Gene Expression In Fish Research Articles

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

Fish respond to salinity stress by transcriptional induction of many genes, but the mechanism of their osmotic regulation is unknown. We developed a reporter assay using cells derived from the brain of the tilapia Oreochromis mossambicus (OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes of Omossambicus Genomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1.1) and myo-inositol phosphate synthase (MIPS), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two in IMPA1.1 and three in MIPS) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named "OSRE1." Additional OSREs that were less effective in conferring salinity-induced trans-activation and do not match the OSRE1 consensus also were identified in both MIPS and IMPA1.1 Although OSRE1 shares homology with the mammalian osmotic-response element/tonicity-responsive enhancer (ORE/TonE) enhancer, the latter is insufficient to confer osmotic induction in fish. Like other enhancers, OSRE1 trans-activates genes independent of orientation. We conclude that OSRE1 is a cis-regulatory element (CRE) that enhances the hyperosmotic induction of osmoregulated genes in fish. Our study also shows that tailored reporter assays developed for OmB cells facilitate the identification of CREs in fish genomes. Knowledge of the OSRE1 motif allows affinity-purification of the corresponding transcription factor and computational approaches for enhancer screening of fish genomes. Moreover, our study enables targeted inactivation of OSRE1 enhancers, a method superior to gene knockout for functional characterization because it confines impairment of gene function to a specific context (salinity stress) and eliminates pitfalls of constitutive gene knockouts (embryonic lethality, developmental compensation).

Linking cortisol response with gene expression in fish exposed to gold nanoparticles.

Fish exposure to environmental stressors (e.g. chemicals, hypoxia, temperature) induce responses enabling them to cope with alterations in their environment. A stress response involves a wide array of changes, from molecular to physiological and behavioural, set to counteract the effect of the stressor and recover homeostatic equilibrium. Among other processes, there is activation of the hypothalamus-pituitary-interrenal (HPI) axis, resulting in stimulation of the steroidogenic pathway and release of cortisol, important mediator of the adaptive response to stress. The purpose of this study was to evaluate if exposure of a marine teleost (gilthead sea bream) to gold nanoparticles (AuNP) could interfere with the HPI axis eliciting an acute stress response and how this response would be linked with alterations in the mRNA levels of target genes in the head kidney, important centre of endocrine response in fish. Fish were exposed via water, for 96h, to four concentrations (0, as control, 4, 80 and 1600μg⋅L-1) of 40nm spherical AuNP, covered with two different types of coatings (citrate and PVP). At the end of the exposure, fish were anesthetized and blood and the head kidney sampled. Results showed that exposure to 1600μg⋅L-1 AuNP-citrate and 80μg⋅L-1 AuNP-PVP increased plasma cortisol levels, compared to controls, but caused no change in glucose levels. AuNP modulated the expression of target genes related to oxidative stress, cell-tissue repair, immune function and apoptosis in the head kidney of fish. The patterns of response were distinct for the two coatings tested. Unlike AuNP-citrate, AuNP-PVP elicited an inverted U-shaped response. Present findings demonstrated that AuNP were able to activate the fish HPI axis and alter a battery of related molecular markers in the head kidney.

Effects of dietary α‐ketoglutarate supplementation on the antioxidant defense system and HSP 70 and HSP 90 gene expression of hybrid sturgeon A cipenser schrenckii ♀ × A . baerii ♂ exposed to ammonia‐N stress

This study was conducted to determine the effects of dietary α-ketoglutarate (AKG) supplementation on the antioxidant defense system and gene expression of heat shock protein (HSP) 70 and HSP 90 in hybrid sturgeons Acipenser schrenckii ♀ × A. baerii ♂ exposed to ammonia-N stress. A 2 × 3 factorial experiment was arranged, in which each diet (0%, 1% AKG) was randomly assigned to 0.25 (control) 5 and 10 mg L−1 ammonia-N groups with three replicate aquaria for each 72 h. The 10 mg L−1 ammonia-N significantly increased serum ammonia concentrations and intestinal Gln concentrations and GS activity compared with the 0.25 or 5 mg L−1 ammonia-N groups. The intestinal Gln concentration and GS activity increased, and the serum ammonia concentration decreased, in fish given dietary supplementation of 1.0% AKG compared with fish given diets without AKG. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity in serum, gills and intestines decreased when fish were exposed to 5 or 10 mg L−1 ammonia-N, and their activity increased in fish given diets with 1% AKG. Catalase in the serum and gills decreased when fish were exposed to 5 or 10 mg L−1 ammonia-N and increased in fish given diets with 1% AKG. The 10 mg L−1 ammonia-N or 1% AKG supplementation increased HSP 70 and HSP 90 gene expression in the liver. The increased activity of antioxidant enzymes, and increased HSP 70 and HSP 90 gene expression in fish fed diets containing 1% AKG suggested higher tolerance to ammonia-N stress.

Expression analysis of Sox9 genes during annual reproductive cycles in gonads and after nanodelivery of LHRH in Clarias batrachus

Transcription factor Sox9 plays a crucial role in determining the fate of several cell types and is a primary factor in regulation of gonadal development. Present study reports full-length cDNA sequence of Sox9a gene and partial coding sequence (cds) of Sox9b (two duplicate orthologs of Sox9 gene) from Clarias batrachus. The coding region of Sox9a gene encoded a peptide of 460 amino acids. The partial cds of Sox9b with the length of 558bp was amplified that codes for 186 amino acids. Quantitative Real-time PCR (qRT-PCR) analysis revealed that Sox9a and Sox9b mRNA expression was significantly higher in gonads and brain tissues. Furthermore Sox9a and Sox9b mRNA expression levels were high during preparatory and pre-spawning phases and decreased gradually with onset of spawning and post-spawning phases of reproductive cycles in gonads. Chitosan nanoconjugated sLHRH (CsLHRH) of particle size 133.0nm and zeta potential of 34.3mV were synthesized and evaluated against naked sLHRH (salmon luteinizing hormone-releasing hormone). The entrapment efficiency of CsLHRH was 63%. CsLHRH nanoparticles increased the expression level of Sox9 transcripts in gonads and steroid hormonal levels in blood of male and female. Thus, our findings clearly indicate that Sox9 genes play essential role during seasonal variation of gonads. Besides, the current study reports that sustained release delivery-system will be helpful for proper gonadal development of fish. To the best of our knowledge, till date no study has been reported on nanodelivery of sLHRH and their effect on reproductive gene expression in fish.

Pleiotropic effect of thyroid hormones on gene expression in fish as exemplified from the blue bream Ballerus ballerus (Cyprinidae): Results of transcriptomic analysis.

A pronounced pleiotropic effect of thyroid hormones on the regulation of gene expression in fish in postembryogenesis was demonstrated for the first time using larvae and juveniles of the blue bream Ballerus ballerus as an example. Genome-wide transcriptome sequencing (RNA-seq) identified 1212 differentially expressed genes in the brain and liver of fish kept in triiodothyronine solution (0.25 ng/mL). Our data show that the regulation of gene expression by thyroid hormones is widespread in nature: it involves not only the structural genes but also the regulatory genes. A significant number of genes under the control of thyroid hormones are involved in the determination of morphological traits.

From Your Body to Theirs: How Exposure to Pharmaceuticals Is Altering Gene Expression in Fishes

FisheriesVolume 40, Issue 6 p. 292-292 Back Page From Your Body to Theirs: How Exposure to Pharmaceuticals Is Altering Gene Expression in Fishes Natalie Sopinka, Natalie Sopinka AFS Contributing Writer natsopinka@gmail.com Search for more papers by this author Natalie Sopinka, Natalie Sopinka AFS Contributing Writer natsopinka@gmail.com Search for more papers by this author First published: 03 June 2015 https://doi.org/10.1080/03632415.2015.1038784Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume40, Issue6June 2015Pages 292-292 RelatedInformation

The treatment with the probiotic Shewanella putrefaciens Pdp11 of specimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.

ISA virus regulates the generation of reactive oxygen species and p47phox expression in a p38 MAPK-dependent manner in Salmo salar

Several viruses, including Orthomyxovirus, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells. However, the role of ROS in early events of viral entry and signal induction has not been elucidated. Here, we show that ISA virus (ISAV) induces ROS production very early during infection of CHSE-214 and SHK-1Ycells, and that production is sustained over the observed 24h post-infection. The mitogen-activated protein kinase (MAPK) family is responsible for important signaling pathways. In this study, we report that ISAV activates ERK and p38 in Salmo salar. In salmonid macrophages, while ERK was required for SOD, GLURED, p47phox expression, p38 regulated the ROS production by the NADPH oxidase complex activation. These results, together with the presence of several consensus target motifs for p38 MAPK in the promoter of the S. salar p47phox gene, suggest that p38 MAPK regulates p47phox gene expression in fish through the activation of this key transcription factor.

Bioavailability of the imidazole antifungal agent clotrimazole and its effects on key biotransformation genes in the common carp (Cyprinus carpio)

Clotrimazole (CTZ) is a persistent imidazole antifungal agent which is frequently detected in the aquatic environment and predicted to bio-concentrate in fish. Common carp (Cyprinus carpio) were exposed to mean measured concentrations of either 1.02 or 14.63μgl−1 CTZ for 4 and 10 days, followed by a depuration period of 4 days in a further group of animals. Following each exposure regimen, plasma and liver CTZ concentrations were measured. Mean measured plasma concentrations of CTZ in animals exposed to the lower concentration of CTZ were 30 and 44μgl−1 on days 4 and 10, respectively, and in the higher concentration were 318 and 336μgl−1. Mean measured liver levels in the same animals were 514, 1725, 2111 and 7017μgl−1 suggesting progressive hepatic accumulation. Measurement of CTZ in plasma after depuration suggested efficient elimination within 4 days, but appreciable levels of CTZ remained in the liver after depuration suggesting a degree of persistence in this tissue. In addition we measured responses of a number of key hepatic detoxification gene targets in the liver associated with the transcription factor pregnane X receptor (PXR); namely cyp450s 2k and 3a, glutathione-S-transferases a and p (gsta and p), and drug transporters multidrug resistance protein1 (mdr1), and MDR-related protein2 (mrp2). CTZ is a potent ligand of the PXR in humans and there is some evidence of PXR activation following exposure to CTZ in fish. The highest concentration of CTZ was adopted to explore the potential for alterations to detoxification gene expression in fish at a pharmacologically relevant dose level, and the lower concentration is within the range reported in effluents from waste water treatment works (WWTW). The genes for all biotransformation enzymes were up-regulated after exposure to the higher concentration of CTZ for 10 days, and alterations in expression occurred for the drug transporter genes mdr1 and mrp2 following exposure to the lower concentration of 1.02μgl−1 CTZ (mean measured concentration). These data support the potential for CTZ to induce alterations in biotransformation and drug transporter genes associated with PXR in fish at concentrations measured in some WWTW effluents.

Crosstalking between the “gut-brain” hormone ghrelin and the circadian system in the goldfish. Effects on clock gene expression and food anticipatory activity

Ghrelin is a potent orexigenic signal mainly synthesized in the stomach and foregut of vertebrates. Recent studies in rodents point out that ghrelin could also act as an input for the circadian system and/or as an output of peripheral food-entrainable oscillators, being involved in the food anticipatory activity (FAA). In this study we pursue the possible interaction of ghrelin with the circadian system in a teleost, the goldfish (Carassius auratus). First, we analyzed if ghrelin is able to modulate the core clock functioning by regulating clock gene expression in fish under a light/dark cycle 12L:12D and fed at 10 am. As expected the acute intraperitoneal (IP) injection of goldfish ghrelin (gGRL[1–19], 44pmol/g bw) induced the expression of hypothalamic orexin. Moreover, ghrelin also induced (∼2-fold) some Per clock genes in hypothalamus and liver. This effect was partially counteracted in liver by the ghrelin antagonist ([D-Lys3]-GHRP-6, 100pmol/g bw). Second, we investigated if ghrelin is involved in daily FAA rhythms. With this aim locomotor activity was studied in response to IP injections (5–10days) of gGRL[1–19] and [D-Lys3]-GHRP-6 at the doses above indicated. Ghrelin and saline injected fish showed similar 24h activity patterns. However, ghrelin antagonist treatment abolished the FAA in schedule fed fish under 24h light, suggesting the involvement of the endogenous ghrelin system in this pre-feeding activity. Altogether these results suggest that ghrelin could be acting as an input for the entrainment of the food-entrainable oscillators in the circadian organization of goldfish.

Fsh controls gene expression in fish both independently of and through steroid mediation.

The mechanisms and the mediators relaying Fsh action on testicular functions are poorly understood. Unlike in mammals, in fish both gonadotropins (Fsh and Lh) are able to efficiently stimulate steroidogenesis, likely through a direct interaction with their cognate receptors present on the Leydig cells. In this context, it is crucial to understand if Fsh effects are mediated through the production of steroids. To address this issue we performed transcriptome studies after in vitro incubations of rainbow trout testis explants in the presence of Fsh alone or in combination with trilostane, an inhibitor of Δ4- steroidogenesis.Trilostane significantly reduced or suppressed the response of many genes to Fsh (like wisp1, testis gapdhs, cldn11, inha, vt1 or dmrt1) showing that, in fish, important aspects of Fsh action follow indirect pathways and require the production of Δ4-steroids. What is more, most of the genes regulated by Fsh through steroid mediation were similarly regulated by Lh (and/or androgens). In contrast, the response to Fsh of other genes was not suppressed in the presence of trilostane. These latter included genes encoding for anti-mullerian hormone, midkine a (pleiotrophin related), angiopoietine-related protein, cyclins E1 and G1, hepatocyte growth factor activator, insulin-like growth factor 1b/3. A majority of those genes were preferentially regulated by Fsh, when compared to Lh, suggesting that specific regulatory effects of Fsh did not depend on steroid production. Finally, antagonistic effects between Fsh and steroids were found, in particular for genes encoding key factors of steroidogenesis (star, hsd3b1, cyp11b2-2) or for genes of the Igf system (igf1b/3). Our study provides the first clear evidence that, in fish, Fsh exerts Δ4-steroid-independent regulatory functions on many genes which are highly relevant for the onset of spermatogenesis.

Induction of genes encoding NADPH oxidase components and activation of IFN regulatory factor-1 by prolactin in fish macrophages

The role played by prolactin (PRL) in fish immunity is scant. We report here that stimulation of the Atlantic salmon monocytic cell line SHK-1 with native salmon PRL resulted in activation of the respiratory burst and induction of the expression of the genes encoding the phagocyte NADPH oxidase components p47phox, p67phox and gp91phox, and the transcription factor IFN regulatory factor-1 (IRF-1). Interestingly, the pharmacologic inhibition of the Jak/Stat signaling pathway with AG490 blocked reactive oxygen species (ROS) production, and the induction of genes encoding the NADPH oxidase components and IRF-1 in PRL-activated SHK-1 cells. In addition, PRL promoted the phosphorylation of Stat and induced the DNA binding activity of IRF-1. These results, together with the presence of several consensus target motifs for Stat and IRF-1 in the promoter of the tilapia p47phox gene, suggest that PRL regulates p47phox gene expression in fish through the activation of these two key transcription factors. Taken together, our results demonstrate that PRL induces the expression of the genes encoding the major phagocyte NADPH oxidase components and ROS production in fish macrophages via the JAK2/Stat/IRF-1 signaling pathway.

Effects of dietary tert-butylhydroquinone on domoic acid metabolism and transcription of detoxification-related liver genes in red sea bream Pagrus major

Domoic acid (DA) is a neuroexcitatory amino acid that is produced by Pseudo-nitzschia during harmful algal blooms (HAB). Accumulation of DA can be transferred through food chain and cause neuronal damage in marine animal and in human. Like other algal toxins, DA was suggested to increase the oxidative stress and increase the detoxification-related gene expression in fish. The widely used food antioxidant, tert-butylhydroquinone (tBHQ), was known to induce a wide range of antioxidative potentials such as elevation of the glutathione levels and glutathione S-transferases (GSTs), via the activation of antioxidant response elements (AREs). In this study, the influences of dietary tBHQ on domoic acid (DA) metabolism and detoxification-related gene transcription were investigated both in vivo and in vitro. Oral administration of tBHQ resulted in significant decreases of DA accumulation of liver tissues in which red sea bream were fed with a single dose of 10 mg DA and 100 mg tBHQ per kg body weight per fish. Real-time PCR further revealed that the mRNA levels of AHR/ARNT/CYP1A1/GSTA1/GSTR were up-regulated in the above liver tissues at 72 h post tBHQ treatment. In consistence, tBHQ exposure also resulted in increased mRNA transcription of GSTA1, GSTA2 and GSTR in cultured red sea bream hepatocytes. Collectively, our findings in this research suggested that the dietary intake of tBHQ accelerated DA metabolism in fish, through mechanisms involving altered transcription of detoxification-related liver genes.

Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: Transient and transgenic analysis of torafugu MYHM86-2 promoter in zebrafish embryos

The myosin heavy chain gene, MYHM86-2, exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYHM86-2 promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614bp 5′-flanking sequences of MYHM86-2 contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. By cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYHM86-2 expression in the fast muscle fibers. The transcriptional mechanism that prevents MYHM86-2 expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYHM86-2 expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYHM86-2 expression.

Total substitution of fish oil by vegetable oils in Senegalese sole (Solea senegalensis) diets: effects on fish performance, biochemical composition, and expression of some glucocorticoid receptor-related genes

To study the substitution of fish oil by vegetable oils in fish diets, juveniles Senegalese sole (Solea senegalensis) were fed diets (56 % crude protein, 12 % crude lipid) containing either linseed (100LO) or soybean (100SO) oils in comparison with a 100 % fish oil-based diet (100FO) for 90 days. Samples of muscle, liver, and intestine were collected for biochemical analysis and for glucocorticoid receptor-related genes, including GR1 and GR2, and the associated heat shock proteins HSP70, HSP90AA, and HSP90AB. Besides, basal levels of plasma cortisol were also determined. After the feeding period, a stress test, consisting on 5 min of net chasing, was applied to a selected population of each dietary group. Total replacement of fish oil by vegetable oils did not induced changes in fish growth and performance, but affected fatty acid profile of muscle, liver, and intestine, reflecting those tissues the characteristic fatty acids of each type of dietary oil. A tendency to conserve the ARA/EPA ratio could be observed in the different tissues, despite of the level of these fatty acids in diet. Chasing stress induced an increase of muscle GR1 and a reduction in intestinal GR2 relative expressions at any of the experimental diets assayed. In liver, chasing stress induced an increase in both GR1 and GR2 gene expression in fish fed fish oil diets. Similarly, chasing stress induced an increase of muscle HSP70 and decrease of HSP90AB in liver at any of the experimental diet assayed. Besides, vegetable oils decreased the expression of HSP70 in intestine, being the relative expression of liver HSP90AA increased by the inclusion of linseed oil in the diet, at any of the experimental conditions assayed.

Does ketoprofen or diclofenac pose the lowest risk to fish?

Ketoprofen and diclofenac are non-steroidal anti-inflammatory drugs (NSAIDs) often used for similar indications, and both are frequently found in surface waters. Diclofenac affects organ histology and gene expression in fish at around 1μg/L. Here, we exposed rainbow trout to ketoprofen (1, 10 and 100μg/L) to investigate if this alternative causes less risk for pharmacological responses in fish. The bioconcentration factor from water to fish blood plasma was <0.05 (4 for diclofenac based on previous studies). Ketoprofen only reached up to 0.6‰ of the human therapeutic plasma concentration, thus the probability of target-related effects was estimated to be fairly low. Accordingly, a comprehensive analysis of hepatic gene expression revealed no consistent responses. In some contrast, trout exposed to undiluted, treated sewage effluents bioconcentrated ketoprofen and other NSAIDs much more efficiently, according to a meta-analysis of recent studies. Neither of the setups is however an ideal representation of the field situation. If a controlled exposure system with a single chemical in pure water is a reasonable representation of the environment, then the use of ketoprofen is likely to pose a lower risk for wild fish than diclofenac, but if bioconcentration factors from effluent-exposed fish are applied, the risks may be more similar.

Evaluation of water quality threats to the endangered Okaloosa darter (Etheostoma okaloosae) in East Turkey Creek on Eglin Air Force Base

The threatened Okaloosa darter (Etheostoma okaloosae) is found almost exclusively on the Eglin Air Force Base in the Choctawhatchee Bay watershed of Florida. Portions of this limited habitat are threatened with soil erosion, altered hydrology, and impaired water quality. In the present study, general water quality parameters (i.e., dissolved oxygen, specific conductance, pH, temperature, relative turbidity, and primary productivity) were characterized in East Turkey Creek, which is a body of water potentially impacted by treated wastewater sprayfields, and Long Creek, an adjacent reference stream that does not border the sprayfields. Water quality was assessed during a 30-day exposure using passive samplers for both non-polar and polar effluent parameters. Because the Okaloosa darter was listed as endangered at the time of sampling we chose a closely related species from the same creeks, the sailfin shiner (Pteronotropis hypseleotris) in which to measure metal body burdens. Additionally, fathead minnows (Pimephales promelas) were used for microarray analysis on gonad and liver tissues after 48h exposures to water collected from the two creeks and brought into the laboratory. Waters from all sites, including reference sites, affected the expression of genes related to various biological processes including transcription and translation, cell cycle control, metabolism, and signaling pathways, suggesting that the sum of anthropogenic compounds in the site waters may cause a generalized stress response in both liver and testis, an effect that could be related to the generally low populations of the Okaloosa darter. Furthermore, effects of site waters on fish gene expression may be related to the impact of human activities other than the wastewater sprayfields, as nearby areas are closed to the public for military testing, training, and administrative activities and due to ordnance contamination.

Feeding Time Synchronizes Clock Gene Rhythmic Expression in Brain and Liver of Goldfish (Carassius auratus)

Little is known about the feeding time dependence of clock gene expression in fish. The aim of the present study was to investigate whether a scheduled feeding time can entrain the rhythmic expression of several clock genes (period and cryptocrome) in the brain and liver of a teleost, the goldfish. Fish maintained under continuous light (LL) conditions were divided into 3 groups. Two groups were fed daily at 1000 h and 2200 h, respectively, and the third group was subjected to a random schedule regime. After 30 days, the fishes under 24-h food deprivation were sacrificed through a 24-h cycle, and clock gene expression in the optic tectum, hypothalamus, and liver was quantified by real-time PCR. The findings pointed to differences between the central and peripheral tissues studied. In the absence of a light-dark cycle (constant light), a scheduled feeding regime was necessary and sufficient to maintain both the rhythmic expression of several clock genes in the optic tectum and hypothalamus, as well as daily rhythms in locomotor activity. In contrast, neither locomotor activity nor clock gene expression in brain tissues was synchronized in randomly fed fish. However, in the liver, most of the clock genes studied presented significant daily rhythms in phase (related to the time of the last meal) in all 3 experimental groups, suggesting that the daily rhythm of clock genes in this organ only depends on the last meal time. The data suggest that, as in mammals, the smooth running of the food entrainable oscillator (FEO) in fish involves the rhythmic expression of several clock genes (Per1 and Cry3) in the central and peripheral structures. The results also indicate that the food anticipatory activity (FAA) in goldfish is not only the result of rhythmic clock gene expression in the liver because rhythmic clock gene expression was observed in randomly fed fishes, while FAA was not observed.

Environmental impact of insecticides applied on biotech soybean crops in relation to the distance from aquatic ecosystems

Aquatic environments located in areas cultivated with biotech soybean were studied. Water and sediment samples were analyzed for insecticides, acute toxicity, genotoxicity, detoxification biomarkers, and fish diversity. Samples were taken in the core area of soybean cultivation in Argentina; all measures were related to the distance between the crops and the streams sampled. Endosulfan (alpha + beta) concentrations as high as 553.33 microg/kg were found in sediments from environments located at 0.15 m from treated fields. Ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P4501A1 (CYP1A1) gene expression in fish showed the highest correlation with the environmental concentration of endosulfan. These biomarkers and mortality of amphipods significantly correlated with the concentration of endosulfan in water and sediment, which correlates inversely with the distance between the crop and streams. The differences with respective controls disappear at distances greater than 5 m. The fish diversity was significantly lower from distances between the margin of the stream and soybean crops, not exceeding 2 m.

Heat shock response of killifish (Fundulus heteroclitus): candidate gene and heterologous microarray approaches

Northern and southern subspecies of the Atlantic killifish, Fundulus heteroclitus, differ in maximal thermal tolerance. To determine whether these subspecies also differ in their heat shock response (HSR), we exposed 20°C acclimated killifish to a 2 h heat shock at 34°C and examined gene expression in fish from both subspecies during heat shock and recovery using real-time quantitative PCR and a heterologous cDNA microarray designed for salmonid fishes. The heat shock proteins Hsp70-1, hsp27, and hsp30 were upregulated to a greater extent in the high temperature-tolerant southern subspecies than in the less tolerant northern subspecies, whereas hsp70-2 (which showed the largest upregulation of all the heat shock proteins) in both gill and muscle and hsp90α in muscle was upregulated to a greater extent in northern than in southern fish. These data demonstrate that differences in the HSR between subspecies cannot be due to changes in a single global regulator but must occur via gene-specific mechanisms. They also suggest that the role, if any, of hsps in establishing thermal tolerance is complex and varies from gene to gene. Heterologous microarray hybridization provided interpretable gene expression signatures, detecting differential regulation of genes known to be involved in the heat shock response in other species. Under control conditions, a variety of genes were differentially expressed in muscle between subspecies that suggest differences in muscle fiber type and could relate to previously observed differences between subspecies in the thermal sensitivity of swimming performance and metabolism.