Embryonic and larval development of yellow perch (Perca flavescens) and its sensitivity to incubation temperature.

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.

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.

Thermal acclimation alters both basal heat shock protein gene expression and the heat shock response in juvenile lake whitefish (Coregonus clupeaformis).

Long-term temperature shifts associated with seasonal variability are common in temperate regions. However, these natural shifts could place significant strain on thermal stress responses of fishes when combined with mean increases in water temperatures predicted by climate change models. We examined the relationship between thermal acclimation, basal expression of heat shock protein (hsp) genes and the activation of the heat shock response (HSR) in lake whitefish (LWF; Coregonus clupeaformis), a cold water species of cultural and commercial significance. Juveniles were acclimated to either 6, 12, or 18°C water for several months prior to the quantification of hsp mRNA levels in the presence or absence of acute heat shock (HS). Acclimation to 18°C increased basal mRNA levels of hsp70 and hsp47, but not hsc70 or hsp90ß in gill, liver and white muscle, while 6°C acclimation had no effect on basal hsp transcription. Fish in all acclimation groups were capable of eliciting a robust HSR following acute HS, as indicated by the upregulation of hsp70 and hsp47. An increase of only 2°C above the 18°C acclimation temperature was required to trigger these transcriptional changes, suggesting that the HSR may be frequently initiated in LWF populations living at mildly elevated temperatures. Collectively, these expression profiles show that environmental temperature influences both basal hsp levels and the HSR in LWF, and indicate that these fish may have a greater physiological and ecological susceptibility to elevated temperatures than to cooler temperatures.

The heat shock response shows plasticity in embryonic lake whitefish (Coregonus clupeaformis) exposed to repeated thermal stress.

We examined the impact of repeated thermal stress on the heat shock response (HSR) of thermally sensitive lake whitefish (Coregonus clupeaformis) embryos. Our treatments were designed to mimic temperature fluctuations in the vicinity of industrial thermal effluents. Embryos were either maintained at control temperatures (3 oC) or exposed to a repeated thermal stress (TS) of 3 or 6 oC above control temperature every 3 or 6 days throughout embryonic development. At 82 days post-fertilisation, repeated TS treatments were stopped and embryos received either a high level TS of 12, 15, or 18 oC above ambient temperature for 1 or 4 h, or no additional TS. These treatments were carried out after a 6 h recovery from the last repeated TS. Embryos in the no repeated TS group responded, as expected, with increases in hsp70 mRNA in response to 12, 15 and 18 oC high-level TS. However, exposure to repeated TS of 3 or 6 °C every 6 days also resulted in a significant upregulation of hsp70 mRNA relative to the controls. Importantly, these repeated TS events and the associated elevations in hsp70 attenuated the upregulation of hsp70 in response to a 1 h, high-level TS of 12 oC above ambient, but not to either longer (4 h) or higher (15 or 18 oC) TS events. Conversely, hsp90α mRNA levels were not consistently elevated in the no repeated TS groups exposed to high-level TS. In some instances, hsp90α levels appeared to decrease in embryos exposed to repeated TS followed by a high-level TS. The observed attenuation of the HSR in lake whitefish embryos demonstrates that embryos of this species have plasticity in their HSR and repeated TS may protect against high-level TS, but the response differs based on repeated TS treatment, high-level TS temperature and duration, and the gene of interest.

Lasting Effects of Low to Non-Lethal Radiation Exposure during Late Gestation on Offspring's Cardiac Metabolism and Oxidative Stress.

Ionizing radiation (IR) is known to cause fetal programming, but the physiological effects of low-dose IR are not fully understood. This study examined the effect of low (50 mGy) to non-lethal (300 and 1000 mGy) radiation exposure during late gestation on cardiac metabolism and oxidative stress in adult offspring. Pregnant C57BL/6J mice were exposed to 50, 300, or 1000 mGy of gamma radiation or Sham irradiation on gestational day 15. Sixteen weeks after birth, 18F-Fluorodeoxyglucose (FDG) uptake was examined in the offspring using Positron Emission Tomography imaging. Western blot was used to determine changes in oxidative stress, antioxidants, and insulin signaling related proteins. Male and female offspring from irradiated dams had lower body weights when compared to the Sham. 1000 mGy female offspring demonstrated a significant increase in 18F-FDG uptake, glycogen content, and oxidative stress. 300 and 1000 mGy female mice exhibited increased superoxide dismutase activity, decreased glutathione peroxidase activity, and decreased reduced/oxidized glutathione ratio. We conclude that non-lethal radiation during late gestation can alter glucose uptake and increase oxidative stress in female offspring. These data provide evidence that low doses of IR during the third trimester are not harmful but higher, non-lethal doses can alter cardiac metabolism later in life and sex may have a role in fetal programming.

The cytochrome P450 (CYP) superfamily in cnidarians.

The cytochrome P450 (CYP) superfamily is a diverse and important enzyme family, playing a central role in chemical defense and in synthesis and metabolism of major biological signaling molecules. The CYPomes of four cnidarian genomes (Hydra vulgaris, Acropora digitifera, Aurelia aurita, Nematostella vectensis) were annotated; phylogenetic analyses determined the evolutionary relationships amongst the sequences and with existing metazoan CYPs. 155 functional CYPs were identified and 90 fragments. Genes were from 24 new CYP families and several new subfamilies; genes were in 9 of the 12 established metazoan CYP clans. All species had large expansions of clan 2 diversity, with H. vulgaris having reduced diversity for both clan 3 and mitochondrial clan. We identified potential candidates for xenobiotic metabolism and steroidogenesis. That each genome contained multiple, novel CYP families may reflect the large evolutionary distance within the cnidarians, unique physiology in the cnidarian classes, and/or different ecology of the individual species.

The gut content microbiome of wild-caught rainbow darter is altered during laboratory acclimation.

An increasing number of laboratory studies are showing that environmental stressors and diet affect the fish gut microbiome. However, the application of these results to wild populations is uncertain as little is known about how the gut microbiome shifts when fish are transitioned from the field to the laboratory. To assess this, intestinal contents (i.e. digesta) of wild-caught rainbow darter (Etheostoma caeruleum) were sampled in the field and in the lab after 14- and 42-days acclimation. In addition, from days 15-42 some fish were exposed to waterborne triclosan, an antimicrobial found in aquatic ecosystems, or to dilutions of municipal wastewater effluents, to determine how these stressors affect the bacterial communities of gut contents. 16S rRNA gene amplicon sequencing was used to determine microbial community composition, alpha, and beta diversity present in the fish gut contents. In total, there was 8,074,658 reads and 11,853 amplicon sequence variants (ASVs) identified. The gut contents of wild fish were dominant in both Proteobacteria (35%) and Firmicutes (27%), while lab fish were dominant in Firmicutes (37-47%) and had lower alpha diversity. Wild fish had greater ASVs per sample (423-1304) compared to lab fish (19-685). Similarly, the beta-diversity of these bacterial communities differed between field and lab control fish; control fish were distinct from the 10% wastewater effluent and 100 ng/L TCS treatment groups. Results indicate that the gut microbiome of wild fish changes with the transition to laboratory environments; hence, prolonged acclimation to new settings may be required to achieve a stable gut content microbiome in wild-caught fish. Research is required to understand the length of time required to reach a stable fish gut microbiome.

Rainbow darter (Etheostoma caeruleum) from a river impacted by municipal wastewater effluents have altered gut content microbiomes.

Municipal wastewater treatment plant (WWTP) effluent contains pharmaceuticals and personal care products known to affect fish health and reproduction. The microbiome is a community of bacteria integral in maintaining host health and is influenced by species, diet, and environment. This study investigated changes in the diversity and composition of the gut content microbiome of rainbow darter (Etheostoma caeruleum) at ten sites on the Grand River, Ontario, Canada. Gut contents were collected in fall 2018 from these fish at sites upstream and downstream of two municipal wastewater treatment plants (WWTPs; Waterloo and Kitchener). 16S rRNA genes were sequenced to determine the composition and diversity (alpha and beta) of microbial taxa present. Gut content bacterial alpha diversity increased downstream of both WWTP outfalls; dominance of bacterial amplicon sequence variants decreased compared to upstream fish. Fish collected at different sites had distinct bacterial communities, with upstream samples dominant in Proteobacteria and Firmicutes, and downstream samples increasingly abundant in Proteobacteria and Cyanobacteria. In mammals, increased abundance of Proteobacteria is indicative of microbial dysbiosis and has been linked to altered health outcomes, but this is not yet known for fish. This research indicates that the fish gut content microbiome was altered downstream of WWTP effluent outfalls and could lead to negative health outcomes.

Unchanged cardiovascular and respiratory outcomes in healthy C57Bl/6 mice after in utero exposure to ionizing radiation.

BACKGROUND: Advancements in medical technologies that utilize ionizing radiation have led to improved diagnosis and patient outcomes, however, the effect of ionizing radiation on the patient is still debated. In the case of pregnancy, the potential effects are not only to the mother but also to the fetus. The aim of this study was to determine if exposure from ionizing radiation during pregnancy alters the development of the cardiovascular and respiratory system of the offspring. MATERIALS AND METHODS: Pregnant C57Bl/6 mice were whole-body irradiated at gestational day 15 with a 137Cs gamma radiation emitting source at 0 mGy (sham), 50 mGy, 300 mGy, or 1000 mGy. Post weaning weight and blood pressure measurements were taken weekly for both male and female pups until euthanasia at 16-17 weeks postnatal age. Immediately following, the trachea was cannulated, and the lungs and heart excised. The lung was then examined to assess respiratory physiological outcomes. RESULTS AND CONCLUSIONS: In utero exposures to 1000 mGy caused significant growth reduction compared to sham irradiated, which remained persistent for both male and female pups. Growth restriction was not observed for lower exposures. There was no significant change in any cardiovascular or respiratory outcomes measured. Overall, intrauterine exposures to ionizing radiation does not appear to significantly alter the development of the cardiovascular and respiratory system in C57Bl/6 pups up to 17 weeks postnatal age.

Prostaglandins prevent acetaminophen induced embryo toxicity in zebrafish (Danio rerio).

Previous research in our laboratory showed that acetaminophen (ACE) induced embryonic mortality and abnormalities in zebrafish. Here, we examined the dose response of ACE (0.05-50 µg L-1) in zebrafish embryos. Concentrations as low as 0.1 µg L-1 significantly increased abnormalities, and all test concentrations significantly increased mortality rates. In mammals, ACE inhibits cyclooxygenase (COX) enzymes to decrease prostaglandin production. Here we report COX activity and expression of the cox-1, cox-2a, and cox-2b genes in zebrafish embryos. COX activity was significantly inhibited by specific mammalian cox-1 (SC-560) and cox-2 (DuP-697) inhibitors in unexposed and ACE-exposed embryos. COX activity declined with development time. Maternal transcripts of all cox genes were found at 1 -h post fertilization and embryonic expression began in gastrulation or early segmentation. Co-exposure of ACE and prostaglandin E2 abolished the ACE-induced effects. This strongly supports that ACE elicits embryo toxicity in zebrafish though the same molecular mechanism of action of their therapeutic effects in mammals.

Zebrafish CYP1A expression in transgenic Caenorhabditis elegans protects from exposures to benzo[a]pyrene and a complex polycyclic aromatic hydrocarbon mixture.

Polycyclic aromatic hydrocarbons (PAHs) are environmental toxicants primarily produced during incomplete combustion; some are carcinogens. PAHs can be safely metabolized or, paradoxically, bioactivated via specific cytochrome P450 (CYP) enzymes to more reactive metabolites, some of which can damage DNA and proteins. Among the CYP isoforms implicated in PAH metabolism, CYP1A enzymes have been reported to both sensitize and protect from PAH toxicity. To clarify the role of CYP1A in PAH toxicity, we generated transgenic Caenorhabditis elegans that express CYP1A at a basal (but not inducible) level. Because this species does not normally express any CYP1 family enzyme, this approach permitted a test of the role of basally expressed CYP1A in PAH toxicity. We exposed C. elegans at different life stages to either the PAH benzo[a]pyrene (BaP) alone, or a real-world mixture dominated by PAHs extracted from the sediment of a highly contaminated site on the Elizabeth River (VA, USA). This site, the former Atlantic Wood Industries, was declared a Superfund site due to coal tar creosote contamination that caused very high levels (in the [mg/mL] range) of high molecular weight PAHs within the sediments. We demonstrate that CYP1A protects against BaP-induced growth delay, reproductive toxicity, and reduction of steady state ATP levels. Lack of sensitivity of a DNA repair (Nucleotide Excision Repair)-deficient strain suggested that CYP1A did not produce significant levels of DNA-reactive metabolites from BaP. The protective effects of CYP1A in Elizabeth River sediment extract (ERSE)-exposed nematodes were less pronounced than those seen in BaP-exposed nematodes; CYP1A expression protected against ERSE-induced reduction of steady-state ATP levels, but not other outcomes of exposure to sediment extracts. Overall, we find that in C. elegans, a basal level of CYP1A activity is protective against the examined PAH exposures.

Chronic exposure of Brown (Hydra oligactis) and green Hydra (Hydra viridissima) to environmentally relevant concentrations of pharmaceuticals.

Low concentrations of pharmaceuticals in the environment (ng/L to µg/L) are an environmental concern. We used the invertebrates, Hydra oligactis and Hydra viridissima, as freshwater models for primary toxicity testing to study effects of chronic low concentrations of pharmaceuticals in the environment. H. oligactis were exposed to three concentrations (0.1, 1.0 and 10 µg/L) of either fluoxetine, carbamazepine, or triclosan; H. viridissima were exposed to three concentrations (0.1, 1.0 and 10 µg/L) of triclosan. Ecologically relevant endpoints including morphology, budding rate, feeding behaviour, and regenerative capacity were examined during the 14 days exposure period. The interstitial:epithelial stem cell ratios was also examined in H. oligactis. There were no significant effects on the morphology, budding rate and feeding behaviour of the H. oligactis across all concentrations of fluoxetine, carbamazepine, and triclosan. However, regenerative capacity significantly decreased in comparison to the controls when H. oligactis was exposed to 10 µg/L of triclosan and fluoxetine, although there was no significant difference when exposed to carbamazepine. Neither fluoxetine nor carbamazepine treatment altered stem cell ratios. Exposure to triclosan at any concentration did not impact H. viridissima morphology, budding rate, regeneration or feeding behaviour. These results show there are limited effects in Hydra after exposure to chronic, low concentrations of fluoxetine, carbamazepine, and triclosan, except for regeneration in H. oligactis. These endpoints can be used effectively (and cost effectively) to study the effects of environmentally relevant concentrations of pharmaceuticals in Hydra species.

Modifying effects of a cobble substrate on thermal environments and implications for embryonic development in lake whitefish (Coregonus clupeaformis).

A laboratory flume was constructed to examine substrate effects on aquatic development. The flume was designed as a once-through system with a submerged cobble-filled corebox. Lake whitefish (Coregonus clupeaformis) embryos and temperature probes were deployed at multiple sites within the cobble and in the open water channel. Embryos were incubated in the flume for two different experimental periods: one to examine substrate impacts during natural lake cooling (37 days: 5 December 2016 to 10 January 2017) and the second to investigate substrate effects while administering a twice weekly 1 h heat shock (51 days: 11 January to 2 March 2017). During incubation, no significant difference was found in the average temperature between locations; however, temperatures were more stable within the cobble. Following both incubation periods, embryos retrieved from the cobble were significantly smaller in both dry mass and body length by up to 20%. These results demonstrate differences between embryos submerged in a cobble substrate and in the open water column, highlighting the need to consider the physical influences from the incubation environment when assessing development effects as part of any scientific study or environmental assessment.

How "simple" methodological decisions affect interpretation of population structure based on reduced representation library DNA sequencing: A case study using the lake whitefish.

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.

Paternal Exposure to Carbamazepine Impacts Zebrafish Offspring Reproduction Over Multiple Generations.

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.

Parental gemfibrozil exposure impacts zebrafish F1 offspring, but not subsequent generations.

Gemfibrozil (GEM) is a fibrate lipid regulator and one of the most commonly occurring fresh water pharmaceuticals. The negative effects of fibrates including GEM on fish reproduction have been frequently reported including effects of F0 GEM exposure on reproduction of the unexposed F1 offspring. We predicted that chronic, direct exposure of zebrafish with low concentrations of GEM would adversely affect parental male reproduction and unexposed offspring for multiple generations. Adult zebrafish were exposed to 10 µg/L GEM for 6 weeks and a range of reproductive indices were analyzed. The F1-F4 offspring were reared in clean water from 3 distinct lineages where only a single or both parents were exposed and compared to a control lineage where parents were unexposed. Reproductive indices were examined in unexposed F1-F4 offspring to test the hypothesis of multi- or trans- generational impacts. Exposure to GEM caused a decline in breeding success and mean embryo production in F0 parents and a reduction in whole body 11-ketotestosterone (11-KT), altered male courtship, aggression and sperm morphology. Our results indicate that paternal exposure alone is sufficient to result in reproductive effects in unexposed male offspring but that effects are mostly limited to F1. We suggest that GEM may act as a reproductive endocrine disruptor in fish and that chronic exposure reduced male reproductive fitness but not over multiple generations.

Waterborne pharmaceutical uptake and toxicity is modified by pH and dissolved organic carbon in zebrafish.

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.

Cardiovascular and growth outcomes of C57Bl/6J mice offspring exposed to maternal stress and ionizing radiation during pregnancy.

Purpose: Developmental programming involves an adverse intrauterine environment which can result in offspring phenotype changes following birth. The developmental programming of hypertension has been reported to possibly involve oxidative stress at the cellular level. Ionizing radiation produces oxidative stress, even at low doses, and irradiation of animals is often coupled with potential sources of maternal stress such as transportation of animals or repeated handling. Materials and methods: Pregnant C57Bl/6J mice were irradiated on gestational day 15 with 5-1000 mGy 137Cs gamma radiation. Post-natal weight, blood pressure (BP) and heart rate (HR) were measured. Radiation had minimal effects at doses ≤300 mGy, but 1000 mGy caused a significant reduction in HR in male pups and growth reduction at 16 weeks of age in both genders. The sham-irradiation protocol included repeated transportation in order to acclimate animals to transport. However, it may have resulted in programming, as sham-irradiation alone resulted in elevated BP measures compared to the offspring of animals that were never transported. Results and conclusions: Overall, there were minimal effects on cardiovascular measures or offspring weight due to irradiation except at 1000 mGy. The presence of maternal stress, a known trigger of developmental programming, may have confounded any potential irradiation effects.

CYP3C gene regulation by the aryl hydrocarbon and estrogen receptors in zebrafish.

Cytochrome P450 (CYPs) enzymes are critical for the metabolism of exogenous and endogenous compounds. In mammals, the CYP3s are arguably the most important xenobiotic metabolizing enzymes and are all contained within the CYP3A subfamily. In fish, CYP3s include CYP3A and multiple subfamilies unique to the teleost lineage. The goal of this study was to provide insight on the regulation of genes in the CYP3C subfamily. Zebrafish, which have 4 CYP3C genes, were exposed to 17ß-estradiol (E2; 0.001-10 µM) or ß-naphthoflavone (ßNF; 0.005-1 µM), prototypical ligands of the estrogen receptor (ER) and the aryl hydrocarbon receptor (AhR), respectively. Gene expression was measured in the liver, intestine and gonads using quantitative PCR. CYP1A and vitellogenin (VTG) gene expression were used as positive controls for AhR and ER regulation, respectively. Exposure to ßNF resulted in the dose-dependant induction of CYP1A and CYP3C genes in the female intestine but not in the liver. E2 exposure resulted in the induction of all CYP3Cs in the male intestine and in the female liver. VTG was induced in both female and male livers. CYP3C3 and CYP3C4 were induced in the testis; CYP3C1 and CYP3C4 were slightly induced in the ovary. The time-course of gene induction was investigated in the liver and intestine after exposure to ßNF (0.5 µM) and E2 (0.1 µM). Inducible genes were up-regulated within 12 h after exposure. These data support a role for the AhR and ER in the regulation of CYP3Cs. Overall, the induction of CYP3Cs by AhR and ER ligands is different from mammalian CYP3A and may suggest a functional role for CYP3Cs that involves planar aromatic hydrocarbons and steroids.

Daily, repeating fluctuations in embryonic incubation temperature alter metabolism and growth of Lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis) utilize overwintering embryonic development (up to 180 days), and such stenothermic, cold-water embryos may be particularly susceptible to thermal shifts. We incubated whitefish embryos in temperature treatments that were constant temperature (2.0 ±â€¯0.1 °C, 5.0 ±â€¯0.1 °C, and 8.0 ±â€¯0.1 °C; mean ±â€¯SD) or variable temperature (VT, mean = 5.0 ±â€¯0.3 °C). In the VT, a daily 2 °C temperature change followed a continuous pattern throughout development: 2-4-6-8-6-4-2 °C. Hatchling survival proportion from fertilization to hatch was significantly impacted by incubation temperature (P < 0.001): 2 °C (0.88 ±â€¯0.01) and 5 °C (0.91 ±â€¯0.01) showed higher survival than both the VT (0.83 ±â€¯0.02) and 8 °C groups (0.15 ±â€¯0.06), which were statistically distinct from each other. Time to hatch (dpf) was significantly different across all treatments (P < 0.001): 8 °C (68 ±â€¯2 dpf), VT (111 ±â€¯4 dpf), 5 °C (116 ±â€¯4 dpf), 2 °C (170 ±â€¯3 dpf). Likewise, hatchling yolk-free dry mass (mg) and total body length (mm) were significantly different across all treatments (P < 0.001): 8 °C (0.66 ±â€¯0.08 mg; 11.1 ±â€¯0.08 mm), VT (0.97 ±â€¯0.06 mg; 11.7 ±â€¯0.05 mm), 5 °C (1.07 ±â€¯0.03 mg; 12.0 ±â€¯0.02 mm), 2 °C (1.36 ±â€¯0.04 mg; 12.8 ±â€¯0.05 mm). Oxygen consumption rate (V̇o2) was significantly affected by the interaction between treatment and measurement temperature (P < 0.001). Hatchling VT whitefish showed mean V̇o2 that was higher compared to the 2 °C group measured at 2 °C, and lower compared to the 2 °C and 5 °C group measured at 8 °C. This study demonstrates that the VT incubation treatment produced fewer (increased mortality), smaller embryos that hatched earlier than 2 °C and 5 °C embryos. The plasticity of V̇o2 for this stenothermic-incubating fish species under variable incubation conditions reveals a metabolic cost to cycling thermal incubation conditions.