Sea Anemone Actinia Equina (Cnidaria: Anthozoa) in Sagami Bay, Japan

BenthosRεsεarchVol。51,No。2:13-20(1996) BENTHOS RESEARCH The Japanese Association of Benthology

Seasonal Cycle of Male Gonad Development of the Intertidal Sea Anemone Actinia equina (Cnidaria: Anthozoa) in Sagami Bay, Japan

Kensuke Yanagi*, Susumu Segawa * and Takashi Okutani** *Tokyo University of Fisheries and ** College of Bioresource Sciences , Nihon University

ABSTRACT The annual cycle of the testicular structure of Actinia equina in Japanese waters was inves- tigated by histological observation. This is the first study concerning the reproductive biology of this species in Japan. Specimens of A. equina were collected once a month from two intertidal rocky shores in eastern Sagami Bay, from January, 1991, to June, 1991, and from April, 1993, to January, 1994, respectively. Most of the specimens were male or non-sexual individuals and there were few females with mature oocytes. Spermatogenesis starts from late fall to early spring and the testicular cysts mature in mid-summer. This indicates that the spawning season of A. equina is mid-summer and sexual reproduction may occur in the summer. Although young are found in the enterons of adults of both sexes all year round, the source of these young remains unknown.

Key words: Actiniaria, Actinia equina, reproductive biology, spermatogenesis, histology

INTRODUCTION anemones in the intertidal zone in temperate Japan (Uchida 1992) . This species has been Developmental pathways of sea anemones ex- thought to be distributed in the Old World hibit a wide variability both sexually and along the Atlantic and Mediterranean coasts of asexually (Fautin 1991) . Over the past few dec- Europe and North Africa (Haylor et al. 1984) ades a considerable number of studies have and over to Japanese waters. Around the Brit- been made on the reproductive biology of sea ish coast, A. equina is dioecious, but through- anemones (Ford 1964; Carter & Thorpe 1979; out the year there are many individuals carry- Gashout & Ormond 1979; Jennison 1979; ing no gonads (Carter & Miles 1989) . In Sebens 1981; Fujii 1991; Bronsdon et al. 1993; addition, it is characteristic of A. equina that a Fukui 1995) . Although sea anemones comprise high proportion of adults, regardless of sex, a large biomass and occupy an important niche are brooding young anemones in the enteron in benthic communities, only a few attempts (Chia & Rostron 1970; Carter & Thorpe 1979; have been made to clarify the reproduction of Carter & Miles 1989) . Young at various devel- Japanese sea anemones (Uchida & Iwata 1954; opmental stages are also observed in the Fujii 1991; Fukui 1995). enteron in most other species of Actinia (Car- Actinia equina is one of the most common sea ter & Miles 1989) . There have been several hy- potheses to explain the origin of the young in Received November 13, 1995 : Accepted March 25, 1996 the enteron (e. g. Chia & Rostron 1970; Gashout

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& Ormond 1979; Orr et al. 1982; Carter & Miles scope and the male gonad-bearing mesenteries 1989; Sole -Cava & Thorpe 1992). The present were checked. Maturity stages of male gonad study describes the seasonal cycle of gonad de- I to VI were assigned on the basis of the fol- velopment of male A. equina in order to eluci- lowing criteria that are modified from Carter & date the developmental pathways of this species Miles (1989) . As the spermatogonia in the in Japan, as one of the approaches to figure out endodermal epithelium depicted by Carter & the puzzling life history of these sea anemones. Miles (1989) were not identifiable in the present observation, the maturation process of the tes- MATERIALS A METHODS ticular cyst was classified into six stages

Actinia equina were collected once a month for I Gamete precursors have entered in to the histological observation from two intertidal mesogloea and started dividing. rocky shores, Okinoshima Islet near the tip of II Gamete precursors have formed solid the Boso Peninsula and Moroiso Cove at the masses of apparently uniform cells in the southwestern tip of the Miura Peninsula (Fig. mesogloea (spermatocytes in testis cysts). 1), during the periods from January, 1991, to III Cells in the testis cysts show some differen- June, 1991, and from April, 1993, to January, tiation (spermatid formation). 1994, respectively. On each sampling occasion, 9 N Differentiation of some spermatozoa with to 27 specimens were collected at random from characteristic tails, with in the testicular each site (Table 1) . Anemones were placed indi- cyst. vidually in small plastic bags to avoid contami- V Testicular cysts with a central lumen; almost nation of the released young. Collected anemo- all gametes are mature spermatozoa. nes were immediately transported to the VI Testicular cyst breakdown and diminution; laboratory and anesthetized in a 1:1 solution of many sperm noticeable among the sur- sea water and 0.5 mol magnesium sulfate. Then, rounding endodermal cells. their wet weight was measured before fixation in Bouin's fixative for 24 hours and preserva- RESULTS tion in 70% ethyl alcohol. Each preserved specimen was cut in half longitudinally and all the The total number of specimens observed was young in the enteron were removed and 100 from Okinoshima and 106 from Moroiso. Of counted. Those adult specimens with brooded those from Okinoshima, the numbers of male, young were termed brooders. One half of each female, and non-sexual individuals were 53, 6, specimen was embedded in paraffin for and 17, respectively. At Moroiso, 53 individuals histological treatment. Sections were cut at 6 to were males and 53 individuals were non-sexual 9 #m and stained with hematoxylin and eosin. animals. Except for the samples of May and These sections were observed with a micro- June, 1991, from Okinoshima, non-sexual specimens were observed in every month. No specimens were determinable to be female at Moroiso. The sex ratios of specimens by each sample by locality are summarized in Table 2. There were great excesses of male specimens at both Okinoshima and Moroiso every month. All the specimens with gonads were male at Moroiso and 89% at Okinoshima. The monthly mean wet weight of all specimens ranged from 1.03 g to 5.77 g (Table 1) . There were 8 individuals of less than 1 g in wet weight, but most of the remaining specimens weighed more than 2 g. The distribution of wet weight by sex is summarized in Fig. 2. Non-sexual specimens

Fig. 1. Sampling localities for Actinia equina. (2.6 g mean wet weight) are lighter than males

14 Spermatogenesis of sea anemone Actinia equina

Table 1. Number and mean wet weight of adult specimens examined of Actinia equina with brooding adult number and mean number of their young.

Table 2. Sex ratio of Actinia equina at two localities.

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Fig. 2. Adult wet weight of Actinia equina by sex.

Fig. 3. Number of young in enteron by sex and locality.

16 Spermatogenesis of sea anemone Actinia equina

Fig. 4. Spermatogenesis in Actinia equina. A, Gonad precursor cells entering the mesogloea and spermatogonia in the mesogloea; B, Relatively uniform cells in testicular cyst as spermatocytes; C, Cells in the central lumen of testicular cyst becoming spermatids; D, Cells in central lumen of testicular cyst becoming spermatozoa with characteristic tails; E, Sec- tion through maturing testicular cysts showing well-developed cells with tails oriented in the same direction; F, Testicu- lar cyst breaking down. Scale bar = 50 #m. Bs, endodermal tissue containing nuclei of breaking down sperm; Ed, endoderm; Gpc, gamete precursor cell; M, mesogloea; Mf, mesenterial filament; S, spermatozoa; Sg, spermatogonia; Sc, spermatocyst; St, spermatid.

(4.4 g) or females (4.6 g). place during the period from September to De- Young in the enterons of adults were discov- cember, when germ cells of 2 - 4 ,um across ered every month except May and June, 1991 came to appear in the mesogloea as individual (Table 1) . The brooders were not only females cells (Fig. 4A), viz. spermatogonia. During and but also males or even non-sexual animals (Fig. after January, the cells began and/ or contin- 3) . The ratio of brooders was high (almost ued to divide to increase in number. Then, the 90%) in October, 1993, and December, 1993 cells, which are relatively uniform in size, (Table 1). appeared in testicular cysts (Fig. 4B) as The onset of the male gametogenic cycle took spermatocytes. From April to June, cells at the

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Table 3. Monthly maturity stage of male gonad.

center of the testicular cyst became spermatids DISCUSSION with distinguishable nuclei (Fig. 4C) . In June the cells at the center of the cyst changed to Although the present study revealed only part spermatozoa with characteristic tails (Fig. of the reproductive pathways of the sea anem- 4D) . In July or August all the cells except for one Actinia equina, some basic knowledge con- those located at the tip of the cyst became sper- cerning the development of the testicular cysts matozoa. The mature cells were oriented in the was obtained. This is the first attempt to same direction (Fig. 4E) . At the same time, record the gonad development of this species in some of the cysts started to break down and Japanese waters. Carter & Miles (1989) sug- large numbers of sperm were observed among gested that A. equina on the British coast is the surrounding cells (Fig. 4F) . dioecious. Among the specimens treated in the An annual cycle in testicular structure was present study, none was an hermaphrodite, apparent (Table 3) . Spermatogenesis started contrary to Chia and Rostron's (1979) sugges- from late fall to early spring (stage I ) and tion. Actinia equina in Japanese waters is con- most specimens examined had no gonads in the sidered to be dioecious. In Moroiso, no individ- fall (Table 2) . From spring to early summer ual could be identified as a female. The testicular cysts developed (stages II to IV) . In population in Moroiso, consisting of only males mid-summer the cysts matured (stage V ) . In and non-sexual individuals, is quite a contrast late summer the cysts broke down (stages VI) . to the A. equina population of almost only fe- The beginning of the subsequent cycles of sper- males with small numbers of non-sexual indi- matogenesis was delayed, and the appearance viduals reported at Filey Brigg, North York- of gonad precursors was never recorded at the shire, England, by Gashout & Ormond (1979). same time as in the previous year. Shick (1991) assumed that such a skewed sex ratio in populations of Haliplanella lineata and Anthopleura elegantissima probably resulted

18 Spermatogenesis of sea anemone Actinia equina from founder effects by asexual reproduction. Carter, M. A. and J. Miles 1989 Gametogenic cycles Males of A. equina exhibited an annual re- and reproduction in the beadlet sea anemone Actinia productive cycle with mid-summer spawning. equina (Cnidaria: Anthozoa) . Biological Journal of This result almost agrees with that in A. equina the Linnean Society, 6: 129-155. in England reported by Carter & Miles (1989). Carter, M. A. and C. H. Thorpe 1979 The reproduction In Japan, Fujii (1991) reported similar seasonal of Actinia equina L. var. mesembryanthemum. Jour- cycles of gametogenesis in Anthopleura nal of the Marine Biological Association of the asiatica, and Fukui (1995) in Haliplanella United Kingdom, 59: 989-1001. lineata. Based on the existence of such an an- Chia, F. S., and M. A. Rostron 1970 Some aspects of nual cycle in the males, it seems reasonable to the reproductive biology of Actinia equina (Cnidaria: suppose that the spawning season of A. equina Anthozoa) . Journal of the Marine Biological Associa- is mid-summer, as was suggested by Carter & tion of the United Kingdom, 50: 253-264. Miles (1989) for British populations, although Fautin, D. G. 1991 Developmental pathways of the present study revealed no evidence for anthozoans. Hydrobiologia, 216/217: 143-149. either female maturity or sexual reproduction. Ford, C. E. Jr. 1964 Reproduction in the aggregating Carter & Miles (1989) concluded that A. sea anemone Anthopleura elegantissima. Pacific Sci- equina lacks a dispersive larval stage. The pre- ence, 18: 138-145. sent study found many non-dispersive off- Fujii, H. 1991 Gonad development of the sea anemone spring in the enteron of A. equina all year Anthopleura asiatica in clonal populations. Hydro- round (Table 1) . Orr et al. (1982) and Sole - biologia, 216/217: 527-532. Cava & Thorpe (1992) suggested that these Fukui, Y. 1995 Seasonal changes in testicular structure young may be produced asexually as a clone of the sea anemone Haliplanella lineata (Coelen- because they are always genetically identical to terata: Actiniaria). Invertebrate Reproduction and the brooding parent. But, the details of the Development, 27: 197-204. mechanism of sexual and asexual reproduction Gashout, S. F. and R. F. G. Oromond 1979 Evidence for of this animal are still unknown (Russo et al. parthenogenetic reproduction in the sea anemone 1994) . In oreder to clarify the reproductive bio- Actinia equina L. Journal of the Marine Biological logy of A. equina in Japan, further investiga- Association of the United Kingdom, 59: 975-987. tions are needed, such as on the female Haylor, G. S., J. P. Thorpe and M. A. Carter 1984 gametogenic cycle and the sexual behavior of Genetical and ecological differentiation between the animal, as well as on the genetic status of sympatric colour morphs of the common intertidal Japanese populations of A. equina in compari- sea anemone Acitnia equina. Marine Ecology son to populations abroad. Progress Series, 16: 281-290. Jennison, B. L. 1979 Gametogenesis and reproductive Acknowledgments cycles in the sea anemone Anthopleura elegantissima (Brandt, 1835). Canadian Journal of Zoology, 57: We express our thanks to Mr. Kenjiro Konno, 403-411. former Lecturer of Tokyo University of Fisher- Orr, J., J. P. Thorpe and M. A. Carter 1982 Biochemi- ies, for giving us the opportunity to undertake cal genetic confirmation of the asexual reproduction this study and providing part of the specimens. of brooded offspring in the sea anemone Actinia We also thank Miss A. Yokoyama and Miss K. equina. Marine Ecology Progress Series, 7: 227-229. Koinuma for their warm cooperation in field Russo, C. A. M., A. M. Sole -Cava and J. P. Thorpe sampling. 1994 Population structure and genetic variation in two tropical sea anemones (Cnidaria, Actinidae) REFERENCES with different reproductive strategies. Marine Bio- logy, 119: 267-276. Bronsdon, S. K. , P. A. Tyler, A. L. Rice, and J. D. Sebens, K. P. 1981 Reproductive ecology of the Gage 1993 Reproductive biology of two epizoic intertidal sea anemones Anthopleura xantho- anemones from the deep North-Eastern Atlantic grammica (Brandt) and A. elegantissima (Brandt) : Ocean. Journal of the Marine Biological Association Body size, habitat, and sexual reproduction. Journal of the United Kingdom, 73: 531-542. of Experimental Marine Biology and Ecology, 54:

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225-250. Uchida, T. and F. Iwata 1954 On the development of a Shick, J. M. 1991 A. Functional Biology of Sea Anemo- broodcaring actinian. Journal of the Faculty of Sci- nes. Chapman and Hall, London, 395pp. ence, Hokkaido University, Series VI, Zoology, 12: Sole -Cava, A. M. and J. O. Thorpe 1992 Genetic diver- 220-224. gence between colour morphs in common intertidal sea anemones Actinia equina and A. prasina Address (Anthozoa: Actiniaria) in the Isle of Man. Marine Kensuke Yanagi (reprint request) and Susumu Segawa: Biology, 112: 243-252. Laboratory of Invertebrate Zoology, Tokyo University Uchida, H., 1992 Cnidaria. In, Guide to seashore ani- of Fisheries, 4-5-7, Konan, Minato-ku, Tokyo 108, mals of Japan with color pictures and keys, vol. 1. Japan. Nishimura, S. (ed.), Hoikusha, Osaka, pp.127-145. (in Takashi Okutani: College of Bioresource Sciences, Japanese). Nihon University, 1866, Kameino-cho, Fujisawa-shi, Kanagawa 252, Japan.