A laboratory investigation into features of morphology and physiology for their potential to predict reproductive success in male frogs

Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological (weight/snout-vent length, nuptial pad colour/size, forelimb width/size) and physiological (nuptial pad/testis histomorphology, plasma hormones, gene expression) features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 μg/L)/linuron (9 or 45 μg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this

Investigating Mechanisms of Environmental Chemical Tolerance and Toxicity in Brown Trout using RNA-seq

Brown trout (Salmo trutta) are an ecologically and economically important native European species, known to be sensitive to environmental stressors. Compared to other model species, there is little information available on the toxicological responses of this species to environmental pollutants. High-throughput RNA-sequencing (RNA-seq) is emerging as a sensitive and accurate tool for conducting transcriptomics, but is yet to be widely used in ecotoxicology. A major advantage of RNA-seq is that it can be used to conduct non-biased, global gene expression analysis in species without existing genomic sequence information. Therefore, during this PhD I set out to investigate global mechanisms of toxicity for a selection of the most environmentally relevant chemicals likely to impact upon natural brown trout populations. By using RNA-seq, I also aimed to demonstrate the potential application of this technology as a valuable tool in ecotoxicology. To address these objectives, I conducted transcriptomic profiling, both on wild brown trout and on those exposed to agricultural pollutants in a laboratory setting. Using RNA-seq in combination with analysis of tissue metal concentration I found evidence of a high degree of metal tolerance in a chronically exposed wild population of brown trout from the river Hayle. The main molecular mechanisms responsible for this metal-tolerance included regulation of metal- and ion-homeostasis pathways. In the laboratory exposures, I found evidence of considerable transcriptomic changes in male brown trout exposed to 34.38 ng/L E2, including up-regulation of typical oestrogen-responsive transcripts (vitellogenins, zona pellucida proteins and estrogen receptor 1), as well as hepatic processes that can be associated with vitellogenesis such as lipid metabolism, cell proliferation and ribosome biogenesis. This concentration is within a range measured in sewage effluent and, more occasionally, in surface waters. I also exposed male brown trout to linuron, a widely used pesticide, and observed a striking down-regulation of enzymes involved in the cholesterol biosynthesis pathway and up-regulation of transcripts involved in cellular stress response following exposure to 250 µg linuron/L. There was also some evidence of similar responses occurring at the lower, environmentally relevant concentration (2.5 µg/L). I then compared the mechanisms of toxicity of glyphosate, the most widely used herbicide in the world, and its commercial formulation Roundup in juvenile brown trout. I found evidence of a cellular stress response consistent with generation of oxidative stress at concentrations of 10 µg/L and above which, importantly, is within the range of concentrations measured in the environment. To investigate the potential reproductive toxicity of these compounds I also conducted an exposure of breeding zebrafish to glyphosate and Roundup, and found evidence of reproductive toxicity, but only at a very high concentration (10 mg/L). This work therefore provides valuable information on the toxicological effects of these environmentally relevant chemicals in brown trout, which can potentially be used to assess the risk they pose to natural populations and therefore contribute to the sustainable management of this species. I have successfully employed RNA-seq to achieve the main objective of this PhD and, in so doing, have demonstrated the value of this technology in ecotoxicology. Specifically, we have demonstrated the ability of RNA-seq to identify conserved responses typically associated with oestrogen exposure. We also highlight the importance of optimising the experimental design and strategy for RNA-seq data analysis to improve the quality of transcript expression analysis. Throughout the course of this work we have benefited from improvements in sequencing technology and the tools available for data analysis. This technology is continuing to develop rapidly, and it is likely that RNA-seq will become the dominant tool for conducting transcriptomics in ecotoxicology in the future.NERCThe Salmon and Trout Associatio

Adaptive capabilities and fitness consequences associated with pollution exposure in fish

Many fish populations are exposed to harmful levels of chemical pollution and selection pressures associated with these exposures have led to the evolution of tolerance. Our understanding of the physiological basis for these adaptations is limited, but they are likely to include processes involved with the absorption, distribution, metabolism and/or excretion of the target chemical. Other potential adaptive mechanisms include enhancements in antioxidant responses, an increased capacity for DNA and/or tissue repair and alterations to the life cycle of fish that enable earlier reproduction. Analysis of single-nucleotide polymorphism frequencies has shown that tolerance to hydrocarbon pollutants in both marine and estuarine fish species involves alteration in the expression of the xenobiotic metabolism enzyme CYP1A. In this review, we present novel data showing also that variants of the CYP1A gene have been under selection in guppies living in Trinidadian rivers heavily polluted with crude oil. Potential costs associated with these adaptations could reduce fitness in unpolluted water conditions. Integrating knowledge of local adaptation to pollution is an important future consideration in conservation practices such as for successful restocking, and improving connectivity within river systems. This article is part of the themed issue ‘Human influences on evolution, and the ecological and societal consequences’

Global transcriptome profiling reveals molecular mechanisms of metal tolerance in a chronically exposed wild population of brown trout.

addresses: Biosciences, College of Life & Environmental Sciences, Geoffrey Pope Building, University of Exeter , Exeter, EX4 4QD. [email protected] © 2013 American Chemical SocietyThis is an open access article that is freely available in ORE or from the publisher's web site. http://pubs.acs.org/doi/abs/10.1021/es401380p Please cite the published versioncontacts: T.M.U.W.: E-mail [email protected], phone +44 (0)1392 724677, fax +44 (0)1392 263434. E.M.S.: E-mail E.Santos@ exeter.ac.uk, phone +44 (0)1392 264607, fax +44 (0)1392 263434.Worldwide, a number of viable populations of fish are found in environments heavily contaminated with metals, including brown trout (Salmo trutta) inhabiting the River Hayle in South-West of England. This population is chronically exposed to a water-borne mixture of metals, including copper and zinc, at concentrations lethal to naïve fish. We aimed to investigate the molecular mechanisms employed by the River Hayle brown trout to tolerate high metal concentrations. To achieve this, we combined tissue metal analysis with whole-transcriptome profiling using RNA-seq on an Illumina platform. Metal concentrations in the Hayle trout, compared to fish from a relatively unimpacted river, were significantly increased in the gills, liver and kidney (63-, 34- and 19-fold respectively), but not the gut. This confirms that these fish can tolerate considerable metal accumulation, highlighting the importance of these tissues in metal uptake (gill), storage and detoxification (liver, kidney). We sequenced, assembled and annotated the brown trout transcriptome using a de novo approach. Subsequent gene expression analysis identified 998 differentially expressed transcripts and functional analysis revealed that metal- and ion-homeostasis pathways are likely to be the most important mechanisms contributing to the metal tolerance exhibited by this population.Natural Environment Research Council (NERC

Shaken not stirred: Mixing semantics into XPDL

Ubiquitous computing requires lightweight approaches to coordinating tasks distributed across smart devices. We are currently developing a semantic workflow modelling approach that blends the proven robustness of XPDL with semantics to support proactive behaviour. We illustrate the potential of the model through an example based on mixing a dry martini

Benefits of the microalgae Spirulina and Schizochytrium in fish nutrition: a meta-analysis

Use of microalgae in fish nutrition can relieve pressure on wild fish stocks, but there is no systematic quantitative evaluation of microalgae benefits. We conducted a metanalysis on the nutritional benefits of Spirulina and Schizochytrium as replacements of fishmeal and fish or plant oil, respectively. We reviewed 50 peer-reviewed studies involving 26 finfish species and 144 control vs microalgae replacement comparisons. Inclusion of Spirulina in the fish diet significantly improved growth compared to controls (SMD = 1.21; 95% CI 0.71–1.70), while inclusion of Schizochytrium maintained the content of omega-3 PUFA of the fish fillet compared to fish fed on fish or plant oils (SMD = 0.62; 95% CI − 0.51–1.76). Benefits were apparent at replacement levels as low as 0.025% in the case of Spirulina and 10% in the case of Schizochytrium oil. Dose-dependent effects were found for Spirulina replacement on growth, but not for Schizochytrium on omega-3 fillet content. Subgroup analysis and meta-regression revealed that ~ 24–27% of variation in effect sizes can be accounted by variation between fish families, the rest likely reflecting variation in experimental conditions. Overall, the evidence indicates that Spirulina and Schizochytrium replacement in aquafeeds can be used to improve fish growth and maintain fillet quality, respectively, but considerable uncertainty exists on the predicted responses. To reduce uncertainty and facilitate the transition towards more sustainable aquafeeds, we recommend that feeding trials using microalgae are conducted under commercially relevant conditions and that greater care is taken to report full results to account for sources of heterogeneity

Data on optimisation of a multiplex HRM-qPCR assay for native and invasive crayfish as well as the crayfish plague in four river catchments

The data presented here corresponds to the research paper “Simultaneous detection of invasive signal crayfish, endangered white-clawed crayfish and the crayfish plague using environmental DNA”. A crayfish-specific assay was designed and optimised using three real-time PCR supermixes (SYBR™ Green, SsoFast™ EvaGreen® and HOT FIREPol® EvaGreen®). Diagnostic high resolution melt (HRM) data from direct application of assay on both ex-situ eDNA water samples and field samples from four catchments (two in Wales, two in England) is presented in this article, displaying positive HRM profiles for invasive signal crayfish (Pacifastacus leniusculus), native white-clawed crayfish (Austropotamobius pallipes) and crayfish plague causal agent (Aphanomyces astaci). Keywords: Pacifastacus leniusculus, Austropotamobius pallipes, Crayfish plague, HRM-qPCR, eDN

Immune-Related Transcriptional Responses to Parasitic Infection in a Naturally Inbred Fish: Roles of Genotype and Individual Variation

Parasites are strong drivers of evolutionary change and the genetic variation of both host and parasite populations can co-evolve as a function of parasite virulence and host resistance. The role of transcriptome variation in specific interactions between host and parasite genotypes has been less studied and can be confounded by differences in genetic variation. We employed two naturally inbred lines of a self-fertilizing fish to estimate the role of host genotype in the transcriptome response to parasite infection using RNA-seq. In addition, we targeted several differentially expressed immune-related genes to further investigate the relative role of individual variation in the immune response using RT-qPCR, taking advantage of the genomic uniformity of the self-fertilizing lines. We found significant differences in gene expression between lines in response to infection both in the transcriptome and in individual gene RT-qPCR analyses. Individual RT-qPCR analyses of gene expression identified significant variance differences between lines for six genes but only for three genes between infected and control fish. Our results indicate that although the genetic background plays an important role in the transcriptome response to parasites, it cannot fully explain individual differences within genetically homogeneous lines, which can be important for determining the response to parasites