Search

A method to evaluate the expression of three hormone responsive genes, vitellogenin (estrogens), spiggin (androgens), and an androgen receptor (ARbeta) using real-time PCR in threespine stickleback is presented. Primers were designed from previously characterised spiggin and ARbeta sequences, while a homology cloning strategy was used to isolate a partial gene sequence for stickleback vitellogenin (Vtg). Spiggin mRNA was significantly higher in kidneys of field-caught males compared to females by greater than five orders of magnitude while ARbeta levels were only 1.4-fold higher in males. Female fish had four order of magnitude higher liver Vtg expression than wild-captured males. To determine the sensitivity of these genes to induction by hormones, male and female sticklebacks were exposed to 1, 10 and 100 ng/L of methyltestosterone (MT) or estradiol (E2) in a flow-through exposure system for 7 days. Spiggin induction in females, and Vtg induction in males were both detectable at 10 ng/L of MT and E2, respectively. MT exposure did not induce ARbeta expression in the kidneys of female stickleback. In vitro gonadal steroid hormones production was measured in testes and ovaries of exposed stickleback to compare gene expression endpoints to an endpoint of hormonal reproductive alteration. Reduction in testosterone production in ovaries at all three MT exposure concentrations, and ovarian estradiol synthesis at the 100 ng/L exposure were the only effects observed in the in vitro steroidogenesis for either hormone exposure. Application of these methods to assess both androgenic, estrogenic, and anti-steroidogenic properties of environmental contaminants in a single fish species will be a valuable tool for identifying compounds causing reproductive dysfunction in fishes.

In a previous cumulative impacts assessments of fish populations along the Waikato River, New Zealand, reduced ovary pigmentation was observed in the common bully (Gobiomorphus cotidianus) with an associated reduction in recruitment in the pulp mill-impacted Kopakorahi Arm of Lake Maraetai compared to an upstream reference site. Carotenoid pigments are typically responsible for the yellow to red colouration of fish eggs and a number of functions for these pigments such as pro-vitamins and antioxidants have been proposed. The purpose of the current study was first to quantify the observed pigment reduction and then to establish possible consequences of carotenoid deficiency. Significantly lower levels of all measured carotenoids were observed in the ovaries of fish from below the mill outfall compared to the reference site. Using in vitro incubations of ovarian follicles, it was also found that these fish had a significantly reduced ability to produce the sex steroids testosterone and estradiol. This study demonstrates changes in ovarian carotenoids associated with pulp mill effluent exposure. Significantly reduced biosynthetic capacity of ovarian follicles demonstrates reproductive endocrine disruption potential associated with exposure to this particular effluent. These results may provide an important link between reproductive dysfunction, oxidative stress and food chain modifications in pulp mill-impacted systems.

In this study, the immune reactions of rainbow trout (Oncorhynchus mykiss) were examined, after exposure to 10, 30 and 70% of tertiary-treated municipal sewage effluent for 27 days. Exposures were conducted concurrently with and without an immune challenge using intraperitoneal injections of inactivated Aeromonas salmonicida salmonicida. Due to the time required to prepare and analyse samples, fish sampling was conducted over two consecutive days. There was no trout mortality for any of the experimental treatments. The exposure to effluent increased in vitro lymphocyte proliferation, decreased circulating lymphocytes and increased degrading erythrocytes in peripheral blood samples. Circulating lymphocytes were only decreased in the sham-injected, but not in the A. salmonicida-injected group. In addition to effluent effects, circulating lymphocytes and lymphocyte proliferation were decreased on day 2 of sampling as compared to day 1. Concentration-dependent degradation of erythrocytes was only observed on day 2 of sampling. Capture and removal of trout on day 1 of sampling presumably caused low-level stress that affected some results on day 2. Oxidative burst, phagocytosis, lysozyme, leucocyte populations other than lymphocytes and A. salmonicida-specific IgM production were not affected by exposure to effluent, and of these parameters, only oxidative burst and total leucocytes showed sampling day effects. From these results it can be observed, that with the exception of oxidative burst, those variables affected by effluent exposure were also significantly changed by the low-level sampling stress imposed by staggered sampling. Elevated liver mixed-function oxygenase activity as measured by 7-ethoxyresorufin-O-deethylase activity, and increased bile polycyclic aromatic hydrocarbon (PAH) metabolites were observed in response to sewage effluent exposure. As both PAHs and stress are known immune suppressors, it is difficult to conclude whether or not changes in immune parameters due to effluent exposure were caused by the direct action of chemicals, or were due to a general stress response.