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Kelly R Munkittrick - One of the best experts on this subject based on the ideXlab platform.
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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
Aquatic Toxicology, 2015Co-Authors: Paulina Bahamonde, Christopher J Martyniuk, Mark E Mcmaster, Mark R Servos, Meghan L M Fuzzen, Charles J Bennett, Gerald R Tetreault, Kelly R MunkittrickAbstract: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).
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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
Aquatic Toxicology, 2015Co-Authors: Paulina Bahamonde, Christopher J Martyniuk, Mark E Mcmaster, Mark R Servos, Kelly R MunkittrickAbstract:Abstract 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.
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Intersex in teleost fish are we distinguishing endocrine disruption from natural phenomena
General and Comparative Endocrinology, 2013Co-Authors: Paulina Bahamonde, Kelly R Munkittrick, Christopher J MartyniukAbstract:Intersex is defined as the simultaneous presence of male and female gonadal tissue in a gonochoristic (fixed-sex) species. The Intersex condition has been documented in both wild and laboratory animals, including fish, amphibians, and reptiles. In aquatic animals, Intersex is often viewed as a signature effect of exposure to endocrine disrupting compounds. At least 37 fish species from 17 families have been identified with Intersex gonads in 54 field survey studies. However, reports of the occurrence of Intersex at reference sites have led to speculation that a baseline level of Intersex is "normal". The objective of this critical review was to assess factors potentially associated with baseline levels of Intersex in fish and to examine the mechanisms involved in the Intersex condition in order to identify priority research areas. Based on current literature, the relationship between Intersex and physiological parameters such as plasma sex steroids and vitellogenin is not well characterized or conclusive. Moreover, the literature is not definitive on whether field studies are distinguishing between natural Intersex and Intersex due to stressors. High throughput transcriptomics will improve understanding of how Intersex condition manifests after exposure to aquatic pollution and it is recommended that studies consider both males with and without Intersex that inhabit the same polluted site in order to differentiate pathways associated with xenobiotic responses versus molecular pathways associated with Intersex. Other experimental design considerations for field studies examining Intersex include data collection on life history (e.g. migratory patterns) and improved reference site characterization.
Charles R Tyler - One of the best experts on this subject based on the ideXlab platform.
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wild Intersex roach rutilus rutilus have reduced fertility
Biology of Reproduction, 2002Co-Authors: Susan Jobling, Nicola Beresford, Geoff Brighty, S Coey, J G Whitmore, D E Kime, K J W Van Look, B G Mcallister, A C Henshaw, Charles R TylerAbstract:Endocrine-disrupting chemicals, known to be present in the environment, have great potential for interfering with reproductive health in wildlife and humans. There is, however, little direct evidence that endocrine disruption has adversely affected fertility in any organism. In freshwater and estuarine fish species, for example, although a widespread incidence of Intersex has been reported, it is not yet known if Intersexuality influences reproductive success. The purpose of this study was, therefore, to determine gamete quality in wild Intersex roach (Rutilus rutilus) by assessing sperm characteristics, fertilization success, and ability to produce viable offspring. The results clearly demonstrate that gamete production is reduced in Intersex roach. A significantly lower proportion of moderately or severely feminized fish (17.4% and 33.3%, respectively) were able to release milt compared with normal male fish from contaminated rivers (in which 97.6% of the males were able to release milt), reference male fish (97.7%), or less severely feminized Intersex fish (experiment 1: 85.8%, experiment 2: 97%). Intersex fish that did produce milt produced up to 50% less (in terms of volume per gram of testis weight) than did histologically normal male fish. Moreover, sperm motility (percentage of motile sperm and curvilinear velocity) and the ability of sperm to successfully fertilize eggs and produce viable offspring were all reduced in Intersex fish compared with normal male fish. Male gamete quality (assessed using sperm motility, sperm density, and fertilization success) was negatively correlated with the degree of feminization in Intersex fish (r = -0.603; P < 0.001) and was markedly reduced in severely feminized Intersex fish by as much as 50% in terms of motility and 75% in terms of fertilization success when compared with either less severely feminized Intersex fish or unaffected male fish. This is the first evidence documenting a relationship between the morphological effects (e.g., Intersex) of endocrine disruption and the reproductive capabilities of any wild vertebrate. The results suggest that mixtures of endocrine-disrupting substances discharged into the aquatic environment could pose a threat to male reproductive health.
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altered sexual maturation and gamete production in wild roach rutilus rutilus living in rivers that receive treated sewage effluents
Biology of Reproduction, 2002Co-Authors: Susan Jobling, Nicola Beresford, M Nolan, Trevor P Rodgersgray, Geoff Brighty, John P Sumpter, Charles R TylerAbstract:Disruption in gonadal development of wild roach living in U.K. rivers receiving large volumes of treated sewage effluent is manifest in a variety of ways, ranging from malformation of the germ cells and/or reproductive ducts to altered gamete production. Intersex fish were also found to have an altered endocrine status and an elevated concentration of plasma vitellogenin. Gonadal growth was inhibited only in severely Intersex fish, whereas progression of spermatogenesis was delayed in a large proportion of all Intersex and exposed male fish. In contrast to the effects observed in the Intersex and exposed male fish, the maturation of ovaries in female fish inhabiting effluent-contaminated rivers appeared to be less obviously affected, although a higher incidence of oocyte atresia was found in the effluent-exposed fish compared with the reference fish. A positive correlation was found between the proportion of female tissue in the gonads of Intersex fish and their plasma vitellogenin concentration, suggesting that vitellogenin can be an indicator for the level of gonadal disruption in Intersex roach. The estradiol-17β concentration in Intersex fish was intermediate between the concentration found in males and females, and the plasma testosterone was between 2- and 3-fold higher in Intersex fish compared with male fish. These data suggest a link between altered endocrine status in Intersex and female fish and gonadal disruption. Spermiation was also affected in roach living in effluent-impacted rivers: a lower proportion of fish were found releasing sperm, and in those Intersex fish that were spermiating, a reduced milt volume and a reduced sperm density were found. All Intersex fish had malformations of the reproductive duct(s), and in severely affected fish, the ducts were occluded, thus preventing release of gametes. In view of the widespread occurrence of Intersexuality in wild fish populations in rivers throughout the United Kingdom, assessment of the reproductive capabilities of these Intersex roach is clearly needed to understand the impact of this phenomenon on roach fertility.
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sexual disruption in a second species of wild cyprinid fish the gudgeon gobio gobio in united kingdom freshwaters
Environmental Toxicology and Chemistry, 2001Co-Authors: Susan Jobling, John P Sumpter, Ronny Van Aerle, Monique Nolan, Lisette B Christiansen, Charles R TylerAbstract:To establish whether the Intersex condition seen in the roach (Rutilus rutilus) in United Kingdom (UK) rivers was species specific or a more general phenomenon in fish, evidence for sexual disruption was sought in a second cyprinid species, the gudgeon (Gobio gobio). Gudgeon were collected from the Rivers Aire and Lea (at locations that receive high-volume discharges of sewage treatment works [STW] effluent and that contain Intersex roach) and from two still waters, and their gonads were examined histologically for evidence of Intersexuality (the simultaneous presence of oocytes and testicular tissue). Intersex gonads were found at all sites, with the highest incidences occurring at one of the still waters (Lakeside Fisheries: 15%) and at sites on the River Aire (Thwaite Weir, Silsden Bridge, and Knostrop: 14, 13, and 12%, respectively). In the River Lea and Longton Park Lake, the incidence of Intersexuality in gudgeon was 6%. In most cases, Intersex gonads were characterized by a few primary oocytes/gonad section in an otherwise normal testis. However, at some sites on the River Aire (Thwaite Weir and Knostrop), the Intersex condition was more severe. At Thwaite Weir, for example, more than half of the gonad in 40% of the Intersex fish was comprised of ovarian tissue. Elevated concentrations of plasma vitellogenin both in male and in Intersex fish indicated that fish had been exposed to estrogen(s). Some of the gudgeon were found at sites several kilometers downstream of any point discharge of STW effluent; therefore, the results likely are representative of this species in wild populations found in typical UK river ecosystems. Together with the findings in the roach, these data on the gudgeon confirm that sexual disruption in fish in UK rivers is not species specific.
Paulina Bahamonde - One of the best experts on this subject based on the ideXlab platform.
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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
Aquatic Toxicology, 2015Co-Authors: Paulina Bahamonde, Christopher J Martyniuk, Mark E Mcmaster, Mark R Servos, Meghan L M Fuzzen, Charles J Bennett, Gerald R Tetreault, Kelly R MunkittrickAbstract: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).
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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
Aquatic Toxicology, 2015Co-Authors: Paulina Bahamonde, Christopher J Martyniuk, Mark E Mcmaster, Mark R Servos, Kelly R MunkittrickAbstract:Abstract 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.
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Intersex in teleost fish are we distinguishing endocrine disruption from natural phenomena
General and Comparative Endocrinology, 2013Co-Authors: Paulina Bahamonde, Kelly R Munkittrick, Christopher J MartyniukAbstract:Intersex is defined as the simultaneous presence of male and female gonadal tissue in a gonochoristic (fixed-sex) species. The Intersex condition has been documented in both wild and laboratory animals, including fish, amphibians, and reptiles. In aquatic animals, Intersex is often viewed as a signature effect of exposure to endocrine disrupting compounds. At least 37 fish species from 17 families have been identified with Intersex gonads in 54 field survey studies. However, reports of the occurrence of Intersex at reference sites have led to speculation that a baseline level of Intersex is "normal". The objective of this critical review was to assess factors potentially associated with baseline levels of Intersex in fish and to examine the mechanisms involved in the Intersex condition in order to identify priority research areas. Based on current literature, the relationship between Intersex and physiological parameters such as plasma sex steroids and vitellogenin is not well characterized or conclusive. Moreover, the literature is not definitive on whether field studies are distinguishing between natural Intersex and Intersex due to stressors. High throughput transcriptomics will improve understanding of how Intersex condition manifests after exposure to aquatic pollution and it is recommended that studies consider both males with and without Intersex that inhabit the same polluted site in order to differentiate pathways associated with xenobiotic responses versus molecular pathways associated with Intersex. Other experimental design considerations for field studies examining Intersex include data collection on life history (e.g. migratory patterns) and improved reference site characterization.
Amir Sagi - One of the best experts on this subject based on the ideXlab platform.
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Intersexuality and behavior in crayfish the de masculinization effects of androgenic gland ablation
Hormones and Behavior, 2006Co-Authors: Assaf Barki, Ilan Karplus, Rivka Manor, Amir SagiAbstract:In crustaceans, male differentiation and primary and secondary characteristics are regulated by the androgenic gland (AG). In gonochoristic crustaceans, the AG is also linked to Intersexuality. Whereas the co-occurrence of various male and female characteristics has been demonstrated in Intersex crustaceans, little is known regarding sexually dimorphic behavior patterns in such individuals. In the present study, we used an Intersex crayfish model to investigate – for the first time in crustaceans – the agonistic and mating behavior of Intersex individuals, and to explore the effects of AG ablation on behavior, morphology and physiology. As was the case for their morphological and physiological reproductive traits, Intersex individuals – despite being genotypically females – generally resembled males in terms of behavior: they engaged in fighting with males and copulated with receptive females. However, fighting durations of Intersex animals were intermediate between those of males and females, and the durations of the copulations were remarkably short. Adult Intersex individuals that had been AG ablated at the juvenile stage were unlikely to engage in fighting with males (similar behavior to females) and did not exhibit any mating behavior with receptive females. AG ablation resulted in feminine morphological and physiological shifts in the treated Intersex individuals and enabled vitellogenin gene transcription and the onset of secondary vitellogenesis. It thus appears that an as-yet-unknown AG-secreted factor(s) regulating maleness also seems to regulate the organization of male behavior in crustaceans.
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sex determination in crayfish are Intersex cherax quadricarinatus decapoda parastacidae genetically females
Genetics Research, 2003Co-Authors: Shmuel Parnes, Isam Khalaila, Gideon Hulata, Amir SagiAbstract:Summary In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae), a gonochoristic species, seven different combinations of Intersex individuals (with both male and female genital openings) have been described. However, to date, the genetic basis for this phenomenon has not been investigated. This study was designed to test a simple chromosome-based sex-determination model for C. quadricarinatus that assumes the male to be the homogametic (ZZ) sex. According to our model, Intersex individuals that are functionally males are genetically females (WZ). Individual crosses were performed between Intersex and female crayfish, with control crosses being performed between normal males and females. The control crosses yielded, in most cases, the expected 1 : 1 sex ratio in the F1 progeny. Crosses between Intersex individuals and females yielded a 1 : 3 (male : female) sex ratio in most crosses. According to our hypothesis, one-third of the females produced in a cross of a female with an Intersex animal should be WW females. The hypothesis was tested by crossing normal males with F1 females, which were progeny of Intersex fathers. These crosses yielded almost 100% females, a finding that conforms to the above-suggested sex determination model for C. quadricarinatus and the female WZ genotype of Intersex individuals.
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endocrine balance between male and female components of the reproductive system in Intersex cherax quadricarinatus decapoda parastacidae
Journal of Experimental Zoology, 1999Co-Authors: Isam Khalaila, Simy Weil, Amir SagiAbstract:Intersex individuals of the crayfish Cherax quadricarinatus are functional males that also possess arrested ovaries. To study the role of eyestalk vs. androgenic gland factors in regulating the functional balance between the different components of the Intersex reproductive system, andrectomy and/or unilateral destalking were performed. Andrectomized Intersex speci- mens had atrophied testes and sperm ducts and showed a reduction in the number of testicular spermatogenic lobules. Large oocytes developed in their ovarian lobes, and the Gonadosomatic Index reached 1.60 ± 0.36 compared with 0.21 ± 0.03 for the control. In the polypeptide profiles of the ovarian lobes from andrectomized individuals, the 177-, 150-, and 106-kDa polypeptides pre- dominated, resembling the profile of the secondary vitellogenic ovary. The andrectomized indi- viduals neither lost their male external characteristics, such as the red patch on the propodus, nor developed ovigerous setae on their male-like pleopods. Unilateral eyestalk ablation did not cause significant differences in the male or the female components of the reproductive system compared with the control group. The maturation of the permanently arrested ovary and the arrest of the testis in andrectomized Intersex individuals illustrated the central role of the androgenic gland in maintaining the endocrine balance in Intersex C. quadricarinatus. J. Exp. Zool. 283:286n294, 1999. © 1999 Wiley-Liss, Inc.
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Intersex red claw crayfish cherax quadricarinatus von martens functional males with pre vitellogenic ovaries
The Biological Bulletin, 1996Co-Authors: Amir Sagi, Isam Khalaila, Assaf Barki, Gideon Hulata, Ilan KarplusAbstract:Intersex individuals, possessing both male and female genital openings, were assessed in two groups-7 and 19 months old-of Australian red claw crayfish (Cherax quadricarinatus). All Intersex individuals inves- tigated were functional males, as suggested by their male- like morphology and the presence of testes, sperm ducts, androgenic glands, and viable spermatozoa. When an ovary was present in an Intersex individual from either group, the gonadosomatic index, the diameter of the oo- cytes, and the ovarian cytosolic polypeptide profile were similar to those of immature, pre-vitellogenic females. We conclude that Intersexuality in C. quadricarinatus does not indicate a case of protandric sequential hermaphro- ditism, as previously suggested. The case of Intersexuality described here presents a unique model for the study of the role of the androgenic gland in the regulation of sex differentiation in crustaceans.
Mark R Servos - One of the best experts on this subject based on the ideXlab platform.
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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
Aquatic Toxicology, 2015Co-Authors: Paulina Bahamonde, Christopher J Martyniuk, Mark E Mcmaster, Mark R Servos, Kelly R MunkittrickAbstract:Abstract 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.
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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
Aquatic Toxicology, 2015Co-Authors: Paulina Bahamonde, Christopher J Martyniuk, Mark E Mcmaster, Mark R Servos, Meghan L M Fuzzen, Charles J Bennett, Gerald R Tetreault, Kelly R MunkittrickAbstract: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).
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occurrence and degree of Intersex testis ova in darters etheostoma spp across an urban gradient in the grand river ontario canada
Environmental Toxicology and Chemistry, 2013Co-Authors: Rajiv N Tanna, Mark E Mcmaster, Charles J Bennett, Gerald R Tetreault, Brendan M Smith, Leslie M Bragg, Ken D Oakes, Mark R ServosAbstract:The variability and extent of the Intersex condition (oocytes in testes, or testis–ova) was documented in fish along an urban gradient in the Grand River, Ontario, Canada, that included major wastewater treatment plant outfalls. A method for rapid enumeration of testis–ova was developed and applied that increased the capacity to quantify Intersex prevalence and severity. Male rainbow darters (Etheostoma caeruleum) sampled downstream of the first major wastewater outfall (Waterloo) had a significant increase, relative to 4 upstream reference sites, in the mean proportion of fish with at least 1 testis–oocyte per lobe of testes (9–20% proportion with ≤ 1 testis–oocyte/lobe vs 32–53% and >1.4 testis–oocyte/lobe). A much higher mean incidence of Intersex proportion and degree was observed immediately downstream of the second wastewater outfall (Kitchener; 73–100% and 8–70 testis–oocyte/lobe); but only 6.3 km downstream of the Kitchener outfall, the occurrence of Intersex dropped to those of the reference sites. In contrast, downstream of a tertiary treated wastewater outfall on a small tributary, Intersex was similar to reference sites. Estrogenicity, measured using a yeast estrogen screen, followed a similar pattern, increasing from 0.81 ± 0.02 ng/L 17b-estradiol equivalents (EEq) (Guelph), to 4.32 ± 0.07 ng/L (Waterloo), and 16.99 ± 0.40 ng/L (Kitchener). Female rainbow darter downstream of the Kitchener outfall showed significant decreases in gonadosomatic index and liver somatic index, and increases in condition factor (k) relative to corresponding reference sites. The prevalence of Intersex and alterations in somatic indices suggest that exposure to municipal wastewater effluent discharges can impact endocrine function, energy use, and energy storage in wild fish. Environ Toxicol Chem. 2013;32:1981–1991. © 2013 SETAC
