Reproductive Cycle of Styela plicata(Urochordata: Ascidiacea) in Yeosu Coastal Waters

Ⅰ. Introduction

Styela plicata belongs to the commonly known as pleated sea squirt, is a species of tunicate in the family Styelidae.

Pleated sea squirt are distributed in the East Sea and the South Sea, and mainly inhabit the subtidal bed at a depth of 5-15 m or attach to aquaculture facilities, and are mainly found in a state where a large number of individuals form a group (Hong, 2006). Compared to Styela clava, S. plicata is more circular and has no tail, and the protrusions on the surface of the shell are thick and have a rather pale color (Park et al., 2011). The incurrent siphon and excurrent siphon do not protrude from the body.

Pleated sea squirt is a useful aquatic resource mainly used for steaming and stews because it has a unique scent and taste. Production rate by cultured breed is as follows. Seaweeds account for 77.5% of the production, Shellfish accounts for 17.8%, Fish accounts for 3.6%, Crustaceans account for 0.4%, and other aquatic animals, including sea squirt of account for 0.7% (MOMAF, 2022). Nevertheless, the domestic production of S. clava is gradually decreasing. Thus the demand for resource enhancement and the development of augmentation and aquaculture technology for the reduced S. clava is increasing.

Pleated sea squirt has been regarded as harmful to the farms of oysters and mussels. Pleated sea squirt are identified as the dominant invasive species in many coastal systems in other countries (Lambert and Lambert 1998; Lambert, 2007). Recently, however, as the production of S. clava has decreased of the demand for S. plicata is steadily increasing. In order to improve the productivity of pleated sea squirt the reproductive cycle and main spawning period should be identified. Through this, efficient aquaculture technology should be developed to increase production. Some of the studies were to Styelidae based on nutritional composition (Lee et al., 1985; Kim et al., 2005; Park et al., 2006; Park et al., 2011) and physiological activity (Kim et al., 2006; Lee et al., 2007; Seo et al., 2009) was actively conducted in the field of food and nutrition. In foreign studies, ecological studies (West and Lambert, 1976; Lambert and Lambert 1998; Lambert, 2007; Thiyagarajan and Quin, 2003) have been reported, but reproductive ecology studies on pleated sea squirt have not been conducted at all.

This study purported to investigate the monthly changes in the main spawning period and egg fertilized egg and reproductive cycle according to the gonad development stage in order to obtain basic data necessary for resource propagation and efficient management of pleated sea squirt living in Gamak Bay, Yeosu. Based on these results, the purpose of this study was to provide basic data for efficient management of pleated sea squirt.

Ⅱ. Material and methods

1. Sampling and measurements

Pleated sea squirt were sampled monthly in the period from March 2008 to February 2009 from the study area of the Garmak bay, Yeosu ([Fig. 1]). For this study, a total of 360 pleated sea squirt were randomly collected from the aquaculture of collection station by the long-line culture for each month. Water temperature and salinity were critical measured at the study area at which the seawater samples were collected using a YSI 556 multiprobe system. The measurements were taken with a vernier caliper to the nearest 0.01 mm. Measurements was performed according to BL, BW, and MW. Abbreviations are: BL=Body Length, BW= Body Width and MW=Meat Weight. Sampling were weighed to the nearest 0.01 g using an electronic balance (MWP-300, CAS).

[Fig. 1]

Map showing the location of the study region.

Ⅲ. Results

1. Water temperature and Salinity

Multiprobe system were record water temperature (℃) and salinity (psu) every month. The average annual water temperature during the survey period was 18.1±7.36℃ in the wrinkled sea squirt habitat area.

The seawater temperature during the investigation period was 18.1±7.36℃. Water temperature was the lowest at 8.1℃ in January and the highest at 26.6℃ in August. The average salinity was 31.5±0.70 psu. Salinity was investigated in the range of 30.6 (July)-33.2 psu (April) ([Fig. 2]).

[Fig. 2]

Monthly variation of water temperature and salinity in sampling sate from March 2008 to February 2009.

2. Condition index(CI)

The spawning period can be estimated by determining whether the proportion of the gonadal weight to the total weight of marine organisms increases. As such, it is possible to indirectly estimate the spawning period using the condition index. The condition index of pleated sea squirt showed the highest value in April (0.30) and the lowest value in August (0.15). The condition index (CI) of the animals was related to the temperature of the seawater. Overall, it rose in spring, reached its maximum value in April, and then declined sharply in summer, a high temperature period, and showed a tendency to recover when the water temperature dropped again ([Fig. 3]).

[Fig. 3]

Monthly variation of condition index (CI) in sampling sate from March 2008 to February 2009.

3. Reproductive cycle

It is difficult to distinguish the immature gonads of S. plicata, but in the mature stage, the ovaries and testes were clearly distinguished. As a result of observing the tissue under a microscope of the wrinkled sea squirt was hermaphroditic.

Specimens were analyzed with the smallest specimen recorded with body length 20.18 mm, and the biggest was 58.11 mm. The histological slides obtained were then examined under an optical microscope to determine the gender of the animal and the stage of the reproductive cycle. The developmental phases for gonad can be divided into five main reproductive cycle: (1) early growing stage (2) growing stage (3) mature stage (4) spawning stage and (5) resting and recovery stage ([Fig. 4]). The criteria for this gonad development stage are as follows.

[Fig. 4]

Gonad transverse sections showing different maturity stages of Styela plicata. a) gonad histological section stained with hematoxyn-eosin (OC: oocyte, MF: male follicle); b, C) female gonad stained with hemotoxylin-eosine (DO: developing oocyte; TC: test cells, FC: follicel cells, N: nucleolus); d, e, f) male follicles stained with hemotoxylin-eosine(d: immature, e: mature, f: Recovery and resting).

1) Early growing stage

In the ovary in the early growth phase, spherical oocytes proliferated along the reproductive epithelium of the ovarian follicle. Early oocytes grew toward the outer wall of the ovary. At this time, the oocyte had a larger nucleus than the cytoplasm and had distinct phosphorus in the nucleus. It was basophilic and heavily stained with hematoxylin and the size of the oocyte was within about 60 μm in diameter. The testis formed testis lobules around the ovaries. At this time, spermatogonial cells were distributed in the reproductive epithelium of the inner layer of the lobules and a group of spermatocytes appeared due to proliferation of spermatogonial cells. The period of emergence of these early juvenile individuals was from February to October. These individuals were observed the most from March to May.

2) Growing stage

Oocytes in the early growth phase gradually enlarged with oil droplets distributed in the cytoplasm and grew to an egg diameter of 80-120 μm. The lobules of the testis gradually grow through the active proliferation of spermatocytes and spermatozoa that have completed metamorphosis appeared along with the sperm cells. The time of emergence of these late-growth individuals was from March to August and the period was from June to August.

3) Mature stage

The mature ovary had a few early growing oocytes, but mature oocytes occupied the majority of the lumen. Mature oocytes had egg yolk granules evenly distributed in the cytoplasm and were about 140-200 μm in diameter at this time. Thereafter, in mature oocytes, many yolk granules were evenly distributed and cytoplasmic test-cells were arranged around the egg membrane. In the double egg membrane, the inner follicle layer was formed between the outer follicle layer and the outer follicle layer as the inner and outer membranes were separated as the interstitial material was filled. In this way, the overwintered with mature ovaries, and individuals with mature oocytes still appeared after the spawning period. The spermatogenesis process was completed and was filled with sperm that had undergone metamorphosis. The mature individuals of gametes were observed from June to September of the following year.

4) Spawning stage

The ovaries in the ejection period progressed from the outer wall toward the lumen, and the mature eggs were released together with the inner follicular epithelium, and only the outer follicular epithelium and basement membrane surrounding the egg were present in the ovary. Simultaneously with egg release, oocytes developed along the ovarian lobular epithelium, and some unreleased oocytes were also present. Unreleased oocytes were gradually degenerated and absorbed. The sperm in the lobules are not completely released, and in the center of the lobules, unreleased spermatozoa and spermatogonial cells developing in the lobule epithelium condense and degenerate and absorb each other. The spawning season was observed in some individuals from March after passing through winter with low water temperature. After that, most of the individuals started laying and laying eggs from April to July. However, it was confirmed that some individuals participated in spawning in November and December.

5) Resting and Recovery stage

In convalescent ovaries, oogonia and early oocytes begin to Recovery stage along the luminal epithelium. The majority of the gametes were used during spawning, but unused gametes quickly disintegrated. Connective tissue begins to fill with lipids and glycogen. They appeared from April to December. The gonads are ready for the resting stage ([Fig. 4F]).

4. Frequency distribution of oocyte diameter

The range of oocyte diameters was determined monthly from March 2008 to February 2009 ([Fig. 5]). Oocytes of various sizes and all stages were present each month. Mature oocytes were 74% of cells larger than 140 μm in April. In May, mature oocytes and early growth stage oocytes (40-60 μm) were also distributed. After that, the growth stage oocytes (60-120 μm) were mainly distributed from June to August, and the mature stage oocytes (140 μm or more) appeared from September to December. Oocytes larger than 200 μm in January showed the highest frequency at 30%. In February and March, anagen-phase oocytes (60-120 μm) appeared mainly, and the frequency of their appearance was 36% and 52%, respectively.

[Fig. 5]

Evolution of the oocyte size-frequency distribution (% abundance per 20 ㎛ size class) of Styela plicata in the sampling sate from March 2008 to February 2009.

Ⅳ. Discussion

Pleated sea squirt has a long and slender body in the shape of a sack, and it lives in a colony by attaching the tip of its body to a rock or an artificial structure. Its body is 5-10 cm long and has a yellowish-brown color, and its outer skin is made of a fibrous material. In the case of Ascidian, such as sea squirts, asian tunicate and pleated sea squirt, the development and proliferation of germ cells are clearly differentiated according to seasonal variations (Sugawara et al., 1968).

It is known that the gonad development and maturation of marine organisms are largely affected by external environmental factors and internal regulatory factors. As external environmental factors, the influence of water temperature has been reported the most (Chung et al., 1991, 1994; Brousseau, 1995; Lee, 1999), food (Maru, 1976; Griffiths, 1977; Jara-millo and Navarro, 1995), photoperiod (Webber and Giese, 1969) and geographic location (Glovani and Diana, 1994) are known to be key factors. Studies have been reported on the development of gonads by factors that are internally controlled by endocrine substances (Boolootian et al., 1962; Euler and Heller, 1963; Fretter, 1984).

In general, the monthly change in the condition index is large during the period when the gonads appear a lot during maturity. In the results of this study, the condition index showed the highest value in spring and winter and the lowest value in summer. Afterwards, the obesity index gradually recovers after the high temperature period and when the water temperature is lowered, which is similar to the study results of Lee (1976; 1999) and Lee et al. (1999) reported that obesity decreased after spawning with maturation of the gonads. In the case of sea squirt, it has been reported that ovaries attached to the body wall have an elongated tubular structure with 2-4 on the left and 5-7 on the right (Lee, 1977). The case of pleated sea squirt, the similar elongated tubular structure with that of sea squirt was identified. The oocytes showed various sizes of 40-200 μm at each stage, and the size of mature oocytes was around 140 μm. The size of the early growth stage oocytes was 40-60 μm, and the size of mature oocytes was relatively large, around 140-200 μm. In addition, it was reported in a study by Lee (1976) and Satoh (1994) that the size of mature oocytes was around 210 μm.

In the case of wrinkled sea squirt, it was confirmed as a hermaphrodite, in which the gonads developed inside the cyst and testes were mixed between the vesicles of the ovary according to the change of season. As such, the development of gonads of marine organisms has a pattern unique to each species and appears constantly. The development and change of gonads in a certain pattern is the reproductive cycle that appears in basic marine organisms. However, the reproductive cycle may appear differently depending on the local population even with the same taxonomic species. Species inhabiting low latitudes have a long reproductive period and species inhabiting high latitudes have a shorter reproductive period (Fretter, 1984). As a result of estimating the spawning season through histological observations of wrinkled sea squirts, the main spawning season was May to June, and the partial spawning season was November to December.

It has been reported that test cells appear in the maturation stage in the process of egg development in chordates such as pleated sea squirt, and that these test cells have various functions such as nutrient supply according to embryogenesis, rupture prevention, and encapsulation(Satoh et al., 1982; Hirose et al., 1996). This study also confirmed that the test cells were located in the cortex of mature oocytes.

Further study on the since development and reproductive cycle of gonads are closely related to changes in water temperature, additional studies on the effects of environmental factors on gonadal maturation should be conducted. In addition, it is judged that artificial seedling technology is needed for stable and continuous aquaculture.

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