Paulina A. Bahamonde

Molecular signatures in rainbow darter (Etheostoma caeruleum) inhabiting an urbanized river reach receiving wastewater effluents

Rainbow darter (Etheostoma caeruleum) is a small benthic fish species found in North America that are abundant and distributed throughout the Grand River watershed, ON, Canada. Rainbow darter exhibit intersex in males at sites adjacent to municipal wastewater effluents (MWWE). In October 2010, female and male rainbow darter were collected at 3 sites (1 upstream reference and 2 downstream exposed sites) in the Grand River near the cities of Kitchener and Waterloo. The primary objectives of this research were (1) to characterize the responses of whole organism endpoints (i.e. condition factor (K), liversomatic (LSI) and gonadosomatic index (GSI), histopathology) to MWWEs and (2) to identify transcripts showing altered steady state abundance with exposure to MWWE in fish inhabiting municipal wastewater effluent-exposed areas. Genes measured in this study included vitellogenin, Sry-box containing protein 9 (sox9), forkhead box L2 (foxl2), doublesex and mab-3 related transcription factor 1 (dmrt1), cytochrome P450, family 11, subfamily A, polypeptide 1 (cyp11a) as well as estrogen (esr1, esrb) and androgen (ar) receptors. There were no changes in condition factor; however, there was a significant increase in LSI and a decrease in GSI in fish inhabiting downstream environments when compared with fish collected from the reference site. Males had a high incidence (∼ 70%) of intersex in downstream sites; characterized by the presence of oocytes within the testis. In the gonad, there were sex specific differences for genes related to sexual differentiation; dmrt1 was only expressed in males whereas foxl2 and sox9 were highly expressed in females compared to males. Expression levels of ar and esr1 were higher in females than males. Conversely, esrb was not differentially expressed between sexes or among sites. There were no differences detected for the genes investigated within sex among sites. This study is the first to report on gene expression changes in the rainbow darter, with emphasis on the differences in transcript abundance between sexes and how these changes relate to exposures to MWWEs. Molecular approaches are being investigated for their potential application to field ecotoxicology, and molecular bioassays for relevant, sentinel species in environmental monitoring programs are required to better understand the impact of anthropogenic impacts on species at risk in river systems.

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.

Whole organism responses and intersex severity in rainbow darter (Etheostoma caeruleum) following exposures to municipal wastewater in the Grand River basin, ON, Canada. Part A.

Municipal wastewater effluents (MWWEs) contain anthropogenic substances that can exhibit endocrine-disrupting activity. These complex mixtures have been observed to exert adverse effects on fish. Rainbow darter (Etheostoma caeruleum, RBD) is a small benthic fish that is widespread throughout the Grand River, Ontario, Canada, and has been previously shown to be adversely affected by MWWE exposure in this watershed. The objectives of this study were to quantify biological responses in this sentinel species and intersex severity in male fish, in relation to the area of urbanization. It focused on RBD populations adjacent to wastewater outfalls in the Grand River watershed. In May 2011, nine sites across the urban gradient were selected to evaluate the impact of MWWEs. Endpoints for energy storage (i.e. condition factor, k; liversomatic index, LSI) as well as reproductive endpoints (i.e. gonadosomatic index, GSI; gonad development, hormone production), and intersex were assessed in the fish. Rainbow darter showed a high incidence of intersex downstream of the wastewater outfalls, especially below the largest treatment plant outfall at Kitchener (∼85%). We applied an intersex index (score from 0 to 7) that considers the number of eggs within the testis and the stage of maturation of the egg. RBD exposed downstream of the largest wastewater outfall at Kitchener had a score of 3.81±0.37 compared to upstream to the urban areas where there were no intersex males found other than a single individual with a score 1 (average intersex score of site 0.06±0.06). In addition, several fish associated with the Kitchener outfall had macroscopic vitellogenic eggs in the testes (intersex scores 5 and 6). The sub-population of fish located at the wastewater outfall also showed a tendency towards skewed sex ratios (greater proportion of females to male fish) compared to the population at the reference sites. Male fish inhabiting the urbanized area of the Grand River also showed reduced levels of testosterone (T) and 11-ketotestosterone (11KT). Intersex males had the lower levels of 11KT relative to the upstream reference fish but could not be distinguished from normal males collected at the exposed sites. Despite the high levels of intersex at these sites, no relationships were evident among intersex severity and other measured endpoints such as GSI, LSI or in vitro steroid production. The effects observed appear to be associated with urbanization and exposure to treated MWWEs in the watershed. Although intersex incidence and severity was a very good indicator of wastewater exposure, intersex could not be directly linked to other effects in this wild population. The effects of MWWEs on transcriptional changes in adult RBD exposed to the effluents are reported in the corresponding report (Part B).

Defining the role of omics in assessing ecosystem health: Perspectives from the Canadian environmental monitoring program

Scientific reviews and studies continue to describe omics technologies as the next generation of tools for environmental monitoring, while cautioning that there are limitations and obstacles to overcome. However, omics has not yet transitioned into national environmental monitoring programs designed to assess ecosystem health. Using the example of the Canadian Environmental Effects Monitoring (EEM) program, the authors describe the steps that would be required for omics technologies to be included in such an established program. These steps include baseline collection of omics endpoints across different species and sites to generate a range of what is biologically normal within a particular ecosystem. Natural individual variability in the omes is not adequately characterized and is often not measured in the field, but is a key component to an environmental monitoring program, to determine the critical effect size or action threshold for management. Omics endpoints must develop a level of standardization, consistency, and rigor that will allow interpretation of the relevance of changes across broader scales. To date, population-level consequences of routinely measured endpoints such as reduced gonad size or intersex in fish is not entirely clear, and the significance of genome-wide molecular, proteome, or metabolic changes on organism or population health is further removed from the levels of ecological change traditionally managed. The present review is not intended to dismiss the idea that omics will play a future role in large-scale environmental monitoring studies, but rather outlines the necessary actions for its inclusion in regulatory monitoring programs focused on assessing ecosystem health.

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.

Estrogenicity and intersex in juvenile rainbow trout (Oncorhynchus mykiss) exposed to Pine/Eucalyptus pulp and paper production effluent in Chile

Pulp and paper mill effluents (PPMEs) have been shown to increase gonad size, cause early maturation, and disrupt hormone functions in native and non-native Chilean fish. In this study, we assessed reproductive (plasma vitellogenin; VTG, gonad development) and metabolic (ethoxyresorufin-O-deethylase activity; EROD) end points, relative liver size (LSI) and condition factor (K) of juvenile female and male rainbow trout exposed to effluents. Unlike previous studies, which have focus either on the specific effects of effluent on fish in laboratory exposures or biotic population statuses downstream of discharge sites, we simultaneously assessed the impacts of PPMES on trout using two approaches: (1) laboratory exposures of tertiary treated PPME produced from processing Eucalyptus globulus or Pinus radiata; and (2) in situ bioassay downstream of the combined discharge of the same pulp mill. Despite an increase in the average gonadosomatic index (GSI) in exposed fish, no statistical differences in gonad size between exposed and unexposed individuals was detected. However, both female and male fish exposed to effluents showed significantly higher concentrations of plasma VTG, so more in fish exposed to Eucalyptus-based effluent when compared to Pinus PPME. In addition, male fish showed intersex characteristics in all exposure assays (Eucaliptus and Pinus) and, despite the low concentration of effluent in the river (<1% [v/v]), similar responses were observed in the caged fish. Finally, EROD activity was induced in both in situ exposures and laboratory assays at the higher PPME concentration (60-85% PPME). This study confirms estrogenic effects in Chilean fish exposed to PPME and the necessity for biological effects monitoring in addition to the assessment of physical-chemical endpoints as required in current government regulations.

Hepatic gene expression profiling in zebrafish (Danio rerio) exposed to the fungicide chlorothalonil.

Chlorothalonil (tetrachloroisophtalonitrile) is a fungicide that is widely used on agricultural crops around the world and as such, it is a ubiquitous aquatic contaminant. Despite high usage, the effects of this fungicide on non-target aquatic organisms have not been fully investigated. The aim of the present study was to (1) determine the effects of chlorothalonil toxicity on adult male zebrafish (Danio rerio) and (2) characterize the effects of chlorothalonil on gene expression patterns in the liver using two different concentrations of the fungicide, 0.007mg/L (environmentally-relevant) and 0.035mg/L (sublethal). These concentrations were selected from range-finding experiments that showed that zebrafish survival was significantly different from control animals at concentrations higher than 0.035mg/L but not below. Male zebrafish in both treatments of chlorothalonil showed a decrease in liversomatic index. A commercial D. rerio microarray (4×44K) was used to determine gene expression profiles in male zebrafish liver following a 96h toxicological assay. Microarray analysis revealed that males exposed to both 0.007mg/L or 0.035mg/L of chlorothalonil showed increased transcriptional sub-networks related to cell division and DNA damage and decreased expression of gene networks associated with reproduction, immunity, and xenobiotic clearance. This study improves knowledge regarding whole animal exposures to chlorothalonil and identifies molecular signaling cascades that are sensitive to this fungicide in the fish liver.

Characterizing Chilean blue whale vocalizations with DTAGs: a test of using tag accelerometers for caller identification

ABSTRACT Vocal behavior of blue whales (Balaenoptera musculus) in the Gulf of Corcovado, Chile, was analysed using both audio and accelerometer data from digital acoustic recording tags (DTAGs). Over the course of three austral summers (2014, 2015 and 2016), seventeen tags were deployed, yielding 124 h of data. We report the occurrence of Southeast Pacific type 2 (SEP2) calls, which exhibit peak frequencies, durations and timing consistent with previous recordings made using towed and moored hydrophones. We also describe tonal downswept (D) calls, which have not been previously described for this population. As being able to accurately assign vocalizations to individual whales is fundamental for studying communication and for estimating population densities from call rates, we further examine the feasibility of using high-resolution DTAG accelerometers to identify low-frequency calls produced by tagged blue whales. We cross-correlated acoustic signals with simultaneous tri-axial accelerometer readings in order to analyse the phase match as well as the amplitude of accelerometer signals associated with low-frequency calls, which provides a quantitative method of determining if a call is associated with a detectable acceleration signal. Our results suggest that vocalizations from nearby individuals are also capable of registering accelerometer signals in the tagged whales DTAG record. We cross-correlate acceleration vectors between calls to explore the possibility of using signature acceleration patterns associated with sounds produced within the tagged whale as a new method of identifying which accelerometer-detectable calls originate from the tagged animal. Summary: Characterization of the Chilean blue whale vocal repertoire and a modified method of using DTAG accelerometers to identify calling blue whales.

Characterizing Transcriptional Networks in Male Rainbow Darter (Etheostoma caeruleum) that Regulate Testis Development over a Complete Reproductive Cycle

Intersex is a condition that has been associated with exposure to sewage effluents in male rainbow darter (Etheostoma caeruleum). To better understand changes in the transcriptome that are associated with intersex, we characterized annual changes in the testis transcriptome in wild, unexposed fish. Rainbow darter males were collected from the Grand River (Ontario, Canada) in May (spawning), August (post-spawning), October (recrudescence), January (developing) and March (pre-spawning). Histology was used to determine the proportion of spermatogenic cell types that were present during each period of testicular maturation. Regression analysis determined that the proportion of spermatozoa versus spermatocytes in all stages of development (R2 ≥ 0.58) were inversely related; however this was not the case when males were in the post-spawning period. Gene networks that were specific to the transition from developing to pre-spawning stages included nitric oxide biosynthesis, response to wounding, sperm cell function, and stem cell maintenance. The pre-spawning to spawning transition included gene networks related to amino acid import, glycogenesis, Sertoli cell proliferation, sperm capacitation, and sperm motility. The spawning to post-spawning transition included unique gene networks associated with chromosome condensation, ribosome biogenesis and assembly, and mitotic spindle assembly. Lastly, the transition from post-spawning to recrudescence included gene networks associated with egg activation, epithelial to mesenchymal transition, membrane fluidity, and sperm cell adhesion. Noteworthy was that there were a significant number of gene networks related to immune system function that were differentially expressed throughout reproduction, suggesting that immune network signalling has a prominent role in the male testis. Transcripts in the testis of post-spawning individuals showed patterns of expression that were most different for the majority of transcripts investigated when compared to the other stages. Interestingly, many transcripts associated with female sex differentiation (i.e. esr1, sox9, cdca8 and survivin) were significantly higher in the testis during the post-spawning season compared to other testis stages. At post-spawning, there were higher levels of estrogen and androgen receptors (esr1, esr2, ar) in the testis, while there was a decrease in the levels of sperm associated antigen 1 (spag1) and spermatogenesis associated 4 (spata4) mRNA. Cyp17a was more abundant in the testis of fish in the pre-spawning, spawning, and post-spawning seasons compared to those individuals that were recrudescent while aromatase (cyp19a) did not vary in expression over the year. This study identifies cell process related to testis development in a seasonally spawning species and improves our understanding regarding the molecular signaling events that underlie testicular growth. This is significant because, while there are a number of studies characterizing molecular pathways in the ovary, there are comparatively less describing transcriptomic patterns in the testis in wild fish.

DTAG studies of blue whales (Balaenoptera musculus) in the Gulf of Corcovado, Chile

This investigation set out to obtain data on the ecology, foraging and acoustic behavior of Chilean blue whales (Balaenoptera musculus) in the Gulf of Corcovado, which is an important feeding ground. We deployed 17 suction cup attached sound and orientation recording tags (DTAGs) on blue whales in 2014-16, for a total duration of 124h 08 min. Acoustic data on the tags revealed a variety of different calls. These included SEP2 (Southeast Pacific) song, previously described in this area, as well as other call types not previously described for Chilean blue whales. Downsweep calls similar to those described for other blue whale populations were observed on several tags, as were various other less stereotyped calls. We are currently working on characterizing these call types and also on using the accelerometers to identify calls from the tagged animal. Tag data will prove useful for interpretation of data collected in this area from passive acoustic monitors (PAM), both for species identification and possibly...