Journal 
Introduction
Climate change alters the water temperature of the ocean, causing changes in the spawning season of marine animals and the timing of larval emergence of fish and invertebrates, thereby inducing changes in the community structure of marine ecosystems (Philippart et al., 2014). Reduction in average body size has been suggested as one of the universal responses to global warming in aquatic ecosystems (Nawrot et al., 2017). It also acts as a potential factor limiting the behavior and survival of bivalves (Rato et al., 2022), leading to changes in reproductive strategies such as changes of reproductive cycle, selection, genetic variation, and hybridization (Oyarzún et al., 2018;Masanja et al., 2023).
The reproductive response of bivalves is one of the important biomarkers for assessing the impact of various stressors, including chemical pollutants and climate change, in the marine environment (Chahouri et al., 2023).
The hermaphroditism of bivalves is divided into simultaneous and sequential hermaphroditism. The sequential hermaphroditism signifies sex reversal in accordance with seasons, as a result (Heller, 1993;Gosling, 2004). Although manifestation of morphological sex and sex ratio change of bivalves are in principle genetically influenced, they are also affected by various environmental factors (Coe, 1943;Guo et al., 1998;Kim et al., 2021).
It is known that change of sex ratio and reproduction of some aquatic animals due to changes in water temperature (Strüssmann et al., 1996;Ribas et al., 2017). However, it is difficult to find studies on the effects of water temperature on sex ratio changes or sex reversal in sequential hermaphroditic bivalves. Therefore, this study aimed to investigate the effects of water temperature, one of the representative environmental factors in aquatic ecosystems, on sex ratio changes in T. granosa, a representative sequential hermaphroditic bivalve.
Materials and methods
1. Experimental animals
Samples of T. granosa were collected from Jangsu Bay (N34°31’51”, E127°25’50”) on the southern coast of Korea, the same as previously reported by Lee et al. (2014) and Jeon et al. (2024). A total of 492 individuals of the 1+ year class (14 months, SL: 28.4 mm) with a sex ratio of approximately 1:1 were used in the study.
2. Rearing of experimental animals
The study was conducted in the following order: 1) collection and acclimation in an indoor aquarium for 2 weeks, 2) rearing in indoor aquarium with various water temperature conditions, 3) acclimation to wild conditions for 2 weeks, 4) rearing in wild, and 5) sex ratio analysis using histological methods (Fig. 1).
2.1. Rearing in indoor aquarium
The rearing period in the indoor aquarium was 5 weeks (35 days) from January 14 to February 17, 2013, corresponding to the gonadal inactive stage, during which sex change is known to occur (Kim et al., 2009;Lee et al., 2014;Jeon et al., 2024). T. granosa was gradually acclimated from natural water temperature to experimental water temperature for 2 weeks before exposure to experimental water temperature. The water temperature conditions were five (wild control, 5.0±0.5, 7.0±0.5, 9.0±0.5, and 11.0±0.5℃), and the salinity was set to the same value of 33.5 psu (Fig. 2).
A 200 L glass aquarium was used for indoor aquaculture, and the bottom of the aquarium was covered with sediment from the cockle collection area, about 10 cm thick. The number of individuals accommodated in each tank was 50, and 100 individuals were used for each water temperature condition in two repetitions. The water temperature was controlled using an automatic water temperature control device (Daeil, Korea). A mixture of phytoplankton (Isochrysis sp. and Chaetoceros sp.) of the clam habitat were fed to the clams in a sufficient amount (106 cells/L) once a day according to the method described by Moon (2005).
2.2. Rearing in wild
After the indoor rearing experiment according to water temperature conditions, the animals were gradually acclimated from the experimental water temperature to wild water temperature for two weeks and then reared in the wild. The rearing site was identical to the location reported in previous studies of Lee et al. (2014) and Jeon et al. (2024) where T. granosa was collected. The rearing period in the wild was 3 months. In order to minimize the escape of experimental individuals, a net was installed in the same way as in Lee et al. (2014).
Data from the KHOA (2013) were used to obtain water temperature and salinity profiles in the study area. The cumulative water temperature was calculated following the method described by Uki and Kikuchi (1984).
3. Sex ratio
The sex ratio was calculated as follows and expressed as the percentage (%) of females.
Sex ratio = Female (n):Male (n)
Female (%) = [Female / Female + Male] × 100
Male (%) = [Male / Female + Male] × 100
4. Histological analysis
In addition to microscopic analysis, histological techniques were also used to confirm the sex of each specimen. Specimen preparation for light microscopy was performed according to Lee et al. (2014). The clams were dissected, and their visceral mass, which included the gonad, was fixed in aqueous Bouin’s solution for 18 h and rinsed in running water for 24 h and then dehydrated through a graded ethanol series (70-100%). The preparations were then embedded in paraplast (McCormick, U.S.A.). Embedded tissues were sectioned at 4-6 μm thickness using a microtome (RM2235, Leica, Germany). Samples were stained with Mayer's hematoxylin-0.5% eosin (H-E) stain.
5. Statistical analysis
Descriptive and comparative statistical analyses were performed using IBM SPSS Statistics 24.0 (IBM Corp., U.S.A.). Sex ratio was assessed by the x2 (Chi-square) t-test. In all cases significance was established at p<0.05.
Results
1. Water temperature and salinity in the study area
The average water temperature at the study area was 14.1℃ (5.8-26.4℃), and displayed trends similar to historical records. The average salinity was 30.3 psu (22.2-33.7 psu).
During the indoor rearing experiment, the average water temperature in the wild was 6.3℃, and the average water temperatures in January and February 2013 were 6.1℃ and 6.8℃, respectively (Fig. 2). During the 5 weeks experimental period in the indoor aquarium, the cumulative water temperature in the wild was 219.3℃ (Table 1).
2. Survival rate
The survival rates of T. granosa reared at four different water temperature conditions (5.0, 7.0, 9.0, and 11.0℃) for 5 weeks in an indoor aquarium were 60.0 (n=60/100), 85.0 (n=85/100), 100 (n=100/100) and 99.0% (n=99/100), respectively. The survival rates of T. granosa reared in the wild for 3 months after experiencing different water temperature conditions in an indoor aquarium were 66.7 (n=40/60), 80.0 (n=68/85), 95.0 (n=95/100) and 99.0% (n=98/99), respectively.
3. Sex ratio
The sex ratio (F:M) in the wild was 1:1.3 (female 43.5%). In experimental groups that experienced different water temperature conditions (5.0, 7.0, 9.0 and 11.0℃) in an indoor aquarium, the sex ratios were 1:0.7 (60.0%), 1:1.2 (45.6%), 1:1.5 (40.0%) and 1:1.5 (40.8%), respectively. The sex ratio showed a higher proportion of males under high temperature conditions than under low temperature conditions (Table 1). A significant difference in the sex ratio was detected at 11.0℃. The correlation between the water temperature and sex ratio was calculated as R2=0.7748 (Fig. 3).
Discussion
In marine bivalves sex determination, sexual maturation and gametogenesis is regulated by exogenous factors, and among the most studied are temperature and food availability in the water. These two variables, together with endogenous factors, such as the genetic and hormonal load, determine the reproductive cycle of an organism, resulting in a pattern of reproduction for a population (Seed, 1976;Giese and Pearse, 1977;MacDonald and Thompson, 1986;Jaramillo and Navarro, 1995;Thorarinsdóttir and Gunnarsson, 2003;Yusa, 2007;Noor et al., 2024).
Studies on the relationship between temperature and sex determination in animals can be found in reptiles. In some species of reptiles, maintaining a certain temperature during embryonic development increases the proportion of one sex, either female or male (Bull and Vogt, 1979;Pieau and Dorizzi, 1981;Vogt and Bull, 1982;McGaugh et al., 2010). Examples of this relationship between temperature and sex ratio can also be found in the fish, Menidia menidia (Conover and Kynard, 1981), Odontesthes bonariensis (Strüssmann et al., 1996) and the zebrafish, Danio rerio (Sfakianakis et al., 2012;Ribas et al., 2017). In reptiles and fish, temperature-dependent effects on sex determination involve the suppression of aromatase activity at elevated temperatures, reducing the conversion of androgens to estrogens and consequently shifts the endocrine environment toward male differentiation (Devlin and Nagahama, 2002;Matsumoto et al., 2013). In contrast, the physiological mechanisms underlying temperature-related sex change in bivalves remain poorly understood, and further research is needed to clarify how thermal conditions influence their sex differentiation processes.
The rise in ocean water temperatures due to climate warming plays an important role in the reproductive cycle and sex determination process of many marine organisms. Many bivalves have sex determination mechanisms that determine sex according to environmental temperature. In general, high temperatures tend to increase the proportion of males, and low temperatures tend to increase the proportion of females. If the ocean water temperature increases due to climate warming, the sex ratio of shellfish may be skewed toward males (Krueger and Janzen, 2023). A decrease in the proportion of females under high-temperature conditions has also been reported in Pinctada margaritifera (Teaniniuraitemoana et al., 2016).
Sex change in the sequential hermaphroditic bivalve T. granosa (Lee et al., 2014) occurs during the gonadal inactive stage following spawning similar to Crassostrea virginica (Thompson et al., 1996), C. gigas (Park et al., 2012) and Ruditapes philippinarum (Lee et al., 2013).
The reproductive phenomena of aquatic animals are influenced by sex hormones, and continuous exposure to water temperature is necessary to maintain the hormonal conditions required for reproduction (Devlin and Nagahama, 2002;Gosling, 2004;Fabioux et al., 2005;Bezault et al., 2007;Sandra and Norma, 2010).
Therefore, the effect of water temperature on the reproduction of aquatic animals is not temporary but continuous over a long period of time, so application of accumulated water temperature is necessary.
In this study, the sex ratio of T. granosa showed a tendency for the proportion of males to increase as the accumulated water temperature increased during the gonadal inactive stage. This result supports the report of Jeon et al. (2024), who suggested that the difference in accumulated water temperature during the gonadal inactive stage was a factor in explaining the annual sex ratio difference of T. granosa.
This study suggests that in sequential hermaphroditic bivalves that change sex during the gonadal inactive stage, rising water temperatures may decrease the proportion of females and ultimately reduce population size. However, further research is needed to determine whether this result applies to dozens of sequential hermaphroditic bivalves (Lee, 2015) in common or to some species including the clam.
