after the breeding season of the year. This characteristic might explain differences in ovarian developmental pattern between species such as P. japonicus and P. trituberculatus. P. trituberculatus females reared at natural tem- peratures began to oviposit from mid–late April when tank temperatures exceeded 12∞C. Females reared under high temperature regimes (21∞C) ovi- posited earlier than those reared at natural tem- peratures. These facts suggest that temperature is Fig. 5 Relationships between the calendar day (Janu- also the prominent environmental factor control- ary 1 = day 1) of beginning times of culture of Portunus ling oviposition in P. trituberculatus, however, final trituberculatus females under high temperature regimes maturation of ovaries and oviposition might be and the number of days to initiation of oviposition (0%<) affected by the photoperiod because the number and the number of days when the cumulative percentage of days to oviposition tended to decrease with of ovipositions first exceeded 50% (50%£) after initiation increasing calendar days of beginning times of of culture trials. Vertical broken lines indicate discrimi- heating culture, but the ovarian developmental nation of months. stage did not change significantly during the over- wintering period; induced ovipositions seem to be easier with increasing photoperiod from c. 12 h in early February to c. 14 h in late April. of P. trituberculatus begins from October – 1 month In this study, ovarian development and induced after the breeding season and 6 months before the ovipositions during overwintering period in P. tri- following breeding season. It is retarded during tuberculatus reared in the laboratory were clari- the overwintering period and resumes from late fied. We discussed the importance of temperature March, 1 month before breeding season, as dem- and photoperiod as environmental factors control- onstrated in both wild4–6 and laboratory females ling ovarian development and oviposition. To (present study). For that reason, the ovarian devel- develop reliable control measures of egg produc- opmental process of P. trituberculatus after breed- tion of P. trituberculatus, we should culture the ing season of one year to the following breeding females under conditions combining various season seems to be unique among these crusta- temperatures and photoperiods in appropriate cean species distributed in the temperature zone. seasons during the reproductive cycle to examine Temperature and photoperiod are important their effects on ovarian development and oviposi- environmental factors controlling the reproduc- tion. For these culture experiments, we would be tive cycle of crustacean species.11 For instance, able to prepare females for which ovarian develop- Matsuda et al. reported that the critical photope- mental conditions are similar by examining their riod at 25∞C for smoothly progressing ovarian oocyte diameters by means of biopsy22 because the development in laboratory-reared P. japonicus was oocyte diameter could represent the GI values. between 12.5 and 14 h, and ovaries grew at all test temperatures (13, 19, 25∞C) under the long photo- period (14 h) and the developmental rate increased ACKNOWLEDGMENTS with increasing temperature.12 Furthermore, they demonstrated that under the short photoperiod We would like to thank the staff of Tamano Station, (10 h), ovarian development progressed slowly at NCSE for their assistance in laboratory work. 13∞C, whereas it was prevented or delayed consid- We also thank B Fast for correcting the English erably at 19 and 25∞C.12 These facts seem to explain manuscript. the phenomenon that the ovaries of wild P. japoni- cus grow rapidly from early spring when both tem- perature and photoperiod increase. Ovaries of P. REFERENCES trituberculatus developed smoothly from October to December at temperatures 23–15∞C even though 1. Miyake S. Japanese Crustacean Decapods and Stomatopods the photoperiod decreased from c. 12.5–11 h and in Color, Vol. II. Brachyura (Crabs). Hoikusha Publishing, showed no signs of development during the over- Osaka, 1983; 82–83. Reproduction of female swimming crab FISHERIES SCIENCE 995

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