Disturbed Sexual Characteristics in Male Mosquitofish (Gambusia Holbrooki) from a Lake Contaminated with Endocrine Disruptors. (Research)

Toft, Gunnar, Edwards, Thea M., Baatrup, Erik, Guillette, Louis J., Jr., Environmental Health Perspectives

Previous laboratory studies have demonstrated that estrogenic and antiandrogenic chemicals can alter several sexual characteristics in male poeciliid fishes. Whether similar disturbances occur under field conditions remains to be confirmed. Lake Apopka, Florida, is contaminated with numerous chemicals, some of which possess endocrine-disrupting activity. Male mosquitofish (Gambusia holbrookl) were collected monthly from December 2000 through May 2001 from Lake Apopka and two nearby reference lakes, Orange Lake and Lake Woodruff National Wildlife Refuge. Selected sexual characteristics were compared temporally and among lakes during the collection period. Male fish from Lake Apopka had slightly shorter gonopodia and on average 32 and 47% fewer sperm cells per milligram testis, when compared with the fish collected from Orange Lake and Lake Woodruff, respectively. The testes weights increased markedly during spring, with significantly smaller testes in fish from Lake Apopka than from Orange Lake, but surprisingly, the smallest testes occurred in males obtained from the Lake Woodruff population. The highest liver weights were found in the Lake Apopka population. Whole-body concentrations of testosterone and estradiol varied among months; the peak testosterone concentration occurred in January and was significantly lower in male fish from Lake Apopka compared with Orange Lake. The intensity of male courtship behavior was highly correlated to body testosterone concentration, but no statistically significant differences in sexual behavior among the lakes were found. We conclude that sexual characteristics of relevance to male reproductive capacity are altered in the Lake Apopka mosquitofish population, and we discuss the presence of chemicals with antiandrogenic effects in Lake Apopka as a possible cause of the observed alterations. Key words: antiandrogen, courtship behavior, endocrine disruption, estradiol, mosquitofish, sex characters, sperm count, testosterone. Environ Health Perspect 111:695-701 (2003). doi:10.1289/ehp.6022 available via http://dx.doi.org/[Online 23 January 2003]

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Reproductive abnormalities have been observed in several wildlife populations living in polluted areas (Guillette et al. 1996; Howell et al. 1980; Jobling et al. 1998). In laboratory studies, it has been confirmed that environmental contaminants with endocrine-disrupting properties (EDCs) can disturb the development and expression of sexual characteristics in fish (Gimeno et al. 1996; Gray and Metcalfe 1997), amphibians (Hayes et al. 2002), reptiles (Crain et al. 1999; Willingham and Crews 1999; Willingham et al. 2000), birds (Feyk and Giesy 1998), and mammals (Gray et al. 1994, Sharpe et al. 1995). However, the extent to which the sexual characteristics and reproductive capabilities of natural populations are impacted by these EDCs is still not well understood.

Because of a chemical spill in 1980, Lake Apopka, Florida, is extensively polluted with dicofol, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and its metabolites 1,1-dichloro-2,2-bis (p-chlorophenyl) ethane (DDD) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE). In addition, various pesticides, such as toxaphene, trans-nonachlor, dieldrin, and aldrin have been released into the lake from the surrounding farmland and are commonly found in the wildlife living in this area. A series of reproductive abnormalities have been reported in alligators living in this lake (Guillette et al. 2000), including reduced clutch size, altered gonadal morphology, smaller penis size, and altered hormone concentrations. Furthermore, elevated levels of environmental contaminants have been found in eggs and plasma of alligators living in Lake Apopka (Guillette et al. 1999; Heinz et al. 1991). It has been suggested that the elevated levels of contaminants in Lake Apopka cause these reproductive abnormalities (Guillette et al. 1994). However, the long generation time and large body size of alligators makes it a protracted and difficult task to verify the effects of chemical exposure in a controlled laboratory setting.

Mosquitofish and other poeciliid fishes have a much shorter generation time, and they are easily kept and handled in the laboratory. Furthermore, poeciliids have several readily measurable sexual characteristics that are affected by exposure to chemicals with estrogenic or antiandrogenic activity (Baatrup and Junge 2001; Bayley et al. 1999; Dreze et al. 2000; Haubruge et al. 2000; Toft and Baatrup 2001). Male mosquitofish have mature sperm cells in their testes year-round (Fraile et al. 1992), and they have an extended reproductive period dependent on photoperiod and temperature (Fraile et al. 1994; Koya et al. 1998). Furthermore, mosquitofish are a very abundant freshwater fish in Florida, which makes it possible to collect specimens and compare the effects on fish living in reference and contaminated lakes. Sperm production and male courtship behavior is generally stimulated by androgens in fish (Borg 1994; Liley and Stacey 1983; Zentel 1988), but estrogens are also likely to influence the expression of these characteristics. Thus, testosterone is aromatized to estradiol locally in the brain and in the testes, where it is believed to be involved in the regulation of male behavior and testis function, respectively (Loomis and Thomas 1999; Nelson 2000; Pasmanik and Callard 1985). In many teleost fish, 11-ketotestosterone (11-KT) is a major androgen. Sperry and Thomas (1999a, 1999b) found two fish androgen receptor subtypes in Atlantic croaker (Micropogonias undulatus) and kelp bass (Paralabrax clathratus), which both have great affinity for testosterone, whereas 11-KT only binds one of these receptors. This indicates a major physiologic role for testosterone. Borg (1994) suggested that the production of 11-KT is very low or absent in poeciliid fishes (including mosquitofish) because very little or no 11-KT is produced in the testis or found in the blood of these fish. The testosterone level is usually similar in male and female teleosts (Borg 1994), but we found a significantly higher testosterone level in male mosquitofish (Toft et al. Unpublished data), suggesting that testosterone could play a key role in the control of male reproductive processes in these fish.

The purpose of this study was to compare male sexual characteristics in mosquitofish (Gambusia holbrooki) living in the contaminated Lake Apopka and several reference lakes. The study was conducted by monthly collections over 6 months to follow possible differences in the characteristics between lakes through a nonreproductive and a reproductive period. Furthermore, we wanted to compare these effects with previous observations of poeciliid fishes exposed to EDCs in the laboratory.

Materials and Methods

Animals and collections. Sexually mature male eastern mosquitofish (Gambusia holbrooki) were collected from three lakes in Florida using a 1-[m.sup.2] dip net. The criterion of hook development on the gonopodium (Angus et al. 2001) was used to determine if the fish were fully mature. The fish were collected monthly from December 2000 through May 2001 from the highly polluted Lake Apopka at the north shore near the mouth of the Apopka Beaudair Canal (28[degrees]67'N, 81[degrees]68'W) and from the reference site Orange Lake (29[degrees]46'N, 82[degrees]37'W). From March through May, Spring Garden Lake in the Lake Woodruff National Wildlife Refuge (29[degrees]12'N, 81[degrees]37'W) was included as a second reference site because of a very low water level in Orange Lake and the risk of dry-out. The selection of the study sites was based on a study by Guillette et al. (1999) demonstrating high plasma concentrations of pesticides in alligators from Lake Apopka and low contaminant levels in animals from Orange Lake and Lake Woodruff National Wildlife Refuge.

At each of the monthly collections, approximately 60 male mosquitofish were collected from each lake. These fish were divided into two subsamples in the collections from January, February, and April, whereas three subsamples were made from the December, March, and May collections. The first subsample, consisting of 10-34 (mode = 12) individuals from each …

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Publication information: Article title: Disturbed Sexual Characteristics in Male Mosquitofish (Gambusia Holbrooki) from a Lake Contaminated with Endocrine Disruptors. (Research). Contributors: Toft, Gunnar - Author, Edwards, Thea M. - Author, Baatrup, Erik - Author, Guillette, Louis J., Jr. - Author. Journal title: Environmental Health Perspectives. Volume: 111. Issue: 5 Publication date: May 2003. Page number: 695+. © 2006 National Institute of Environmental Health Sciences. COPYRIGHT 2003 Gale Group.

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