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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., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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.

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.

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.

Background: In 2014, Western Africa experienced an unanticipated explosion of Ebola virus infections. What distinguishes fatal from non-fatal outcomes remains largely unknown, yet is key to optimising personalised treatment strategies. We used transcriptome data for peripheral blood taken from infected and convalescent recovering patients to identify early stage host factors that are associated with acute illness and those that differentiate patient survival from fatality., Results: The data demonstrate that individuals who succumbed to the disease show stronger upregulation of interferon signalling and acute phase responses compared to survivors during the acute phase of infection. Particularly notable is the strong upregulation of albumin and fibrinogen genes, which suggest significant liver pathology. Cell subtype prediction using messenger RNA expression patterns indicated that NK-cell populations increase in patients who survive infection. By selecting genes whose expression properties discriminated between fatal cases and survivors, we identify a small panel of responding genes that act as strong predictors of patient outcome, independent of viral load., Conclusions: Transcriptomic analysis of the host response to pathogen infection using blood samples taken during an outbreak situation can provide multiple levels of information on both disease state and mechanisms of pathogenesis. Host biomarkers were identified that provide high predictive value under conditions where other predictors, such as viral load, are poor prognostic indicators. The data suggested that rapid analysis of the host response to infection in an outbreak situation can provide valuable information to guide an understanding of disease outcome and mechanisms of disease.

Advances in genomic and transcriptome sequencing are revealing the massive scale of previously unrecognised alterations occurring during neoplastic transformation. Breast cancers are genetically and phenotypically heterogeneous. Each of the three major subtypes [ERBB2 amplified, estrogen receptor (ESR)-positive and triple-negative] poses diagnostic and therapeutic challenges. Here we show, using high-resolution next-generation transcriptome sequencing, that in all three breast cancer subtypes, but not matched controls, there was significant overexpression of transcripts from intronic and untranslated regions in addition to exons from specific genes, particularly amplified oncogenes and hormone receptors. For key genes ERBB2 and ESR1, we demonstrate that overexpression is linked to the production of highly modified and truncated splice variants in tumours, but not controls, correlated with tumour subtype. Translation of these tumour-specific splice variants generates truncated proteins with altered subcellular locations and functions, modifying the phenotype, affecting tumour biology, and targeted antitumour therapies. In contrast, tumour suppressors TP53, BRCA1/2 and NF1 did not show intronic overexpression or truncated splice variants in cancers. These findings emphasize the detection of intronic as well as exonic changes in the transcriptional landscapes of cancers have profound therapeutic implications.

The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population, and evolutionary studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. When compared against the N2 reference, the CB4856 genome has 327,050 single nucleotide variants (SNVs) and 79,529 insertion-deletion events that result in a total of 3.3 Mb of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 but not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, which have a greatly elevated SNV density, ranging from 2 to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors, suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes., (Copyright © 2015 by the Genetics Society of America.)

Crucian carp are unusual among vertebrates in surviving extended periods in the complete absence of molecular oxygen. During this time cardiac output is maintained though these mechanisms are not well understood. Using a high-density cDNA microarray, we have defined the genome-wide gene expression responses of cardiac tissue after exposing the fish at two temperatures (8 and 13 °C) to one and seven days of anoxia, followed by seven days after restoration to normoxia. At 8 °C, using a false discovery rate of 5%, neither anoxia nor re-oxygenation elicited appreciable changes in gene expression. By contrast, at 13 °C, 777 unique genes responded strongly. Up-regulated genes included those involved in protein turnover, the pentose phosphate pathway and cell morphogenesis while down-regulated gene categories included RNA splicing and transcription. Most genes were affected between one and seven days of anoxia, indicating gene regulation over the medium term but with few early response genes. Re-oxygenation for 7 days was sufficient to completely reverse these responses. Glycolysis displayed more complex responses with anoxia up-regulated transcripts for the key regulatory enzymes, hexokinase and phosphofructokinase, but with down-regulation of most of the non-regulatory genes. This complex pattern of responses in genomic transcription patterns indicates divergent cardiac responses to anoxia, with the transcriptionally driven reprogramming of cardiac function seen at 13 °C being largely completed at 8 °C.

Organismal development is the most dynamic period of the life cycle, yet we have only a rough understanding of the dynamics of gene expression during adolescent transition. Here we show that adolescence in Caenorhabditis elegans is characterized by a spectacular expression shift of conserved and highly polymorphic genes. Using a high resolution time series we found that in adolescent worms over 10,000 genes changed their expression. These genes were clustered according to their expression patterns. One cluster involved in chromatin remodelling showed a brief up-regulation around 50 h post-hatch. At the same time a spectacular shift in expression was observed. Sequence comparisons for this cluster across many genotypes revealed diversifying selection. Strongly up-regulated genes showed signs of purifying selection in non-coding regions, indicating that adolescence-active genes are constrained on their regulatory properties. Our findings improve our understanding of adolescent transition and help to eliminate experimental artefacts due to incorrect developmental timing.

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.

Ecological speciation occurs with the adaptation of populations to different environments and concurrent evolution of reproductive isolation. Phenotypic plasticity might influence both ecological adaptation and reproductive traits. We examined environment-specific gene expression and male mating success in cactophilic Drosophila mojavensis using transcriptome sequencing. This species exhibits cactus-dependent mating success across different species of host plants, with genotype-by-environment interactions for numerous traits. We cultured flies from egg to eclosion on two natural cactus hosts and surveyed gene expression in adult males that were either successful or unsuccessful in achieving copulation in courtship trials. We identified gene expression differences that included functions involved with metabolism, most likely related to chemical differences between host cactus species. Several epigenetic-related functions were identified that might play a role in modulating gene expression in adults due to host cactus effects on larvae, and mating success. Cactus-dependent mating success involved expression differences of genes implicated in translation, transcription, and nervous system development. This suggests a role of neurological function genes in the mating success of D. mojavensis males. Together, these results suggest that the influence of environmental variation on mating success via regulation of gene expression might be an important aspect of ecological speciation., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

Extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and adaptations that promote their economical use. However, little is known regarding the molecular and evolutionary underpinnings of the high muscle myoglobin concentration phenotype of divers. We used ancestral sequence reconstruction to trace the evolution of this oxygen-storing protein across a 130-species mammalian phylogeny and reveal an adaptive molecular signature of elevated myoglobin net surface charge in diving species that is mechanistically linked with maximal myoglobin concentration. This observation provides insights into the tempo and routes to enhanced dive capacity evolution within the ancestors of each major mammalian aquatic lineage and infers amphibious ancestries of echidnas, moles, hyraxes, and elephants, offering a fresh perspective on the evolution of this iconic respiratory pigment.

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.

In 2000, fishermen reported the appearance of deformed reproductive organs in whitefish (Coregonus spp.) from Lake Thun, Switzerland. Despite intensive investigations, the causes of these abnormalities remain unknown. Using gene expression profiling, we sought to identify candidate genes and physiological processes possibly associated with the observed gonadal deformations, in order to gain insights into potential causes. Using in situ-synthesized oligonucleotide arrays, we compared the expression levels at 21,492 unique transcript probes in liver and head kidney tissue of male whitefish with deformed and normally developed gonads, respectively. The fish had been collected on spawning sites of two genetically distinct whitefish forms of Lake Thun. We contrasted the gene expression profiles of 56 individuals, i.e., 14 individuals of each phenotype and of each population. Gene-by-gene analysis revealed weak expression differences between normal and deformed fish, and only one gene, ictacalcin, was found to be up-regulated in head kidney tissue of deformed fish from both whitefish forms, However, this difference could not be confirmed with quantitative real-time qPCR. Enrichment analysis on the level of physiological processes revealed (i) the involvement of immune response genes in both tissues, particularly those linked to complement activation in the liver, (ii) proteolysis in the liver and (iii) GTPase activity and Ras protein signal transduction in the head kidney. In comparison with current literature, this gene expression pattern signals a chronic autoimmune disease in the testes. Based on the recent observations that gonad deformations are induced through feeding of zooplankton from Lake Thun we hypothesize that a xenobiotic accumulated in whitefish via the plankton triggering autoimmunity as the likely cause of gonad deformations. We propose several experimental strategies to verify or reject this hypothesis.

Theoretical and empirical studies have sought to explain the formation and maintenance of social relationships within groups. The resulting dominance hierarchies have significant fitness and survival consequences dependent upon social status. We hypothesised that each position or rank within a group has a distinctive brain gene expression profile that correlates with behavioural phenotype. Furthermore, transitions in rank position should determine which genes shift in expression concurrent with the new dominance status. We used a custom cDNA microarray to profile brain transcript expression in a model species, the rainbow trout, which forms tractable linear hierarchies. Dominant, subdominant and submissive individuals had distinctive transcript profiles with 110 gene probes identified using conservative statistical analyses. By removing the dominant, we characterised the changes in transcript expression in sub-dominant individuals that became dominant demonstrating that the molecular transition occurred within 48 hours. A strong, novel candidate gene, ependymin, which was highly expressed in both the transcript and protein in subdominants relative to dominants, was tested further. Using antibody injection to inactivate ependymin in pairs of dominant and subdominant zebrafish, the subdominant fish exhibited a substantial increase in aggression in parallel with an enhanced competitive ability. This is the first study to characterise the molecular signatures of dominance status within groups and the first to implicate ependymin in control of aggressive behaviour. It also provides evidence for indirect genetic effect models in which genotype/phenotype of an individual is influenced by conspecific interactions within a group. The variation in the molecular profile of each individual within a group may offer a new explanation of intraspecific variation in gene expression within undefined groups of animals and provides new candidates for empirical study.

Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free system-wide view of toxicological effects and modes-of-action of chemicals and ecotoxicants. However, early applications of microarray technology have identified relatively small groups of responding genes with which to define new targets for analysis by conventional means. We have trialled a more intensive approach to the design and interpretation of array experiments incorporating a balanced interwoven ANOVA design with higher levels of biological replication, a more thorough analysis of errors and false discovery rates, and an analysis of response patterns using gene network models. Zebrafish embryos were exposed from 1.5 h post-fertilization for 72 h to ecotoxicants representing different classes--2,4-dichlorophenol, 3,4-dichloroaniline, pentachlorophenol, and cadmium chloride--at low concentrations producing a developmental disturbance to 10% of embryos and half of this dose. Extracted whole embryo RNA was then analyzed on microarrays. Analysis revealed responses of 3000-5000 genes, which is 10-1000 times greater than previously reported, with significance at lower levels of fold change. Some gene responses were common to multiple toxicants, and others were restricted to just one or two toxicants. The gene expression profiles for the different toxicants were distinctive, and analysis using network-based models provided a high level of detail of affected processes, some of which were novel. This approach provides a more highly refined view of toxic effects, from which meaningful patterns of response can be discerned and related to functional deficits and from which more reliable indicators of toxicological effect can be predicted.

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.

The Van Gogh-like 2 (vangl2) gene is typically associated with planar cell polarity pathways, which is essential for correct orientation of epithelial cells during development. The encoded protein of this gene is a transmembrane protein and is highly conserved through evolution. Van Gogh-like 2 was selected for further study on the basis of consistent regulation after a nociceptive stimulus in adult common carp and rainbow trout in a microarray study. An in situ hybridisation was conducted in the brain of mature common carp (Cyprinus carpio), 1.5 and 3 h after a nociceptive stimulus comprising of an acetic acid injection to the lips of the fish and compared with a saline-injected control. The vangl2 gene was expressed in all brain regions, and particularly intensely in neurons of the telencephalon and in ependymal cells. In the cerebellum, a greater number (P=0.018) of Purkinje cells expressed vangl2 after nociception (n=7) compared with controls (n=5). This regulation opens the possibility that vangl2 is involved in nociceptive processing in the adult fish brain and may be a novel target for central nociception in vertebrates.

Background: Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted., Methodology/principal Findings: In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-daylength photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABA(A) gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays., Conclusions/significance: Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development.

Myoglobin (Mb) is famous as a muscle-specific protein--yet the common carp expresses the gene (cMb1) encoding this protein in a range of non-muscle tissues and also expresses a novel isoform (cMb2) in the brain. Using a homologous antibody and riboprobes, we have established the relative amounts and cellular sites of non-muscle Mb expression in different tissues. The amounts of carp myoglobin (cMb) in supernatants of different tissues were just 0.4-0.7% relative to that of heart supernatants and were upregulated by two-to-four fold in liver, gill and brain following 5 days of hypoxic treatment. Brain exhibited both cMb proteins in western analysis, whereas all other tissues had only cMb1. We have also identified cells expressing cMb protein and cMb mRNA using immunohistology and RNA in situ hybridisation (RNA-ISH), respectively. Mb was strongly expressed throughout all cardiac myocytes and a subset of skeletal muscle fibres, whereas it was restricted to a small range of specific cell types in each of the non-muscle tissues. These include pillar and epithelial cells in secondary gill lamellae, hepatocytes, some neurones, and tubular epithelial cells in the kidney. Capillaries and small blood vessels in all tissues exhibited Mb expression within vascular endothelial cells. The cMb2 riboprobe located expression to a subset of neurones but not to endothelial cells. In zebrafish, which possesses only one Mb gene, a similar expression pattern of Mb protein and mRNA was observed. This establishes a surprisingly cell-specific distribution of Mb within non-muscle tissues in both carp and zebrafish, where it probably plays an important role in the regulation of microvascular, renal and brain function.

The mechanical properties of the axial muscles vary along the length of a fish's body. This variation in performance correlates with the expression of certain muscle proteins. Parvalbumin (PARV) is an important calcium binding protein that helps modulate intracellular calcium levels which set the size and shape of the muscle calcium transient. It therefore has a central role in determining the functional properties of the muscle. Transcript data revealed eight specific isoforms of PARV in common carp (Cyprinus carpio) skeletal muscle which we classified as alpha1 and beta1-7. This study is the first to show expression of all eight skeletal muscle PARV isoforms in carp at the protein level and relate regional differences in expression to performance. All of the PARV isoforms were characterised at the protein level using 2D-PAGE and tandem mass spectrometry. Comparison of carp muscle from different regions of the fish revealed a higher level of expression of PARV isoforms beta4 and beta5 in the anterior region, which was accompanied by an increase in the rate of relaxation. We postulate that changes in specific PARV isoform expression are an important part of the adaptive change in muscle mechanical properties in response to varying functional demands and environmental change.

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.

Goldfish (Carassius auratus) are excellent model organisms for the neuroendocrine signaling and the regulation of reproduction in vertebrates. Goldfish also serve as useful model organisms in numerous other fields. In contrast to mammals, teleost fish do not have a median eminence; the anterior pituitary is innervated by numerous neuronal cell types and thus, pituitary hormone release is directly regulated. Here we briefly describe the neuroendocrine control of luteinizing hormone. Stimulation by gonadotropin-releasing hormone and a multitude of classical neurotransmitters and neuropeptides is opposed by the potent inhibitory actions of dopamine. The stimulatory actions of gamma-aminobutyric acid and serotonin are also discussed. We will focus on the development of a cDNA microarray composed of carp and goldfish sequences which has allowed us to examine neurotransmitter-regulated gene expression in the neuroendocrine brain and to investigate potential genomic interactions between these key neurotransmitter systems. We observed that isotocin (fish homologue of oxytocin) and activins are regulated by multiple neurotransmitters, which is discussed in light of their roles in reproduction in other species. We have also found that many novel and uncharacterized goldfish expressed sequence tags in the brain are also regulated by neurotransmitters. Their sites of production and whether they play a role in neuroendocrine signaling and control of reproduction remain to be determined. The transcriptomic tools developed to study reproduction could also be used to advance our understanding of neuroendocrine-immune interactions and the relationship between growth and food intake in fish.

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.

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.

The common carp (Cyprinus carpio) has a well-developed capacity to modify muscle properties in response to changes in temperature. Understanding the mechanisms underpinning this phenotypic response at the protein level may provide fundamental insights into the molecular basis of adaptive processes in skeletal muscle. In this study, common carp were subjected to a cooling regimen and soluble extracts of muscle homogenates were separated by 1-D SDS-PAGE and 2-DE. Proteins were identified using MALDI-TOF-MS and de novo peptide sequencing using LC-MS/MS. The 2-D gel was populated with numerous protein spots that were fragments of all three muscle isoforms (M1, M2 and M3) of carp creatine kinase (CK). The accumulation of the CK fragments was enhanced when the carp were cooled to 10 degrees C. The protein changes observed in the skeletal muscle of carp subjected to cold acclimation were compared to changes described in a previous transcript analysis study. Genes encoding CK isoforms were downregulated and the genes encoding key proteins of the ubiquitin-proteasome pathway were upregulated. These findings are consistent with a specific cold-induced enhancement of proteolysis of CK.

The life cycle of the European eel (Anguilla anguilla) includes two long migratory periods, when the newly hatched leptocephali larvae drift on ocean currents from the Sargasso Sea to the shores of Western Europe and then again up to 30 years later when adult eels swim back to their place of birth for reproductive purposes. Prior to the migration from fresh water (FW) to sea water (SW) adult yellow eels undergo various anatomical and physiological adaptations (silvering) which promote sexual development and aid the transition to increased environmental salinities. The aim of this study was to identify and characterise changes in gene expression within the major osmoregulatory tissues of the eel which enable these fish to make the physiological adaptations required for transfer to SW environments. In particular, changes in the expression of the FW-adapting hormone prolactin were correlated with differential expression of known osmoregulatory important genes within the gill, intestine and kidney following the acclimation of eels to SW. Various tissues were sampled from individual fish at selected intervals over a 5-month period following FW/SW transfer and RNA was isolated. Suppressive subtractive hybridization (SSH) was used for enrichment of differentially expressed genes. Microarrays comprising 6144 cDNAs spotted in triplicate, from brain, gill, intestine and kidney libraries (1536 randomly selected clones per tissue library), were hybridized with appropriate targets and analysed. Microarray results were validated using known genes implicated in osmoregulation, such as prolactin, growth hormone, Na, K-ATPase and some unknown genes, the role of which in osmoregulation needs to be elucidated.

Protection of poikilothermic animals from seasonal cold is widely regarded as being causally linked to changes in the unsaturation of membrane phospholipids, yet in animals this proposition remains formally untested. We have now achieved this by the genetic manipulation of lipid biosynthesis of Caenorhabditis elegans independent of temperature. Worms transferred from 25 degrees C to 10 degrees C develop over several days a much-increased tolerance of lethal cold (0 degrees C) and also an increased phospholipid unsaturation, as in higher animal models. Of the three C. elegans Delta9-desaturases, transcript levels of fat-7 only were up-regulated by cold transfer. RNAi suppression of fat-7 caused the induction of fat-5 desaturase, so to control desaturase expression we combined RNAi of fat-7 with a fat-5 knockout. These fat-5/fat-7 manipulated worms displayed the expected negative linear relationship between lipid saturation and cold tolerance at 0 degrees C, an outcome confirmed by dietary rescue. However, this change in lipid saturation explains just 16% of the observed difference between cold tolerance of animals held at 25 degrees C and 10 degrees C. Thus, although the manipulated lipid saturation affects the tolerable thermal window, and altered Delta9-desaturase expression accounts for cold-induced lipid adjustments, the effect is relatively small and none of the lipid manipulations were sufficient to convert worms between fully cold-sensitive and fully cold-tolerant states. Critically, transfer of 10 degrees C-acclimated worms back to 25 degrees C led to them restoring the usual cold-sensitive phenotype within 24 h despite retaining a lipid profile characteristic of 10 degrees C worms. Other nonlipid mechanisms of acquired cold protection clearly dominate inducible cold tolerance.

The increase of environmental temperature at physiological range can cause oxidative stress in exotherms, which in most cases leads to activation of the antioxidant enzyme, manganese-1 superoxide dismutase (Mn-SOD). This work is aimed to evaluate the changes in Mn-SOD enzyme activity and mRNA levels in the common carp, Cyprinus carpio, during transition from lower (15 degrees C) to higher (25 and 30 degrees C) temperatures. In liver, 25 degrees C exposure elicited little effect, but at 30 degrees C there was a significant increase in both Mn-SOD enzyme activity and mRNA levels. In brain enzyme activity was maximal at 25 degrees C and surprisingly, this increased activity was accompanied by a decrease in mRNA levels. This work suggests that the activity of Mn-SOD in carp is regulated by environmental conditions through transcriptional, translational and post-translational mechanisms, the particular mechanism used being dependent upon the tissue type.

A simple and novel electrochemical biosensor is described for differentiating between differing gene sequences on the basis of DNA hybridization events. Polyethylenimine-modified screen-printed carbon electrodes were used to immobilize single-stranded PCR fragments from plasmid DNA from the gene for pyruvate kinase. AC impedimetric measurements were first performed on these systems in buffer and then upon exposure to single-stranded DNA. When the electrode and solution DNA were complementary, a large drop in impedance was measured. Complementary DNA could be clearly detected at concentrations down to 1 fg/mL. Higher concentrations gave faster hybridization with saturation occurring at levels above 1 ng/mL. Responses were much lower upon exposure to noncomplementary DNA, even at higher concentrations, with the sensor showing a high degree of selectivity. This sensor format offers great promise for many DNA hybridization applications and lends itself to mass fabrication.

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.

Since the decoding of the human genome, the quest to obtain more and more molecular information from smaller and smaller samples is intensifying. Today the burden of this challenge is being borne by planar arrays, but the quality of the data provided by this approach is limited by variations in performance between different arrays. Suspension arrays of encoded microspheres provide higher quality data, but the amount of molecular information that can be acquired with them is limited by the number of codes that can be distinguished in the same sample. New methods of preparing encoded particles promise to alleviate this problem, but in the face of a growing number of new technologies it is sometimes difficult to decide which, if any, will succeed. Herein we appraise these new forms of encoded particle critically, and ask if they can deliver the necessary multiplexing power and whether they will perform well in multiplexed assays.

Myoglobin (Myg) is an oxygen-binding hemoprotein that is widely thought to be expressed exclusively in oxidative skeletal and cardiac myocytes, where it plays a key role in coping with chronic hypoxia. We now show in a hypoxia-tolerant fish model, that Myg is also expressed in a range of other tissues, including liver, gill, and brain. Moreover, expression of Myg transcript was substantially enhanced during chronic hypoxia, the fold-change induction being far greater in liver than muscle. By using 2D gel electrophoresis, we have confirmed that liver expresses a protein corresponding to the Myg-1 transcript and that it is significantly up-regulated during hypoxia. We have also discovered a second, unique Myg isoform, distinct from neuroglobin, which is expressed exclusively in the neural tissue but whose transcript expression was unaffected by environmental hypoxia. Both observations of nonmuscle expression and a brain-specific isoform are unprecedented, indicating that Myg may play a much wider role than previously understood and that Myg might function in the protection of tissues from deep hypoxia and ischemia as well as in reoxygenation and reperfusion injury.

Hibernation is a seasonally entrained and profound phenotypic transition to conserve energy in winter. It involves significant biochemical reprogramming, although our understanding of the underpinning molecular events is fragmentary and selective. We have conducted a large-scale gene expression screen of the golden-mantled ground squirrel, Spermophilus lateralis, to identify transcriptional responses associated specifically with the summer-winter transition and the torpid-arousal transition in winter. We used 112 cDNA microarrays comprising 12,288 probes that cover at least 5,109 genes. In liver, the profiles of torpid and active states in the winter were almost identical, although we identified 102 cDNAs that were differentially expressed between winter and summer, 90% of which were downregulated in the winter states. By contrast, in cardiac tissue, 59 and 115 cDNAs were elevated in interbout arousal and torpor, respectively, relative to the summer active condition, but only 7 were common to both winter states, and during arousal none was downregulated. In brain, 78 cDNAs were found to change in winter, 44 of which were upregulated. Thus transcriptional changes associated with hibernation are qualitatively modest and, since these changes are generally less than twofold, also quantitatively modest. Unbiased Gene Ontology profiling of the transcripts suggests a winter switch to beta-oxidation of lipids in liver and heart, a reduction in metabolism of toxic compounds and the urea cycle in liver, and downregulated electron transport in the brain. We identified just one strongly winter-induced transcript common to all tissues, namely an RNA-binding protein, RBM3. This analysis clearly differentiates responses of the principal tissues, identifies a large number of new genes undergoing regulation, and broadens our understanding of affected cellular processes that, in part, account for the winter-adaptive hibernating phenotype.

Microarrays, or gene chips, are transforming the way that gene expression is measured by allowing us to determine the expression of thousands of genes from a sample. This gives immense power to examine gene expression on a global scale within individual animals and between animals. The scope for analysing complex animal functions at the molecular level is within our grasp. Relatively few studies have examined complex behaviours and correlated them with gene expression in the central nervous system. Here, we review the use of microarray technology in the dissection of behaviour and focus specifically on dominance status. A cDNA library using suppression subtraction hybridisation on rainbow trout Oncorhynchus mykiss of differing status has been produced to enrich the cDNA library for genes that are differentially expressed between individuals of different dominance status. A preliminary analysis demonstrated that there were 1,165 genes that differed between fish of different dominance status. Therefore, there is the potential of correlating gene expression profile with rank position within dominance hierarchies, thus identifying targets for candidate gene approaches.

We report a microarray format for the detection of proteins and protein functionality (kinase activity) based on marking either specific antibody-protein binding or peptide phosphorylation events by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The attachment of the gold nanoparticles is achieved by standard avidin-biotin chemistry. The detection principle is resonance light scattering. Highly selective recognition of standard proteins (proteins A and G) down to 1 pg/mL for proteins in solution and 10 fg for proteins on the microarray spots is demonstrated. Enzyme activity of the kinase (PKA) is detected with high specificity down to a limit of 1 fg for an established peptide substrate (kemptide) on the microarray spots. Kinase inhibition by the inhibitor (H89) is shown, demonstrating the potential for high-throughput screening for inhibitors.

Fish offer important advantages for defining the organism-environment interface and responses to natural or anthropogenic stressors. Genomic approaches using fish promise increased investigative power, and have already provided insights into the mechanisms that underlie short-term and long-term environmental adaptations. The range of fish species for which genomic resources are available is increasing, but will require significant further expansion for the optimal application of fish environmental genomics.

We have reconstructed the events that led to the evolution of a key physiological innovation underpinning the large adaptive radiation of fishes, namely their unique ability to secrete molecular oxygen (O2). We show that O2 secretion into the swimbladder evolved some 100 million years after another O2-secreting system in the eye. We unravel the likely sequence in which the functional components of both systems evolved. These components include ocular and swimbladder countercurrent exchangers, the Bohr and Root effects, the buffering power and surface histidine content of hemoglobins, and red blood cell Na+/H+ exchange activity. Our synthesis reveals the dynamics of gains and losses of these multiple traits over time, accounting for part of the huge diversity of form and function in living fishes.

How do organisms respond adaptively to environmental stress? Although some gene-specific responses have been explored, others remain to be identified, and there is a very poor understanding of the system-wide integration of response, particularly in complex, multitissue animals. Here, we adopt a transcript screening approach to explore the mechanisms underpinning a major, whole-body phenotypic transition in a vertebrate animal that naturally experiences extreme environmental stress. Carp were exposed to increasing levels of cold, and responses across seven tissues were assessed by using a microarray composed of 13,440 cDNA probes. A large set of unique cDNAs (approximately 3,400) were affected by cold. These cDNAs included an expression signature common to all tissues of 252 up-regulated genes involved in RNA processing, translation initiation, mitochondrial metabolism, proteasomal function, and modification of higher-order structures of lipid membranes and chromosomes. Also identified were large numbers of transcripts with highly tissue-specific patterns of regulation. By unbiased profiling of gene ontologies, we have identified the distinctive functional features of each tissue's response and integrate them into a comprehensive view of the whole-body transition from one strongly adaptive phenotype to another. This approach revealed an expression signature suggestive of atrophy in cooled skeletal muscle. This environmental genomics approach by using a well studied but nongenomic species has identified a range of candidate genes endowing thermotolerance and reveals a previously unrecognized scale and complexity of responses that impacts at the level of cellular and tissue function.

The DNA microarray is a powerful, high throughput technique for assessing gene expression on a system-wide genomic scale. It has great potential in pain research for determining the network of gene regulation in different pain conditions, and also for producing detailed gene expression maps in anatomical areas that process nociceptive stimuli. However, for the potential of this high throughput technology to be realised in pain research, microarrays need to be combined with other technologies. Laser capture microdissection is capable of isolating small populations of homogenous cells, allowing distinct areas involved in nociceptive processing to be examined. In combination with sophisticated PCR-based amplification protocols this technique provides sufficient amounts of messenger RNA (mRNA) for application to microarrays. Aside from the technological issues, a difficult task in any microarray study is the analysis of the resulting enormous data set to reveal the key genes, whose regulation is central to the phenotypic changes observed. For this to be achieved, the methods of data analysis, pattern searching and feature recognition, and bioinformatics have to be properly deployed all within the context of an appropriate statistical design. These issues are especially relevant to pain research where interindividual and interpopulation variation is likely to be high, and where polymorphisms can greatly affect nociceptive sensitivity and susceptibility to pain conditions. Methods for assessing the function of new candidate genes identified in microarray screening experiments are also discussed.

The beta-adrenergic stress response in red blood cells (RBCs) of rainbow trout shows seasonal changes in expression. We have explored the mechanisms underpinning this response by following, over a period of 27 months, changes in beta-adrenergic receptor (beta-AR) binding characteristics, beta-adrenergically stimulated RBC Na(+)/H(+) exchanger (betaNHE) activity, together with beta-AR and betaNHE mRNA levels and plasma steroid hormone and lactate levels. These parameters were measured at approximately monthly intervals in a single population of fish held under semi-natural conditions. Membrane-bound, high-affinity beta-ARs were present in RBCs at all sampling times, varying from 668+/-112 receptors cell(-1) to 2654+/-882 receptors cell(-1) (mean +/- S.E.M.; N=8). betaNHE activity, however, was reduced by 57% and 34% in December 1999 and February 2001, respectively, compared with an otherwise sustained influx that averaged 110.4+/-2.3 mmol l(-1) RBCs h(-1) (N=119). Only one reduction coincided with a spawning period but both were preceded by transient increases in circulating testosterone. betaNHE activity measured under standard conditions was not correlated with the number or affinity of beta-ARs nor with water temperature, but both beta-AR numbers and betaNHE activity were positively related to their respective mRNA levels (P=0.005 and 0.038, respectively). Pharmaceutical intervention in the transduction cascade linking the beta-AR and betaNHE failed to indicate any failure of the transduction elements in RBCs displaying low betaNHE activity. Similarly, we failed to demonstrate any link between seasonal cortisol fluctuations and seasonally reduced betaNHE activity. However, the betaNHE activity of age-separated RBC fractions showed that younger RBCs had a significantly higher betaNHE response than older RBCs, consistent with the seasonal reductions in betaNHE being linked to turnover of RBCs and erythropoiesis. Testosterone is known to induce erythropoiesis and we conclude that seasonal reductions in betaNHE are not caused by changes in beta-AR numbers but may be linked to testosterone-induced erythropoiesis.

Carp respond to cold by the upregulated expression of Delta9-acyl-CoA desaturase. Here we report the cloning and characterization of Cds2, a second Delta9-acyl CoA-desaturase expressed in carp liver. Both Cds1 and Cds2 complemented the ole1 mutation in Saccharomyces cerevisiae, permitting the synthesis of delta9-monounsaturates, confirming their identity as delta9-desaturases. We demonstrate that under a standard feeding regime it is the Cds2, and not Cds1, transcript that is transiently upregulated during the first few days of cooling from 30 degrees C to 10 degrees C, the period when cold-induced membrane restructuring occurs. Cds2 exists as two differentially spliced transcripts, differing by a small segment from the 3'-untranslated region, the ratio of which varies with temperature. Feeding a diet enriched in saturated fats produced a fourfold increase in Cds1 transcript levels, which was blocked by cooling to 15 degrees C. Cds2 transcript levels, however, showed no substantial response to the saturated diet. Thus carp liver uniquely expresses two isoforms of delta9-acyl CoA desaturase, possibly formed by a recent duplication event, that are differentially regulated by cooling and dietary treatment.

DNA microarray technology is revolutionizing many aspects of biological research, allowing the expression of many thousands of gene transcripts to be monitored simultaneously. This provides powerful tools for the genome-wide correlation of gene transcript levels with physiological responses and alterations in physiological states. To date, microarray analyses have been applied almost exclusively to a few model species for which the abundant gene sequence data permit the fabrication of whole-genome microarrays. However, many interesting physiological traits and responses are poorly expressed or absent in model species and may be better illustrated in nonmodel organisms. Comparative approaches to understanding function traditionally focus on species that by virtue of their unusual adaptations, lifestyles, and phylogeny are particularly suited to address a specific biological process or problem. In this review, we show that microarray technology can be successfully applied to these nonmodel species and used to generate new insights of comparative and evolutionary significance into animal function.

Cold acclimation of carp from 30 degrees C to 10 degrees C causes a restructuring of liver microsomal phospholipids characterised by increased proportions of monounsaturated fatty acid in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Here, we have used electrospray ionisation mass spectrometry (ESI-MS) to determine the patterns of alteration to individual molecular species compositions of PC, PE and phosphatidylinositol (PI) in response to gradually decreasing temperature. The results demonstrate that cold induces precise changes to a limited number of phospholipid species, and that these changes are distinct and different for each phospholipid class. The major change for PC was increased 16:1/22:6, but for PE the species that increased was 18:1/22:6. By contrast, the PI species that increased during cold acclimation were characterised by an sn-1 monounsaturated fatty acid in combination with arachidonoyl or eicosapentaenoyl fatty acid at the sn-2 position. Analysis of acyl distribution indicates that cold only caused the accumulation of monounsaturated fatty acids at the sn-1 and not at the sn-2 position of phospholipids. These results highlight the tight and restricted range of modifications that membranes make to their phospholipid composition in response to thermal stress.

All organisms respond to environmental challenge by adaptive responses, although, in many cases, the underlying molecular mechanisms are not understood. In the case of membranes, the physical structure of membrane phospholipids is conserved in the face of cold, rigidifying conditions by the elevated proportions of unsaturated fatty acids. We have observed a clear positional specificity in this substitution and head group preferences in carp liver membranes. We have also demonstrated changes in the activity of lipid desaturases that mediate the unsaturation response, caused by both transcriptional and post-translational mechanisms. Another hepatic isoform has recently been discovered with sensitivity, not to cooling, but to dietary variations. Finally, we are testing the importance of desaturase inductions in the inducible cold tolerance of the whole animal.