Model Fish Species Research Articles

Wild eel microbiome reveals that skin mucus of fish could be a natural niche for aquatic mucosal pathogen evolution

BackgroundFish skin mucosal surfaces (SMS) are quite similar in composition and function to some mammalian MS and, in consequence, could constitute an adequate niche for the evolution of mucosal aquatic pathogens in natural environments. We aimed to test this hypothesis by searching for metagenomic and genomic evidences in the SMS-microbiome of a model fish species (Anguilla Anguilla or eel), from different ecosystems (four natural environments of different water salinity and one eel farm) as well as the water microbiome (W-microbiome) surrounding the host.ResultsRemarkably, potentially pathogenic Vibrio monopolized wild eel SMS-microbiome from natural ecosystems, Vibrio anguillarum/Vibrio vulnificus and Vibrio cholerae/Vibrio metoecus being the most abundant ones in SMS from estuary and lake, respectively. Functions encoded in the SMS-microbiome differed significantly from those in the W-microbiome and allowed us to predict that successful mucus colonizers should have specific genes for (i) attachment (mainly by forming biofilms), (ii) bacterial competence and communication, and (iii) resistance to mucosal innate immunity, predators (amoeba), and heavy metals/drugs. In addition, we found several mobile genetic elements (mainly integrative conjugative elements) as well as a series of evidences suggesting that bacteria exchange DNA in SMS. Further, we isolated and sequenced a V. metoecus strain from SMS. This isolate shares pathogenicity islands with V. cholerae O1 from intestinal infections that are absent in the rest of sequenced V. metoecus strains, all of them from water and extra-intestinal infections.ConclusionsWe have obtained metagenomic and genomic evidence in favor of the hypothesis on the role of fish mucosal surfaces as a specialized habitat selecting microbes capable of colonizing and persisting on other comparable mucosal surfaces, e.g., the human intestine.

Genotyping live fish larvae: Non-lethal and noninvasive DNA isolation from 3-5 day old hatchlings.

Genotyping fish larvae is a valuable technique for numerous fields of study. While methods for collecting DNA from early stage larvae have been published, a non-lethal, non-invasive genotyping protocol for hatchlings that is amenable to high-throughput approaches is desirable. Here, we describe a method to individually genotype live, free-swimming, early fish larvae by characterizing their environmental DNA (eDNA). We demonstrate the utility of the method by assigning parentage to a sample (n = 50) of 3-5-day-old sheepshead minnow (Cyprinodon variegatus) larvae hatchlings, with very high rates of genotyping success (98%) and survival (92%) using mitochondrial and microsatellite DNA data. This method could be easily adapted to characterize early fish larvae from other model and non-model fish species, such as Danio rerio (zebrafish) and Medaka medaka.

Evaluation of chlorpyrifos effects, alone and combined with lipopolysaccharide stress, on DNA integrity and immune responses of the three-spined stickleback, Gasterosteus aculeatus

Organism immune defences might be weakened by pollutants, largely detected in aquatic ecosystems, leading to the facilitation for opportunistic pathogens to infect organisms. In this context, destabilization of fish non-specific immune parameters and erythrocyte DNA integrity was tested, on a model fish species, the three-spined stickleback (Gasterosteus aculeatus), after exposure to chlorpyrifos (CPF). Alone, pesticide exposure induced a genotoxic potential (chlorpyrifos at 1.75 and 0.88µg/L) in addition to a decrease in phagocytosis capacity and a stimulation of respiratory burst. Then, to mimic pathogenic infection, fish exposure to chlorpyrifos was combined with lipopolysaccharides (LPS) stress. In this second experiment, an increase of DNA damage was observed in fish exposed to a lower concentration of chlorpyrifos and LPS. Moreover, at the higher concentration of chlorpyrifos, an early destabilization of innate immunity was observed as suggested by the absence of an increase of lysosomal presence in fish injected with LPS. This study highlighted the usefulness of stress on stress responses to better understand the impact of contaminants on the organism's health.

On the use of climate covariates in aquatic species distribution models: are we at risk of throwing out the baby with the bath water?

Species distribution models (SDMs) in river ecosystems can incorporate climate information by using air temperature and precipitation as surrogate measures of instream conditions or by using independent models of water temperature and hydrology to link climate to instream habitat. The latter approach is preferable but constrained by the logistical burden of developing water temperature and hydrology models. We therefore assessed whether regional scale, freshwater SDM predictions are fundamentally different when climate data versus instream temperature and hydrology are used as covariates. Maximum entropy (MaxEnt) SDMs were built for 15 freshwater fishes using one of two covariate sets: 1) air temperature and precipitation (climate variables) in combination with physical habitat variables; or 2) water temperature, hydrology (instream variables) and physical habitat. Three procedures were then used to compare results from climate vs instream models. First, equivalence tests assessed average pairwise differences (site‐specific comparisons throughout each species’ range) among climate and instream models. Second, ‘congruence’ tests determined how often the same stream segments were assigned high habitat suitability by climate and instream models. Third, Schoener's D and Warren's I niche overlap statistics quantified range‐wide similarity in predicted habitat suitability from climate vs instream models. Equivalence tests revealed small, pairwise differences in habitat suitability between climate and instream models (mean pairwise differences in MaxEnt raw scores for all species < 3 × 10–4). Congruence tests showed a strong tendency for climate and instream models to predict high habitat suitability at the same stream segments (median congruence = 68%). D and I statistics reflected a high margin of overlap among climate and instream models (median D = 0.78, median I = 0.96). Overall, we found little support for the hypothesis that SDM predictions are fundamentally different when climate versus instream covariates are used to model fish species’ distributions at the scale of the Columbia Basin.

Molecular Mechanisms of Crude Oil Developmental Toxicity in Fish.

With major oil spills in Korea, the United States, and China in the past decade, there has been a dramatic increase in the number of studies characterizing the developmental toxicity of crude oil and its associated polycyclic aromatic compounds (PACs). The use of model fish species with associated tools for genetic manipulation, combined with high throughput genomics techniques in nonmodel fish species, has led to significant advances in understanding the cellular and molecular bases of functional and morphological defects arising from embryonic exposure to crude oil. Following from the identification of the developing heart as the primary target of crude oil developmental toxicity, studies on individual PACs have revealed a diversity of cardiotoxic mechanisms. For some PACs that are strong agonists of the aryl hydrocarbon receptor (AHR), defects in heart development arise in an AHR-dependent manner, which has been shown for potent organochlorine agonists, such as dioxins. However, crude oil contains a much larger fraction of compounds that have been found to interfere directly with cardiomyocyte physiology in an AHR-independent manner. By comparing the cellular and molecular responses to AHR-independent and AHR-dependent toxicity, this review focuses on new insights into heart-specific pathways underlying both acute and secondary adverse outcomes to crude oil exposure during fish development.

Assessing how uncertainty and stochasticity affect the dispersal of fish in river networks

Species dispersal and population dynamics determine the spatio-temporal patterns of species spread and thus, species invasions and recolonization following habitat restorations. However, the effects of stochasticity and spatial habitat heterogeneity on species spread are poorly understood.By coupling a fish dispersal model and a population growth model in GIS, we simulated the spread of a model fish species (Salmo trutta) over five years in river networks of varying habitat heterogeneity. Replicated model runs and a sampling-based sensitivity analysis allowed us to disentangle the uncertainty in the rate of spread related to (i) specific model input parameters and (ii) stochasticity inherent to the dispersal process.Our results revealed the spread of the model species being particularly sensitive to the choice of parameters determining fish dispersal (e.g. fish size) and less to demographic parameters (e.g. reproductive rate). Moreover, the spread of fish is strongly affected by stochasticity inherent to dispersal and the availability and spatial arrangement of suitable habitats patches. Stochastic effects cause considerable variations in the predictability of up to 120% of the median dispersal distance whereof 52% is solely attributed to stochasticity inherent to dispersal decisions.Our findings demonstrate that process-based models combining individual dispersal, population dynamics and spatial habitat heterogeneity as well as carefully selected model input parameters are crucial for reasonably simulating the spread of fishes in river networks. Moreover, we suggest considering stochasticity inherent to dispersal in models of species spread as this contributes to a considerable variation in the predictability of movement distances.

Coenzyme Q10 protects against statin-induced myotoxicity in zebrafish larvae (Danio rerio)

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the rate-limiting enzyme of the mevalonic acid pathway and is required for cholesterol biosynthesis and the synthesis of Coenzyme Q10 (CoQ10). Statins inhibit HMGCR, thus inhibiting the downstream products of this pathway including the biosynthesis of decaprenyl-pyrophosphate that is critical for the synthesis of Coenzyme Q10 (CoQ10). We show that zebrafish (Danio rerio) larvae treated in tank water with Atorvastatin (ATV; Lipitor) exhibited movement alterations and reduced whole body tissue metabolism. The ATV-inhibition of HMGCR function altered transcript abundance of muscle atrophy markers (atrogen-1, murf) and the mitochondrial biogenesis marker (pgc-1α). Furthermore, ATV-induced reduction in larval response to tactile stimuli was reversed with treatment of CoQ10. Together, the implication of our results contributes to the understanding of the mechanisms of action of the statin-induced damage in this model fish species.

Application of Passive Sampling to Characterise the Fish Exometabolome.

The endogenous metabolites excreted by organisms into their surrounding environment, termed the exometabolome, are important for many processes including chemical communication. In fish biology, such metabolites are also known to be informative markers of physiological status. While metabolomics is increasingly used to investigate the endogenous biochemistry of organisms, no non-targeted studies of the metabolic complexity of fish exometabolomes have been reported to date. In environmental chemistry, Chemcatcher® (Portsmouth, UK) passive samplers have been developed to sample for micro-pollutants in water. Given the importance of the fish exometabolome, we sought to evaluate the capability of Chemcatcher® samplers to capture a broad spectrum of endogenous metabolites excreted by fish and to measure these using non-targeted direct infusion mass spectrometry metabolomics. The capabilities of C18 and styrene divinylbenzene reversed-phase sulfonated (SDB-RPS) Empore™ disks for capturing non-polar and polar metabolites, respectively, were compared. Furthermore, we investigated real, complex metabolite mixtures excreted from two model fish species, rainbow trout (Oncorhynchus mykiss) and three-spined stickleback (Gasterosteus aculeatus). In total, 344 biological samples and 28 QC samples were analysed, revealing 646 and 215 m/z peaks from trout and stickleback, respectively. The measured exometabolomes were principally affected by the type of Empore™ (Hemel Hempstead, UK) disk and also by the sampling time. Many peaks were putatively annotated, including several bile acids (e.g., chenodeoxycholate, taurocholate, glycocholate, glycolithocholate, glycochenodeoxycholate, glycodeoxycholate). Collectively these observations show the ability of Chemcatcher® passive samplers to capture endogenous metabolites excreted from fish.

Effects of dietary methylmercury on the dopaminergic system of adult fathead minnows and their offspring.

Mercury (Hg) is a ubiquitous environmental contaminant and potent neurotoxin, which may be transformed by bacteria in aquatic ecosystems to methylmercury (MeHg), an organic form which bioaccumulates and biomagnifies. Consequently, long-lived organisms at the top of the food web are at risk of dietary MeHg exposure, which can be actively transferred from mother to offspring. Exposure during neurodevelopment can lead to serious, irreversible neurological dysfunction, associated with a variety of cognitive and motor abnormalities. At low dietary concentrations, MeHg exposure has been associated with deficits in attention and hyperactivity in multiple species. Pathways associated with cognitive function and motor activity are primarily associated with the dopaminergic system. The present study used a model fish species, Pimephales promelas, to examine the effects of MeHg exposure on dopamine concentrations and monoamine oxidase activity in embryos and adult brains. Adult fatheads were exposed for 30 d to either a control or a treated diet (0.72 ppm Hg). Embryonic and larval exposures were a result of maternal transfer of dietary MeHg. The authors confirmed hyperactive behaviors in embryos and detected significant changes in embryonic dopamine concentrations. Similar effects on dopamine concentrations were seen in the telencephalon of adult brains. Exposure to MeHg also corresponded with a significant decrease in monoamine oxidase activity in both embryos and brain tissue. Collectively, these results suggest that current exposure scenarios in North America are sufficient to induce alterations to this highly conserved neurochemical pathway in offspring, which may have adverse effects on fish behavior and cognition. Environ Toxicol Chem 2017;36:1077-1084. © 2016 SETAC.

In vivo micronucleus screening in zebrafish by flow cytometry.

Micronucleus (MN) assay is extensively used to biomonitor aneuploidy and clastogenicity of genotoxic compounds. However, the suitability of this assay for early life stages of model fish species is still poorly documented. In this study, the determination of MN using flow cytometry was successfully applied for the first time to zebrafish (Danio rerio) larvae. Mitomycin C (MMC), etoposide (ETO), cyclophosphamide, demecolcine (COL), benzo[a]pyrene (BP) and dibenzo[def,p]chrysene (DBC) were selected as model genotoxicants. The method was first confirmed in human HepG2 liver cells and then applied in vivo on isolated cells from exposed 4 days post fertilisation zebrafish larvae. All tested compounds induced MN formation. The flow cytometry results were validated by a strong correlation with results from a standard MN microscopy analysis (P = 0.002). Moreover, flow cytometry analysis enabled the detection of an up to 3.7-fold increase of hypodiploidy in zebrafish exposed to MMC, COL, BP or DBC. MMC, COL and DBC induced more than a 2-fold MN increase by flow cytometry and were therefore considered as the most suitable positive controls for in vivo zebrafish MN determination. These findings make important contribution by proposing a new reliable and sensitive method for using zebrafish as a model for genotoxicity monitoring.

Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review

Protocols for the cryopreservation of fish gametes have been developed for many different fish species, in special, freshwater salmonids and cyprinids. Methods for sperm freezing have progressed during the last decades due to the increasing number of potential applications: aquaculture (genetic improvement programs, broodstock management, helping with species having reproductive problems), biotechnology studies using model fish species (preservation of transgenic or mutant lines), cryobanking of genetic resources from endangered species, etc.This mini-review tries to give an overview of the present situation of this area of research, identifying the main challenges and perspectives, redirecting the reader to more in-depth reviews and papers.

Factors associated with parasite dominance in fishes from Brazil.

The present study used regression models to evaluate the existence of factors that may influence the numerical parasite dominance with an epidemiological approximation. A database including 3,746 fish specimens and their respective parasites were used to evaluate the relationship between parasite dominance and biotic characteristics inherent to the studied hosts and the parasite taxa. Multivariate, classical, and mixed effects linear regression models were fitted. The calculations were performed using R software (95% CI). In the fitting of the classical multiple linear regression model, freshwater and planktivorous fish species and body length, as well as the species of the taxa Trematoda, Monogenea, and Hirudinea, were associated with parasite dominance. However, the fitting of the mixed effects model showed that the body length of the host and the species of the taxa Nematoda, Trematoda, Monogenea, Hirudinea, and Crustacea were significantly associated with parasite dominance. Studies that consider specific biological aspects of the hosts and parasites should expand the knowledge regarding factors that influence the numerical dominance of fish in Brazil. The use of a mixed model shows, once again, the importance of the appropriate use of a model correlated with the characteristics of the data to obtain consistent results.

An Empirical Test of Metabolic Effects and Adaptive Potential in Response to Global Ocean Warming in a Model Fish Species, the Threespine Stickleback

Climate change has had a significant influence on the world's oceans, which have shown constant increases in mean water temperature over the last 50 years. The biota of Arctic environments, ectotherms in particular, may be particularly threatened by this change: whilst southern animals have the possibility of migrating north, those adapted to colder northern climes have nowhere left to move and may be faced with the ‘choice’ of adapt or die. A key factor in adaptation to the changing thermal environment is the physiological response. Metabolic scope (the difference between minimum and maximum oxygen consumption rate) is likely a key fitness related trait because it defines energy available for locomotion, growth and reproduction. The unifying aim of our research is to develop a deeper understanding of the constraints and/or adaptive potential in metabolic scope with respect to thermal changes predicted due to climate change.As obligate ectotherms, threespine stickleback (Gasterosteus aculeatus) populations adapted to localized thermal conditions provide an excellent and logistically tractable model for study of thermal and metabolic evolution in a changing environment. We collected broodstock from anadromous Barents Sea fish and bred them under controlled conditions for two generations to standardize environmental & maternal sources of variance. The multi‐generational pedigree and large numbers of families (N=180, 40 families) permits estimation of additive genetic variance, whereas repeated measurements in multiple environments allows us to estimate direct environmental variance on metabolism and swimming performance. Maximum and minimum metabolic rates, in addition to critical swimming performance, were measured with the same individuals at three different temperatures: (i) contemporary summer temperature for Barents Sea coastal waters (12°C), (ii) extreme predictions under global warming (17°C), and (iii) stressful temperature (80% of CTmax). Individual critical temperature in observed population varied between 27.2 and 32.1°C. The preliminary results showed that minimum and maximum metabolic rates increased with the temperature rise: 61.6 ± 10.1 and 126.2 ± 36.1 mg O2/kg/hour at 12°C; 70.19 ± 11.8 and 144.9 ± 38.8 mg O2/kg/hour at 17°C; 114.0 ± 15.7 and 183.7 ± 32.0 mg O2/kg/hour at individual stressful temperature, respectively. However, the highest aerobic metabolic scope and swimming performance were observed at 17°C. Quantitative genetic analyses are ongoing, but point to the broad potential for adaptation to environmental change.Support or Funding InformationSM: Alfred Cordelin Foundation, Otto A. Malm Foundation, Oscar Öflund Foundation, LUOVA doctoral programme; RJSM, JM, TM: Academy of Finland

Comparative Evaluation of Genotoxic Effects Induced by CuO Bulk and Nano-Particles in Nile Tilapia, Oreochromis niloticus

Metal oxide nano-particles have definitely unique toxicological properties than currently investigated oxides. Therefore, this study was aimed to comparatively evaluate the genotoxicity of nano-CuO and bulk CuO particles on a model fish species Oreochromis niloticus. Fish were exposed to two selected concentrations (1/10 and 1/20 of the LC50/96 h) of both nano-CuO and bulk CuO for 30 days. Genotoxic effects associated with DNA ladder formation and chromosomal damage were investigated using DNA fragmentation and micronucleus techniques. Based on DNA fragmentation of fish hepatocytes, the two selected concentrations of bulk and nano-CuO were found to induce DNA damage. Analysis of the DNA fragments initiated by bulk CuO on agarose gel revealed DNA ladder pattern, which is commonly considered as a hallmark of apoptosis, while fish exposed to nano-CuO particles showed a molecular hallmark of necrosis which is the fragmentation of the nuclear DNA into a smear-like pattern. Also, DNA damage was further confirmed quantitatively using the image analysis software Image J. In this context, nano-CuO-treated groups exhibited a maximum DNA damage especially at the lower concentration (1/20 LC50/96 h). To ensure CuO genotoxicity, micronucleus and other nine nuclear abnormalities were studied in peripheral erythrocytes and significant (p < 0.05) elevation was observed in nano-CuO-exposed groups at the lower concentration followed by a decrease in extent of chromosomal damage at the higher concentration, while fish groups treated with bulk CuO showed a more or less dose-dependent effect.

Gene map of large yellow croaker (Larimichthys crocea) provides insights into teleost genome evolution and conserved regions associated with growth.

The genetic map of a species is essential for its whole genome assembly and can be applied to the mapping of important traits. In this study, we performed RNA-seq for a family of large yellow croakers (Larimichthys crocea) and constructed a high-density genetic map. In this map, 24 linkage groups comprised 3,448 polymorphic SNP markers. Approximately 72.4% (2,495) of the markers were located in protein-coding regions. Comparison of the croaker genome with those of five model fish species revealed that the croaker genome structure was closer to that of the medaka than to the remaining four genomes. Because the medaka genome preserves the teleost ancestral karyotype, this result indicated that the croaker genome might also maintain the teleost ancestral genome structure. The analysis also revealed different genome rearrangements across teleosts. QTL mapping and association analysis consistently identified growth-related QTL regions and associated genes. Orthologs of the associated genes in other species were demonstrated to regulate development, indicating that these genes might regulate development and growth in croaker. This gene map will enable us to construct the croaker genome for comparative studies and to provide an important resource for selective breeding of croaker.

Transcriptome Analysis of Blunt Snout Bream (Megalobrama amblycephala) Reveals Putative Differential Expression Genes Related to Growth and Hypoxia.

The blunt snout bream (Megalobrama amblycephala) is an important freshwater aquaculture species, but it is sensitive to hypoxia. No transcriptome data related to growth and hypoxia response are available for this species. In this study, we performed de novo transcriptome sequencing for the liver and gills of the fast-growth family and slow-growth family derived from ‘Pujiang No.1’ F10 blunt snout bream that were under hypoxic stress and normoxia, respectively. The fish were divided into the following 4 groups: fast-growth family under hypoxic stress, FH; slow-growth family under hypoxic stress, SH; fast-growth family under normoxia, FN; and slow-growth family under normoxia, SN. A total of 185 million high-quality reads were obtained from the normalized cDNA of the pooled samples, which were assembled into 465,582 contigs and 237,172 transcripts. A total of 31,338 transcripts from the same locus (unigenes) were annotated and assigned to 104 functional groups, and 23,103 unigenes were classified into seven main categories, including 45 secondary KEGG pathways. A total of 22,255 (71%) known putative unigenes were found to be shared across the genomes of five model fish species and mammals, and a substantial number (9.4%) of potentially novel genes were identified. When 6,639 unigenes were used in the analysis of differential expression (DE) genes, the number of putative DE genes related to growth pathways in FH, SH, SN and FN was 159, 118, 92 and 65 in both the liver and gills, respectively, and the number of DE genes related to hypoxic response was 57, 33, 23 and 21 in FH, FN, SH and SN, respectively. Our results suggest that growth performance of the fast-growth family should be due to complex mutual gene regulatory mechanisms of these putative DE genes between growth and hypoxia.

Early life exposure to PCB126 results in delayed mortality and growth impairment in the zebrafish larvae

The occurrence of chronic or delayed toxicity resulting from the exposure to sublethal chemical concentrations is an increasing concern in environmental risk assessment. The Fish Embryo Toxicity (FET) test with zebrafish provides a reliable prediction of acute toxicity in adult fish, but it cannot yet be applied to predict the occurrence of chronic or delayed toxicity. Identification of sublethal FET endpoints that can assist in predicting the occurrence of chronic or delayed toxicity would be advantageous. The present study characterized the occurrence of delayed toxicity in zebrafish larvae following early exposure to PCB126, previously described to cause delayed effects in the common sole. The first aim was to investigate the occurrence and temporal profiles of delayed toxicity during zebrafish larval development and compare them to those previously described for sole to evaluate the suitability of zebrafish as a model fish species for delayed toxicity assessment. The second aim was to examine the correlation between the sublethal endpoints assessed during embryonal and early larval development and the delayed effects observed during later larval development. After exposure to PCB126 (3–3000ng/L) until 5 days post fertilization (dpf), larvae were reared in clean water until 14 or 28 dpf. Mortality and sublethal morphological and behavioural endpoints were recorded daily, and growth was assessed at 28 dpf. Early life exposure to PCB126 caused delayed mortality (300ng/L and 3000ng/L) as well as growth impairment and delayed development (100ng/L) during the clean water period. Effects on swim bladder inflation and cartilaginous tissues within 5 dpf were the most promising for predicting delayed mortality and sublethal effects, such as decreased standard length, delayed metamorphosis, reduced inflation of swim bladder and column malformations. The EC50 value for swim bladder inflation at 5 dpf (169ng/L) was similar to the LC50 value at 8 dpf (188 and 202ng/L in two experiments). Interestingly, the patterns of delayed mortality and delayed effects on growth and development were similar between sole and zebrafish. This indicates the comparability of critical developmental stages across divergent fish species such as a cold water marine flatfish and a tropical freshwater cyprinid. Additionally, sublethal effects in early embryo-larval stages were found promising for predicting delayed lethal and sublethal effects of PCB126. Therefore, the proposed method with zebrafish is expected to provide valuable information on delayed mortality and delayed sublethal effects of chemicals and environmental samples that may be extrapolated to other species.

Evaluation of whole-mount in situ hybridization as a tool for pathway-based toxicological research with early-life stage fathead minnows

Early-life stage fish can be more sensitive to toxicants than adults, so delineating mechanisms of perturbation of biological pathways by chemicals during this life stage is crucial. Whole-mount in situ hybridization (WISH) paired with quantitative real-time polymerase chain reaction (QPCR) assays can enhance pathway-based analyses through determination of specific tissues where changes in gene expression are occurring. While WISH has frequently been used in zebrafish (Danio rerio), this technology has not previously been applied to fathead minnows (Pimephales promelas), another well-established small fish model species. The objective of the present study was to adapt WISH to fathead minnow embryos and larvae, and use the approach to evaluate the effects of estrone, an environmentally-relevant estrogen receptor (ER) agonist. Embryos were exposed via the water to 0, 18 or 1800ngestrone/L (0, 0.067 and 6.7nM) for 3 or 6 days in a solvent-free, flow-through test system. Relative transcript abundance of three estrogen-responsive genes, estrogen receptor-α (esr1), cytochrome P450-aromatase B (cyp19b), and vitellogenin (vtg) was examined in pooled whole embryos using QPCR, and the spatial distribution of up-regulated gene transcripts was examined in individual fish using WISH. After 3 days of exposure to 1800ngestrone/L, esr1 and cyp19b were significantly up-regulated, while vtg mRNA expression was not affected. After 6 days of exposure to 1800ngestrone/L, transcripts for all three genes were significantly up-regulated. Corresponding WISH assays revealed spatial distribution of esr1 and vtg in the liver region, an observation consistent with activation of the hepatic ER. This study clearly demonstrates the potential utility of WISH, in conjunction with QPCR, to examine the mechanistic basis of the effects of toxicants on early-life stage fathead minnows.

Influence of polyethylene microplastic beads on the uptake and localization of silver in zebrafish (Danio rerio)

This study aimed to determine whether the uptake and localization of Ag in zebrafish was affected by the presence of polyethylene microplastic beads (PE MPBs). Zebrafish were exposed to 1 μg Ag L−1 (radiolabelled with 110mAg) for 4 and 24 h in the presence or absence of PE MPBs (10, 100 or 1000 MPBs mL−1), and one treatment in which MPBs (1000 MPBs mL−1) were incubated with Ag to promote adsorption. The presence of MPBs, at any of the tested doses, had no effect on the uptake or localization of Ag. However, exposure to the Ag-incubated MPBs (∽75% of the Ag bound to MPBs) significantly reduced Ag uptake at both time points and also significantly increased the proportion of intestinal Ag. This study demonstrates that microplastics can alter the bioavailability and uptake route of a metal contaminant in a model fish species.

Comparative Analysis of Transgene Copy Numbers and Expression Characteristics across Multiple Transgenic Marine Medaka Oryzias dancena Strains carrying the β-Actin Promoter-Driven GFP Reporter

【Several transgenic marine medaka Oryzias dancena strains harboring a green fluorescent protein (GFP) reporter construct regulated by an endogenous ${\beta}$ -actin promoter were established and their expression characteristics in relation to transgene copy numbers were examined in 21 transgene genotypes. Most of the transgenic strains displayed transgene insertion patterns typical of microinjection-mediated introduction of foreign DNA into fish embryos, characterized by the random integration of multiple transgene copies (ranging from 1 - 282 copies per cell), often accompanied by the formation of concatemer(s), as assessed by genomic Southern blot hybridization analysis and qPCR. Transgenic strains showed ubiquitous and continued temporal and spatial expression patterns of the transgenic GFP during most of their life cycle, from the embryonic stage to adulthood, enabling assessment of the expression pattern of the endogenous ${\beta}$ -actin gene. However, a comparative evaluation of transgene copy numbers and expression levels showed that copy number-dependent expression, the stability of the ubiquitous distribution and expression efficiency per transgene copy varied among the transgenic strains. Fluorescence expression levels were positively correlated with absolute transgene copy numbers, whereas the expression efficiency per transgene copy was inversely related to the number of transgene integrant copies. Data from this study will guide the selection of potentially desirable transgenic strains with ubiquitous expression of a fluorescent transgene, not only in this marine medaka species but also in other related model fish species.】