The Cytochrome P450 super family (CYP) is responsible for a wide range of functions in metazoans, having roles in both exogenous and endogenous substrate metabolism. Annelids are known to metabolize ...polycyclic aromatic hydrocarbons (PAHs) and produce estrogen. CYPs are postulated to be key enzymes in these processes in annelids. In this study, the CYP complement (CYPome) of the annelid Capitella teleta has been robustly identified and annotated with the genome assembly available. Phylogenetic analyses were performed to understand the evolutionary relationships between CYPs in C. teleta and other species. Predictions of which CYPs are potentially involved in both PAH metabolism and steroidogensis were made based on phylogeny. Annotation of 84 full length and 12 partial CYP sequences predicted a total of 96 functional CYPs in C. teleta. A further 13 CYP fragments were found but these may be pseudogenes. The C. teleta CYPome contained 24 novel CYP families and seven novel CYP subfamilies within existing families. A phylogenetic analysis identified that the C. teleta sequences were found in 9 of the 11 metazoan CYP clans. Two CYPs, CYP3071A1 and CYP3072A1, did not cluster with any metazoan CYP clans. We found xenobiotic response elements (XREs) upstream of C. teleta CYPs related to vertebrate CYP1 (CYP3060A1, CYP3061A1) and from families with reported transcriptional upregulation in response to PAH exposure (CYP4, CYP331). C. teleta had a CYP51A1 with ∼65% identity to vertebrate CYP51A1 sequences and has been predicted to have lanosterol 14 α-demethylase activity. CYP376A1, CYP3068A1, CYP3069A1, and CYP3070A1 were the most appropriate candidates for steroidogenesis genes based on their phylogeny and warrant further analyses, though no specific aromatase (estrogen synthesis) candidates were found. Presence of XREs upstream of C. teleta CYPs may indicate a functional aryl hydrocarbon receptor in C. teleta and candidate CYPs for studies of PAH metabolism.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•pH altered sertraline, fluoxetine and diclofenac uptake and toxicity, but not EE2.•DOC reduced sertraline uptake and toxicity, but not EE2 uptake.•pH and DOC impacts appear to be predictable based ...on drug properties (e.g. pKa).•Environmental drug toxicity may depend on the chemistry of the receiving water body.
Human and veterinary pharmaceuticals have been observed in natural aquatic environments around the world, and many have been shown to impact fish health. Presently, we examined the influence of pH, dissolved organic carbon (DOC) and Na+ or Ca2+ on the bioavailability and toxicity of waterborne pharmaceuticals in larval zebrafish. Drugs included sertraline (selective serotonin reuptake inhibitor; SSRI), fluoxetine (SSRI), diclofenac (nonsteroidal anti-inflammatory drug) and ethinyl estradiol (estrogen; EE2). The 96 h-LC50s for sertraline, fluoxetine and diclofenac were influenced by pH over an environmentally relevant range (pH 5.8–8.2). Toxicity was related to the predicted concentration of non-ionized compounds, which more readily cross cell membranes than ionized compounds. For example, sertraline was 4.1-fold more toxic (as measured by 96 h-LC50s) at pH 8.2 compared to pH 5.8, while the predicted amount of non-ionized sertraline was also greater at pH 8.2 (based on previously reported pKa values). Experiments with radiolabelled drugs demonstrated that sertraline uptake was also 5.4-fold higher at pH 8.2 compared to pH 5.8. Terrigenous and autochthonous DOC samples (as low as 1 mg/L) protected against sertraline uptake and toxicity, although they were more effective at lower (environmentally relevant) drug concentrations. In contrast, the uptake of EE2, which was principally non-ionized in all water chemistries tested, was not altered by pH or DOC. There was no change in sertraline toxicity with the addition of 12 mM Na+ or 3 mM Ca2+. In conclusion, the influence of pH and DOC on drug uptake and toxicity in fish appears to be predictable based on the physicochemical properties of the drug (e.g. pKa, polar surface area). The influence of water chemistry on drug bioavailability in fish is likely relevant to all aquatic life.
► We exposed zebrafish to a mixture of 4 pharmaceuticals and wastewater effluent. ► Exposures were chronic and environmentally relevant concentrations. ► Both mixtures impacted reproduction and ...histology of the kidney. ► Both mixtures increased developmental abnormalities.
Pharmaceuticals and personal care products (PPCPs) are discharged in municipal wastewater. Effects in aquatic organisms exposed to individual pharmaceuticals in the laboratory have raised concerns regarding the environmental impacts of PPCPs, yet environmental exposures are always to complex mixtures. In this study, adult zebrafish (Danio rerio) showed significantly decreased embryo production after a 6 week exposure to a pharmaceutical mixture (MIX; 0.5 and 10μgL−1) of acetaminophen, carbamazepine, gemfibrozil and venlafaxine and to diluted wastewater effluent (WWE; 5% and 25%). Atretic oocytes and altered ovarian histology were significantly increased in female zebrafish exposed to both concentrations of MIX or WWE, which indicates a direct effect on oocyte development that may account for reduced embryo production. Apoptosis within the thecal and granulosa cell layers was identified in female zebrafish with atresia. Exposures to MIX or WWE at both concentrations severely altered kidney proximal tubule morphology, but no histological impacts on other organs were observed. Exposure of embryos to MIX or WWE at the high concentration significantly increased the incidence of developmental abnormalities. Embryo mortality was elevated with exposure to the high concentration of MIX. These studies indicate that chronic exposure of fish to pharmaceutical mixtures and wastewater impacts reproduction and induces histopathological changes, similar to what we have previously seen with single compound exposures. These data suggest that fish populations exposed to pharmaceuticals discharged in wastewater are at risk of negative impacts to reproductive capacity and health.
Reduced representation (RRL) sequencing approaches (e.g., RADSeq, genotyping by sequencing) require decisions about how much to invest in genome coverage and sequencing depth, as well as choices of ...values for adjustable bioinformatics parameters. To empirically explore the importance of these "simple" methodological decisions, we generated two independent sequencing libraries for the same 142 individual lake whitefish (Coregonus clupeaformis) using a nextRAD RRL approach: (1) a larger number of loci at low sequencing depth based on a 9mer (library A); and (2) fewer loci at higher sequencing depth based on a 10mer (library B). The fish were selected from populations with different levels of expected genetic subdivision. Each library was analyzed using the STACKS pipeline followed by three types of population structure assessment (FST, DAPC and ADMIXTURE) with iterative increases in the stringency of sequencing depth and missing data requirements, as well as more specific a priori population maps. Library B was always able to resolve strong population differentiation in all three types of assessment regardless of the selected parameters, largely due to retention of more loci in analyses. In contrast, library A produced more variable results; increasing the minimum sequencing depth threshold (-m) resulted in a reduced number of retained loci, and therefore lost resolution at high -m values for FST and ADMIXTURE, but not DAPC. When detecting fine population differentiation, the population map influenced the number of loci and missing data, which generated artefacts in all downstream analyses tested. Similarly, when examining fine scale population subdivision, library B was robust to changing parameters but library A lost resolution depending on the parameter set. We used library B to examine actual subdivision in our study populations. All three types of analysis found complete subdivision among populations in Lake Huron, ON and Dore Lake, SK, Canada using 10,640 SNP loci. Weak population subdivision was detected in Lake Huron with fish from sites in the north-west, Search Bay, North Point and Hammond Bay, showing slight differentiation. Overall, we show that apparently simple decisions about library construction and bioinformatics parameters can have important impacts on the interpretation of population subdivision. Although potentially more costly on a per-locus basis, early investment in striking a balance between the number of loci and sequencing effort is well worth the reduced genomic coverage for population genetics studies. More conservative stringency settings on STACKS parameters lead to a final dataset that was more consistent and robust when examining both weak and strong population differentiation. Overall, we recommend that researchers approach "simple" methodological decisions with caution, especially when working on non-model species for the first time.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•Carbamazepine and gemfibrozil decreased 11-ketotestosterone in zebrafish.•Both drugs lowered ex vivo testicular 11-ketotestosterone production.•The biochemical processes for steroid production ...appears unimpacted by gemfibrozil.•Gemfibrozil alters the movement of cholesterol into the mitochondrial membrane.•Carbamazepine exposure impacts steroidogenic enzyme expression or function.
Gemfibrozil (GEM) and carbamazepine (CBZ) are two environmentally relevant pharmaceuticals and chronic exposure of fish to these compounds has decreased androgen levels and fish reproduction in laboratory studies. The main focus of this study was to examine the effects of GEM and CBZ on testicular steroid production, using zebrafish as a model species. Chronic water borne exposures of adult zebrafish to 10 μg/L of GEM and CBZ were conducted and the dosing was confirmed by chemical analysis of water as 17.5 ± 1.78 and 11.2 ± 1.08 μg/L respectively. A 67 day exposure led to reduced reproductive output and lowered whole body, plasma, and testicular 11-ketotestosterone (11-KT). Testicular production of 11-KT was examined post exposure (42 days) using ex vivo cultures to determine basal and stimulated steroid production. The goal was to ascertain the step impaired in the steroidogenic pathway by each compound. Ex vivo 11-KT production in testes from males chronically exposed to GEM and CBZ was lower than that from unexposed males. Although hCG, 25-OH cholesterol, and pregnenolone stimulation increased 11-KT production in all treatment groups over basal levels, hCG stimulated 11-KT production remained significantly less in testes from exposed males compared to controls. 25-OH cholesterol and pregnenolone stimulated 11-KT production was similar between GEM and control groups but the CBZ group had lower 11-KT production than controls with both stimulants. We therefore propose that chronic GEM and CBZ exposure can reduce production of 11-KT in testes through direct effects independent of mediation through HPG axis. The biochemical processes for steroid production appear un-impacted by GEM exposure; while CBZ exposure may influence steroidogenic enzyme expression or function.
The ontogenetic development in teleost fish is sensitive to temperature, and the developmental rate has a direct relationship with the environmental temperature within a species' thermal tolerance ...limit. Temperature determines time to and survival at hatching. Yellow perch is a North American species of ecological and commercial importance, and its phenology is vulnerable to climate change. The embryonic development of yellow perch was comparable to closely related members of the family Percidae. Developmental progression was fastest at 18°C and slowest at 12°C, with medial progression at 15°C. Time to hatch and swim-up, feeding onset, and exogenous feeding phases were different across all incubation temperatures regardless of a gradual post-hatch warming of the 12 and 15°C groups to a common garden temperature of 18°C. Incubation temperature may lower the rate of survival to hatch at 15°C and had complex impacts on developmental abnormalities. Temperature had significant effects on the development rate, time of hatch, survival, and incidence of developmental abnormalities. Early ontogenetic thermal history in ectotherms is an important factor determining phenotypic variation. It will be important to link the thermally induced changes in development described here to the physiological and morphological differences and to link the developmental abnormalities to functional performance.
Chronic low-concentration chemical exposures may have both direct health outcomes on adults and indirect effects on their offspring. Using zebrafish, we examined the impacts of chronic, ...low-concentration carbamazepine (CBZ) exposure on a suite of male reproductive endpoints in the parents and four generations of offspring reared in clean water. CBZ is one of the most frequently detected pharmaceutical residues in water, is a histone deacetylase inhibitor in mammals, and is reported to lower androgens in mammals and fish. Exposure of adult zebrafish to 10 μg/L CBZ for 6 weeks decreased reproductive output, courtship and aggressive behaviors, 11-ketotestosterone (11KT), and sperm morphology but did not impact milt volume or sperm swimming speed. Pairwise breeding generated lineages of offspring with both parents exposed and two lineages where only one parent was exposed; the control lineage had unexposed parents. Reproductive output and male reproductive indices were assessed in F1–F4 offspring to determine whether parental CBZ exposure had transgenerational impacts. The offspring of CBZ-exposed males had lower 11KT, reproductive output, altered courtship, aggression, and sperm morphology compared to the lineage from unexposed parents. Our results indicate that parental carbamazepine exposure history impacts the unexposed progeny up to the F4 generations and that paternal, but not maternal, exposure is most important for the reproductive health of male offspring.
Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next‐generation DNA sequencing (RADSeq, GBS) is ...unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double‐digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0‐, 12‐, 48‐ and 96‐h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12–48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96‐h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2‐ to 16‐fold), and additional read‐length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.
•Rainbow trout were exposed to 0, 10 and 30 μgL−1 acetaminophen for 4 weeks.•Acetaminophen impacted kidney, gill and liver histology.•Acetaminophen impacted kidney, gill and liver ...function.•Acetaminophen altered urine ion, glucose and protein concentrations.•Exposure limited oxygen consumption and decreased critical swimming speed.
In this study, we examined if rainbow trout chronically exposed to acetaminophen (10 and 30 μgL−1) showed histological changes that coincided with functional changes in the kidney, gill and liver. Histological changes in the kidney included movement and loss of nuclei, non-uniform nuclei size, non-uniform cytoplasmic staining, and loss of tubule integrity. Histological effects were more severe at the higher concentration and coincided with concentration dependent increases in urine flow rate and increased urinary concentrations of sodium, chloride, potassium, calcium, urea, ammonia, glucose, and protein. Yet, glomerular filtration rate was not altered with acetaminophen exposure. In the gill, filament end swelling, whole filament swelling, and swelling of the lamellae were observed in exposed fish. Lamellar spacing decreased in both exposure groups, but lamellar area decreased only with 30 μgL−1 exposure. At faster swimming speeds, oxygen consumption was limited in acetaminophen exposed fish, and critical swimming speed was also decreased in both exposure groups. The liver showed decreased perisinusoidal spaces at 10 and 30 μgL−1 acetaminophen, and decreased cytoplasmic vacuolation with 30 μgL−1 acetaminophen. A decrease in liver glycogen was also observed at 30 μgL−1. There was no change in plasma concentrations of sodium, chloride, potassium, calcium, magnesium, and glucose with exposure, suggesting compensation for urinary loss. Indeed, an increase in Na+-K+-ATPase activity in the gills was found with 30 μgL−1 acetaminophen exposure. Chronic exposure of rainbow trout to the environmentally relevant pharmaceutical acetaminophen, alters both histology and function of organs responsible for ion and nutrient homeostasis.
•Parental exposure to GEM and CBZ at 10μgL−1 impacts F1 courtship and fecundity.•Reciprocal crosses amongst the F1 indicate that males are more severely impacted.•Exposed F1 males behave differently ...and court exposed or control females poorly.•Sperm of GEM and CBZ F1 males saw faster and was morphologically different from CTLs.•Chronic, low dose pharmaceutical exposure of parents can cause impacts in F1.
In this study we explored how parental exposure to pharmaceuticals influences reproduction in offspring. Adult zebrafish (Danio rerio) were exposed for 6 weeks to 10μgL−1 of carbamazepine (CBZ) and gemfibrozil (GEM), two commonly prescribed drugs. Embryos were collected, reared in clean water until sexual maturity and then assessed for reproductive output, courtship, sperm function and organ histology. While 34% of the control pairs produced clutches, only 11% of the fish with CBZ exposed parents or 17% of the fish with GEM exposed parents produced clutches. Reciprocal crosses indicated that exposure in males had more profound reproductive effects. When a control F1 male was crossed with either a F1 female whose parents were CBZ or GEM exposed; no differences were observed in embryo production compared to controls. However, when a control F1 female was crossed with either a CBZ or GEM F1 male, 50% less embryos were produced. Male courtship was reduced in both CBZ and GEM F1 fish but the deficits in courtship displays were drug specific. Compared to control males, the sperm from GEM F1 males had shorter head lengths and midpieces whereas sperm from CBZ F1 males had longer midpeices. Although it remains unclear how specifically these morphological differences influenced sperm velocity, the sperm from GEM F1 males and from CBZ F1 males swam faster than the sperm of control F1 at 20s post activation. No significant differences were observed in the histology of the liver, kidney and gonads across treatment groups. These data are important as they show that chronic, low dose pharmaceutical exposure of parental fish is sufficient to cause significant reproductive effects in offspring.
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