Nascent transcription reveals regulatory changes in extremophile fishes inhabiting hydrogen sulfide-rich environments.

Regulating transcription allows organisms to respond to their environment, both within a single generation (plasticity) and across generations (adaptation). We examined transcriptional differences in gill tissues of fishes in the Poecilia mexicana species complex (family Poeciliidae), which have colonized toxic springs rich in hydrogen sulfide (H2S) in southern Mexico. There are gene expression differences between sulfidic and non-sulfidic populations, yet regulatory mechanisms mediating this gene expression variation remain poorly studied. We combined capped-small RNA sequencing (csRNA-seq), which captures actively transcribed (i.e. nascent) transcripts, and messenger RNA sequencing (mRNA-seq) to examine how variation in transcription, enhancer activity, and associated transcription factor binding sites may facilitate adaptation to extreme environments. csRNA-seq revealed thousands of differentially initiated transcripts between sulfidic and non-sulfidic populations, many of which are involved in H2S detoxification and response. Analyses of transcription factor binding sites in promoter and putative enhancer csRNA-seq peaks identified a suite of transcription factors likely involved in regulating H2S-specific shifts in gene expression, including several key transcription factors known to respond to hypoxia. Our findings uncover a complex interplay of regulatory processes that reflect the divergence of extremophile populations of P. mexicana from their non-sulfidic ancestors and suggest shared responses among evolutionarily independent lineages.

Exaggerated heterochiasmy in a fish with sex-linked male coloration polymorphisms.

It is often stated that polymorphisms for mutations affecting fitness of males and females in opposite directions [sexually antagonistic (SA) polymorphisms] are the main selective force for the evolution of recombination suppression between sex chromosomes. However, empirical evidence to discriminate between different hypotheses is difficult to obtain. We report genetic mapping results in laboratory-raised families of the guppy (Poecilia reticulata), a sexually dimorphic fish with SA polymorphisms for male coloration genes, mostly on the sex chromosomes. Comparison of the genetic and physical maps shows that crossovers are distributed very differently in the two sexes (heterochiasmy); in male meiosis, they are restricted to the termini of all four chromosomes studied, including chromosome 12, which carries the sex-determining locus. Genome resequencing of male and female guppies from a population also indicates sex linkage of variants across almost the entire chromosome 12. More than 90% of the chromosome carrying the male-determining locus is therefore transmitted largely through the male lineage. A lack of heterochiasmy in a related fish species suggests that it originated recently in the lineage leading to the guppy. Our findings do not support the hypothesis that suppressed recombination evolved in response to the presence of SA polymorphisms. Instead, a low frequency of recombination on a chromosome that carries a male-determining locus and has not undergone genetic degeneration has probably facilitated the establishment of male-beneficial coloration polymorphisms.

microRNA expression variation as a potential molecular mechanism contributing to adaptation to hydrogen sulphide.

microRNAs (miRNAs) are post-transcriptional regulators of gene expression and can play an important role in modulating organismal development and physiology in response to environmental stress. However, the role of miRNAs in mediating adaptation to diverse environments in natural study systems remains largely unexplored. Here, we characterized miRNAs and their expression in Poecilia mexicana, a species of small fish that inhabits both normal streams and extreme environments in the form of springs rich in toxic hydrogen sulphide (H2 S). We found that P. mexicana has a similar number of miRNA genes as other teleosts. In addition, we identified a large population of mature miRNAs that were differentially expressed between locally adapted populations in contrasting habitats, indicating that miRNAs may contribute to P. mexicana adaptation to sulphidic environments. In silico identification of differentially expressed miRNA-mRNA pairs revealed, in the sulphidic environment, the downregulation of miRNAs predicted to target mRNAs involved in sulphide detoxification and cellular homeostasis, which are pathways essential for life in H2 S-rich springs. In addition, we found that predicted targets of upregulated miRNAs act in the mitochondria (16.6% of predicted annotated targets), which is the main site of H2 S toxicity and detoxification, possibly modulating mitochondrial function. Together, the differential regulation of miRNAs between these natural populations suggests that miRNAs may be involved in H2 S adaptation by promoting functions needed for survival and reducing functions affected by H2 S. This study lays the groundwork for further research to directly demonstrate the role of miRNAs in adaptation to H2 S. Overall, this study provides a critical stepping-stone towards a comprehensive understanding of the regulatory mechanisms underlying the adaptive variation in gene expression in a natural system.

Molecular insights into post-mating immune response in a fish with internal fertilization.

The tight connection between immunity and reproduction has been studied for decades. However, basic knowledge at the molecular level of the effect of mating on immune function is still lacking in many taxa. Determining whether and how the immune system is engaged after mating is a crucial step in understanding post-mating mechanisms of reproduction and sexual selection. Here, we study the transcriptional changes in immunity-related genes caused by the ejaculate in the female reproductive tract using a model species for sexual selection studies, the guppy Poecilia reticulata. To study changes triggered by the ejaculate only, rather than caused by mating, we used artificial inseminations to transfer ejaculate into females. We then compared gene expression in the reproductive tract (gonoduct and ovary) of females artificially inseminated either with ejaculate or with a control solution, after 1 hr and after 6 hr. Overall, contact with ejaculate caused short-term changes in the expression of immune-related genes in the female reproductive tract, with a complex pattern of up- and down-regulation of immune-related pathways, but with clear indication of a marked down-regulation of the immune system shortly after ejaculate contact. This suggests a link between immune function and processes occurring between mating and fertilization in this species.

Impacts of the biocide chlorothalonil on biomarkers of oxidative stress, genotoxicity, and sperm quality in guppy Poecilia vivipara.

Chlorothalonil is a fungicide present in antifouling paints and other formulations used in agriculture, although studies have shown this chemical to be toxic to fish species. To clarify the deleterious effects of chlorothalonil for these non-target organisms, the present study evaluated the toxic effects of this biocide for the estuarine guppy Poecilia vivipara in terms of an acute mortality test (96 h) and the analysis of biomarkers of oxidative stress, genotoxicity, and sperm quality. The LC50 calculated for P. vivipara was 40.8 µg/L of chlorothalonil. For the analysis of biomarkers, fish were exposed (96 h) to 1 and 10 µg/L of chlorothalonil. It was observed that chlorothalonil alters the levels of pro- and antioxidants towards oxidative stress. In the gills, a negative effect on total antioxidant capacity (ACAP) was detected, while there was a reduction in the activity of glutathione S-transferase (GST) in the liver. However, levels of glutathione (GSH) and the activity and glutamate-cysteine-ligase (GCL) increased in both tissues, as a possible detoxification response. Following chlorothalonil exposure, oxidative damage measured by lipoperoxidation (LPO) significantly increased at the cellular level only (red blood cells (RBCs) and sperm cells). An increase in fluidity of membranes, reactive oxygen species concentration and micronuclei (MNs) incidence were also seen in RBCs. In sperm cells, LPO increased, while membrane and mitochondrial functionality as well as sperm motility decreased. Based on these results, chlorothalonil can be considered as a toxic compound for fish, causing genotoxicity and affecting the RBCs physiology and the fertility of males of P. vivipara.

Triphenyltin exposure affects mating behaviors and attractiveness to females during mating in male guppies (Poecilia reticulata).

The impacts of triphenyltin (TPT) on ecological health have been of great concern due to their widespread use and ubiquity in aquatic ecosystems. However, little is known about the effects of TPT on the reproductive behaviors of fishes. Therefore, the present study was conducted to investigate the effects of TPT at environmentally relevant concentrations (0, 1 and 10 ng Sn/L) on the mating behaviors and the attractiveness to females during mating in male guppies (Poecilia reticulata). The results showed that TPT exposure disturbed the mating behaviors; the TPT-exposed male fish performed more sneaking attempts, but no changes in sigmoid courtship were displayed. The increases in sneaking attempts might be related to increases in testosterone levels induced by TPT exposure. In the context of a competing male, the TPT-exposed males were less attractive to females during mating. The decreases in attractiveness might be related to decreases in carotenoid-based coloration, shown as decreases in caudal fin redness values and skin carotenoid contents. In addition, TPT-induced total antioxidant capacities, the activities of superoxide dismutase and catalase, and the contents of malondialdehyde in liver and intestinal tissues indicated increases in oxidative stress. Both oxidative stress and coloration are linked to carotenoids. Thus, we speculated that the TPT-exposed males might use carotenoids to cope with increases in oxidative stress at the expense of carotenoid-based coloration. The disruption of mating behaviors and the decrease in attractiveness to females in male fish could result in reproductive failure. The present study underscores the importance of using behavioral tests as a sensitive tool in assessing the impact of pollutants present in aquatic environments.

Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ.

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

The gonadal transcriptome of the unisexual Amazon molly Poecilia formosa in comparison to its sexual ancestors, Poecilia mexicana and Poecilia latipinna.

BACKGROUND: The unisexual Amazon molly (Poecilia formosa) originated from a hybridization between two sexual species, the sailfin molly (Poecilia latipinna) and the Atlantic molly (Poecilia mexicana). The Amazon molly reproduces clonally via sperm-dependent parthenogenesis (gynogenesis), in which the sperm of closely related species triggers embryogenesis of the apomictic oocytes, but typically does not contribute genetic material to the next generation. We compare for the first time the gonadal transcriptome of the Amazon molly to those of both ancestral species, P. mexicana and P. latipinna. RESULTS: We sequenced the gonadal transcriptomes of the P. formosa and its parental species P. mexicana and P. latipinna using Illumina RNA-sequencing techniques (paired-end, 100 bp). De novo assembly of about 50 million raw read pairs for each species was performed using Trinity, yielding 106,922 transcripts for P. formosa, 115,175 for P. latipinna, and 133,025 for P. mexicana after eliminating contaminations. On the basis of sequence similarity comparisons to other teleost species and the UniProt databases, functional annotation, and differential expression analysis, we demonstrate the similarity of the transcriptomes among the three species. More than 40% of the transcripts for each species were functionally annotated and about 70% were assigned to orthologous genes of a closely related species. Differential expression analysis between the sexual and unisexual species uncovered 2035 up-regulated and 564 down-regulated genes in P. formosa. This was exemplary validated for six genes by qRT-PCR. CONCLUSIONS: We identified more than 130 genes related to meiosis and reproduction within the apomictically reproducing P. formosa. Overall expression of these genes seems to be down-regulated in the P. formosa transcriptome compared to both ancestral species (i.e., 106 genes down-regulated, 29 up-regulated). A further 35 meiosis and reproduction related genes were not found in the P. formosa transcriptome, but were only expressed in the sexual species. Our data support the hypothesis of general down-regulation of meiosis-related genes in the apomictic Amazon molly. Furthermore, the obtained dataset and identified gene catalog will serve as a resource for future research on the molecular mechanisms behind the reproductive mode of this unisexual species.

Individual variation in ACTH-induced cortisol levels in females of a livebearing fish at different gestational stages.

Individuals vary in their baseline levels of stress hormones (predictive homeostasis) and in their stress responses (reactive homeostasis). Variation in normal reactive scope, both predictive and reactive homeostasis, may be important for understanding how endocrine traits respond to selection. Reactive homeostasis is the increase in glucocorticoid (GCs) hormones above baseline. Individuals at different life history stages, such as gestation in females, may show variation in normal reactive scope. We performed an adrenocorticotropic hormone (ACTH) challenge and measured changes in circulating GCs to estimate the reactive scope of female sailfin mollies (Poecilia latipinna) at different gestational states. We measured cortisol, primary GC in teleost fishes, to obtain baseline release rates prior to injection with either ACTH or saline control. Using water-borne hormones, we measured cortisol release rates at four time intervals post-injection. Females were then sacrificed to determine the developmental stage of embryos, if present, and the number of developing embryos or mature ova. We found that ACTH-injected females had significant increases in cortisol releases rates, whereas cortisol release rates of control females did not change during the 4 h post-injection period. We found high repeatability in predictive homeostasis of cortisol and moderate repeatability in reactive homeostasis and a phenotypic correlation between predictive and reactive homeostasis. Gestational state did not affect female predictive or reactive homeostasis. We applied the reactive scope model to P. latipinna and gained a further understanding of how among- and within-individual variation in both predictive and reactive homeostasis are partitioned and how these traits vary under certain life-history conditions.

Physiological and behavioural responses to acid and osmotic stress and effects of Mucuna extract in Guppies.

Variation in pH (acidification) and salinity conditions have severe impact at different levels of biological organization in fish. Present study focused to assess the effects of acidification and salinity changes on physiological stress responses at three different levels of function: i) hormonal and oxidative response, ii) osmoregulation and iii) reproduction, in order to identify relevant biomarkers. Second objective of the study was to evaluate the efficacy of plant (Mucuna pruriens) extract for alleviating pH and salinity related stress. Guppies (Poecilia reticulata) were exposed to different pH (6.0, 5.5, 5.0) and salinity (1.5, 3.0, 4.5 ppt) for 7, 14 and 21 days. Following exposure to stress for respective duration, fish were fed diet containing methanol extract of Mucuna seeds (dose 0.80 gm/kg feed) for 7, 14 and 21 days to measure their possible recovery response. Stress hormone (cortisol), hepatic oxidative stress parameters [superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRd), glutathione peroxidise (GPx), glutathione S-transferase (GST), malondialdehyde (MDA), glutathione (GSH)], gill osmoregulatory response (Na+-K+ATPase activity), sex steroid profiles and mating behaviours (gonopodial thrust and gestation period) were estimated. Cortisol and MDA levels increased with dose and duration of acid and salinity stress, and cortisol levels were higher in males than in females. Effect on Na+-K+ATPase activity was more intense by salinity stress rather than pH induced stress. Both acid and salinity stress reduced sex steroid levels, and mating response was highly affected by both stresses in a dose- and duration-dependent manner. Mucuna treatment reduced stress-induced alteration of cortisol, MDA, Na+-K+ATPase activity and reproductive parameters. Dietary administration of Mucuna seed extract decreased the intensity of environmental stressors at all three functional levels. Mucuna treatment was more effective against salinity stress than acid stress. Thus, cortisol, oxidative stress marker MDA and Na+-K+ATPase could be effective indicators for acid and salinity stress in wild and domestic fish populations. Dietary administration of Mucuna extract may limit the detrimental effects of acidification and salinity variations that are the inevitable outcomes expected under global climate change conditions.

Cholinergic alterations by exposure to pesticides used in control vector: Guppies fish (Poecilia reticulta) as biological model.

Spinosad and temephos are two of the most used pesticides in Mexico for the control of vector causing disease such as dengue, chikungunya and Zika. The aim of this study was to compare the neurotoxic effects of these two pesticides using guppy fish (Poecilia reticulata) as a model organism. Guppies were exposed for 7 and 21 days to technical grade temephos and spinosad at 1.0 and 0.07 g/L, respectively, (10 and 0.5 mg/L of active substance; concentrations recommended by the Ministery of Health of the State (Secretaría de Salud de Nayarit (SSN) Mexico)). Subsequently, acetylcholinesterase activity (AChE) and acetylcholine concentrations (ACh) in muscle tissue were determined. Temephos exposure decreased AChE activity and increased ACh concentration, whereas exposure to spinosad only increased ACh concentration. Though cholinergic alterations were more severe in fish exposed to temephos, both pesticides were equally lethal during the first seven days after exposure. Nonetheless, temephos was more lethal after 21 days.

Mechanisms Underlying Adaptation to Life in Hydrogen Sulfide-Rich Environments.

Hydrogen sulfide (H2S) is a potent toxicant interfering with oxidative phosphorylation in mitochondria and creating extreme environmental conditions in aquatic ecosystems. The mechanistic basis of adaptation to perpetual exposure to H2S remains poorly understood. We investigated evolutionarily independent lineages of livebearing fishes that have colonized and adapted to springs rich in H2S and compared their genome-wide gene expression patterns with closely related lineages from adjacent, nonsulfidic streams. Significant differences in gene expression were uncovered between all sulfidic and nonsulfidic population pairs. Variation in the number of differentially expressed genes among population pairs corresponded to differences in divergence times and rates of gene flow, which is consistent with neutral drift driving a substantial portion of gene expression variation among populations. Accordingly, there was little evidence for convergent evolution shaping large-scale gene expression patterns among independent sulfide spring populations. Nonetheless, we identified a small number of genes that was consistently differentially expressed in the same direction in all sulfidic and nonsulfidic population pairs. Functional annotation of shared differentially expressed genes indicated upregulation of genes associated with enzymatic H2S detoxification and transport of oxidized sulfur species, oxidative phosphorylation, energy metabolism, and pathways involved in responses to oxidative stress. Overall, our results suggest that modification of processes associated with H2S detoxification and toxicity likely complement each other to mediate elevated H2S tolerance in sulfide spring fishes. Our analyses allow for the development of novel hypotheses about biochemical and physiological mechanisms of adaptation to extreme environments.

Differential sensitivity to estrogen-induced opsin expression in two poeciliid freshwater fish species.

The sensory system shapes an individual's perception of the world, including social interactions with conspecifics, habitat selection, predator detection, and foraging behavior. Sensory signaling can be modulated by steroid hormones, making these processes particularly vulnerable to environmental perturbations. Here we examine the influence of exogenous estrogen manipulation on the visual physiology of female western mosquitofish (Gambusia affinis) and sailfin mollies (Poecilia latipinna), two poeciliid species that inhabit freshwater environments across the southern United States. We conducted two experiments to address this aim. First, we exposed females from both species to a one-week dose response experiment with three treatments of waterborne ß-estradiol. Next, we conducted a one-week estrogen manipulation experiment with a waterborne estrogen (ß-Estradiol), a selective estrogen receptor modulator (tamoxifen), or combination estrogen and tamoxifen treatment. We used quantitative PCR (qPCR) to examine the expression of cone opsins (SWS1, SWS2b, SWS2a, Rh2, LWS), rhodopsin (Rh1), and steroid receptor genes (ARα, ARß, ERα, ERß2, GPER) in the eyes of individual females from each species. Results from the dose response experiment revealed estradiol-sensitivity in opsin (SWS2a, Rh2, Rh1) and androgen receptor (ARα, ARß) gene expression in mosquitofish females, but not sailfins. Meanwhile, our estrogen receptor modulation experiments revealed estrogen sensitivity in LWS opsin expression in both species, along with sensitivity in SWS1, SWS2b, and Rh2 opsins in mosquitofish. Comparisons of control females across experiments reveal species-level differences in opsin expression, with mosquitofish retinas dominated by short-wavelength sensitive opsins (SWS2b) and sailfins retinas dominated by medium- and long-wavelength sensitive opsins (Rh2 and LWS). Our research suggests that variation in exogenous levels of sex hormones within freshwater environments can modify the visual physiology of fishes in a species-specific manner.

Gender-specific histopathological response in guppies Poecilia reticulata exposed to glyphosate or its metabolite aminomethylphosphonic acid.

Ecotoxicity of glyphosate (GLY) and its metabolite aminomethylphosphonic acid (AMPA) was investigated in guppies, Poecilia reticulata. We tested the effects of these chemicals on the gills and liver of both male and female guppies using qualitative and quantitative histopathological analyses associated with histopathological condition indexes. Both genders showed similar median lethal concentration (LC50 ) at 96 h for GLY (68.78 and 70.87 mg l-1 ) and AMPA (180 and 164.32 mg l-1 ). However, the histopathological assessment of both fish organs exposed to sublethal concentrations of GLY (35 mg l-1 ) and AMPA (82 mg l-1 ) for 96 h showed a tissue- and gender-specific histopathological response. In both exposure assays, fish presented mainly progressive changes, such as proliferation of the interlamellar epithelium, partial and total fusion of secondary lamellae. The liver showed mainly regressive changes, such as steatosis, pyknotic nuclei and high distribution of collagen fibers. Unusually large hepatocytes as degenerated cells were also detected. Histopathological changes in gills were similar for the males and females, but the liver response was different between the genders. The hepatic inflammatory changes were more common in males. The increase in the area of hepatocyte vacuoles is gender dependent with higher values in the male compared to the female guppies exposed to GLY and AMPA. Multiparametric analysis indicated that the male guppies are more sensitive than females, particularly in the presence of AMPA. Our study shows that the histopathological assessment associated with gender-specific response can be successfully used in ecotoxicological assessment of GLY and the metabolite AMPA. Copyright © 2017 John Wiley & Sons, Ltd.

Extremophile Poeciliidae: multivariate insights into the complexity of speciation along replicated ecological gradients.

BACKGROUND: Replicate population pairs that diverge in response to similar selective regimes allow for an investigation of (a) whether phenotypic traits diverge in a similar and predictable fashion, (b) whether there is gradual variation in phenotypic divergence reflecting variation in the strength of natural selection among populations, (c) whether the extent of this divergence is correlated between multiple character suites (i.e., concerted evolution), and (d) whether gradual variation in phenotypic divergence predicts the degree of reproductive isolation, pointing towards a role for adaptation as a driver of (ecological) speciation. Here, we use poeciliid fishes of the genera Gambusia and Poecilia that have repeatedly evolved extremophile lineages able to tolerate high and sustained levels of toxic hydrogen sulfide (H2S) to answer these questions. RESULTS: We investigated evolutionary divergence in response to H2S in Gambusia spp. (and to a lesser extent Poecilia spp.) using a multivariate approach considering the interplay of life history, body shape, and population genetics (nuclear miscrosatellites to infer population genetic differentiation as a proxy for reproductive isolation). We uncovered both shared and unique patterns of evolution: most extremophile Gambusia predictably evolved larger heads and offspring size, matching a priori predictions for adaptation to sulfidic waters, while variation in adult life histories was idiosyncratic. When investigating patterns for both genera (Gambusia and Poecilia), we found that divergence in offspring-related life histories and body shape were positively correlated across populations, but evidence for individual-level associations between the two character suites was limited, suggesting that genetic linkage, developmental interdependencies, or pleiotropic effects do not explain patterns of concerted evolution. We further found that phenotypic divergence was positively correlated with both environmental H2S-concentration and neutral genetic differentiation (a proxy for gene flow). CONCLUSIONS: Our results suggest that higher toxicity exerts stronger selection, and that divergent selection appears to constrain gene flow, supporting a scenario of ecological speciation. Nonetheless, progress toward ecological speciation was variable, partially reflecting variation in the strength of divergent selection, highlighting the complexity of selective regimes even in natural systems that are seemingly governed by a single, strong selective agent.

Expression change in Angiopoietin-1 underlies change in relative brain size in fish.

Brain size varies substantially across the animal kingdom and is often associated with cognitive ability; however, the genetic architecture underpinning natural variation in these key traits is virtually unknown. In order to identify the genetic architecture and loci underlying variation in brain size, we analysed both coding sequence and expression for all the loci expressed in the telencephalon in replicate populations of guppies (Poecilia reticulata) artificially selected for large and small relative brain size. A single gene, Angiopoietin-1 (Ang-1), a regulator of angiogenesis and suspected driver of neural development, was differentially expressed between large- and small-brain populations. Zebra fish (Danio rerio) morphants showed that mild knock down of Ang-1 produces a small-brained phenotype that could be rescued with Ang-1 mRNA. Translation inhibition of Ang-1 resulted in smaller brains in larvae and increased expression of Notch-1, which regulates differentiation of neural stem cells. In situ analysis of newborn large- and small-brained guppies revealed matching expression patterns of Ang-1 and Notch-1 to those observed in zebrafish larvae. Taken together, our results suggest that the genetic architecture affecting brain size in our population may be surprisingly simple, and Ang-1 may be a potentially important locus in the evolution of vertebrate brain size and cognitive ability.

Determining high-quality critical body residues for multiple species and chemicals by applying improved experimental design and data interpretation concepts.

Ecotoxicological effect data are generally expressed as effective concentrations in the external exposure medium and do thus not account for differences in chemical uptake, bioavailability, and metabolism, which can introduce substantial data variation. The Critical Body Residue (CBR) concept provides clear advantages, because it links effects directly to the internal exposure. Using CBRs instead of external concentrations should therefore reduce variability. For compounds that act via narcosis even a constant CBR has been proposed. Despite the expected uniformity, CBR values for these compounds still show large variability, possibly due to biased and inconsistent experimental testing. In the present study we tested whether variation in CBR data can be substantially reduced when using an improved experimental design and avoiding confounding factors. The aim was to develop and apply a well-defined test protocol for accurately and precisely measuring CBR data, involving improved (passive) dosing, sampling, and processing of organisms. The chemicals 1,2,4-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, 2,3,4-trichloroaniline, 2,3,5,6-tetrachloroaniline, 4-chloro-3-methylphenol, pentylbenzene, pyrene, and bromophos-methyl were tested on Lumbriculus variegatus (California blackworm), Hyalella azteca (scud), and Poecilia reticulata (guppy), which yielded a high-quality database of 348 individual CBR values. Medians of CBR values ranged from 2.1 to 16.1 mmol/kg wet weight (ww) within all combinations of chemicals and species, except for the insecticide bromophos-methyl, for which the median was 1.3 mmol/kg ww. The new database thus covers about one log unit, which is considerably less than in existing databases. Medians differed maximally by a factor of 8.4 between the 7 chemicals but within one species, and by a factor of 2.6 between the three species but for individual chemicals. Accounting for the chemicals' internal distribution to different partitioning domains and relating effects to estimated concentrations in the target compartment (i.e., membrane lipids) was expected to but did not decrease the overall variability, likely because the surrogate partition coefficients for membrane lipid, storage lipid, protein, and carbohydrate that were used as input parameters did not sufficiently represent the actual partitioning processes. The results of this study demonstrate that a well-designed test setup can produce CBR data that are highly uniform beyond chemical and biological diversity.

Enzymatic Alterations and Genotoxic Effects Produced by Sublethal Concentrations of Organophosphorous Temephos in Poecilia reticulata.

The responses of biochemical and genetic parameters were evaluated in tissues of Poecilia reticulata exposed to sublethal and environmentally relevant concentrations of 0.005, 0.01, or 0.02 mg/L of the organophosphorous (OP) pesticide temephos (TE) for 168 h. Activities of enzymes brain acetylcholinesterase (AChE) and liver carboxylesterase (CbE) were determined. Nuclear abnormalities (NA) and micronucleus (MN) frequency in gill erythrocytes were also measured. No mortality was observed over the experimental period; however, brain AChE activities were decreased significantly in guppies in all TE treatment groups after 72 h of exposure. Hepatic CbE activities of fish were increased in all TE treatment groups at 96, 120, and 144 h of exposure. The frequencies of MN and NA in fish gill erythrocytes displayed a marked rise after 168 h of exposure to concentrations of 0.01 or 0.02 mg/L TE. Thus, determination of these parameters may be employed as potential indices of exposure to TE using this sentinel organism for monitorining.

Accumulation of heavy metals in the fish, Oreochromis niloticus and Poecilia latipinna and their concentration in water and sediment of dam lake of Wadi Namar, Saudi Arabia.

The present study reports the accumulation of heavy metals like Cu, Hg, Cd, Pb and Cr in different tissues viz. liver, kidney, gills and muscles of Oreochromisniloticus and Poecilia latipinna from two sites in dam lake of Wadi Namar. Water and sediment samples were also collected from two sites for heavy metal analysis. Metal concentration in water and sediment samples of both the sites were observed in the following order: Cu>Cr>Pb>Cd>Hg; however, their concentration was found to be more at site 2 as compared to site 1. The order of metal accumulation in different tissues of O. niloticus and P. latipinna was in the following order: Cu>Cr>Pb>Cd>Hg at both the sites, while liver accumulated maximum amounts of metals followed by kidney, gills and muscles. The results showed the site 2 was more polluted by metals than Site 1 and O. niloticus accumulated greater amount of metals than P. latipinna.

Olfactory receptor OR52N2 for PGE2 in mediation of guppy courtship behaviors.

Prostaglandins (PGs) are a type of physiologically active unsaturated fatty acids. As an important sex pheromone, PGs play a vital role in regulating the reproductive behaviors of species by mediating nerve and endocrine responses. In this study, guppy (Poecilia reticulate) was used as the model specie to detect the function of PGE2 in inducing the onset of courtship behaviors. Our results showed that adding PGE2 into the water environment could activate the courtship behavior of male guppy, indicating that the peripheral olfactory system mediated the PGE2 function. Thereafter, the open reading frame (ORF) of olfactory receptor or52n2 was cloned, which was 936 bp in length, coding 311 amino acids. As a typical G protein-coupled receptor, OR52N2 had a conservative seven α-helix transmembrane domains. To confirm the regulatory relationship between OR52N2 and PGE2, dual-luciferase reporter assay was employed to verify the activation of downstream CREB signaling pathways. Results showed that PGE2 significantly enhanced CRE promoter activity in or52n2 ORF transient transfected HEK-293 T cells. Finally, localization of or52n2 mRNA were observed in ciliated receptor cells of the olfactory epithelium using in situ hybridization. Our results provide a novel insight into sex pheromone signaling transduction in reproductive behavior.