The Society for Integrative and Comparative Biology
Sexual Disruption in the Freshwater Crab (Geothelphusa dehaani)1
1 Nagasaki Prefectural University, 123 Kawashimo-cho, Sasebo City, 858-8580, Nagasaki, Japan
2 Sasebo Public Health Center, 5-17 Takasago-cho, Sasebo City, 857-0042, Nakasaki, Japan
3 Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto City 862-8502, Kumamoto, Japan
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SYNOPSIS |
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To clarify the effect of environmental endocrine disruptors on the induction of secondary sexual characteristics in wild animals, we observed 1,527 male and 1,847 female specimens of sexually mature Japanese freshwater crabs, Geothelphusa dehaani, collected at 12 sample points from 10 rivers in Sasebo City, Nagasaki Prefecture, Japan. The results obtained were as follows:
- Abnormal males, with external appearance of a complete male, but having female genital openings, were observed at frequencies of 8–32% of males collected at all sample points. In contrast to this, all females collected were normal.
- The frequency of abnormal males increased significantly with the increase in individual carapace width.
This species inhabits mountain streams in Japan, which are free from tributyltin that is known to induce the sexual mosaic phenomenon in mollusks. The streams are most likely polluted by some agricultural chemicals and chemicals present in waste gases from cars and especially particulate materials from diesel engines. The clear relationship of the frequencies of abnormal males to size (or age) suggests that the sexual mosaic phenomenon is induced not only at early stages of male development but after sexual maturation, in response to long-term exposure to chemical(s) during the whole life cycle of males. The definitive causal factors and mechanisms inducing sexual mosaics in this species are still unclear.
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INTRODUCTION |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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Recently, a number of studies have been performed worldwide that have examined endocrine disruptors (EDs) and their interaction with the development and function of various systems in animals and humans (Colborn et al., 1993
, 1996; Jobling et al., 1998). As invertebrates account for roughly 95% of the known species of animals on our planet (DeFur et al., 1999), evaluation of the adverse effects of ED exposure on invertebrates is important, in order to develop systems to test the health of any given environment. Field studies on imposex and organotin (butyltin and phenyltin) contamination in Japanese molluscs (the rock shell, Thais clavigera, and the Pacific oyster, Crassostrea gigas) conducted along the coast of both the Seto Inland Sea and in the Sanriku region, Japan, suggested that the percentage occurrence of imposex in the rock shell, T. clavigera, was 100% at all sites surveyed (Horiguchi et al., 1998). During a one-year survey, beginning in September 1990, six abnormal Geothelphusa dehaani were found out of 263 individuals collected in the Taniyama river, a small stream in north Kyushu, Fukuoka, Japan (Araki and Matsuura, 1995). The imposex phenomenon has been found to occur in crabs which inhabit tributyltin-uncontaminated mountain streams in Japan (Takahashi et al., 2000). To clarify the effect of EDs on the induction of secondary sexual characteristics of wild animals, we observed 1,527 males and 1,847 females of sexually mature Japanese freshwater crabs, G. dehaani, collected at 12 sample points from 10 rivers in Sasebo City, Nagasaki Prefecture, Japan. Crabs with the female genital opening gonopore, with incomplete male genitals were categorized as masculinized females; in addition, crabs possessing complete male genitals with the female genital opening gonopore were categorized as feminized males.
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MATERIALS AND METHODS |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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We observed 1,527 males and 1,847 females of sexually mature Japanese freshwater crabs, G. dehaani, collected at 12 sample points (Point A–L) from 10 rivers (Sasebo City, Nagasaki Prefecture, Japan) between August 2000 and August 2001. A map depicting the research sites is shown in Figure 1. Among the collection sites, Yunoki Bridge on the main stream of the Ainoura River (Point D), Takao River, a tributary of the Ainoura River (Point F), and the Hiji River, a tributary of the Komori River (Point J) are located on the outskirts of Sasebo city where the rivers run through a residential area. In particular, samples collected from points D and F were found to be foul smelling, which indicated that these sites were eutrophic. The other 8 points are located in the mountains, in the upper reaches of rivers. Paddies and farms are located upstream of every river. A fruit farm (tangerine) is located upstream of the Horito River (Point K), a tributary of the Miyamura River and also located upstream of the Eshimo River (Point L). Golf courses are located in the foothills of Mt. Iimori, close to the Ogawachi River (Point A), a tributary of the Ainoura River, and at an area several hundred meters away to the northwest of the Komori River (Point I and Point J). An industrial waste disposal facility is located upstream of the Kubonita River (Point E). Points B and C are located in the upper reaches of the Ainoura River and points G and H are located in the upper reaches of the Sasebo River. A few private houses are dotted within the inter-mountain agricultural community area (Points B, C, G and H).
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Observation of abnormality in crabs: The carapace sizes of collected crabs were larger than 10.9 mm and thus considered to be sexually mature. The external genital organs of crabs were checked for abnormality under a microscope. Crabs with the female genital opening gonopore, with incomplete male genitals were categorized as masculinized females. Crabs possessing complete male genitals with the female genital opening gonopore were categorized as feminized males. The individual crabs were released back into their original territory to avoid destruction of natural conditions as a result of this field study.
Differences were considered significant at P < 0.05. All statistical analyses were performed using SPSS10.0J (SPSS Ltd. Japan).
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RESULTS AND DISCUSSION |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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The results concerning the observation of 1,527 males and 1,847 females of sexually mature Japanese freshwater crabs (the carapace sizes of collected crabs are larger than 10.9 mm) from the river of Sasebo city are summarized in Table 1. Although all females collected were normal, a number of male freshwater crabs with the female genital opening gonopore were found to have sexual disruption (Fig. 2). These abnormal males that looked externally like a complete male, but having female genital openings, were observed at a frequencies of 8–32% for males at all sample points (Table 1). Significant differences were observed between the frequency of abnormal males and the sites where they were collected (P < 0.05).
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As crabs were collected from a confined geographical area, such as the Sasebo city area in this field study, it was thought that environmental factors such as temperature were approximately equal and thus growth rates were considered to be uniform. Given this factor, the relationship between the frequency of abnormal males and their carapace width was evaluated (Table 2). The results indicated that the frequency of abnormal males increased with their growth (P < 0.05).
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The intersex phenomenon is not attributed as a normal character for this species, but as a causal factor. Rhizocephalan or epicaridean parasites sometimes feminize the male brachyuran host; e.g., the male chelipeds become smaller, and the abdominal width becomes larger (parasitic castration). However, no parasites were detected in the present population. Takahashi et al. (2000)
reported that no abnormal individuals were found in unpolluted sites such as the Shiratani river, about 1 km outside of the World Heritage Forest, Yakushima Island, Japan. The abnormality of crabs in our study site may be caused by changes in external conditions such as chemical pollution. This species inhabits mountain streams in Japan. The points in this field study are also located in the inter mountain basin and this area is free from tributyltin, known to induce the sexual mosaic phenomenon in mollusks. These areas are related to the diversified environment where chemicals can be delivered from places such as agricultural fields, fruits farm, golf courses, industrial waste disposal facilities and traffic-congested roads. The results indicate that these streams are probably polluted by certain agricultural chemicals and chemicals found in waste gases from cars and most likely in particulate materials from diesel engines. The effects seen in the crabs may not be caused by a single factor, but may be caused by synergistic effects of multiple agents. Unlike common brachyurans, G. dehaani progress through their larval stages in the capsule of the egg and hatch out as a juvenile crab. As the growth of G. dehaani is rather slow (it takes 3 years to reach maturation size), some chemicals may accumulate in the yolk of the large egg (>3 mm in diameter), which is over 1,000 times as large as that of a similar size of sea shore crab, Hemigrapsus sanguinenus. This may be why this phenomenon is observed in this species.
The clear relationship of the frequencies of abnormal males to size (or age) suggests that the sexual mosaic is induced, not only at early stages of male development, but after sexual maturation in response to long-term exposure to chemical(s) during the whole life cycle of males. The male crab may start their feminization (gynandromorphism, sex-mosaic) after some chemical concentrations in the crab crosses a critical level (even if these events are caused after their sex maturation). However, the definitive causal factors and mechanisms inducing sexual mosaics in this species are unclear. Effects of EDs concerning reproduction by these abnormal males are also unclear.
In this study, the frequency of abnormality in male crabs from Sasebo city was checked for uniformity. We also conducted a test of independence between the occurrence of abnormal males and body size. It was found that the frequency of abnormal males was not uniform from Sasebo city at a significance level of 5% (Table 1), and that there were size dependent differences (Table 2). The frequency of abnormal males increased significantly with the increase in individual carapace width. Concerning future research, water samples will be taken from the points sampled in this study for chemical analysis. Freshwater crabs in other areas will also be checked for sexual disruption.
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FOOTNOTES |
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1 From the Symposium EcoPhysiology and Conservation: The Contribution of Endocrinolgy and Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
2 E-mail: arizono{at}pu-kumamoto.ac.jp
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References |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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Araki, A., and S. Matsuura. 1995. Gynandromorph of a freshwater crab, Geothelphusa dehaani. Sci. Bull. Fac. Agr. Kyushu Univ. 49:133–137.
Colborn, T., D. Dumanoshi, and J. P. Myers. 1996. Our stolen future. Dutton, New York, USA.
Colborn, T., F. S. vom Saal, and A. M. Soto. 1993. Developmental effects of EDCs in wildlife and humans. Environ. Health Perspect, 101:378-384.[ISI][Medline]
DeFur, P. L., M. Crane, C. Ingershold, and L. Tattersfield. 1999. Endocrine disruption in invertebrates: Endocrinology, testing, and assessment. SETAC, Pensacola, Forida, USA.
Jobling, S., M. Nolan, C. R. Tyler, G. Brighty, and J. P. Sumpter. 1998. Widespread sexual disruption in wild fish. Environ. Toxicol. Chem, 19:2812-2820.[CrossRef]
Horiguchi, T., C. Hyeon-Seo, H. Shiraishi, Y. Shibata, M. Soma, M. Morita, and M. Shimizu. 1998. Field studies on imposex and organotin accumulation in the rock shell, Thais clavigera, from the Seto Inland Sea and the Sanriku region, Japan. Sci. Total Environ, 18:214:65-70.
Takahashi, T., A. Araki, Y. Nomura, M. Koga, and K. Arizono. 2000. The occurrence of dual-gender imposex in Japanese freshwater crab. J. Health Sci, 46:376-379.
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