Exploring Yersinia ruckeri (O1 Biotype 2) infection in three early life-stages of rainbow trout.

Enteric redmouth disease (ERM) caused by the enterobacterium Yersinia ruckeri poses a significant threat to salmonid aquaculture globally. Despite decades of experimental infection studies, key knowledge gaps remain regarding the onset of disease susceptibility and mechanisms of immunity during early developmental stages, undermining disease management efforts in all susceptible life-stages. In this study, a series of immersion challenges were conducted, challenging and re-challenging rainbow trout Oncorhynchus mykiss (Walbaum) at 7, 14 and 51 d post-hatch (dph; mean weights = 0.085, 0.1 and 2.0 g respectively) to high concentrations (1.72 × 107-1.1 × 108 CFU) of Y. ruckeri at 15°C. This study indicates the hitherto unknown initial point of susceptibility to infection as the time of first ingestion of exogenous food (14 dph), and shows that individuals surviving primary challenge at 14 dph are significantly more likely to survive re-challenge at 51 dph compared with naive individuals (hazard ratio = 1.446, p = 0.032). Other key findings include large variation in mortality between different development-stages, from 21.1% at 14 dph to 81.2% at 51 dph, and novel age-dependent symptoms not reported previously. Results from this study enhance our understanding of ERM in juvenile rainbow trout and inform the development of improved aquatic animal health management strategies, thereby contributing to the productivity and sustainability of salmonid aquaculture into the future.

Novel atypical Aeromonas salmonicida bath challenge model for juvenile ballan wrasse (Labrus bergylta, Ascanius).

Atypical Aeromonas salmonicida (aAs) is currently one of the most routinely recovered bacterial pathogens isolated during disease outbreaks in farmed cleaner fish, ballan wrasse (Labrus bergylta, Ascanius). Vibrionaceae family bacteria have also been isolated from ballan wrasse in Scotland. This study determined the infectivity, pathogenicity and virulence of aAs and Vibrionaceae isolates in juvenile farmed ballan wrasse (n = 50; approx. 2 g) using a bath challenge, and fish were monitored for a period of 16 days. Atypical As caused significant mortalities in contrast to Vibrionaceae isolates. Notably, differential virulence was observed between two aAs vapA type V strains at similar challenge doses. Diseased fish exhibited a systemic infection where aAs was detected in all analysed tissues (liver, spleen and kidney) by PCR and qPCR. Macroscopically, moribund and survivor fish exhibited hepatomegaly and splenomegaly. In moribund and surviving fish, histopathology showed granulomatous hepatitis with eosinophilic granular cells surrounding bacterial colonies and endocarditis along with splenic histiocytosis. This is the first report of a successful aAs bath challenge model for juvenile ballan wrasse which provides an important tool for future studies on vaccine efficacy and immunocompetence.

Dying for change: A roadmap to refine the fish acute toxicity test after 40 years of applying a lethal endpoint.

The fish acute toxicity test (TG203; OECD, 2019) is frequently used and highly embedded in hazard and risk assessment globally. The test estimates the concentration of a chemical that kills 50% of the fish (LC50) over a 96 h exposure and is considered one of the most severe scientific procedures undertaken. Over the years, discussions at the Organisation for Economic Co-operation and Development (OECD) have resulted in changes to the test which reduce the number of fish used, as well as the development of a (potential) replacement test (TG236, OECD, 2013). However, refinement of the mortality endpoint with an earlier (moribundity) endpoint was not considered feasible during the Test Guideline's (TG) last update in 2019. Several stakeholders met at a UK-based workshop to discuss how TG203 can be refined, and identified two key opportunities to reduce fish suffering: (1) application of clinical signs that predict mortality and (2) shortening the test duration. However, several aspects need to be addressed before these refinements can be adopted. TG203 has required recording of major categories of sublethal clinical signs since its conception, with the option to record more detailed signs introduced in the 2019 update. However, in the absence of guidance, differences in identification, recording and reporting of clinical signs between technicians and laboratories is likely to have generated piecemeal data of varying quality. Harmonisation of reporting templates, and training in clinical sign recognition and recording are needed to standardise clinical sign data. This is critical to enable robust data-driven detection of clinical signs that predict mortality. Discussions suggested that the 96 h duration of TG203 cannot stand up to scientific scrutiny. Feedback and data from UK contract research organisations (CROs) conducting the test were that a substantial proportion of mortalities occur in the first 24 h. Refinement of TG203 by shortening the test duration would reduce suffering (and test failure rate) but requires a mechanism to correct new results to previous 96 h LC50 data. The actions needed to implement both refinement opportunities are summarised here within a roadmap. A shift in regulatory assessment, where the 96 h LC50 is a familiar base for decisions, will also be critical.

Tributyltin: Advancing the Science on Assessing Endocrine Disruption with an Unconventional Endocrine-Disrupting Compound.

Tributyltin (TBT) has been recognized as an endocrine disrupting chemical (EDC) for several decades. However, only in the last decade, was its primary endocrine mechanism of action (MeOA) elucidated-interactions with the nuclear retinoid-X receptor (RXR), peroxisome proliferator-activated receptor γ (PPARγ), and their heterodimers. This molecular initiating event (MIE) alters a range of reproductive, developmental, and metabolic pathways at the organism level. It is noteworthy that a variety of MeOAs have been proposed over the years for the observed endocrine-type effects of TBT; however, convincing data for the MIE was provided only recently and now several researchers have confirmed and refined the information on this MeOA. One of the most important lessons learned from years of research on TBT concerns apparent species sensitivity. Several aspects such as the rates of uptake and elimination, chemical potency, and metabolic capacity are all important for identifying the most sensitive species for a given chemical, including EDCs. For TBT, much of this was discovered by trial and error, hence important relationships and important sensitive taxa were not identified until several decades after its introduction to the environment. As recognized for many years, TBT-induced responses are known to occur at very low concentrations for molluscs, a fact that has more recently also been observed in fish species. This review explores the MeOA and effects of TBT in different species (aquatic molluscs and other invertebrates, fish, amphibians, birds, and mammals) according to the OECD Conceptual Framework for Endocrine Disruptor Testing and Assessment (CFEDTA). The information gathered on biological effects that are relevant for populations of aquatic animals was used to construct Species Sensitivity Distributions (SSDs) based on No Observed Effect Concentrations (NOECs) and Lowest Observed Effect Concentrations (LOECs). Fish appear at the lower end of these distributions, showing that they are as sensitive as molluscs, and for some species, even more sensitive. Concentrations in the range of 1 ng/L for water exposure (10 ng/g for whole-body burden) have been shown to elicit endocrine-type responses, whereas mortality occurs at water concentrations ten times higher. Current screening and assessment methodologies as compiled in the OECD CFEDTA are able to identify TBT as a potent endocrine disruptor with a high environmental risk for the original use pattern. If those approaches had been available when TBT was introduced to the market, it is likely that its use would have been regulated sooner, thus avoiding the detrimental effects on marine gastropod populations and communities as documented over several decades.

Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus.

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-ß) and luteinizing hormone (lh-ß) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR. Ovarian weights peaked in "ready to spawn" females, dropped after spawning, before again progressively increasing from 6 to 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-ß mRNA levels showed a peak at 48hps, while fsh-ß did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.

Microarray analysis of di-n-butyl phthalate and 17α ethinyl-oestradiol responses in three-spined stickleback testes reveals novel candidate genes for endocrine disruption.

Phthalate esters are plasticizers frequently found in wastewater effluents. Previous studies on phthalates have reported anti-androgenic activity in mammals, causing concerns of their potential effects on the reproduction of aquatic organisms. Another group of environmental endocrine disrupters, steroidal estrogens, are known to inhibit steroid biosynthesis in the gonads, but the effects related to spermatogenesis are not well understood in fish. In this study, three-spined sticklebacks were exposed to di-n-butyl phthalate (DBP) and 17α ethinyl-oestradiol (EE2) at nominal concentrations 35µg/L and 40ng/L, respectively, for four days. The aim of the study was to obtain insight into the acute transcriptional responses putatively associated with endocrine disruption. RNA samples from eight individual male fish per treatment (including controls) were used in microarray analysis, covering the expression of approximately 21,000 genes. In the EE2 treatment the results show transcriptional downregulation of genes associated with steroid biosynthesis pathway and up-regulation of genes involved in pathways related to epidermal growth factor signaling and xenobiotic metabolism. The transcriptional response to DBP was in general weaker than to EE2, but based on enrichment analysis, we suggest adverse effects on retinoid metabolism, creatine kinase activity and cell adhesion. Among the genes showing highest fold changes after DBP treatment compared to control was the teleost fish -specific cytochrome P450 17A2. Overall, this study promotes our understanding on molecular responses to anti-androgens and estrogens in fish testes.

Seasonal profile of common pharmaceuticals in edible bivalve molluscs.

Pharmaceuticals are recognised as environmental contaminants of emerging concern (CECs) due to their increasing presence in the aquatic environment, along with high bioactivity linked to their therapeutic use. Therefore, information on environmental levels is urgently required. This study examined the presence of a range of common pharmaceuticals in oysters and mussels intended for human consumption from England and Wales using stable isotope dilution tandem mass spectrometry. A range of compounds were detected in bivalve tissue, with the Selective Serotonin Reuptake Inhibitor antidepressant sertraline being most abundant, reaching a maximum concentration of 22.1 ng/g wet weight shellfish tissue. Levels of all pharmaceuticals showed seasonal and geographical patterns. A dietary risk assessment revealed that the levels of pharmaceuticals identified in bivalve molluscs represent a clear hazard, but not a risk for the consumer. This study highlights the requirement for further monitoring of the presence of pharmaceuticals and other CECs in bivalve molluscs.

Assessment of reproductive biomarkers in three-spined stickleback (Gasterosteus aculeatus) from sewage effluent recipients.

The aim of this study was to examine the occurrence of endocrine disruption close to sewage treatment plant effluent discharges along the Finnish Baltic Sea coast using a set of reproductive biomarkers present in adult three-spined stickleback (Gasterosteus aculeatus). Possible variation and sensitivity of the biomarkers during an entire reproductive period were also examined. The analysis of vitellogenin (VTG) for estrogenic activity and spiggin for androgenic activity, together with histopathological analysis indicated that sticklebacks were exposed to estrogenic loads sufficient to cause inappropriate production of VTG and to disrupt normal testicular structure in adult male sticklebacks. No androgenic disruption was observed. The results emphasize the need of a combination of several reproductive biomarkers in fish and repeated sampling for the detection of potential endocrine modulating substances under field condition.

Piscine follicle-stimulating hormone triggers progestin production in gilthead seabream primary ovarian follicles.

Ovarian growth (vitellogenesis) in most lower vertebrates is mediated by estradiol-17beta (E2) secreted by the follicles in response to follicle-stimulating hormone (Fsh), whereas oocyte maturation and ovulation are mediated by progestins, such as 17alpha,20beta-dihydroxypregn-4-en-3-one (17,20beta-P), produced in response to luteinizing hormone (Lh). In teleosts, follicular synthesis of 17,20beta-P at the time of maturation is due primarily to up-regulation of the enzymes P450c17-II (Cyp17a2) and 20beta-hydroxysteroid dehydrogenase (Cbr1). Here, we show that follicular cells associated with primary growth (previtellogenic) oocytes of the gilthead seabream also express cyp17a2 and cbr1, in addition to P450c17-I (cyp17a1) and aromatase (cyp19a1), enzymes required for E2 synthesis. Ovaries containing only oogonia and early primary ovarian follicles had a 60-fold higher concentration of 17,20beta-P than ovaries in the succeeding stages and had a higher expression of cbr1 and Fsh receptor (fshra). Stimulation of explants of primary follicles in vitro with recombinant piscine Fsh (rFsh), which specifically activates the seabream Fshra, promoted a rapid accumulation of 17,20beta-P, and synthesis was sustained by an external supply of 17alpha-hydroxyprogesterone. In the presence of Cbr1 inhibitors, rFsh-mediated 17,20beta-P production was reduced, with a concomitant increase in testosterone and E2 synthesis. In primary explants, rFsh up-regulated cyp17a2 and cbr1 transcription and simultaneously down-regulated cyp17a1 and cyp19a1 steady-state mRNA levels within 24 h. In contrast, in explants containing vitellogenic follicles, rFsh had no effect on cyp17a2 and cbr1 expression, but increased that of cyp17a1 and cyp19a1. These data suggest a functional Fshra-activated Cyp17a2/Cbr1 steroidogenic pathway in gilthead seabream primary ovarian follicles triggering the production of 17,20beta-P.

Towards a system level understanding of non-model organisms sampled from the environment: a network biology approach.

The acquisition and analysis of datasets including multi-level omics and physiology from non-model species, sampled from field populations, is a formidable challenge, which so far has prevented the application of systems biology approaches. If successful, these could contribute enormously to improving our understanding of how populations of living organisms adapt to environmental stressors relating to, for example, pollution and climate. Here we describe the first application of a network inference approach integrating transcriptional, metabolic and phenotypic information representative of wild populations of the European flounder fish, sampled at seven estuarine locations in northern Europe with different degrees and profiles of chemical contaminants. We identified network modules, whose activity was predictive of environmental exposure and represented a link between molecular and morphometric indices. These sub-networks represented both known and candidate novel adverse outcome pathways representative of several aspects of human liver pathophysiology such as liver hyperplasia, fibrosis, and hepatocellular carcinoma. At the molecular level these pathways were linked to TNF alpha, TGF beta, PDGF, AGT and VEGF signalling. More generally, this pioneering study has important implications as it can be applied to model molecular mechanisms of compensatory adaptation to a wide range of scenarios in wild populations.

Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17ß-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

Clarification of early end-points for refinement of animal experiments, with specific reference to fish.

Appropriate end-points are integral to the refinement of laboratory animal experiments. Our recent experience has highlighted that ambiguity around end-points is hampering their adoption in experiments that cause severe suffering to fish. In toxicology, the term endpoint (single word) refers to the response variable to the treatment that is measured and analysed. This differs to usage within laboratory animal experimentation, where end-point (hyphenated) refers to the time-point when exposure of the animal(s) to the treatment (and suffering) ends. Within laboratory animal experimentation, standardised terminology is needed for different types of early end-point which are based on the condition of the animal(s) or progress of the experiment. We propose that those involved in regulating and conducting animal experiments consider seven distinct types of early end-point (aim, technical error, biological error, mortality, moribundity, prognostic humane, non-prognostic humane) in addition to the planned experimental end-point (i.e. maximum duration). Moribundity (not morbidity) refers to an animal in a severely debilitated state close to death. Moribundity in fish is not yet defined, so we propose identification via a lack of response to external stimuli, loss of equilibrium (i.e. loss of righting reflex), and a slow opercular ventilation rate. As these clinical signs equate to those of deep/surgical anaesthesia, this moribundity end-point cannot be considered a humane end-point as the fish is likely to be unconscious and have passed the point of maximum suffering. We believe that identification of earlier humane end-points based on clinical signs and wider recognition of other types of early end-point can reduce suffering in experiments.

The Uptake of Ethinyl-Estradiol and Cortisol From Water by Mussels (Mytilus spp.).

Previous toxicokinetic studies have shown that mussels (Mytilus spp.) can readily absorb the three main mammalian sex steroids, estradiol (E2), testosterone (T) and progesterone (P) from water. They also have a strong ability to store E2 and the 5α-reduced metabolites of T and P in the form of fatty acid esters. These esters were shown to have half-lives that were measured in weeks (i.e. they were not subject to fast depuration). The present study looked at the toxicokinetic profile of two other common steroids that are found in water, the potent synthetic oestrogen, (ethinyl-estradiol) (EE2; one of the two components of 'the pill'), and cortisol, a natural stress steroid in vertebrates. In the first three hours of uptake, tritiated EE2 was found to be taken up at a similar rate to tritiated E2. However, the levels in the water plateaued sooner than E2. The ability of the animals to both esterify and sulphate EE2 was found to be much lower than E2, but nevertheless did still take place. After 24 h of exposure, the majority of radiolabelled EE2 in the animals was present in the form of free steroid, contrary to E2, which was esterified. This metabolism was reflected in a much lower half-life (of only 15 h for EE2 in the mussels as opposed to 8 days for E2 and >10 days for T and P). Intriguingly, hardly any cortisol (in fact none at all in one of the experiments) was absorbed by the mussels. The implications of this finding in both toxicokinetic profiling and evolutionary significance (why cortisol might have evolved as a stress steroid in bony fishes) are discussed.

Modeling the metabolic profile of Mytilus edulis reveals molecular signatures linked to gonadal development, sex and environmental site.

The monitoring of anthropogenic chemicals in the aquatic environment including their potential effects on aquatic organisms, is important for protecting life under water, a key sustainable development goal. In parallel with monitoring the concentrations of chemicals of concern, sentinel species are often used to investigate the biological effects of contaminants. Among these, bivalve molluscs such as mussels are filter-feeding and sessile, hence an excellent model system for measuring localized pollution. This study investigates the relationship between the metabolic state of the blue mussel (Mytilus edulis) and its physiology in different environments. We developed a computational model based on a reference site (relatively unpolluted) and integrated seasonal dynamics of metabolite relative concentrations with key physiological indicators and environmental parameters. The analysis of the model revealed that changes in metabolite levels during an annual cycle are influenced by water temperature and are linked to gonadal development. This work supports the importance of data-driven biology and its potential in environmental monitoring.

Hypoxia modifies the response to flutamide and linuron in male three-spined stickleback (Gasterosteus aculeatus).

Hypoxia is a major stressor in aquatic environments and it is frequently linked with excess nutrients resulting from sewage effluent discharges and agricultural runoff, which often also contain complex mixtures of chemicals. Despite this, interactions between hypoxia and chemical toxicity are poorly understood. We exposed male three-spined stickleback during the onset of sexual maturation to a model anti-androgen (flutamide; 250 µg/L) and a pesticide with anti-androgenic activity (linuron; 250 µg/L), under either 97% or 56% air saturation (AS). We assessed the effects of each chemical, alone and in combination with reduced oxygen concentration, by measuring the transcription of spiggin in the kidney, as a marker of androgen signalling, and 11 genes in the liver involved in some of the molecular pathways hypothesised to be affected by the exposures. Spiggin transcription was strongly inhibited by flutamide under both AS conditions. In contrast, for linuron, a strong inhibition of spiggin was observed under 97% AS, but this effect was supressed under reduced air saturation, likely due to interactions between the hypoxia inducible factor and the aryl hydrocarbon receptor (AhR) pathways. In the liver, hypoxia inducible factor 1α was induced following exposure to both flutamide and linuron, however this was independent of the level of air saturation. This work illustrates the potential for interactions between hypoxia and pollutants with endocrine or AhR agonist activity to occur, with implications for risk assessment and management.

Skin swabbing is a refined technique to collect DNA from model fish species.

Model fish species such as sticklebacks and zebrafish are frequently used in studies that require DNA to be collected from live animals. This is typically achieved by fin clipping, a procedure that is simple and reliable to perform but that can harm fish. An alternative procedure to sample DNA involves swabbing the skin to collect mucus and epithelial cells. Although swabbing appears to be less invasive than fin clipping, it still requires fish to be netted, held in air and handled-procedures that can cause stress. In this study we combine behavioural and physiological analyses to investigate changes in gene expression, behaviour and welfare after fin clipping and swabbing. Swabbing led to a smaller change in cortisol release and behaviour on the first day of analysis compared to fin clipping. It also led to less variability in data suggesting that fewer animals need to be measured after using this technique. However, swabbing triggered some longer term changes in zebrafish behaviour suggesting a delayed response to sample collection. Skin swabbing does not require the use of anaesthetics and triggers fewer changes in behaviour and physiology than fin clipping. It is therefore a more refined technique for DNA collection with the potential to improve fish health and welfare.

Insights into the development of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from UK estuaries.

Hepatocellular fibrillar inclusions (HFI) are an unusual pathology of unknown aetiology affecting European flounder (Platichthys flesus), particularly from estuaries historically impacted by pollution. This study demonstrated that the HFI prevalence range was 6-77% at several UK estuaries, with Spearman rank correlation analysis showing a correlation between HFI prevalence and sediment concentrations of ∑PBDEs and ∑HBCDs. The data showed that males exhibit higher HFI prevalence than females, with severity being more pronounced in estuaries exhibiting higher prevalence. HFI were not age associated indicating a subacute condition. Electron microscopy confirmed that HFI were modified proliferating rough endoplasmic reticulum (RER), whilst immunohistochemistry provided evidence of VTG production in HFI of male P. flesus. Despite positive labelling of aberrant VTG production, we could not provide additional evidence of xenoestrogen exposure. Gene transcripts (VTG/CHR) and plasma VTG concentrations (>1 µg ml-1), were only considered elevated in four male fish showing no correlation with HFI severity. Further analysis revealed that reproductively mature female P. flesus i.e. >3-year-old, did not exhibit HFI, whereas males of all ages were affected. This, combined with previous reports that estradiol (E2) can impair mixed function oxygenase activity, supports a hypothesis that harmful chemical metabolites (following phase 1 metabolism of their parent compounds) are potentially responsible for HFIs observed in male and ≤ 3-year-old female fish. Consequently, HFI and xenoestrogenic induced VTG production could be independent of each other resulting from different concurrent toxicopathic mechanisms, although laboratory exposures will likely be the only way to determine the true aetiology of HFI.

Estrogenic and androgenic effects of municipal wastewater effluent on reproductive endpoint biomarkers in three-spined stickleback (Gasterosteus aculeatus).

Municipal wastewater treatment plants have been associated with the release of endocrine-disrupting chemicals, which consequently lead to alterations of reproductive function in aquatic organisms. The three-spined stickleback (Gasterosteus aculeatus) has quantifiable biomarkers for assessment of both estrogen (vitellogenin) and androgen (spiggin) activity, which makes this species very valuable in the research of endocrine disruption. The estrogenic and androgenic biomarkers were used for evaluating exposure effects of municipal wastewater effluent. We evaluated the effects of 17alpha-ethinylestradiol (EE2), 17alpha-methyltestosterone (MT), and wastewater effluents on induction of vitellogenin and spiggin production, gonadosomatic index, hepatosomatic index, nephrosomatic index, plasma steroid levels, and histopathology. Adult female and male sticklebacks were exposed to 20 ng/L of EE2, 10 microg/L of MT, and wastewater effluent (10, 50, and 80% of original concentration) in a flow-through system for an exposure of one week and an extended exposure of four weeks. Chemical analyses of the steroids were done for verification of exposure concentrations and presence in the used wastewater. Our results show that municipal wastewater effluent exerts estrogenic action on three-spined stickleback as observed by elevated vitellogenin levels in exposed fish, corresponding to the effect seen in fish exposed to EE2. Furthermore, wastewater and EE2 exerted similar histopathological effects on testis of exposed fish. Although domestic effluent is suspected to have a high content of natural androgens, no obvious androgenic effect of wastewater was observed in the present study.

The organophosphorous pesticide, fenitrothion, acts as an anti-androgen and alters reproductive behavior of the male three-spined stickleback, Gasterosteus aculeatus.

Fenitrothion (FN) is a widely used organophosphorous pesticide that has structural similarities with the clinical anti-androgen flutamide. The potential for FN to act as an anti-androgen (at exposures of 1, 50, and 200 microg FN/l over a 26-day period) was assessed in male three-spined sticklebacks, Gasterosteus aculeatus, by measuring kidney spiggin concentration, nest-building, and courtship behavior. Spiggin is the glue protein that male sticklebacks use to build their nests and is directly controlled by androgens. FN exposure significantly reduced spiggin production as well as nest-building activity. It also adversely affected courtship--especially the 'zigzag dance' and biting behavior of the males. FN thus appears to have anti-androgenic effects on both the physiology and behavior of the male stickleback.

Unravelling paralogous gene expression dynamics during three-spined stickleback embryogenesis.

Development requires the implementation of a plethora of molecular mechanisms, involving a large set of genes to ensure proper cell differentiation, morphogenesis of tissues and organs as well as the growth of the organism. Genome duplication and resulting paralogs are considered to provide the raw genetic materials important for new adaptation opportunities and boosting evolutionary innovation. The present study investigated paralogous genes, involved in three-spined stickleback (Gasterosteus aculeatus) development. Therefore, the transcriptomes of five early stages comprising developmental leaps were explored. Obtained expression profiles reflected the embryo's needs at different stages. Early stages, such as the morula stage comprised transcripts mainly involved in energy requirements while later stages were mostly associated with GO terms relevant to organ development and morphogenesis. The generated transcriptome profiles were further explored for differential expression of known and new paralogous genes. Special attention was given to hox genes, with hoxa13a being of particular interest and to pigmentation genes where itgb1, involved in the melanophore development, displayed a complementary expression pattern throughout studied stages. Knowledge obtained by untangling specific paralogous gene functions during development might not only significantly contribute to the understanding of teleost ontogenesis but might also shed light on paralogous gene evolution.