Seasonal Gametogenesis of Host Sea Anemone (Entacmaea quadricolor) InhabitingHong Kong Waters

BI Ying, ZHANG Bin, ZHANG Zhifeng, *, and QIU Jianwen



Seasonal Gametogenesis of Host Sea Anemone () InhabitingHong Kong Waters

BI Ying1), ZHANG Bin1), ZHANG Zhifeng1), *, and QIU Jianwen2), *

1),,266003,2),,,

Studying gonadal development of annual cycle can reveal the process of gametogenesis and reproductive period, and evaluate fertility and source utilization of a species. Host sea anemones are conspicuous members of tropical and subtropical reef ecosystems, but little is known about its biology including reproductive seasonality. Here we reported a one-year study on the gametogenesis and reproduction of host sea anemone () inhabiting Hong Kong waters.tissues were sampled in 12 occasions from 5m and 15m depths of water, respectively. Histological sectioning of the tissues showed thatwas dioecious, and populational ratio of female to male was 1:1.6. The gonadal development was asynchronous within an annual cycle, which included proliferating, growing, maturing, spawning, and resting stages. The spawning occurred between August and October when surface seawater temperature reached the annual maximum (28℃), suggesting that temperature is an important factor modulating the gonadal development and mature of.

; gonad; annual cycle; histology; seawater temperature

1 Introduction

Host sea anemones are conspicuous members inhabiting the shallow water of Indo-Pacific coastal ecosystems (Fautin and Allen, 1992). Though they usually live as solitary individuals among coral ecosystems, they can develop into large populations (Mariscal, 1970; Richardson., 1997; Brolund., 2004; Scott., 2011). Their population development can drastically change the ecosystem structure and function due to their multiple roles,., the host of symbiotic anemone fish and shrimp (Fautin., 1995; Porat and Chadwick-Furman, 2004), the competitor of corals, sponges and macroalgae (Chad- wick and Morrow, 2011), and the predator of mussels, crabs and small fish (Fautin and Allen, 1992).

Recent studies have identified two major threats to host sea anemone. Bleaching, caused by elevated sea temperature, has been reported to reduce the abundance of host sea anemone (Jones., 2008; Saenz-Agudelo., 2011). Extensive collection for aquarium trade is another one, which has also reduced the abundance of host sea anemone (Shuman., 2005; Jones, 2008). Despite its ecological importance and the threats it faced, little is known about its critical biological processes, such as sexual reproduction, clonal formation and population development (Scott and Harrison, 2009).

Host sea anemone(Rüppell and Leuckart, 1828) inhabits the shallow waters of Red Sea, Indian Ocean, Southeast Asia, and Australia (Fautin and Allen, 1992; Richardson., 1997; Chadwick and Arvedlund, 2005). It can reproduce sexually and asexually. However, little is known about its reproductive periodicity and how environmental factors modulate this critical life-history event that is essential to maintain the local population and the relationship among different populations. The only studiedpopulation inhabits the Solitary Islands, Australia, where the anemone spawns in austral summer and autumn when the water temperature is the highest (Scott and Harrison, 2009). Here we report the gametogenetic cycle and reproduction of two populations ofinhabiting Hong Kong waters.

2 Material and Methods

2.1 Studying Area and Sampling Location

Hong Kong, southern China, is surrounded by Pearl River Estuary in west and South China Sea in south and east (Fig.1). Due to the freshwater discharge from Pearl River, Hong Kong waters can be roughly divided into an estuarine zone in west, a transitional zone in middle, and an oceanic zone in east (Morton and Morton, 1983).is common in the oceanic zone and develops into one of the largest populations in Tsim Chau (22˚24´7´´N, 114˚23´11´´E), which inhabited depths from 4m to 16m and occupied an area of 40m×80m (Lee., 2014).

Fig.1 A map of Hong Kong showing Tsim Chau (filled circle), the sampling location in the eastern waters, and Environmental Protection Department (EPD)’s water quality monitoring station (MM15, open circle) closest to the sea anemone population.

2.2 Tissue Sampling

Sixty tissue samples were collected from two water depths (40 samples from 5m, 20 from 15m) by SCUBA divers monthly from May 2011 to April 2012. Only anemone individuals with an oral disk diameter greater than 10cm were chosen to increase the chance of collecting the gonadal mesenteries for histological sectioning, and they were at least 1m apart to avoid sampling clones that were of the same sex and developmental stage. Each sample was collected from one individual (polyp), using a stainless steel punch connected to a clinical syringe (Scott and Harrison, 2009).

Like many sea anemones, the mesenteries ofare located from the basal disc to the middle of polyp (Pei, 1998), therefore tissue sampling was conducted by inserting the stainless punch into the middle section of polyp and at least 0.6cm3gonad tissues were extracted with the syringe. The content of the syringe containing anemone tissues with several mesenteries was transferred into a numbered ziplock bag underwater. Upon finishing underwater sampling, the samples were transported to the diving boat where extra seawater was removed and formalin was added to a final concentration of 10%.

2.3 Laboratory Procedures

Visual inspection of the samples was conducted to select tissues containing mesenteries for histological sectioning. For each sample, two to eight gonad-like tissue subsamples were sectioned. Each subsample was dehydrated through a graded ethanol series, cleared with xylene, and embedded in wax. The embedded samples were sectioned at 4–5μm thickness using a Reichert Histostart 820, stained with Mallory’s trichrome stain, observed and photographed using a Nikon E80i microscope.

Anemone sex was determined by the histological sections. Individuals lacking gametes were considered as non-reproductive. Individuals with oocytes and spermato- genic vesicles in the mesenteries were scored as females and males, respectively. In females, the diameter of each nucleated oocyte was determined as the maximum oocyte diameter (=8) measured using an ocular micrometer fitted to a compound microscope. Oocytes were assigned to size classes representing different reproductive stages. Male anemones were classified into five developmental stages of spermeries as described in Scott and Harrison (2009).

Since temperature is known to affect the reproductive seasonality of marine invertebrates including sea anemone (Wedi and Dunn, 1983; Scott and Harrison, 2009; Lombardi and Lesser, 2010), seawater monitoring data taken from Environmental Protection Department, Hong Kong’s regular water quality monitoring station closest to our study site (Fig.1; Station MM15, 7km from Tsim Chau; http://www.epd.gov.hk/epd/english/environmentinhk/water/marine_quality/mwq_home.html) were used to determine the timing ofreproductive events in relation to seawater temperature.

3 Results

3.1 Gonadal Structure

was dioecious and the mesenteries connected to the gastrodermis at the lower half of the gastrovascular cavity. Reproductive mesenteries were composed of a gastrodermis enclosing mesoglea embedded with follicles containing germ cells. The spermeries had a mean diameter of 150μm, and contained several follicles (spermatogenic vesicles) at different developmental stages (Fig.2A), and each follicle was filled with a large number of germ cells. Female mesenteries were similar in size with spermeries, but each follicle (ovarian lobule) contained an oocyte only (Fig.2B).

3.2 Gonadal Development

3.2.1 Proliferating stage

The gonadal structure at the proliferating stage was characterized by the occurrence of germ cells in gastrodermis of reproductive mesenteries, and germ cell proliferation and migration into the mesoglea. Germ cells were distinguished based on their shape and clustering pattern. In males, many spermatogenia were clustered near the mesoglea (Figs.3A, B). They were small, with a mean diameter of 1.8μm. The nuclei were conspicuous and stained red, but the cytoplasm was thin and only lightly stained (Fig.3C).

In females, oogonia had a mean diameter of 10μm and were present individually or in small clusters of 2–4 cells; the nuclei were stained light purple; the cytoplasm was abundant and stained lighter than nuclei (Figs.4A, B). As the female mesenteries formed, each lobule contained only one cell, and the oogonia began to develop into oocytes (Fig.4C).

Fig.2 Reproductive mesentaries of E. quadricolor. A, Male mesenteries; B, Female mesenteries. G: gastrodermis; RM: reproductive mesenteries; M: mesoglea; SV: spermatogenic vesicle; OL: ovarian lobule.

Fig.3 Annual cycle of testis development of E. quadricolor. A–C, proliferating stage; D–G, growing stage; H, maturing stage; I–K, spawning stage, arrow showing sperm being released; L, resting stage; M, mesoglea; G, gastrodermis; Mg, germ cell close to the mesoglea; Gm, germ cell in mesoglea; Gg, migratory sperm cell; SV, spermatogenic vesicle; Sg, spermatogenium; Sc, spermatocyte; St, sperm cell; Sz, sperm; Stl, sperm tail.

Fig.4 Annual cycle of ovarian development of E. quadricolor. A–C, proliferating stage; B is a magnification of box in A; D–H, growing stage; E, F and H is a magnification of box in D, E and G, respectively; I–K, maturing and spawning stage; J is a magnification of box in J; L, resting stage; M, mesoglea; G, gastrodermis; Oo, oogonium; Oc, oocyte; OL, ovarian lobule; N, nucleus; Nu, nucleolus.

3.2.2 Growing stage

The reproductive mesenteries at growing stage were characterized by the prominent presence and development of reproductive lobules. In males, spermatogenic vesicles became larger and more numerous (Figs.3D, F). Cells in spermatogenic vesicles began to differentiate. Spermatocytes (diameter 2.5μm) became more abundant than spermatogenia, stained light purple, and the nuclei were not as red as those of spermatogenia. Spermatids could be distinguished from the spermatogonia and spermatocytes by their small size (diameter 2μm) and bright-red nuclei (inset of Fig.3D). As the spermatogenic vesicles grew further to around 100–150μm in diameter and their number became more abundant, they occupied more than half of the reproductive mesenteries (Figs.3E– G). Spermatocytes and spermatids became more abundant, and a small amount of sperms with tails were present in the spermatogenic vesicles (inset of Figs.3E, G).

In females, the ovarian lobules became larger and more abundant in reproductive mesenteries, but each ovarian lobule contained only one oocyte. The oocyte was initially small, with a diameter of about 100μm; its cytoplasm contained a small amount of vitellogen, which was stained purple (Fig.4C). As the oocyte developed, its size increased and more vitellogen was accumulated (Figs.4D–G).

3.2.3 Maturing stage

In the maturing stage the reproductive lobules of both males and females were fully grown. In males, sperm abundance increased substantially to occupy almost the whole spermatogenic vesicle, and the sperm arranged in a bouquet-like fashion with sperm tails facing the edge of the spermatogenic vesicle (Fig.3H).

In females, the oocyte grew to a size of 400μm, with a large amount of vitellogen, and the nucleus was now located near the cell membrane, being far from the middle of the cell; the ovarian lobules were at the edges of the reproductive mesentaries because of the growth and en- largement in size (Figs.4I–K).

3.2.4 Spawning stage

In males, the spermatogenic vesicles in the reproductive mesenteries decreased quickly to about 100μm in diameter (Fig.3I) as the sperms were released (Fig.3J). At this stage, spermatogenic vesicles with broken walls and other cellular remains could be seen in the mesoglea (Fig.3K). In females, the mesentaries no longer contained oocytes.

3.2.5 Resting stage

After the release of the reproductive products, the reproductive mesenteries began to reduce in both size and number. By the end of the reproductive season, the anemone entered the resting stage. No reproductive cells could be found in the mesenteries of either male (Fig.3L) or female (Fig.4L).

3.3 Sex Ratio

Of 720 samples collected and sectioned, only 47 females and 74 males were identified. The overall female to male ratio was 1 to 1.6.

3.4 Spermatogenesis Cycle

From the histology results,exhibited a clear annual cycle of spermatogenesis, but the development was asynchronous within an individual in most months (Fig.5). Spermatogenic vesicles were first observed in Tsim Chau population in March and reached the spawning stage in September. There was no obvious difference in the time of gametogenesis between samples collected from shallow and deep waters, therefore all samples were pooled.

Fig.5 Composition of spermatogenic vesicles in E. quadricolor collected from Tsim Chau. Stage 1, proliferating stage; Stage 2, growing stage; Stage 3, maturing stage; Stage 4, spawning stage. No spermatogenic vesicles were found in samples collected from November to February. Data above bars represent the number of specimens with identifiable spermatogernic vesicles.

3.5 Oogenesis Cycle

population exhibited an annual cycle of oogenesis, but oocyte development was asynchronous within an individual in most months (Fig.6). Oocytes were first observed in the mesoglea in March, but oocytes of 450µm in diameter and larger presented from March to October. No reproductive lobules were observed from November to January.

Fig.6 Monthly oocyte size distribution in E. quadricolor collected from Tsim Chau. No oocyte was found in samples collected from November to February. The figure was plotted using the number of oocytes with a measurable nucleus (left number above bar). Total number of females is shown as the right number above bar.

3.6 Relationship Between Annual Change of Seawater Temperature andReproduction

There was a remarkable seasonal change in surface seawater temperature, ranging from approximately 15℃ in February–March to 28℃ in June–October (Fig.7). Gametogenesis was initiated when water temperature rose to around 17℃ in March, and reached the maturation stage when water temperature rose to around 28℃ in June. The decreasing temperature from 28℃ in September to 15℃ in February corresponded to the post-spawning resting stage.

Fig.7 Monthly change in surface water temperature at MM 15 (EPA water quality monitoring station close to Tsim Chau).

4 Discussion

Our data are consistent with those of a previous study conducted in North Solitary Island, Australia, showing that gametogenesis inis similar to the common pattern observed in other sea anemones (Wedi and Dunn, 1983; Carter and Miles, 1989; Lombardi and Lesser, 2010). Gametes of both sexes appeared first in the endoderm, and then migrated into the mesoglea where the majority of growth and maturation took place. Our results were also consistent with those of Scott and Harrison (2009) who found thatwas doecious and its oocyte development was asynchronous. The asynchronous oocyte development might be due to a slow, yet continued production of oocytes throughout most of year except for the coldest months, or asynchronous rate of oocyte development (Scott and Harrison, 2009).

Spermatogenesis ofinhabiting Hong Kong coast was asynchronous. Spermatogonia at different stages presented in the same individual or different individuals collected from the same month in a year, while no reproductive mesenterie was presented in the coldest winter months. For example, spermeries at both proliferating and growing stages were found in samples collected from May to July 2011, whereas spermeries at maturing and spawning stages were found in samples collected from September to October 2011. This result was consistent with that of, a species of sea anemone, from Maine, US (Lombardi and Lesser, 2010) andfromCalifornia, US(Wedi and Dunn, 1983) where the spermatogenesis was asynchronous, but different from the findings infrom North Solitary Island where the spermatogenesis was synchronous (Scott and Harrison, 2009).

In general, temperature is an important factor for the gonadal development of aquatic organisms. In the present study, the temporary gonads ofbegan to develop at around 17℃ and spawning occurred at around 28℃, which indicated that this species reached reproductive quiescence in the coldest winter months and matured in the hottest summer months. However, the range of mean monthly temperature experienced byin North Solitary Island (19.2℃ to 25.0℃) is narrower than that in Hong Kong (15℃ to 28℃). The lowest month temperature might have contributed to the long quiescent period after spawning infrom Hong Kong (November to February). At North Solitary Island, samples collected from 16 occasions from January 2003 to February 2005 could be identified at developmental stages 1 to 4 (., no reproductive quiescence) as the sea anemone resumed gametogenesis within one month of spawning (Scott and Harrison, 2009).

Acknowledgements

This study was supported by Environment and Conservation Fund, Hong Kong (Project number: ECF 2009/29).We thank Dr. Anna Scott (Southern Cross University, Australia) for her advice on sampling anemone, and Drs. Raymond Man and James Li (Ocean Sky Divers, Hong Kong) for their help in underwater work.

Brolund, T.M., Tychsen, A., Nielsen, L.E., and Arvedlund, M., 2004. An assemblage of the host anemonein the northern Red Sea, and distribution of the resident anemonefish., 84: 671-674.

Carter, M. A., and Miles, J., 1989. Gametogenic cycles and reproduction in the beadlet sea anemone(Cnidaria: Anthozoa).,, 36: 129-155.

Chadwick, N.E., and Arvedlund, M., 2005. Abundance of giant sea anemones and patterns of association with anemonefish in the northern Red Sea., 85:1287-1292.

Chadwick, N. E., and Morrow, K. M., 2011. Competition among sessile organisms on coral reefs. In:. Dubinsky, Z., and Stambler, N., eds., Springer, New York, 347-371.

Fautin, D. F., and Allen, G. R., 1992.. Western Australian Museum, Perth, 160pp.

Fautin, D.G., Guo, C.C., and Hwang, J.S., 1995. Costs and benefits of the anemone shrimpsand its host., 129:77-84.

Jones, A. M., Gardner, S., and Sinclair, W., 2008. Losing ‘Nemo’: Bleaching and collection appear to reduce inshore populations of anemonefishes., 73: 753-761.

Lee, K. M., Xie, J. Y., Sun, Y. N., and Qiu, J. W., 2014. Four dense assemblages of the bulb-tentacle sea anemoneand its associated clownfish in Hong Kong.,DOI: http://dx.doi.org/10.1017/S0025315414001192.

Lombardi, M. R., and Lesser, M. P., 2010. The annual gametogenic cycle of the sea anemonefrom the Gulf of Maine., 390: 58-64.

Mariscal, R. N., 1970. The nature of symbiosis between Indo- Pacific anemone fishes and sea anemones., 6: 58-65.

Morton, B., and Morton, J., 1983.. Hong Kong University Press, Hong Kong, 350pp.

Pei, Z. N., 1998.,Scientific Press, Beijing, 308pp (in Chinese).

Porat, D., and Chadwick-Furman, N. E., 2004. Effects of anem- onefish on giant sea anemones: Expansion behavior, growth, and survival.530-531: 513-520.

Richardson, D.L., Harriott, V.J., and Harrison, P.L., 1997. Distribution and abundance of giant sea anemones (Actinaria) in subtropical eastern Australian waters., 48:59-66.

Scott, A., Malcolm, H. A., Damiano, C., and Richardson, D. L., 2011. Long-term increase in abundance of anemonefish and their host sea anemones in an Australian marine protected area., 62: 187-196.

Shuman, C. S., Hodgson, G., and Ambrose, R. F., 2005. Population impacts of collecting sea anemones and anemonefish for the marine aquarium trade in the Philippines., 24: 564-573.

Wedi, S.E., and Dunn, D.F., 1983. Gametogenesis and reproductive periodicity of the subtidal sea anemone(Coelenterata: Actiniaria) in California., 165:458-472.

(Edited by Qiu Yantao)

DOI 10.1007/s11802-015-2349-y

ISSN 1672-5182, 2015 14 (1): 143-148

© Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

(April 2, 2013; revised May 14, 2013; accepted November 5, 2014)

* Corresponding authors. E-mail: zzfp107@ouc.edu.cn qiujw@hkbu.edu.hk