Experimental sexual selection reveals rapid evolutionary divergence in sex-specific transcriptomes and their interactions following mating.

Post copulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these processes to rapid transcriptomic evolution in sex-specific tissues and substantial transcriptomic post mating responses in females, patterns of which are altered when mating between reproductively isolated species. Here, we tested multiple predictions arising from sexual selection and conflict theory about the evolution of sex-specific and tissue-specific gene expression and the post mating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, we found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the post mating response, particularly in the female reproductive tract - the main arena for sexual conflict - compared to ovaries. This effect was asymmetric with monandry females typically showing more post mating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the post mating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. Our microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.

Acute and chronic stress prevents responses to pain in zebrafish: evidence for stress-induced analgesia.

The state of an animal prior to the application of a noxious stimulus can have a profound effect on their nociceptive threshold and subsequent behaviour. In mammals, the presence of acute stress preceding a painful event can have an analgesic effect whereas the presence of chronic stress can result in hyperalgesia. While considerable research has been conducted on the ability of stress to modulate mammalian responses to pain, relatively little is known about fish. This is of particular concern given that zebrafish (Danio rerio) are an extensively used model organism subject to a wide array of invasive procedures where the level of stress prior to experimentation could pose a major confounding factor. This study, therefore, investigated the impact of both acute and chronic stress on the behaviour of zebrafish subjected to a potentially painful laboratory procedure, the fin clip. In stress-free individuals, those subjected to the fin clip spent more time in the bottom of the tank, had reduced swimming speeds and less complex swimming trajectories; however, these behavioural changes were absent in fin-clipped fish that were first subject to either chronic or acute stress, suggesting the possibility of stress-induced analgesia (SIA). To test this, the opioid antagonist naloxone was administered to fish prior to the application of both the stress and fin-clip procedure. After naloxone, acutely stressed fin-clipped zebrafish exhibited the same behaviours as stress-free fin-clipped fish. This indicates the presence of SIA and the importance of opioid signalling in this mechanism. As stress reduced nociceptive responses in zebrafish, this demonstrates the potential for an endogenous analgesic system akin to the mammalian system. Future studies should delineate the neurobiological basis of stress-induced analgesia in fish.

Contribution of trans regulatory eQTL to cryptic genetic variation in C. elegans.

BACKGROUND: Cryptic genetic variation (CGV) is the hidden genetic variation that can be unlocked by perturbing normal conditions. CGV can drive the emergence of novel complex phenotypes through changes in gene expression. Although our theoretical understanding of CGV has thoroughly increased over the past decade, insight into polymorphic gene expression regulation underlying CGV is scarce. Here we investigated the transcriptional architecture of CGV in response to rapid temperature changes in the nematode Caenorhabditis elegans. We analyzed regulatory variation in gene expression (and mapped eQTL) across the course of a heat stress and recovery response in a recombinant inbred population. RESULTS: We measured gene expression over three temperature treatments: i) control, ii) heat stress, and iii) recovery from heat stress. Compared to control, exposure to heat stress affected the transcription of 3305 genes, whereas 942 were affected in recovering animals. These affected genes were mainly involved in metabolism and reproduction. The gene expression pattern in recovering animals resembled both the control and the heat-stress treatment. We mapped eQTL using the genetic variation of the recombinant inbred population and detected 2626 genes with an eQTL in the heat-stress treatment, 1797 in the control, and 1880 in the recovery. The cis-eQTL were highly shared across treatments. A considerable fraction of the trans-eQTL (40-57%) mapped to 19 treatment specific trans-bands. In contrast to cis-eQTL, trans-eQTL were highly environment specific and thus cryptic. Approximately 67% of the trans-eQTL were only induced in a single treatment, with heat-stress showing the most unique trans-eQTL. CONCLUSIONS: These results illustrate the highly dynamic pattern of CGV across three different environmental conditions that can be evoked by a stress response over a relatively short time-span (2 h) and that CGV is mainly determined by response related trans regulatory eQTL.

Molecular basis of chill resistance adaptations in poikilothermic animals.

Chill and freeze represent very different components of low temperature stress. Whilst the principal mechanisms of tissue damage and of acquired protection from freeze-induced effects are reasonably well established, those for chill damage and protection are not. Non-freeze cold exposure (i.e. chill) can lead to serious disruption to normal life processes, including disruption to energy metabolism, loss of membrane perm-selectivity and collapse of ion gradients, as well as loss of neuromuscular coordination. If the primary lesions are not relieved then the progressive functional debilitation can lead to death. Thus, identifying the underpinning molecular lesions can point to the means of building resistance to subsequent chill exposures. Researchers have focused on four specific lesions: (i) failure of neuromuscular coordination, (ii) perturbation of bio-membrane structure and adaptations due to altered lipid composition, (iii) protein unfolding, which might be mitigated by the induced expression of compatible osmolytes acting as 'chemical chaperones', (iv) or the induced expression of protein chaperones along with the suppression of general protein synthesis. Progress in all these potential mechanisms has been ongoing but not substantial, due in part to an over-reliance on straightforward correlative approaches. Also, few studies have intervened by adoption of single gene ablation, which provides much more direct and compelling evidence for the role of specific genes, and thus processes, in adaptive phenotypes. Another difficulty is the existence of multiple mechanisms, which often act together, thus resulting in compensatory responses to gene manipulations, which may potentially mask disruptive effects on the chill tolerance phenotype. Consequently, there is little direct evidence of the underpinning regulatory mechanisms leading to induced resistance to chill injury. Here, we review recent advances mainly in lower vertebrates and in arthropods, but increasingly in genetic model species from a broader range of taxa.

Nitrite regulates hypoxic vasodilation via myoglobin-dependent nitric oxide generation.

BACKGROUND: Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for >100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO·) and thereby contribute to cardiomyocyte NO· signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO·. METHODS AND RESULTS: We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO· from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO·, and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice. CONCLUSIONS: Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO· via the heme globin myoglobin enhances blood flow and matches O(2) supply to increased metabolic demands under hypoxic conditions.

Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus.

Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this 'nonmodel' species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator-activated receptor (PPAR) pathways. Application of rosiglitazone, a well-known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT-induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR-responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption.

Functional differentiation of myoglobin isoforms in hypoxia-tolerant carp indicates tissue-specific protective roles.

Because of a recent whole genome duplication, the hypoxia-tolerant common carp and goldfish are the only vertebrates known to possess two myoglobin (Mb) paralogs. One of these, Mb1, occurs in oxidative muscle but also in several other tissues, including capillary endothelial cells, whereas the other, Mb2, is a unique isoform specific to brain neurons. To help understand the functional roles of these diverged isoforms in the tolerance to severe hypoxia in the carp, we have compared their O(2) equilibria, carbon monoxide (CO) and O(2) binding kinetics, thiol S-nitrosation, nitrite reductase activities, and peroxidase activities. Mb1 has O(2) affinity and nitrite reductase activity comparable to most vertebrate muscle Mbs, consistent with established roles for Mbs in O(2) storage/delivery and in maintaining nitric oxide (NO) homeostasis during hypoxia. Both Mb1 and Mb2 can be S-nitrosated to similar extent, but without oxygenation-linked allosteric control. When compared with Mb1, Mb2 displays faster O(2) and CO kinetics, a lower O(2) affinity, and is slower at converting nitrite into NO. Mb2 is therefore unlikely to be primarily involved in either O(2) supply to mitochondria or the generation of NO from nitrite during hypoxia. However, Mb2 proved to be significantly faster at eliminating H(2)O(2,) a major in vivo reactive oxygen species (ROS), suggesting that this diverged Mb isoform may have a specific protective role against H(2)O(2) in the carp brain. This property might be of particular significance during reoxygenation following extended periods of hypoxia, when production of H(2)O(2) and other ROS is highest.

ExprAlign--the identification of ESTs in non-model species by alignment of cDNA microarray expression profiles.

BACKGROUND: Sequence identification of ESTs from non-model species offers distinct challenges particularly when these species have duplicated genomes and when they are phylogenetically distant from sequenced model organisms. For the common carp, an environmental model of aquacultural interest, large numbers of ESTs remained unidentified using BLAST sequence alignment. We have used the expression profiles from large-scale microarray experiments to suggest gene identities. RESULTS: Expression profiles from approximation 700 cDNA microarrays describing responses of 7 major tissues to multiple environmental stressors were used to define a co-expression landscape. This was based on the Pearsons correlation coefficient relating each gene with all other genes, from which a network description provided clusters of highly correlated genes as 'mountains'. We show that these contain genes with known identities and genes with unknown identities, and that the correlation constitutes evidence of identity in the latter. This procedure has suggested identities to 522 of 2701 unknown carp ESTs sequences. We also discriminate several common carp genes and gene isoforms that were not discriminated by BLAST sequence alignment alone. Precision in identification was substantially improved by use of data from multiple tissues and treatments. CONCLUSION: The detailed analysis of co-expression landscapes is a sensitive technique for suggesting an identity for the large number of BLAST unidentified cDNAs generated in EST projects. It is capable of detecting even subtle changes in expression profiles, and thereby of distinguishing genes with a common BLAST identity into different identities. It benefits from the use of multiple treatments or contrasts, and from the large-scale microarray data.

Application of ESTs in microarray analysis.

Microarray analyses provide information on the relative expression levels of large numbers of gene products (transcripts). As such they have been widely used to examine differences in gene expression across a variety of samples such as tissues and life-cycle stages. Due to a previous lack of sequence data, microarray analyses have typically centred on the study of well-characterised model organisms. However, the recent availability of large sets of expressed sequence tags (ESTs) generated for the purpose of gene discovery offers the opportunity to consider designing and applying microarray technology to a larger and more diverse set of species. Here we outline the array-design process involving the generation of an optimised set of oligoprobes from a minimally redundant but maximally representative list of sequences from raw EST data. We illustrate these principles by showing how we designed and fabricated a high-density oligoarray for the rainbow trout, a non-model species for which large numbers of ESTs, and a non-redundant assembly is available. This approach brings array technology within the reach of all investigators, even those with limited budgets.

Host selectively contributes to shaping intestinal microbiota of carnivorous and omnivorous fish.

Fish production is increasingly important to global food security. A major factor in maintaining health, productivity and welfare of farmed fish is the establishment and promotion of a stable and beneficial intestinal microbiota. Understanding the effects of factors such as host and environment on gut microbial community structure is essential for developing strategies for stimulating the establishment of a health-promoting gut-microbiota. We compared intestinal microbiota of common carp and rainbow trout, two fish with different dietary habits, sourced from various farm locations. There were distinct differences in the gut microbiota of carp and trout intestine. The microbiota of carp was dominated by Fusobacteriia and Gammaproteobacteria, while the trout microbiota consisted predominantly of Mollicutes and Betaproteobacteria. The majority of bacterial sequences clustered into a relatively low number of operational taxonomic units (OTUs) revealing a comparatively simple microbiota, with Cetobacterium, Aeromonas and Mycoplasma being highly abundant. Within each species, fish from different facilities were found to have markedly similar predominant bacterial populations despite distinctly different rearing environments, demonstrating intra-species uniformity and significant influence of host selectivity. This study demonstrates that in fish the host species imparts substantial impact in shaping the community structure of the intestinal microbiota.

Automated monitoring of behaviour in zebrafish after invasive procedures.

Fish are used in a variety of experimental contexts often in high numbers. To maintain their welfare and ensure valid results during invasive procedures it is vital that we can detect subtle changes in behaviour that may allow us to intervene to provide pain-relief. Therefore, an automated method, the Fish Behaviour Index (FBI), was devised and used for testing the impact of laboratory procedures and efficacy of analgesic drugs in the model species, the zebrafish. Cameras with tracking software were used to visually track and quantify female zebrafish behaviour in real time after a number of laboratory procedures including fin clipping, PIT tagging, and nociceptor excitation via injection of acetic acid subcutaneously. The FBI was derived from activity and distance swum measured before and after these procedures compared with control and sham groups. Further, the efficacy of a range of drugs with analgesic properties to identify efficacy of these agents was explored. Lidocaine (5 mg/L), flunixin (8 mg/L) and morphine (48 mg/L) prevented the associated reduction in activity and distance swum after fin clipping. From an ethical perspective, the FBI represents a significant refinement in the use of zebrafish and could be adopted across a wide range of biological disciplines.

Ancient and modern duplication events and the evolution of stearoyl-CoA desaturases in teleost fishes.

Stearoyl-CoA desaturases (SCDs) are key enzymes of fatty acid biosynthesis whose regulation underpins responses to dietary, thermal, and hormonal treatment. Although two isoforms are known to exist in the common carp and human and four in mouse, there is no coherent view on how this gene family evolved to generate functionally diverse members. Here we identify numerous new SCD homologs in teleost fishes, using sequence data from expressed sequence tag (EST) and cDNA collections and genomic model species. Phylogenetic analyses of the deduced coding sequences produced only partially resolved molecular trees. The multiple SCD isoforms were, however, consistent with having arisen by an ancient gene duplication event in teleost fishes together with a more recent duplication in the tetraploid carp and possibly also salmonid lineages. Critical support for this interpretation comes from comparison across all vertebrate groups of the gene order in the genomic environments of the SCD isoforms. Using syntenically aligned chromosomal fragments from large-insert clones of common carp and grass carp together with those from genomically sequenced model species, we show that the ancient and modern SCD duplication events in the carp lineage were each associated with large chromosomal segment duplications, both possibly linked to whole genome duplications. By contrast, the four mouse isoforms likely arose by tandem duplications. Each duplication in the carp lineage gave rise to differentially expressed SCD isoforms, either induced by cold or diet as previously shown for the recent duplicated carp isoforms or tissue specific as demonstrated here for the ancient duplicate zebrafish isoforms.

Effects of fluoxetine on the reproductive axis of female goldfish (Carassius auratus).

We investigated the effects of fluoxetine, a selective serotonin reuptake inhibitor, on neuroendocrine function and the reproductive axis in female goldfish. Fish were given intraperitoneal injections of fluoxetine twice a week for 14 days, resulting in five injections of 5 microg fluoxetine/g body wt. We measured the monoamine neurotransmitters serotonin, dopamine, and norepinephrine in addition to their metabolites with HPLC. Homovanillic acid, a metabolite in the dopaminergic pathway, increased significantly in the hypothalamus. Plasma estradiol levels were measured by radioimmunoassay and were significantly reduced approximately threefold after fluoxetine treatment. We found that fluoxetine also significantly reduced the expression of estrogen receptor (ER)beta1 mRNA by 4-fold in both the hypothalamus and the telencephalon and ERalpha mRNA by 1.7-fold in the telencephalon. Fluoxetine had no effect on the expression of ERbeta2 mRNA in the hypothalamus or telencephalon. Microarray analysis identified isotocin, a neuropeptide that stimulates reproductive behavior in fish, as a candidate gene affected by fluoxetine treatment. Real-time RT-PCR verified that isotocin mRNA was downregulated approximately sixfold in the hypothalamus and fivefold in the telencephalon. Intraperitoneal injection of isotocin (1 microg/g) increased plasma estradiol, providing a potential link between changes in isotocin gene expression and decreased circulating estrogen in fluoxetine-injected fish. Our results reveal targets of serotonergic modulation in the neuroendocrine brain and indicate that fluoxetine has the potential to affect sex hormones and modulate genes involved in reproductive function and behavior in the brain of female goldfish. We discuss these findings in the context of endocrine disruption because fluoxetine has been detected in the environment.

Novel candidate genes identified in the brain during nociception in common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

Recent studies have demonstrated that teleost fish possess nociceptors that detect potentially painful stimuli and that the physiological properties of these fibres are markedly similar to those found in mammals. This finding led to suggestions of possible pain perception in fish, contrary to the view that the sensory response in these animals is limited to the spinal cord and hindbrain and as such is reflexive. Therefore, the aim of this study was to determine if the brain is active at the molecular level by using a microarray analysis of gene expression in the forebrain, midbrain and hindbrain of two fish species. A comparison between the two species at different time points showed that many genes were differentially regulated in response to a noxious stimulus compared with controls. A number of genes that are involved in mammalian nociception, such as brain-derived neurotrophic factor (BDNF) and the cannabinoid CB1 receptor were regulated in the fish brain after a nociceptive event. Novel candidates that showed significant regulation in both species were also identified. In particular, the Van Gogh-like 2 gene, was regulated in both carp and trout and should be pursued to establish its precise role in nociception.

Cutaneous immune responses in the common carp detected using transcript analysis.

In order to detect new immune-related genes in common carp (Cyprinus carpio L.) challenged by an ectoparasitic infection, two cDNA libraries were constructed from carp skin sampled at 3 and 72h after infection with Ichthyophthirius multifiliis. In a total of 3500 expressed sequence tags (ESTs) we identified 82 orthologues of genes of immune relevance previously described in other organisms. Of these, 61 have never been described before in C. carpio, thus shedding light on some key components of the defence mechanisms of this species. Among the newly described genes, full-length molecules of prostaglandin D2 synthase (PGDS), the CC chemokine molecule SCYA103, and a second gene for the carp beta(2)-microglobulin (beta(2)m), beta(2)m-2, were described. Transcript amounts of the genes PGDS, interferon (IFN), SCYA103, complement factor 7 (C7), complement factor P (FP), complement factor D (FD) and beta(2)m-2 were evaluated by real-time quantitative PCR (RQ-PCR). Samples from skin, blood and liver from fish challenged with I. multifiliis were taken at 3, 12, 24, 36 and 48h post infection. Higher expression levels of most of these transcripts were observed in skin from uninfected fish, compared to the transcript levels detected in blood and liver from the same animals. Also, there was significant down-regulation of the genes PGDS and beta(2)m-2 in skin, whilst significant up-regulation was observed for the C7 and SCYA103 genes in liver of fish infected with the parasite. These results confirm the active role of fish skin in the immune response against infections, acting as an important site of expression of immune-related molecules.

One step visual detection of PCR products with gold nanoparticles and a nucleic acid lateral flow (NALF) device.

Specific PCR products are detected with an antibody-free lateral-flow device by sandwiching them between reporter oligonucleotides covalently attached to gold nanoparticles (GNPs) and capture oligonucleotides covalently attached to a nitrocellulose chromatographic strip.

Beyond the lipid hypothesis: mechanisms underlying phenotypic plasticity in inducible cold tolerance.

The physiological adjustment of organisms in response to temperature variation is a crucial part of coping with environmental stress. An important component of the cold response is the increase in membrane lipid unsaturation, and this has been linked to an enhanced resistance to the debilitating or lethal effects of cold. Underpinning the lipid response is the upregulation of fatty acid desaturases (des), particularly those introducing double bonds at the 9-10 position of saturated fatty acids. For plants and microbes there is good genetic evidence that regulation of des genes, and the consequent changes in lipid saturation, are causally linked to generation of a cold-tolerant phenotype. In animals, however, supporting evidence is almost entirely limited to correlations of saturation with cold conditions. We describe our recent attempts to provide a direct test of this relationship by genetic manipulation of the nematode Caenorhabditis elegans. We show that this species displays a strong cold tolerant phenotype induced by prior conditioning to cold, and that this is directly linked to upregulated des activity. However, whilst genetic disruption of des activity and lipid unsaturation significantly reduced cold tolerance, animals retained a substantial component of their stress tolerant phenotype produced by cold conditioning. This indicates that mechanisms other than lipid unsaturation play an important role in cold adaptation.

Mating system manipulation and the evolution of sex-biased gene expression in Drosophila.

Sex differences in dioecious animals are pervasive and result from gene expression differences. Elevated sexual selection has been predicted to increase the number and expression of male-biased genes, and experimentally imposing monogamy on Drosophila melanogaster has led to a relative feminisation of the transcriptome. Here, we test this hypothesis further by subjecting another polyandrous species, D. pseudoobscura, to 150 generations of experimental monogamy or elevated polyandry. We find that sex-biased genes do change in expression but, contrary to predictions, there is usually masculinisation of the transcriptome under monogamy, although this depends on tissue and sex. We also identify and describe gene expression changes following courtship experience. Courtship often influences gene expression, including patterns in sex-biased gene expression. Our results confirm that mating system manipulation disproportionately influences sex-biased gene expression but show that the direction of change is dynamic and unpredictable.