A candidate sex determination locus in amphibians which evolved by structural variation between X- and Y-chromosomes.

Most vertebrates develop distinct females and males, where sex is determined by repeatedly evolved environmental or genetic triggers. Undifferentiated sex chromosomes and large genomes have caused major knowledge gaps in amphibians. Only a single master sex-determining gene, the dmrt1-paralogue (dm-w) of female-heterogametic clawed frogs (Xenopus; ZW♀/ZZ♂), is known across >8740 species of amphibians. In this study, by combining chromosome-scale female and male genomes of a non-model amphibian, the European green toad, Bufo(tes) viridis, with ddRAD- and whole genome pool-sequencing, we reveal a candidate master locus, governing a male-heterogametic system (XX♀/XY♂). Targeted sequencing across multiple taxa uncovered structural X/Y-variation in the 5'-regulatory region of the gene bod1l, where a Y-specific non-coding RNA (ncRNA-Y), only expressed in males, suggests that this locus initiates sex-specific differentiation. Developmental transcriptomes and RNA in-situ hybridization show timely and spatially relevant sex-specific ncRNA-Y and bod1l-gene expression in primordial gonads. This coincided with differential H3K4me-methylation in pre-granulosa/pre-Sertoli cells, pointing to a specific mechanism of amphibian sex determination.

Endocrine disruption in teleosts and amphibians is mediated by anthropogenic and natural environmental factors: implications for risk assessment.

Environmental variation in the Anthropocene involves several factors that interfere with endocrine systems of wildlife and humans, presenting a planetary boundary of still unknown dimensions. Here, we focus on chemical compounds and other impacts of anthropogenic and natural origins that are adversely affecting reproduction and development. The main sink of these endocrine disruptors (EDs) is surface waters, where they mostly endanger aquatic vertebrates, like teleost fish and amphibians. For regulatory purposes, EDs are categorized into EATS modalities (oestrogenic, androgenic, thyroidal, steroidogenesis), only addressing endocrine systems being assessable by validated tests. However, there is evidence that non-EATS modalities-and even natural sources, such as decomposition products of plants or parasitic infections-can affect vertebrate endocrine systems. Recently, the disturbance of natural circadian light rhythms by artificial light at night (ALAN) has been identified as another ED. Reviewing the knowledge about EDs affecting teleosts and amphibians leads to implications for risk assessment. The generally accepted WHO-definition for EDs, which focuses exclusively on 'exogenous substances' and neglects parasitic infections or ALAN, seems to require some adaptation. Natural EDs have been involved in coevolutionary processes for ages without resulting in a general loss of biodiversity. Therefore, to address the 'One Health'-principle, future research and regulatory efforts should focus on minimizing anthropogenic factors for endocrine disruption. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Insect-based fish feed in decoupled aquaponic systems: Effect on lettuce production and resource use.

The utilisation of insect meal-based fish feed as a substitute for conventional fish meal-based fish feed is considered as a promising innovative alternative to boost circularity in aquaculture and aquaponics. Basic research on its use in aquaponics is limited. So far, no reports on the effects of fish waste water, derived from a recirculating aquaculture system using Black Soldier Fly (BSF) meal-based diets, were available on the growth performance of lettuce. Therefore, this study aimed to compare the effect of reusing fish waste water from tilapia culture (as a base for the nutrient solution) fed with a fish meal-based diet (FM) and a BSF meal-based diet on resource use and lettuce growth in decoupled aquaponic systems. A conventional hydroponics nutrient solution (HP) served as control, and inorganic fertilisers were added to all nutrient solutions to reach comparable target concentrations. The experiment was conducted in a controlled climate chamber in nine separate hydroponics units, three per treatment. Lettuce fresh and dry weight, number of leaves, relative leaf chlorophyll concentration, water consumption, and the usage of inorganic fertilisers were measured. Micro- and macronutrients in the nutrient solutions were monitored in time series. Similar lettuce yield was seen in all treatments, with no significant effects on fresh and dry weight, the number of leaves, and relative chlorophyll values. Water use per plant was also similar between treatments, while the amount of total inorganic fertiliser required was 32% lower in FM and BSF compared to HP. Higher sodium concentrations were found in the FM nutrient solutions compared to BSF and HP. The results confirm that BSF-based diet is a promising alternative to FM-based diet in aquaponics with no negative effects on lettuce growth. Additionally, BSF-based diet might be beneficial in intensive, professional aquaponics applications due to the lower sodium concentration in the nutrient solution.

Multi-genome comparisons reveal gain-and-loss evolution of the anti-Mullerian hormone receptor type 2 gene, an old master sex determining gene, in Percidae.

The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplications (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome-18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variants (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in testis than ovary. Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Modification of the chemically induced inflammation assay reveals the Janus face of a phenol rich fulvic acid.

Inflammation is an essential process as a reaction towards infections or wounding. Exposure to hazardous environmental pollutants can lead to chronic inflammations, where the resolving phase is delayed or blocked. Very contradictory studies have been reported on the pro- and anti-inflammatory effects of humic substances (HSs) leading to significant disagreements between researchers. To a certain extent, this can be attributed to the chemical heterogeneity of this group of xenobiotics. Here we show for the first time that pro- and anti-inflammatory effects can occur by one HSs. We adapted an assay that uses green fluorescence-labeled zebrafish larvae and CuSO4 to indue an inflammation. In wild-type larvae, exposure to 50 µM CuSO4 for 2 h activated the production of reactive oxygen species, which can be monitored with a fluorescence dye (H2DCFDA) and a microplate reader. This allows not only the use of wild-type fish but also a temporal separation of copper exposure and inflammatory substance while retaining the high throughput. This modified assay was then used to evaluate the inflammatory properties of a fulvic acid (FA). We found, that the aromatic structure of the FA protects from inflammation at 5 and 50 mg C/L, while the persistent free radicals enhance the copper-induced inflammation at ≥ 300 mg C/L.

A duplicated copy of id2b is an unusual sex-determining candidate gene on the Y chromosome of arapaima (Arapaima gigas).

Arapaima gigas is one of the largest freshwater fish species of high ecological and economic importance. Overfishing and habitat destruction are severe threats to the remaining wild populations. By incorporating a chromosomal Hi-C contact map, we improved the arapaima genome assembly to chromosome-level, revealing an unexpected high degree of chromosome rearrangements during evolution of the bonytongues (Osteoglossiformes). Combining this new assembly with pool-sequencing of male and female genomes, we identified id2bbY, a duplicated copy of the inhibitor of DNA binding 2b (id2b) gene on the Y chromosome as candidate male sex-determining gene. A PCR-test for id2bbY was developed, demonstrating that this gene is a reliable male-specific marker for genotyping. Expression analyses showed that this gene is expressed in juvenile male gonads. Its paralog, id2ba, exhibits a male-biased expression in immature gonads. Transcriptome analyses and protein structure predictions confirm id2bbY as a prime candidate for the master sex-determiner. Acting through the TGFß signaling pathway, id2bbY from arapaima would provide the first evidence for a link of this family of transcriptional regulators to sex determination. Our study broadens our current understanding about the evolution of sex determination genetic networks and provide a tool for improving arapaima aquaculture for commercial and conservation purposes.

A 180 Myr-old female-specific genome region in sturgeon reveals the oldest known vertebrate sex determining system with undifferentiated sex chromosomes.

Several hypotheses explain the prevalence of undifferentiated sex chromosomes in poikilothermic vertebrates. Turnovers change the master sex determination gene, the sex chromosome or the sex determination system (e.g. XY to WZ). Jumping master genes stay main triggers but translocate to other chromosomes. Occasional recombination (e.g. in sex-reversed females) prevents sex chromosome degeneration. Recent research has uncovered conserved heteromorphic or even homomorphic sex chromosomes in several clades of non-avian and non-mammalian vertebrates. Sex determination in sturgeons (Acipenseridae) has been a long-standing basic biological question, linked to economical demands by the caviar-producing aquaculture. Here, we report the discovery of a sex-specific sequence from sterlet (Acipenser ruthenus). Using chromosome-scale assemblies and pool-sequencing, we first identified an approximately 16 kb female-specific region. We developed a PCR-genotyping test, yielding female-specific products in six species, spanning the entire phylogeny with the most divergent extant lineages (A. sturio, A. oxyrinchus versus A. ruthenus, Huso huso), stemming from an ancient tetraploidization. Similar results were obtained in two octoploid species (A. gueldenstaedtii, A. baerii). Conservation of a female-specific sequence for a long period, representing 180 Myr of sturgeon evolution, and across at least one polyploidization event, raises many interesting biological questions. We discuss a conserved undifferentiated sex chromosome system with a ZZ/ZW-mode of sex determination and potential alternatives. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

Fulvic acid accelerates hatching and stimulates antioxidative protection and the innate immune response in zebrafish larvae.

Aquaculture plays a pivotal role in covering dietary animal protein demands and restocking endangered fish populations. However, high mortality takes place at the earliest life stages: prior and immediately after hatching. Improving growth and health parameters by immunostimulants is widely used in older fish, but rarely studied in larvae. Fulvic acids (FAs) are natural substances found in soil and water. Using zebrafish as a model organism, we evaluated the effects of exposure to a FA at concentrations ranging from 1 to 500 mg C/L (mg dissolved organic carbon per liter) on embryonic development. Furthermore, the concentration of reactive oxygen species (ROS) inside the larvae as well as the molecular mechanisms involved in growth, immune response, and antioxidative protection were determined at 5, 50, and 500 mg C/L. 20 to 200 mg C/L accelerated the hatching, which was mediated by increased expression of ifg-1, gh, and he1-α. Furthermore, lyz and mpx were significantly increased at 5 and 50 mg C/L. A concentration of 500 mg C/L induced genes involved in the protection against ROS (nrf-2, keap-1, cat, sod-1), increased the concentration of ROS inside the larvae and caused tissue damage and mortality. Interestingly, 50 mg C/L activated ROS protection as well (nrf-2, sod-2), while no increase of ROS was found in the larvae. Our results show, that FA at low to medium concentrations can increase the health of larvae, but becomes detrimental at higher concentrations.

The Role of Behavioral Ecotoxicology in Environmental Protection.

For decades, we have known that chemicals affect human and wildlife behavior. Moreover, due to recent technological and computational advances, scientists are now increasingly aware that a wide variety of contaminants and other environmental stressors adversely affect organismal behavior and subsequent ecological outcomes in terrestrial and aquatic ecosystems. There is also a groundswell of concern that regulatory ecotoxicology does not adequately consider behavior, primarily due to a lack of standardized toxicity methods. This has, in turn, led to the exclusion of many behavioral ecotoxicology studies from chemical risk assessments. To improve understanding of the challenges and opportunities for behavioral ecotoxicology within regulatory toxicology/risk assessment, a unique workshop with international representatives from the fields of behavioral ecology, ecotoxicology, regulatory (eco)toxicology, neurotoxicology, test standardization, and risk assessment resulted in the formation of consensus perspectives and recommendations, which promise to serve as a roadmap to advance interfaces among the basic and translational sciences, and regulatory practices.

Misbalance of thyroid hormones after two weeks of exposure to artificial light at night in Eurasian perch Perca fluviatilis.

Artificial light at night (ALAN) can affect the physiology and behavior of animals because it alters the natural rhythm of light and darkness. Thyroid hormones (TH) are partially regulated by the light information of photoperiod and are involved in metabolic adjustments to daily and seasonal changes in the environment, such as larval and juvenile development, somatic growth and reproduction. ALAN can change photoperiodic information and might thereby lead to changes in thyroid metabolism, but so far research on this topic is scarce. Therefore, we tested in two different experiments the effects of nocturnal illumination at a wide range of light intensities on TH in plasma of Eurasian perch (Perca fluviatilis). Total 3,3',5-triiodo-L-thyronine (T3) was significantly affected by ALAN and reduced at the highest tested intensity of 100 lx after only two weeks of exposure. Although total L-thyroxine (T4) was not significantly affected, the ratio of T3 to T4 tended to slightly decrease at 100 lx. In a second low-light experiment ALAN did not have clear effects on T3, T4 or the ratio of T3 to T4 at intensities between 0.01 lx and 1 lx. The results show first signs of endocrine disruption in thyroid metabolism after a relatively short ALAN exposure of two weeks under high-intensity streetlight conditions. Misbalanced thyroidal status can have serious implications for metabolic rates as well as developmental and reproductive processes.

Innate immunity, oxidative stress and body indices of Eurasian perch Perca fluviatilis after two weeks of exposure to artificial light at night.

Artificial light at night (ALAN) can disrupt biological rhythms of fish and other vertebrates by changing the light information of the nocturnal environment. Disrupted biorhythms can impair the immune system of vertebrates as it has been shown for conditions with continuous illumination or long-day photoperiod in many vertebrates, including fish. Nonetheless, this has not been shown so far for typical ALAN scenarios with high light intensities during day and low light intensities at night. Therefore, in this study, proxies for the innate immune system and oxidative stress as well as body indices of Eurasian perch Perca fluviatilis were measured under a wide range of intensities of nocturnal illumination. The authors found no changes in parameters of the innate immune system and no significant changes in proxies for oxidative stress after 2-week exposures to nocturnal illuminance ranging from 0.01 lx to 1 lx in one experiment or from 1 lx to 100 lx in a second experiment. A decrease in the hepato-somatic index at the highest tested light intensity of 100 lx compared to the dark control was the only significant difference in all parameters among treatments. After 2 weeks of exposure, ALAN does not seem to seriously challenge the innate immune system and seems to cause less oxidative stress than expected. The results of this study contradict the findings from other studies applying continuous illumination or long-day photoperiod and highlight the importance of further research in this field. Because ALAN represents a sustained modulation of the environment that may have cumulative effects over time, long-term studies are required for a better understanding of how ALAN modulates the health of fish.

Phenol-rich fulvic acid as a water additive enhances growth, reduces stress, and stimulates the immune system of fish in aquaculture.

Aquaculture has become imperative to cover the demands for dietary animal protein. Simultaneously, it has to overcome prejudices from excessive use of antibiotics and environmental impacts. Natural supplements are traditionally applied orally. In this study, we demonstrated another pathway: the gills. Humic substances are immunostimulants and a natural part of every aquatic ecosystem, making them ideal to be used as bath stimulants. Five and 50 mg C/L of a fulvic acid-rich humic substance was added for 28 days to the water of juvenile rainbow trout (Oncorhynchus mykiss). This fulvic acid is characterized by a high content of phenolic moieties with persistent free radicals and a high electron exchange capacity. The high concentration of the fulvic acid significantly increased growth and reduced the food conversion ratio and the response to a handling-stressor. Phagocytosis and potential killing activity of head kidney leukocytes were increased, as well as the total oxyradical scavenging capacity (TOSC) and lysozyme activity in the gills. In conclusion, immunostimulation via gills is possible with our fulvic acid, and the high phenolic content improved overall health and stress resistance of fish.

Ion-rich potash mining effluents affect sperm motility parameters of European perch, Perca fluviatilis, and impair early development of the common roach, Rutilus rutilus.

Secondary salinization of freshwater ecosystems is of increasing global concern. One of the main causes are the effluents of the potash mining industry containing high concentrations of major ions (Cl-, Na+, Mg2+, K+). In Germany, the ongoing discharge of effluents into the River Werra led to a strong impoverishment of the biodiversity and abundance of local species. Young cohorts of many freshwater fish are completely absent suggesting reproductive failure under these conditions. Therefore, the aim of the study was to experimentally investigate the effects of high concentrations and imbalances of ions that are prevalent in potash mining effluents on reproductive traits of native freshwater teleosts. Sperm motility parameters of the common roach, Rutilus rutilus, and European perch, Perca fluviatilis, were assessed as well as fertilization rate, egg size, hatching, malformations and mortality of embryonic and larval stages of roach. Concentrations of the permitted thresholds (HT) and future thresholds (LT) as well as three ion solutions containing high Mg2+ (Mg), high K+ (K) and both in combination (Mg + K) were tested. Curvilinear velocity and linearity of perch spermatozoa were elevated with potentially adverse effects on fertilization success. Sperm motility parameters and fertilization rate of roach were not affected. However, egg sizes of roach were increased in all groups due to the osmotic action of ions and in LT, premature hatch was observed. Furthermore, all groups comprised a higher number of malformations including pericardial edema and spine curvatures and group HT exhibited a higher mortality rate compared to control. The results clearly demonstrated that particularly the sum of high concentrations of ions, as prevalent in HT and LT, rather than individual ion species exerts detrimental effects on early development of roach potentially increasing overall mortality under natural conditions. These results emphasize that currently permitted and future thresholds are exceeding tolerated ion concentrations.

The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization.

Sturgeons seem to be frozen in time. The archaic characteristics of this ancient fish lineage place it in a key phylogenetic position at the base of the ~30,000 modern teleost fish species. Moreover, sturgeons are notoriously polyploid, providing unique opportunities to investigate the evolution of polyploid genomes. We assembled a high-quality chromosome-level reference genome for the sterlet, Acipenser ruthenus. Our analysis revealed a very low protein evolution rate that is at least as slow as in other deep branches of the vertebrate tree, such as that of the coelacanth. We uncovered a whole-genome duplication that occurred in the Jurassic, early in the evolution of the entire sturgeon lineage. Following this polyploidization, the rediploidization of the genome included the loss of whole chromosomes in a segmental deduplication process. While known adaptive processes helped conserve a high degree of structural and functional tetraploidy over more than 180 million years, the reduction of redundancy of the polyploid genome seems to have been remarkably random.

ERGO: Breaking Down the Wall between Human Health and Environmental Testing of Endocrine Disrupters.

ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded research and innovation action, that aims to break down the wall between mammalian and non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying, developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of effects between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed. The AOP development will be based on existing and new data from in vitro and in vivo experiments with fish, amphibians and mammals, using a battery of different THDs. This will provide the scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower vertebrates, predictive of human health outcomes. These assays will be prioritized for validation at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think ED testing strategies from in silico methods to in vivo testing and develop, optimize and validate existing in vivo and early life-stage OECD guidelines, as well as new in vitro protocols for THD. This strategy will reduce requirements for animal testing by preventing duplication of testing in mammals and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be tested for ED properties.

Can skyglow reduce nocturnal melatonin concentrations in Eurasian perch?

Artificial light at night (ALAN) changes the natural rhythm of light and darkness and can impair the biorhythms of animals, for example the nocturnal melatonin production of vertebrates, which serves as a proxy for daily physiological rhythms. Freshwater fish are exposed to ALAN in large urban and suburban areas in the form of direct light or in the form of skyglow, a diffuse brightening of the night sky through the scattered light reflected by clouds, atmospheric molecules, and particles in the air. However, investigations on the sensitivity of melatonin production of fish towards low intensities of ALAN in the range of typical skyglow are rare. Therefore, we exposed Eurasian perch (Perca fluviatilis) to nocturnal illumination levels of 0.01 lx, 0.1 lx and 1 lx and a control group with dark nights and daylight intensities of 2900 lx in all groups. After ten days of exposure to the experimental conditions, tank water was non-invasively sampled every 3 h over a 24 h period and melatonin was measured by ELISA. Melatonin was gradually reduced in all treatments with increasing intensity of ALAN whereas rhythmicity was maintained in all treatment groups although at 1 lx not all evaluated parameters confirmed rhythmicity. These results show a high sensitivity of Eurasian perch towards ALAN indicating that low light intensities of 0.01 lx and 0.1 lx as they occur in urban and suburban areas in the form of skyglow can affect the physiology of Eurasian perch. Furthermore, we highlight how this may impact perch in their sensitivity towards lunar rhythms and the role of skyglow for biorhythms of temperate freshwater fish.

The potential of VKORC1 polymorphisms in Mustelidae for evolving anticoagulant resistance through selection along the food chain.

In response to strong selection, new mutations can arise quickly and sweep through populations, particularly, if survival and reproduction depend on certain allele copies for adaptation to rapidly changing environments, like resistance against deadly diseases or strong toxins. Since the 1950s, resistance to anticoagulant rodenticides in several rodents has emerged through single nucleotide mutations in the vitamin-K-epoxid-reductase-complex-subunit-1 (VKORC1) gene, often located in its exon 3. Detection of high prevalence and concentrations of anticoagulant rodenticides in non-target vertebrates, including carnivorous Mustelidae, let us assume that secondary exposure by feeding on poisoned prey may also cause selection along the food chain and we hypothesized that VKORC1-based resistance might also have evolved in rodents' predators. Using newly-developed mustelid-specific primers for direct sequencing of genomic DNA, we studied VKORC1-DNA-polymorphisms in 115 mustelids of five species (Martes martes, M. foina, Mustela nivalis, M. erminea, M. putorius), obtained from northern Denmark, yielding six sites with nonsynonymous and several synonymous amino acid polymorphisms in exon 3. Comparison of these VKORC1-genotypes with hepatic rodenticide residues (obtained by HPLC combined with fluorescence or mass spectrometry) in 83 individuals (except M. martes), using generalized linear models, suggested that anticoagulant levels depended on species and specific polymorphisms. Although most VKORC-1 polymorphisms may present standing genetic variation, some are situated in resistance-mediating membrane parts of the VKORC1-encoded protein, and might be a result of selection due to exposure to anticoagulant poisons. Our new molecular markers might allow detecting indirect effects of anticoagulant rodenticides on rodent predator populations in the future.

Potash mining effluents induce moderate effects on histopathological and physiological endpoints of adult zebrafish (Danio rerio).

Stress in fish can be caused by a variety of factors and has the potential to evoke stress responses leading to a reduction of physical condition and of health. The river Werra (Germany) presents a severe case of secondary salinisation caused by potash mining activities. The model organism Danio rerio was exposed to different ion-concentrations depicting current (HT) and future (LT) threshold values of the Werra, as well as to solutions with single-exceeding ions (Mg2+ + K+ (KMg), Mg2+ (Mg) and K+ (K)). After a six-week exposure period, cortisol levels, growth and weight were measured, gills and gonads were histologically analysed and mRNA expression of follicle stimulating hormone (FSH), luteinising hormone (LH), growth hormone (GH) and prolactin (PRL) were determined. Cortisol was still elevated in fish in the HT and K group, indicating moderate stress. However, gills revealed structural changes in zebrafish in all exposure groups, size of oocytes differed in the LT and K group, male FSH mRNA levels were elevated in the HT and LT group whereas PRL mRNA levels were lower in HT and LT for both, male and female fish. These results suggest that ion-stress induces moderate effects on a variety of biological parameters that mainly serve to adapt to elevated ion concentrations. For these reasons current and even future thresholds should be reconsidered, including thresholds for total as well as single ion concentrations. Future research looking at the effects on local fish species is needed, along with regular and long-term monitoring of environmental conditions, species abundance and diversity.

Impacts of the synthetic androgen Trenbolone on gonad differentiation and development - comparisons between three deeply diverged anuran families.

Using a recently developed approach for testing endocrine disruptive chemicals (EDCs) in amphibians, comprising synchronized tadpole exposure plus genetic and histological sexing of metamorphs in a flow-through-system, we tested the effects of 17ß-Trenbolone (Tb), a widely used growth promoter in cattle farming, in three deeply diverged anuran families: the amphibian model species Xenopus laevis (Pipidae) and the non-models Bufo(tes) viridis (Bufonidae) and Hyla arborea (Hylidae). Trenbolone was applied in three environmentally and/or physiologically relevant concentrations (0.027 µg/L (10-10 M), 0.27 µg/L (10-9 M), 2.7 µg/L (10-8 M)). In none of the species, Tb caused sex reversals or masculinization of gonads but had negative species-specific impacts on gonad morphology and differentiation after the completion of metamorphosis, independently of genetic sex. In H. arborea and B. viridis, mounting Tb-concentration correlated positively with anatomical abnormalities at 27 µg/L (10-9 M) and 2.7 µg/L (10-8 M), occurring in X. laevis only at the highest Tb concentration. Despite anatomical aberrations, histologically all gonadal tissues differentiated seemingly normally when examined at the histological level but at various rates. Tb-concentration caused various species-specific mortalities (low in Xenopus, uncertain in Bufo). Our data suggest that deep phylogenetic divergence modifies EDC-vulnerability, as previously demonstrated for Bisphenol A (BPA) and Ethinylestradiol (EE2).

Potash mining effluents and ion imbalances cause transient stress in adult common roach, Rutilus rutilus.

A present ecological issue causing secondary salinization in different countries is the discharge of effluents by the potash mining industry. In Germany, the River Werra is used as a sink for potash mining discharges containing high concentrations of ions, predominantly Cl-, K+, Na+, and Mg2+ resulting in a strong decline of the biodiversity and abundance of local species. However, hardly anything is known about the acute and chronic physiological effects of high concentrations and imbalances of ions being prevalent in potash mining effluents in fish. Therefore, the stress response and selected immune and growth parameters were investigated in standardized laboratory experiments. A native freshwater fish species, Rutilus rutilus, was exposed to concentrations of the high currently allowed (HT) and lowered future thresholds (LT) and three different ion solutions (containing high Mg2+ (Mg), high K+ (K) and high Mg2++K+ (Mg+K) concentrations) for four different exposure times (24 h, 7 d, 21 d, 8 wk). Tank water (additionally after 9 and 12 h) and plasma cortisol, glucose and protein, hematocrit and hemoglobin were determined after each exposure time. Furthermore, plasma lysozyme and head kidney leucocyte respiratory burst activity (only after 21 d) were evaluated as well as growth parameters. A transient stress response was induced in almost all groups. Tank water cortisol was elevated after 9 h in HT, LT and Mg+K and in HT after 12 h, whereas glucose concentrations increased after 24 h in all exposure groups except K. HT led to enhanced hematocrit and hemoglobin content after 24 h. Plasma protein, immune system and growth were not affected in any group. None of the ion solutions induced acute toxicity but most triggered typical acute stress reactions. Rather the sum of high ion concentrations than single ions challenged the fish. Even though the effects observed in adult roach were only transient and indicate acclimatization under laboratory conditions, adverse effects observed in the river are evident and further research on physiological endpoints including reproductive parameters and impacts on younger life stages seem to be needed to scientifically base protective thresholds.