Dev. Reprod. Vol. 14, No. 1, 7-11 (2010) 7

Germ Cell Aspiration (GCA) Method as a Non-fatal Technique for Sex Identification in Two Bivalves (Gomphina veneriformis and Tegillaca granosa)

† Jung Sick Lee1, Sun Mi Ju1, Ji Seon Park1, Young Guk Jin2, Yun Kyung Shin3 and Jung Jun Park4 1Dept. of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Korea 2South Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Yeosu 556-823, Korea 3Aquaculture Management Division, National Fisheries Research and Development Institute, Busan 619-705, Korea 4Pathology Division, National Fisheries Research and Development Institute, Busan 619-705, Korea

ABSTRACT : This study attempted to verify the possibility of using germ cell aspiration (GCA) method as a non-fatal technique in studying the life-history of equilateral venus, Gomphina veneriformis () and granular ark, granosa (Arcidae). Using twenty-six gauge 1/2" (12.7㎜) needle, GCA was carried out in equilateral venus through external ligament. In granular ark, GCA was performed by preventing closure of the shells by inserting a tongue depressor between the shells while still open. The success rate of sex identification using the GCA method was 95.6% for the equilateral venus (n=650/680) and 94.3% for the granular ark (n=707/750). Mortality of equilateral venus, which spent 33 days under wild conditions, was 13.8% (n=90/650) while the mortality of granular ark, which spent 390 days under wild conditions, was 2.4% (n=17/707). Although we believe that GCA does not appear to cause death in equilateral venus or granular ark, the success rate in employing of this methodology may differ depending on the level of proficiency of the researcher and reproductive stage of the bivalve. This study concludes that GCA is a convenient non-fatal methodology, which can be employed to identify sex and investigate gonadal maturity in Gomphina veneriformis and Tegillarca granosa. Key words : Germ cell aspiration, Gomphina veneriformis, Tegillarca granosa, Sex identification, Gonadal maturity.

INTRODUCTION (Wright & Lindberg, 1979), Mytilus edulis (Jabbar & Davies, 1987; Burton et al., 1996) and Mytilus galloprovincialis Information on sex, gonadal maturity and spawning (Mikailov et al., 1995). periodicity are equally important in artificial seed production Equilateral venus, Gomphina veneriformis and granular of these organisms. Reproductive history and sexual matu- ark, Tegillarca granosa were selected as the case-study ration of bivalves differ in accordance with various environ- species for this investigation. These are ecologically dominant mental factors, including water temperature (Eversole, 2001). bivalves in eastern and southern coastal waters of Korea, Sex identification and gonadal maturity of bivalves are, in addition to being economically valuable. This study in general, have been assessed through histological and attempted to verify the possibility of using germ cell microscopic analysis. However, such methods cannot be aspiration (GCA) method as a non-fatal technique in studying applied without sacrificing the organism. Previous studies the life-history and reproduction of bivalves. have attempted to investigate the possibility of sex deter- mination without killing the organisms in patellacean gastropods MATERIALS AND METHODS

† Corresponding author: Jung Jun Park, National Fisheries Research 1. Experimental Bivalves and Development Institute, Pathology Division, 152-1 Haean-ro, Gijang- Equilateral venus, Gomphina veneriformis (: eup, Gijang-gun, Busan 619-705, Korea. Tel: +82-51-720-2487, Fax: Veneridae) and granular ark, Tegillarca granosa (Bivalvia: +82-51-720-2498, E-mail: [email protected] 8 Lee JS, Ju SM, Park JS, Jin YG, Shin YK, Park JJ Dev. Reprod. Vol. 14, No. 1 (2010)

(Figs. 2G, H and 3G, H).

2. Syringe Disposable syringes were employed in this experiment. Two needle sizes were used; 29 gauge 1/2" (12.7 ㎜) (Becton Dickinson, U.S.A.) and 26 gauge 1/2" (12.7 ㎜) (Sung Shim Medical Co., Ltd., Korea). The size of the syringe needle was identified with considerations to size, inner diameter and length of the needle. Consequently, we identified that 29 gauge 1/2" (12.7 ㎜) needle was not appropriate for extraction of oocyte due to its narrow inner diameter. Accordingly, the 26 gauge 1/2" (12.7 ㎜) needle Fig. 1. Sampling area of the experimental bivalves. was used in most cases.

Arcidae) were used in the experiment. Equilateral venus 3. Germ Cell Aspiration was collected from the coastal region of Goseong on the Equilateral venus, Gomphina veneriformis has a well- eastern coast, while granular ark was collected from developed dorsal external ligament in the posterior portion Jangsu Bay in Yeosu on the southern coast of Korea (Fig. of the umbo (Fig. 2B, C), which enables the needle to be 1). In relation to appropriate organism size and experi- inserted into the mantle cavity. Insert the syringe needle mental timing the methodology of Park et al. (2003) and into the gonad of the visceral mass through the external Lee (1997) were followed. Adults of 25.0-34.9 ㎜ in shell ligament of the hinge line in a single thrust at an angle of length were used (Figs. 2A, 3A), while experiments began approximately 45° (Fig. 2D). The shells of the granular in July to coincide with the ripe and spawning season ark, Tegillarca granosa are normally opened within the

Fig. 2. Equilateral venus, Gomphina veneriformis. (A) and (B) External morphology and external ligament (El). (C) Morphology of socket and teeth arrangement. (D) Syringe needle insertion through the external ligament. (E) Aspirated oocytes by syringe needle aspiration. (F) Aspirated spermatozoa by syringe needle aspiration. (G) Ovary of ripe stage. (H) Testis of ripe stage. Ct, cardinal tooth; Lt, lateral tooth; Oc, oocyte; S, socket; Sp, sperm. Dev. Reprod. Vol. 14, No. 1 (2010) Sex Identificatioin by Germ Cell Aspiration (GCA) Method in Bivalves 9

Fig. 3. Granular ark, Tegillarca granosa. (A) and (B) External morphology and external ligament (El). (C) Morphology of socket and teeth arrangement. (D) Syringe needle insertion through between shells. (E) Aspirated oocytes by syringe needle aspiration. (F) Aspirated spermatozoa by syringe needle aspiration. (G) Ovary of ripe stage. (H) Testis of ripe stage. Oc, oocyte; S, socket; Sp, sperm; Sr, syringe; T, tooth; Td, tongue depressor. aquarium (Fig. 3A). Although the external ligament of the observed over 390 days (July 7, 2006-August 1, 2007). granular ark is explicit and located in the umbo (Fig. 3B), The disparity in the experimental period between the two needle insertion is prohibited by teeth which are regularly species was as a result of damage to the equilateral venus arranged in clusters (Fig. 3C). Accordingly, insert the tongue cage brought on by heavy wave action, thus making depressor into the area near the boundary of the shell for further continuation impractical. the organisms with their shells opened in order to prevent the shell from closing. Insert the syringe needle into the 5. Histological Technique gonad of the visceral mass through the opened shells with For light microscopy, the tissues were fixed in aqueous a single thrust (Fig. 3D). Bouin’s solution for 18 hours and rinsed in running water for 36 hours. The tissues were dehydrated in ascending 4. Mortality grades of ethyl alcohols and then embedded in paraplast Experimental organisms were reared in an indoor plastic (McCormick, USA). Embedded tissues were transversely aquarium for 3 days, where the 500ℓ seawater capacity sectioned at 5 ㎛ thickness and routinely stained with was adequately filtered, aerated and kept at a salinity of Mayer's hematoxylin - 0.5% eosin. 32.5‰ and a temperature of 20.5℃. Subsequently, the Morphological and anatomical terminologies of Stachowitsch rearing experiment was carried out under wild conditions (1992) were used. close to the collection area of the organisms. A cage where the bivalves could be prevented from escaping was installed. RESULTS The bottom of the cage was securely inserted into the sediment in order to allow the organisms to be able to 1. Success Rate of Sex Identification using Germ burrow. Equilateral venus was reared for 33 days (August Cell Aspiration (GCA) Method 20, 2006-September 22, 2006), while granular ark was The success rate of sex identification using the GCA 10 Lee JS, Ju SM, Park JS, Jin YG, Shin YK, Park JJ Dev. Reprod. Vol. 14, No. 1 (2010)

Table 1. Success rate of sex identification and mortality with GCA

Sex identification Mortality Species Success rate No. of No. of Indoor Wild (n) inactive stage spent stage aquarium condition Gomphina veneriformis 95.6% (n=650/680) 2 28 0% (n=0/650) 13.8% (n=90/650) Tegillarca granosa 94.3% (n=707/750) 11 32 0% (n=0/707) 2.4% (n=17/707) method was 95.6% for the equilateral venus (n=650/680) was employed in this study as a possible alternative non- and 94.3% for the granular ark (n=707/750). Results of intrusive technique. histological analysis of the 30 equilateral venus, for which It is generally very difficult to identify sex in organisms sex identification was uncertain, revealed that 2 were in for which sexual dimorphism is not definitive, without inactive reproductively while the remaining 28 were in the having to sacrifice the subject. Previous studies have spent stage. Among the 43 granular ark for which sex attempted to investigate the possibility of sex identification identification was uncertain, 11 were inactive while the in shelled molluscs without killing the organisms. Wright remaining 32 were spent reproductively (Table 1). and & Lindberg (1979) verified the sex of patellacean gastropods by extracting germ cells through syringe insertion 2. Mortality with GCA into the internal organs. For these gastropods, the abdominal In order to find out the effect of GCA on the death of portion gets exposed when detached from the substratum, these bivalves, experimental organisms were reared in an thus making insertion of the syringe easier in comparison indoor plastic aquarium and wild condition. to bivalves. Furthermore, insertion locations for bivalves Mortality for both species, after having been cultured are complicated by their shape and consequent linkage of for 3 days in the indoor aquarium, was 0%. Mortality of the hinge at the apex of the shell. equilateral venus, which spent 33 days under wild conditions, The equilateral venus has a well-developed dorsal external was 13.8% (n=90/650) while the mortality of granular ark, ligament in the posterior portion of the umbo, on the basis which spent 390 days under wild conditions, was 2.4% of the right valve. The arrangement of teeth on the inner (n=17/707) (Table 1). socket portion of apex of the shell is a heterodont type displaying definitive distinction between cardinal and DISCUSSION lateral teeth. The external ligament covers the empty space between the rear portions of the lateral teeth, thus making Extraction of tissues from the internal organs while the it possible to insert the needle into the mantle cavity. organism is still alive, is very difficult, especially for molluscs The external ligament of the granular ark is located such as gastropods or bivalves with thick exoskeleton. in the umbo, and is wide and definitive. However, the Currently, fine needle aspiration (FNA) is applied widely arrangement of teeth on the inner socket portion of the as a methodology in pathological diagnosis of human beings. external ligament is a taxodont type. With the regularly However, there are technical difficulties and consequent arranged teeth in clusters, insertion of the syringe needle cost-effectiveness issues associated with non-proficient is problematic. Accordingly, needle was inserted after researchers that maybe working with a large number of having prevented closure of the shells by inserting a subjects. The germ cell aspiration (GCA) method using syringe tongue depressor between the shells while still open. Dev. Reprod. Vol. 14, No. 1 (2010) Sex Identificatioin by Germ Cell Aspiration (GCA) Method in Bivalves 11

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(received 15 January 2009, received in revised form 28 February 2010, accepted 2 March 2010)