Effect of elevated embryonic incubation temperature on the temperature preference of juvenile lake (Coregonus clupeaformis) and round whitefish (Prosopium cylindraceum).

Anthropogenic impacts can lead to increased temperatures in freshwater environments through thermal effluent and climate change. Thermal preference of aquatic organisms can be modulated by abiotic and biotic factors including environmental temperature. Whether increased temperature during embryogenesis can lead to long-term alterations in thermal preference has not been explicitly tested in native freshwater species. Lake (Coregonus clupeaformis) and round (Prosopium cylindraceum) whitefish were incubated at natural and elevated temperatures until hatching, following which, all groups were moved to common garden conditions (15°C) during the post-hatching stage. Temperature preference was determined at 8 months (Lake whitefish only) and 12 months of age (both species) using a shuttle box system. Round whitefish preferred a cooler temperature when incubated at 2 and 6°C compared with 0.5°C. Lake whitefish had similar temperature preferences regardless of age, weight and incubation temperature. These results reveal that temperature preference in freshwater fish can be programmed during early development, and that round whitefish may be more sensitive to incubation temperature. This study highlights the effects that small increases in temperature caused by anthropogenic impacts may have on cold-adapted freshwater fish.

Intersex manifestation in the rainbow darter (Etheostoma caeruleum): Are adult male fish susceptible to developing and recovering from intersex after exposure to endocrine active compounds?

For over a decade, intersex has been observed in rainbow darter (RD) (Etheostoma caeruleum) populations living downstream wastewater treatment plants (WWTPs) in the Grand River, Ontario, Canada. To further our understanding of intersex development in adult male fish, the current study addressed three objectives: i) can intersex be induced in adult male fish, ii) is there a specific window of exposure when adult male fish are more susceptible to developing intersex, and iii) can pre-exposed adult male fish recover from intersex? To assess intersex induction in adult male fish, wild male RD were exposed in the laboratory for 22 weeks (during periods of spawning, gonadal regression, and gonadal recrudescence) to environmentally relevant concentrations of 17α-ethinylestradiol (EE2) including nominal 0, 1, and 10 ng/L. Intersex rates and severity at 10 ng/L EE2 were similar to those observed historically in adult male populations living downstream WWTPs in the Grand River and confirmed previous predictions that 1-10 ng/L EE2 would cause these adverse effects. To assess a window of sensitivity in developing intersex, male RD were exposed to nominal 0, 1 or 10 ng/L EE2 for 4 weeks during three different periods of gonadal development, including (i) spawning, (ii) early recrudescence and (iii) late recrudescence. These short-term exposures revealed that intersex incidence and severity were greater when RD were exposed while gonads were fully developed (during spawning) compared to periods of recrudescence. To assess if RD recover from intersex, wild fish were collected downstream WWTPs in the Grand River and assessed for intersex both before and after a 22-week recovery period in clean water that included gonadal regression and recrudescence. Results showed that fish did not recover from intersex, with intersex rates and severity similar to those both before and after the transition to clean water. This study further advances our knowledge on intersex manifestation in adult male fish including their sensitivity to endocrine active compounds during different periods of their annual reproductive cycle and their limited ability to recover from intersex after onset of the condition.

Wild fish responses to wastewater treatment plant upgrades in the Grand River, Ontario.

Municipal wastewater treatment plant (WWTP) effluent is one of several point sources of contaminants (nutrients, pharmaceuticals, estrogens, etc.) which can lead to adverse responses in aquatic life. Studies of WWTP effluent impacts on rainbow darter (Etheostoma caeruleum) collected downstream of WWTPs in the Grand River, Ontario have reported disruption at multiple levels of biological organization, including altered vitellogenin gene expression, lower levels of in vitro steroid production, and high frequency of intersex. However, major upgrades have occurred at treatment plants in the central Grand River over the last decade. Treatment upgrades to the Waterloo WWTP were initiated in 2009 but due to construction delays, the upgrades came fully on-line in 2017/2018. Responses in rainbow darter have been followed at sites associated with the outfall consistently over this entire time period. The treatment plant upgrade resulted in nitrification of effluent, and once complete there was a major reduction in effluent ammonia, selected pharmaceuticals, and estrogenicity. This study compared several key responses in rainbow darter associated with the Waterloo WWTP outfall prior to and post upgrades. Stable isotopes signatures in fish were used to track exposure to effluent and changed dramatically over time, corresponding to the effluent quality. Disruptions in in vitro steroid production and intersex in the darters that had been identified prior to the upgrades were no longer statistically different from the upstream reference sites after the upgrades. Although annual variations in water temperature and flow can potentially mask or exacerbate the effects of the WWTP effluent, major capital investments in wastewater treatment targeted at improving effluent quality have corresponded with the reduction of adverse responses in fish in the receiving environment.

Corticotropin-releasing factor regulates caspase-3 and may protect developing zebrafish from stress-induced apoptosis.

The corticotropin-releasing factor (CRF) system is expressed in the earliest stages of zebrafish development, long before its canonical function in the endocrine stress response is realized, and yet its function during embryogenesis is unknown. We tested the hypothesis that CRF protects embryos from stress-induced apoptosis. Here we confirm that a 1 h heat shock applied at either 6 h post-fertilization (hpf) or 30 hpf elicits an increase in caspase-3 activity, a key effector of apoptosis. Temporal changes in the expression of crf and its binding protein (crf-bp) during recovery from heat shock indicate that the CRF system is responsive to stressors experienced as early as gastrulation. Next, we heat shocked embryos that were microinjected with crf mRNA, and showed that caspase-3 induction is significantly reduced in embryos that overexpress CRF relative to control embryos. In addition, incubating embryos in the presence of the CRF receptor type 1 (CRF-R1) antagonist, antalarmin, during recovery from heat shock significantly increased caspase-3 activity, suggesting that CRF regulates caspase-3 via a CRF-R1-dependent pathway. Finally, we show that most heat shock-induced mortality occurred during the first hour of recovery, long before a significant increase in caspase-3 activity was detected. Indeed, the delayed caspase-3 induction coincided with a mortality plateau, and neither CRF overexpression nor antalarmin treatment altered heat shock induced mortality, supporting previous in vitro evidence that CRF-mediated cytoprotection occurs through the slow and tightly controlled apoptotic pathway. This study provides novel in vivo evidence that CRF regulates stress-induced apoptosis in a vertebrate model species, and demonstrates for the first time a function for the CRF system in early development that precedes its role in the endocrine stress response.

Modeling the exposure of wild fish to endocrine active chemicals: Potential linkages of total estrogenicity to field-observed intersex.

Decades of studies on endocrine disruption have suggested the need to manage the release of key estrogens from municipal wastewater treatment plants (WWTP). However, the proposed thresholds are below the detection limits of most routine chemical analysis, thereby restricting the ability of watershed managers to assess the environmental exposure appropriately. In this study, we demonstrated the utility of a mechanistic model to address the data gaps on estrogen exposure. Concentrations of the prominent estrogenic contaminants in wastewaters (estrone, estradiol, and ethinylestradiol) were simulated in the Grand River in southern Ontario (Canada) for nine years, including a period when major WWTP upgrades occurred. The predicted concentrations expressed as total estrogenicity (E2 equivalent concentrations) were contrasted to a key estrogenic response (i.e., intersex) in rainbow darter (Etheostoma caeruleum), a wild sentinel fish species. A predicted total estrogenicity in the river of ≥10 ng/L E2 equivalents was associated with high intersex incidence and severity, whereas concentrations <0.1 ng/L E2 equivalents were associated with minimal intersex expression. Exposure to a predicted river concentration of 0.4 ng/L E2 equivalents, the environmental quality standard (EQS) proposed by the European Union for estradiol, was associated with 34% (95% CI:30-38) intersex incidence and a very low severity score of 0.6 (95% CI:0.5-0.7). This exposure is not predicted to cause adverse effects in rainbow darter. The analyses completed in this study were only based on the predicted presence of three major estrogens (E1, E2, EE2), so caution must be exercised when interpreting the results. Nevertheless, this study illustrates the use of models for exposure assessment, especially when measured data are not available.

Assessing recovery of in vitro steroid production in male rainbow darter (Etheostoma caeruleum) in response to municipal wastewater treatment plant infrastructure changes.

The present study examined in vitro 11-ketotestosterone and testosterone production by the testes of rainbow darter (Etheostoma caeruleum) collected from selected reference sites and downstream of 2 municipal wastewater treatment plants (MWWTPs; Waterloo and Kitchener) on the central Grand River (Ontario, Canada), over a 6-yr period (2011-2016). The main objective was to investigate if infrastructure upgrades at the Kitchener MWWTP in 2012 resulted in a recovery of this response in the post-upgrade period (2013-2016). Two supporting studies showed that the fall season is appropriate for measuring in vitro sex steroid production because it provides stable detection of steroid patterns, and that the sample handling practiced in the present study did not introduce a bias. Infrastructure upgrades of the Kitchener MWWTP resulted in significant reductions in ammonia and estrogenicity. After the upgrades, 11-ketotestosterone production by MWWTP-exposed fish increased in 2013 and it continued to recover throughout the study period of 2014 through 2016, returning to levels measured in reference fish. Testosterone production was less sensitive and it lacked consistency. The Waterloo MWWTP underwent some minor upgrades but the level of ammonia and estrogenicity remained variable over time. The production of 11-ketotestosterone and testosterone in rainbow darter below the Waterloo MWWTP was variable and without a clear recovery pattern over the course of the present study. The results of the present study demonstrated that measuring production of sex steroids (especially 11-ketotestosterone) over multiple years can be relevant for assessing responses in fish to environmental changes such as those resulting from major infrastructure upgrades. Environ Toxicol Chem 2018;37:501-514. © 2017 SETAC.

Returning to normal? Assessing transcriptome recovery over time in male rainbow darter (Etheostoma caeruleum) liver in response to wastewater-treatment plant upgrades.

The present study measured hepatic transcriptome responses in male rainbow darter (Etheostoma caeruleum) exposed to 2 municipal wastewater-treatment plants (MWWTPs; Kitchener and Waterloo) over 4 fall seasons (2011-2014) in the Grand River (Ontario, Canada). The overall goal was to determine if upgrades at the Kitchener MWWTP (in 2012) resulted in transcriptome responses indicative of improved effluent quality. The number of differentially expressed probes in fish downstream of the Kitchener outfall (904-1223) remained comparable to that downstream of Waterloo (767-3867). Noteworthy was that year and the interaction of year and site explained variability in more than twice the number of transcripts than site alone, suggesting that year and the interaction of year and site had a greater effect on the transcriptome than site alone. Gene set enrichment analysis revealed a gradual reduction in the number of gene ontologies over time at exposure sites, which corresponded with lower contaminant load. Subnetwork enrichment analysis revealed that there were noticeable shifts in the cell pathways differently expressed in the liver preupgrade and postupgrade. The dominant pathways altered preupgrade were related to genetic modifications and cell division, whereas postupgrade they were associated with the immune system, reproduction, and biochemical responses. Molecular pathways were dynamic over time, and following the upgrades, there was little evidence that gene expression profiles in fish collected from high-impact sites postupgrade were more similar to those in fish collected from reference site. Environ Toxicol Chem 2017;36:2108-2122. © 2017 SETAC.

Reduction of Intersex in a Wild Fish Population in Response to Major Municipal Wastewater Treatment Plant Upgrades.

Intersex in fish downstream of municipal wastewater treatment plants (MWWTPs) is a global concern. Consistent high rates of intersex in male rainbow darter (Etheostoma caeruleum) have been reported for several years in the Grand River, in southern Ontario, Canada, in close proximity to two MWWTPs. The larger MWWTP (Kitchener) recently underwent upgrades that included the conversion from a carbonaceous activated sludge to nitrifying activated sludge treatment process. This created a unique opportunity to assess whether upgrades designed to improve effluent quality could also remediate the intersex previously observed in wild fish. Multiple years (2007-2012) of intersex data on male rainbow darter collected before the upgrades at sites associated with the MWWTP outfall were compared with intersex data collected in postupgrade years (2013-2015). These upgrades resulted in a reduction from 70 to 100% intersex incidence (preupgrade) to <10% in postupgrade years. Although the cause of intersex remains unknown, indicators of effluent quality including nutrients, pharmaceuticals, and estrogenicity improved in the effluent after the upgrades. This study demonstrated that investment in MWWTP upgrades improved effluent quality and was associated with an immediate change in biological responses in the receiving environment. This is an important finding considering the tremendous cost of wastewater infrastructure.

An Assessment of the Spatial and Temporal Variability of Biological Responses to Municipal Wastewater Effluent in Rainbow Darter (Etheostoma caeruleum) Collected along an Urban Gradient.

Municipal wastewater effluent (MWWE) and its constituents, such as chemicals of emerging concern, pose a potential threat to the sustainability of fish populations by disrupting key endocrine functions in aquatic organisms. While studies have demonstrated changes in biological markers of exposure of aquatic organisms to groups of chemicals of emerging concern, the variability of these markers over time has not been sufficiently described in wild fish species. The aim of this study was to assess the spatial and temporal variability of biological markers in response to MWWE exposure and to test the consistency of these responses between seasons and among years. Rainbow darter (Etheostoma caeruleum) were collected in spring and fall seasons over a 5-year period in the Grand River, Ontario, Canada. In addition to surface water chemistry (nutrients and selected pharmaceuticals), measures were taken across levels of biological organization in rainbow darter. The measurements of hormone production, gonad development, and intersex severity were temporally consistent and suggested impaired reproduction in male fish collected downstream of MWWE outfalls. In contrast, ovarian development and hormone production in females appeared to be influenced more by urbanization than MWWE. Measures of gene expression and somatic indices were highly variable between sites and years, respectively, and were inconclusive in terms of the impacts of MWWE overall. Robust biomonitoring programs must consider these factors in both the design and interpretation of results, especially when spatial and temporal sampling of biological endpoints is limited. Assessing the effects of contaminants and other stressors on fish in watersheds would be greatly enhanced by an approach that considers natural variability in the endpoints being measured.

Severe intersex is predictive of poor fertilization success in populations of rainbow darter (Etheostoma caeruleum).

Municipal wastewater effluent (MWWE) contains emerging contaminants such as pharmaceuticals and personal care products (PPCPs) that have estrogenic properties. PPCPs are thought to be responsible for feminization of male fish in heavily urbanized areas around the globe. While many observations of feminized male fish have been made, the impact of feminization on reproductive success is not well understood. To address this lack of knowledge of the impacts of feminization, we examined the reproductive fitness of rainbow darter (Etheostoma caeruleum), a fish that is also known to have been feminized in some reaches of the Grand River, Kitchener-Waterloo, Ontario, Canada. In order to assess their reproductive health, somatic indices, gonadal steroid production, fecundity, and histological severity of intersex were measured in male rainbow darter collected through an urban gradient. Reproductive fitness was assessed by stripping milt and eggs from wild spawning fish, fertilizing eggs manually, and rearing embryos to hatch. The fertilization success and survival of embryos to hatch were compared among sites. In this study, it was found that rainbow darter collected at sites near a large municipal wastewater treatment plant (MWWTP) had decreased gonad size, increased severity of intersex, and decreased androgen production relative to other sites. Fish collected near the largest MWWTP also had lower fertilization success and survival to hatch. In contrast, fish collected near a second MWWTP farther upstream had comparable fertilization success, but lower survival to hatch relative to the upstream rural reference site. Intersex severity was negatively correlated with fertilization success, but not survival to hatch, suggesting that intersex is a good indicator of a population's fertilization success. Further investigation is required in order to determine if feminization will impact the sustainability of wild populations of fish.

Ontogeny of the corticotropin-releasing factor system in rainbow trout and differential effects of hypoxia on the endocrine and cellular stress responses during development.

To further our understanding of the development of the stress axis and the responsiveness of embryonic and larval fish to environmental stressors, this study examined the ontogeny of whole-body cortisol levels and of the corticotropin-releasing factor (CRF) system in rainbow trout, as well as the endocrine and cellular stress responses to hypoxia. After depletion of a maternal deposit, de novo synthesis of cortisol increased exponentially between the 'eyed' stage and first feeding. Whole body CRF mRNA levels dominated over those of the related peptide urotensin I (UI) from hatch through complete yolk sac absorption. The mRNA levels of CRF-binding protein (CRF-BP) closely paralleled those of CRF and UI throughout ontogeny except at first feeding when an increase in CRF gene expression was not matched by change in CRF-BP transcript abundance. In the hypoxia challenge, fish were exposed to 15% O(2) saturation for either 90 min or 24h at three key developmental stages: hatch, swim up and first feeding. While the embryos were unaffected, chronic hypoxia elicited a transient 2-fold increase in whole-body cortisol levels in the larval stages. The hypoxia challenge also generally suppressed the mRNA levels of CRF and CRF-BP, had no effect on the expression of UI, but had a marked stimulatory effect on heat shock protein 70 (Hsp70) gene expression. Taken together, these results suggest a role for the CRF system in the ontogenic regulation of corticosteroidogenesis and show that hypoxia has developmental stage-specific effects on the endocrine and cellular stress responses in rainbow trout.

Differential effects of 17ß-estradiol and 11-ketotestosterone on the endocrine stress response in zebrafish (Danio rerio).

Sexually dimorphic stress responses are present in species across all vertebrate taxa and it has been suggested that these effects are mediated by circulating sex steroids. While a few species of fish have been identified as having a sexually dimorphic stress response, there is conflicting evidence as to the effects of sex steroids on the stress axis. In this study, we tested whether zebrafish exhibit a sexually dimorphic cortisol stress response and whether 17ß-estradiol (E2) or 11-ketotestosterone (11KT) modulate the activity of the hypothalamic-pituitary-interrenal (HPI) axis. To accomplish this, we quantified the whole body cortisol response to a physical stressor, cortisol release in vitro, and the expression of key HPI axis regulating genes of control and E2- or 11KT-exposed zebrafish. Under control conditions no dimorphisms in the HPI axis were apparent at rest or in response to a standardized stressor. In contrast, E2-exposure blunted the cortisol response of male fish in vivo and in vitro and as well as corticotropin-releasing factor (crf) expression in the pre-optic area (POA) of the brain. While the expression of some interrenal genes was suppressed by E2-exposure, these changes occurred in both male and female zebrafish. 11KT-exposure increased whole-body cortisol of males at rest and vortex-exposed females, but had no impact on the rate of cortisol synthesis in vitro or on POA crf expression. Therefore, while we found no evidence that zebrafish exhibit a sexually dimorphic cortisol stress response, both E2 and 11KT can modulate the activity of the HPI axis in this species and do so via different mechanisms.

Stirring up new ideas about the regulation of the hypothalamic-pituitary-interrenal axis in zebrafish (Danio rerio).

The dynamic relationships between the changes in cortisol synthesis during and after a stressor and the expression pattern of the key genes that regulate the different levels of the hypothalamic-pituitary-interrenal (HPI) stress axis are poorly understood. This study established a novel vortex stressor and characterized its impact at all levels of the HPI axis in adult zebrafish. Exposure to a moderate vortex speed for 60 min was associated with a marked 18-fold increase in whole-body cortisol after 10 min followed by a gradual return to basal values 30 min poststress. The changes in whole-body cortisol were paralleled by increases in the expression of preoptic area corticotropin-releasing factor, pituitary prohormone convertase 1, and interrenal melanocortin 2 receptor, steroid acute regulatory protein, 11ß-hydroxylase and 11ß-hydroxysteroid dehydrogenase 2. The response to the vortex stressor also included delayed increases in preoptic area urotensin I and pituitary pro-opiomelanocortin mRNA levels but no change in the expression of other putative HPI axis regulators. Notably, the expression of several genes was depressed below control values 30 min poststress. These findings suggest that multiple genes at all levels of the HPI axis play an active role in the stimulation and termination of the cortisol stress response in zebrafish.