Molecular pathways associated with the intersex condition in rainbow darter (Etheostoma caeruleum) following exposures to municipal wastewater in the Grand River basin, ON, Canada. Part B.

Rainbow darter (Etheostoma caeruleum; RBD) is a small benthic fish found in North America. This species is sensitive to sewage effluent, and intersex is found in up to 80% of males in near-field areas in the Grand River, Ontario, Canada. To learn more about the molecular signaling cascades associated with intersex, a developed customized oligonucleotide microarray (4×180 K) using next generation sequencing was developed to characterize the transcriptome in the gonad of male and female RBD. Gene expression profiling was performed in males and females from both a reference site and a polluted site. Males with and without intersex condition from the areas closest to effluent outfalls were compared to males and females from a reference site. Microarray analysis revealed that there was increased mRNA abundance for genes associated with oogenesis in intersex males (i.e. the presence of eggs within the testis), and a decrease in mRNA abundance for genes associated with spermatid development. In females exposed to effluent, cell processes related with hatching and ovulation were down-regulated, and genes involved in immune responses were increased in abundance. In the non-intersex males exposed to effluent, cell processes such as sperm cell adhesion were decreased at the transcript level relative to males from the reference site. Microarray analysis revealed that heat shock proteins (HSP) were significantly increased in non-intersex males exposed to effluent; however, HSPs were not differentially expressed in intersex males exposed to the effluent. Genes involved in sex differentiation (sox9, foxl2 and dmrt1) and reproduction (esr1, esrb, ar, vtg, cyp19a1 and cyp11a) were measured in males, females, and intersex individuals. Consistent with the intersex condition, many transcripts showed an intermediate expression level in intersex males when compared to phenotypic males and females. This study improves our knowledge regarding the molecular pathways that underlie the intersex condition and develops a suite of qPCR bioassays in RBD that are able to discriminate pollutant-exposed males without intersex from those males with intersex. Part A of this study reports on the effects of municipal wastewater effluents (MWWEs) on RBD in the Grand River and demonstrates that there are disruptions in higher level endpoints that include altered steroid levels. Here we develop a new tool for assessing and monitoring the intersex condition in RBD in polluted natural environments and begin to characterize gene networks that are associated with the condition.