Dev. Reprod. Vol. 16, No. 3, 177-183 (2012) 177

Indirect Evidence on Sex Reversal with Sex Ratio of granosa (: Arcidae) and Ruditapes philippinarum (Bivalvia: )

Jung Sick Lee1, Kayeon Ku1, Hyejin Kim1, Ji Seon Park1, Jung Jun Park2, Yun Kyung Shin2 and Mi Ae Jeon1,†

1Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Korea 2Aquaculture Management Division, Research Institute, NFRDI, Busan 619-902, Korea

ABSTRACT : This study attempts to propose the possibility of the sex reversal in Tegillarca granosa and Ruditapes philippinarum by confirming the changes in the sex ratio with the shell length (SL) in the same population level. For analysis of sex ratio with SL, 1500 individuals of T. granosa (SL 10.1-45.0 mm) and 712 individuals of R. philippinarum (SL 15.1-70.0 mm) were used. Sex was analyzed histologically. The average sex ratios (F:M) of T. granosa and R. philippinarum were 1:1.22 and 1:0.96, respectively. However, sex ratio was found to differ when the were divided into groups according to SL in 5.0 mm intervals. Both species displayed the tendency of increase in the proportion of female with increase in SL. In this study, changes in the sex ratio in accordance with the growth of T. granosa and R. philippinarum are determined to be indirect evidence that signifies their sex reversal. Key words : T. granosa, R. philippinarum, Sex ratio, Sex reversal

INTRODUCTION of bivalves have an inactive stage during the reproductive cycle and classification of morphological sex is impossible Sex of bivalves is classified into gonochorism and herma- as the gonad tissue is completely degenerated during this phroditism, and hermaphroditism is further divided into period (inactive stage) (Chung et al., 1994; García-Domínguez synchronous (simultaneous or functional) and asynchronous et al., 1994; Behzadi et al., 1997; Lee, 1997; Villalejo- (sequential) hermaphroditism. The asynchronous hermaphrodi- Fuerte & García-Domínguez, 1998; Park et al., 2003; Wi tism signifies sex reversal in accordance with seasons, et al., 2003). In the case of these bivalves, there is possi- resultantly (Heller, 1993; Gosling, 2004). bility that they may develop as different sex when the next Indirect evidence of sex reversal in bivalves is a change reproductive cycle begins. in the sex ratio of a population with size (Orton, 1933; This study attempted to present indirect evidence on the Galtsoff, 1964; Guo et al., 1998; Eversole, 2001; Gosling, sex reversal of Tegillarca granosa and Ruditapes philippinarum 2004). Sex reversal of bivalves has been reported in the by examining the changes in sex ratio with shell length Ostreidae (Orton, 1933; Galtsoff, 1964; Thompson et al., and also to suggest basic information to definitively inve- 1996; Guo et al., 1998; Gosling, 2004), Pectinidae (Osanai, stigate their sex. 1975; Ventilla, 1982) and Veneridae (Eversole, 2001). Another indication of indirect evidence is the fact that large number MATERIALSAND METHODS

1. Materials †Corresponding author: Jung Sick Lee, Dept. of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Korea. Tel: +82-61- Tegillarca granosa used for sex ratio analysis was 659-7172, Fax: +82-61-659-7170, E-mail: [email protected] collected from the Jangsu Bay of Yeosu on the southern 1 7 8 JS Lee, K Ku, H Kim, JS Park, JJ Park, YK Shin, MA Jeon Dev. Reprod. Vol. 16, No. 3 (2012) coast of Korea from April 2007 to June 2011. Total number preparation for light microscopy was performed according of T. granosa used for sex ratio analysis was 1,500 (shell to the methodology of Drury & Wallington (1980). The length 10.1-45.0 mm) (Table 1). Ruditapes philippinarum study clams were dissected, and their visceral mass, which used for sex ratio analysis was collected from the intertidal included the gonad, was fixed in aqueous Bouin`s solution zone of Goheung on the southern coast of Korea for 18 h. and rinsed in running water for 24 h. and then from June 2009 to August 2011. Total number of R. dehydrated through a graded ethanol series (70-100%). philippinarum used for sex ratio analysis was 712 (shell The tissues were then embedded in paraplast (McCormick, length 15.1-70.0 mm) (Table 2). USA). Embedded tissues were sectioned at 4-6 um thick- ness using a microtome (RM2235, Leica, Germany). Samples 2. Histological Analysis were stained with Mayer's hematoxylin-0.5% eosin (H-E). Histological techniques were also used to confirm the sex and gonadal development of each specimen. Specimen 3. Statistical Analysis

Table 1. Sex ratio with shell size of Tegillarca granosa

Shell length Number Sex ratio χ2 P-value (mm) Total Female Male (F:M) 10.1-15.0 20 2 18 1:9.00 12.800 0.000 15.1-20.0 75 25 50 1:2.00 8.333 0.004 20.1-25.0 73 23 50 1:2.17 9.986 0.002 25.1-30.0 537 219 318 1:1.45 18.251 0.000 30.1-35.0 598 277 321 1:1.16 3.237 0.072 35.1-40.0 175 118 57 1:0.48 21.263 0.000 40.1-45.0 22 13 9 1:0.69 0.727 0.394 Total 1500 677 823 1:1.22 14.211 0.000

Table 2. Sex ratio with shell size of Ruditapes philippinarum

Shell length Number Sex ratio χ2 P-value (mm) Total Female Male (F:M) 15.1-20.0 2 - 2 - - - 20.1-25.0 69 29 40 1:1.38 1.754 0.185 25.1-30.0 191 87 104 1:1.20 0.005 0.942 30.1-35.0 104 46 58 1:1.26 1.385 0.239 35.1-40.0 103 56 47 1:0.84 0.786 0.375 40.1-45.0 119 64 55 1:0.86 0.681 0.409 45.1-50.0 45 27 18 1:0.67 1.800 0.180 50.1-55.0 18 13 5 1:0.38 3.556 0.059 55.1-60.0 23 18 5 1:0.27 7.348 0.007 60.1-65.0 32 20 12 1:0.60 2.000 0.157 65.1-70.0 6 4 2 1:0.50 0.667 0.414 Total 712 364 348 1:0.96 0.369 0.549 Dev. Reprod. Vol. 16, No. 3 (2012) Indirect Evidence on Sex Reversal of Bivalves 179

Statistical analyses were performed using SPSS 18.0 inactive stage (Figs. 1 and 2). (SPSS Inc., Microsoft Co. WA). Sex ratio was assessed by Chi-square (χ2) test. In all cases significance was established DISCUSSION at p<0.05. There is still a paucity of data on sex differentiation and RESULTS sex reversal in many bivalves, which makes sex identification of some species difficult. In general, in the case of gono- 1. Sex Ratio chorism, manifestation of sex as male or female, after having identified the morphological sex of bivalve, is 1) Tegillarca granosa limited to a prescribed time period of the life history of the Sex ratio (F:M) for the entire population of T. granosa, given individual. In order to accurately identify their sex, shell length (SL) 10.1-45.0 mm (n=1500) was 1:1.22. How- continuous tracing of changes in the sex by tagging the sex ever, sex ratio was found to differ when the clams were of each individual is necessary. However, this is also divided into groups according to SL in 5.0 mm intervals. problematic since classification of bivalves into males or Sex ratio displayed the results of the tendency of increase females is difficult macroscopically (Coe, 1943). in the proportion of female as SL increased in SL There was a case in which sexual dimorphism in bivalves 10.1-35.0 mm but increase in the proportion of male in was reported in Dysnomia capsaeformis and D. brevidens above 35.1 mm in SL (Table 1). (Mackie, 1984). However, since it is difficult to distinguish sex macroscopically, histological analysis of gonads remains 2) Ruditapes philippinarum the only conclusive method. However, this method cannot Sex ratio (F:M) for the entire population of R. phili- be carried out without killing the organism (Gosling, 2004). ppinarum, SL 15.1-70.0 mm (n=712) was 1:0.96. However, Although the sex of the majority of bivalves cannot be sex ratio was found to differ when the clams were divided identified externally, the ratio of females to males is the into groups according to SL in 5.0 mm intervals. Although same and they are generally gonochoristic (Gosling, 2004). there was tendency of increase in the proportion of female Hermaphroditism (synchronous or asynchronous) in bivalves as SL increased in SL 15.1-60.0 mm, increase in the was recorded in 13 families among 117 analyzed families proportion of male in above 60.0 mm in SL was observed (Heller, 1993). (Table 2). Synchronous hermaphroditism is the simultaneous release of eggs and sperm by one organism during the same season. 2. Gonadal Histology Asynchronous hermaphrodities function first as one sex, The sexes of the study clams were separate. Both ovary then as another. Asynchronous hermaphrodities can be pro- and testis were composed of many gametogenic follicles, tandrous, protogynous or can alternate sexuality. In pro- and it was possible to observe the developmental process tandry, the individual assumes the sex of male initially and of the germ cells within the follicle. The developmental then sex is reversed to female. The opposite happens in the stages of gonads were divided into five stages of inactive, case of protogyny. Alternating sexuality is the functioning early active, late active, ripe and degenerative stage. From of an organism first as one sex then as another, repeatedly the result of histological analysis, histological characteristics (Heller, 1993). of the gonad that illustrate the process of sex reversal were Synchronous hermaphroditism in bivalves has been re- not observed at either of the reproductive season and at the ported in Anodonta grandis (Van Der Schalie & Locke, 1 8 0 JS Lee, K Ku, H Kim, JS Park, JJ Park, YK Shin, MA Jeon Dev. Reprod. Vol. 16, No. 3 (2012)

Fig. 1. Gonadal development stage of Tegillarca granosa. A, Fig. 2. Gonadal development stage of Ruditapes philippinarum. Inactive stage; B, D, F and H, Female; C, E, G and I, A, Inactive stage; B, D, F and H, Female; C, E, G and Male; B and C, Early active stage; D and E, Late active I, Male; B and C, Early active stage; D and E, Late active stage; F and G, Ripe stage; H and I, Degenerative stage. stage; F and G, Ripe stage; H and I, Degenerative stage.

1941) and Elliptio (Heard, 1979) of Unionidae; Argopecten (Sastry, 1979; Gosling, 2004), C. virginica (Mackie, 1984; irradians (Sastry, 1979), Chlamys opercularis (Sastry, 1979) Gosling, 2004) of Ostreidae and Mercenaria mercenaria and Pecten latiauritus (Mackie, 1984) of Pectinidae; Fulvia (Sastry, 1979; Gosling, 2004) of Veneridae. mutica of Cardiidae (Chang & Lee, 1982); Corbicula Although sex reversal has been reported for other bivalves, fluminea of Corbiculidae (Britton & Morton, 1982); and such as Patinopecten yessoensis (Osanai, 1975; Ventilla, Gemma spp. of Veneridae (Mackie, 1984). 1982), sex reversal studies have traditionally been conducted Asynchronous hermaphroditism in bivalves has been on . Historically, the literature points to the higher reported in rivularis, C. madrasensis, Saccostrea proportion of males at the early stage, with increasing glomerata and S. cucculata (Asif, 1979), proportion of females arising through sex reversal at older Dev. Reprod. Vol. 16, No. 3 (2012) Indirect Evidence on Sex Reversal of Bivalves 181 stages of oysters (Orton, 1933; Coe, 1934; Galtsoff, 1964; gonad through all the stages of the reproductive cycle of Thompson et al., 1996; Guo et al., 1998). T. granosa and R. philippinarum, no observation could be European , O. edulis exhibits between 10-16% male made of the histological characteristics of the gonad that to female sex reversal in the first year, and approximately illustrated the process of sex reversal at any stage of the 50% male to female sex reversal in the second year, reproductive cycle. Therefore, it is determined that the sex respectively (Orton, 1933). They mature as males initially reversal in the study clams occurs during the inactive stage and undergo sex reversal into females after the first discharge following the spawning season. of sperm, while sex reversal is repeated throughout their Two factors, namely genetic and environmental, are life cycle (Walne, 1974). Quahog , M. mercenaria, involved in the changes in sex ratio and sex determination become sexually mature at less than one year old, deve- of bivalves. In the case of Pomacea canaliculata, C. gigas loping first as males but changing to an equal sex ratio in and Mytilus sp., an oligogenic sex determination method the second year (Menzel, 1989). was used, which is a mechanism that results in highly Sex of T. granosa has been reported to be gonochoristic diversified sex ratio and sex determination by small number (Lee, 1997). Chung et al. (1994) also reported the sex of of genes (Guo et al., 1998; Yusa, 2007). Factors such as R. philippinarum to be gonochoristic. However, in this temperature, food availability and day length are involved study, sex reversal is forecasted for T. granosa and R. in environmental sex determination of bivalves (Yusa, 2007). philippinarum based on the results of analysis of changes Changes in sex ratio due to exposure to pollutants, such as in the sex ratio with shell sizes. Therefore, the possibility EDCs and heavy metals during the inactive season, have that the sex of these species is asynchronous herma- also been reported in Mya arenaria (Gagné et al., 2005), phroditism is very high. Gomphina veneriformis (Lee & Park, 2007; Ju et al., 2009) These conclusions on the sex of T. granosa (Lee, 1997) and Scapharca broughtonii (Lee et al., 2009). and R. philippinarum (Chung et al., 1994) may have been As the result of this study, although the sex reversal of reached because of the limited investigation undertaken by T. granosa and R. philippinarum is predicted, additional these authors some stage of the life cycle of T. granosa researches are necessary for presentation of direct evi- and R. philippinarum rather than continuous tracking of dences and factors of sex reversal in these species. the reproductive life-history over a long period. Crassostrea virginica is a protandric species with an ACKNOWLEDGEMENTS increase in the proportion of females through sex reversal as it ages. Sex reversal normally occurs when the gonad is This research was supported by Basic Science Research undifferentiated between spawning seasons (Thompson et Program through the National Research Foundation of al., 1996). Although the sex of the Patinopecten Korea (NRF) funded by the Ministry of Education, Science yessoensis, is male during the young stage of less than one and Technology (2012-0004670). year, sex is thereafter reversed to female. Histological analysis showed that their sex is not reversed during the REFERENCES inactive stage of their reproductive cycle, but is rather reversed through continuous stages during the reproductive Asif M (1979) Hermaphroditism and sex reversal in the cycle (Osanai, 1975). four common oviparous species of oysters from the From the results of Lee (1997), Chung et al. (1994) and coast of Karachi. 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