Benthos Research, 45 : 29-41, Aug., 1993 Reproductive Cycle, Copulating Activity and Dispersion Pattern of the Dogwhelk Nucella freycineti (DESHAYES) KEI KAWAI and SHIGERU NAKAO Laboratory of Mariculture. Faculty of Fisheries, Hokkaido University チ ヂ ミボ ラ Nucella freycineti (DESHAYES)の 生 殖 周 期 と交 尾 頻度 お よび 分布 の集 中度 河 合 渓 ・中 尾 繁 Abstract KAWAI, KEI and SHIGERU NAKAO (Faculty of Fisheries, Hokkaido University). 1993. Reproductive cycle, copulating activity and dispersion pattern of the dogwhelk Nucella freycineti (DESHAYES). Benthos Research, 45 : 29-41. The reproductive cycle of Nucella freycineti was studied based on samples collected at monthly intervals from September 1987 to December 1988 and from May 1990 to July 1991 in Usujiri, southern Hokkaido, Japan. The developmental processes of the gonads and the seminal vesicle were classified into four and three successive stages, respectively. The development of the gonads and the seminal vesicle exhibits a distinct annual cycle. In both sexes, remarkable gonadal development was observed during the warm season from June to October. More than 90% of the males were mature in October. However, oogenesis was observed through the winter until the next breeding season in April and May. The annual cycle of the ovary is not synchronous with that of the testis but with that the seminal vesicle. Size at sexual maturity was 19+ mm and 15+ mm shell length for femalesof and males, respectively. MORISITA index (I8) showed that these snails aggregated all year round. Strongest aggregation was observed during the colder and coldest seasons from November to May, when high copulation and breeding activities successively occurred. During warmer period from June to September, the index was relatively low but high foraging activity was observed. stones and in crevices in a way similar to its con- INTRODUCTION familial species Thais (=Nucella) lamellosa (SPIGH- cella freycineti Nu (DESHAYES) is a common ,1977) and Nucella crassilabrum (GALLARD0,1979).T prosobranch gastropod found on low intertidal espite the abundance of Nucella freycinetiD in north- cky shores from Touhoku to Hokkaido,ro Japan. ern Japan, neither its reproductive biology nor its This species deposits egg capsules on boulders, behavior has been examined yet. Some studies on the reproductive biology of Received January 5, 1993; Accepted May 21, 1993. cky shore muricidae have been reported.ro KON et 29 No.45 Benthos Research Aug., 1993 al. (1966) described the seasonal changes in the gonads of Purpura (= Thais) clavigera. FEARE (1970) studied the reproductive cycle of the dog- whelk Nucella lapillus. Factors affecting reproduc- tion were also studied in neogastropods. HOUSTON (1971) discussed the relationship of gonadal devel- opment to temperature, salinity, photoperiod and lunar period while describing the reproductive biol- ogy of Thais (= Nucella) emarginata and T. canaliculata. SPIGHT (1975) examined temperature affecting the embryonic development of T. (= Nucella) lamellosa. MARTEL et al. (1986) studied the reproductive cycle and copulating activity of Buc- cinum undatum and also mentioned the rela- tionship between feeding and reproduction. TAKA- HASHI et al. (1972), TAKAMARU& FUJI (1981), and FUJINAGA(1985) made histological observations on the gonad of Neptunea arthritica and investigated seasonal relationship between body and gonad in weight. The present study aimed to determine the annual reproductive cycle of .N freycineti based on Fig. 1 Map showing the study area and approxi- histological examination, gonad index and size at mate height above M. T. L. in meters. sexual maturity. The relationship between sea Black circles represent the sampling sta- water temperature and gonadal development, tions. copulating period, foraging activity and dispersion pattern was also examined. tissue, the gonad together with the digestive gland MATERIALS AND METHODS were separated as one component and dehydrated The study area covered the intertidal rocky in an ethyl alcohol series. The gonad-digestive shore from 53 cm to 145 cm tide levels, at Usujiri, gland complex was embedded in paraffin with Hokkaido (42° 21' N ; 140° 57' E) (Fig. 1). butyl alcohol, sectioned at 5-8,am, stained with Chthamalus challengeri, Littorina brevicula and DELAFIELD'Shematoxylin, and counterstained with Septifer virgatus were dominant in the platform eosin. from 43 cm to 141 cm tide levels near the study area To determine size at sexual maturity, 230 (FUJI & NOMURA,1990). Snails larger than 20 mm snails of shell length ranging from 7 to 29 mm were shell length were collected from area A (Fig. l) at randomly sampled from area A in the field (Fig, 1) monthly intervals from September 1987 to Septem- on 25 April 1988. After removing the shell, the ber 1988. To determine the seasonal gametogenic ovary and the seminal vesicle were dissected out cycle, histological preparations were done. The and embedded using the same method described length of the shell was measured, the shell was above. Decision of sexual maturity size was foll- cracked, and the sex was checked by the body owed HARVEY & VINCENT (1989). components. And the entire visceral masses (Fig. 2 Because the gonad and digestive gland are -A) from 40 snails of each sex were removed and interconnected and difficult to separate from each fixed in aqueous Bouin's solution. From this fixed other, the following method was used to determine 30 Reproductive biology of Nucella freycineti. time of the first spring tide, from November 1987 to December 1988. An index (18)derived by MORISITA (1959) was calculated to measure the dispersion pattern of N. freycineti in the field. To know the copulating period, the number of snails in each quadrat and the number of copulating pairs were counted once a month from May 1990 to July 1991. The copulation was distinguished by insertion of a penis into the female. To learn the foraging activity, the number of foraging snails in each quadrat was counted once a month from April 1990 to June 1991. The foraging snails were checked by insertion of the proboscis into the prey. Surface sea water temperatures were monitor- ed every morning at Usujiri throughout the study period. Fig. 2 Diagram of Nucella freycineti showing the RESULTS visceral mass (A), and the cross-sections of the gonad-digestive gland complex (B). G) OOGENESIS gonad ; S) seminal vesicle ; D) digestive The developmental process of female germ gland cells was divided into five stages following TA- KAMARU& FUJI (1981) : the gonad index (G. L). The ovary-digestive gland 1) 4ogonia : These cells were oval, measuring 8-12 complex for the female and the testis-digestive p m in diameter with a thin cytoplasmic layer. The gland-seminal vesicle complex for the male were nucleus contained a few nucleoli and chromatin- divided into three parts : anterior, middle and pos- like granules (Fig. 3-A). terior (Fig. 2--A). Each part was serially sectioned 2) Previtellogenic oocytes : These cells were also and the G. L was calculated as follows : oval and measured 15-30 pm. The cytoplasm was N scarce, stained basophilically and bulged from the G. L(%)= N x 3 G a2l+Sat1+Da1 follicle epithelium. The nucleus was large and had an enlarged nucleolus and dispersed chromatin (Fig. + Gm Ami + Api x 100 r+Smr+Dmi Gpi+Spt+Dpz 3-B). where N : total number of individuals, Aai : area 3) Early-vitellogenic oocytes : These oocytes were of gonad or seminal vesicle (only for male) in the irregular in shape, about 40-100 pm across, and had anterior part of individual Z,Gat : area of gonad in a round nucleus 35-40pm in diameter. Small yolk the anterior part of individual i, Dai : area of granules were present in the cytoplasm (Fig. 3-C). digestive gland in the anterior part of individual i, 4) Vitellogenic oocytes : At this stage, the oocytes Sat: area of seminal vesicle in the anterior part of were projected well into the follicles from 50 pm to individual i (only for males), mi and pr are the 230 pm. The yolk granules in the cytoplasm mea- respective areas of the middle and posterior parts sured 2-3pm in diameter (Fig. 3-D). of individual i (Fig. 2-B). 5) Mature oocytes : These included pre-mature Sixty-f our fixed quadrats (1 m x 1 m) were set and mature oocytes. The former were about 200- up in the study area (Fig. 1). The number of snails 300 pm and their cytoplasm was filled with in each quadrat was counted once a month at the various-sized yolk granules (Fig. 3-E). The latter 31 t No.45 Benthos Research Aug., 1993 Fig. 3 Gametogenesis in Nucella freycineti. (A) Oogonia ; (B) Previtellogenic oocyte ; (C) Early-vitellogenic oocyte ; (D) Vitellogenic oocyte ; (E) Pre-mature oocyte ; (F) Mature oocyte ; (G) Spermatogonia ; (H) Primary spermatocytes ; (I) Primary spermatocytes in the first meiotic division ; (J) Secondary spermatocytes ; (K) Spermatids ; (L) Spermatozoa. Scale bars=50 jcm. 32 Reproductive biology of Nucella freycineti. were 200-400 um. They could be distinguished visible at the center of the cells (Fig. 3-I). After the from the pre-mature ones by the presence of minute primary meiotic division, the chromosomes in the yolk granules about 1 pm across surrounding the secondary spermatocytes (Fig. 3-J) appeared again, nucleus while larger granules about 10 pm in diam- and at this time they were scattered in the nucleus. eter filled the outer cytoplasm (Fig. 3-F). 3) Spermatids : The chromatids of these cells gath- SPERMATOGENESIS ered on one side of the nucleus, forming a meniscus The developmental process of the male germ or a bar (Fig. 3-K). cells was divided into four stages following FEARE 4) Spermatozoa : The head of the spermatozoon (1970) : was stained by hematoxylin and the tail by eosin 1) Spermatogonia : Each spermatogonium had an (Fig. 3-L).