Stress and interpopulation variation in glycolytic enzyme activity and expression in a teleost fish Fundulus heteroclitus

Northern populations of Fundulus heteroclitus have twofold greater activity of lactate dehydrogenase-B (LDH-B) than southern populations, but exposure to stress increases LDH-B in southern populations, abolishing this difference. To test whether differences in the activity of other hepatic glycolytic enzymes between populations are sensitive to stress, we injected fish with a pharmacological dose of cortisol in coconut oil (400 microg g(-1)) or exposed them to handling stress and measured the activities of all the glycolytic enzymes. At rest, liver phosphofructokinase (PFK) and aldolase (ALD) activities were greater in southern fish, whereas LDH-B activity was greater in northern fish. No other glycolytic enzymes differed in activity between populations in control fish. Cortisol injection and handling stress decreased PFK and ALD and increased LDH activities in the southern but not the northern population, such that the populations no longer differed in the activity of any enzyme following treatment. Unlike Ldh-B mRNA, Pfk and Ald mRNA levels did not parallel enzyme activity, suggesting complex kinetics or regulation at multiple levels. Plasma cortisol did not differ between populations at rest but was significantly different between populations in treated fish. These data suggest that differences in liver enzyme activity may be related to differences in stress hormone physiology between populations.     

Microsatellite analysis of the phylogeography, Pleistocene history and secondary contact hypotheses for the killifish, Fundulus heteroclitus

The mummichog, Fundulus heteroclitus, exhibits extensive latitudinal clinal variation in a number of physiological and biochemical traits, coupled with phylogeographical patterns at mitochondrial and nuclear DNA loci that suggest a complicated history of spatially variable selection and secondary intergradation. This species continues to serve as a model for understanding local and regional adaptation to variable environments. Resolving the influences of historical processes on the distribution of genetic variation within and among extant populations of F. heteroclitus is crucial to a better understanding of how populations evolve in the context of contemporary environments. In this study, we analysed geographical patterns of genetic variation at eight microsatellite loci among 15 populations of F. heteroclitus distributed throughout the North American range of the species from Nova Scotia to Georgia. Genetic variation in Northern populations was lower than in Southern populations and was strongly correlated with latitude throughout the species range. The most common Northern alleles at all eight loci exhibited concordant latitudinal clinal patterns, and the existence of an abrupt transition zone in allele frequencies between Northern and Southern populations was similar to that observed for mitochondrial DNA and allozyme loci. A significant pattern of isolation by distance was observed both within and between northern and southern regions. This pattern was unexpected, particularly for northern populations, given the recent colonization history of post-Pleistocene habitats, and was inconsistent with either a recent northward population expansion or a geographically restricted northern Pleistocene refugium. The data provided no evidence for recent population bottlenecks, and estimates of historical effective population sizes suggest that post-Pleistocene populations have been large throughout the species distribution. These results suggest that F. heteroclitus was broadly distributed throughout most of its current range during the last glacial event and that the abrupt transition in allele frequencies that separate Northern and Southern populations may reflect regional disequilibrium conditions associated with the post-Pleistocene colonization history of habitats in that region.     

Investigating the molecular basis of local adaptation to thermal stress: population differences in gene expression across the transcriptome of the copepod Tigriopus californicus

ABSTRACT: BACKGROUND: Geographic variation in the thermal environment impacts a broad range of biochemical and physiological processes and can be a major selective force leading to local population adaptation. In the intertidal copepod Tigriopus californicus, populations along the coast of California show differences in thermal tolerance that are consistent with adaptation, i.e., southern populations withstand thermal stresses that are lethal to northern populations. To understand the genetic basis of these physiological differences, we use an RNA-seq approach to compare genome-wide patterns of gene expression in two populations known to differ in thermal tolerance. RESULTS: Observed differences in gene expression between the southern (San Diego) and the northern (Santa Cruz) populations included both the number of affected loci as well as the identity of these loci. However, the most pronounced differences concerned the amplitude of upregulation of genes producing heat shock proteins (Hsps) and genes involved in ubiquitination and proteolysis. Among the hsp genes, orthologous pairs show markedly different thermal responses as the amplitude of hsp response was greatly elevated in the San Diego population, most notably in members of the hsp70 gene family. There was no evidence of accelerated evolution at the sequence level for hsp genes. Among other sets of genes, cuticle genes were up-regulated in SD but down-regulated in SC, and mitochondrial genes were down-regulated in both populations. CONCLUSIONS: Marked changes in gene expression were observed in response to acute sub-lethal thermal stress in the copepod T. californicus. Although some qualitative differences were observed between populations, the most pronounced differences involved the magnitude of induction of numerous hsp and ubiquitin genes. These differences in gene expression suggest that evolutionary divergence in the regulatory pathway(s) involved in acute temperature stress may offer at least a partial explanation of population differences in thermal tolerance observed in Tigriopus.     

Environmental adaptations as windows on molecular evolution

Changes in gene regulation may play an important role in adaptive evolution, particularly during adaptation to a changing environment. However, little is known about the molecular mechanisms underlying adaptively significant variation in gene regulation. To address this question, we are using environmental adaptations in populations of a fish, Fundulus heteroclitus as a window into the molecular evolution of gene regulation. F. heteroclitus are found along the East Coast of North America, with populations distributed along a steep thermal gradient. At the extremes of the species range, populations have undergone local adaptation to their habitat temperatures. A variety of genes differ in their regulation between these populations. We have determined the mechanism responsible for changes in lactate dehydrogenase-B (Ldh-B) gene regulation. A limited number of mutations in the regulatory sequence of this gene result in changes in its expression. Both the phenotypic (increased LDH activity) and genotypic (changes in Ldh-B regulatory sequences) differences between populations have been shown to be affected by natural selection, rather than genetic drift. Therefore, even a small number of mutations within important regulatory sequences can provide evolutionarily significant variation and have an impact on environmental adaptation.     

Population‐specificity of heat stress gene induction in northern and southern eelgrass Zostera marina populations under simulated global warming

Summer heat waves have already resulted in mortality of coastal communities, including ecologically important seagrass meadows. Gene expression studies from controlled experiments can provide important insight as to how species/genotypes react to extreme events that will increase under global warming. In a common stress garden, we exposed three populations of eelgrass, Zostera marina, to extreme sea surface temperatures, simulating the 2003‐European heat wave. Populations came from locations widely differing in their thermal regime, two northern European locations [Ebeltoft (Kattegat), Doverodde (Limfjord, Baltic Sea)], and one southern population from Gabicce Mare (Adriatic Sea), allowing to test for population specificity in the response to a realistic heat stress event. Eelgrass survival and growth as well as the expression of 12 stress associated candidate genes were assessed during and after the heat wave. Contrary to expectations, all populations suffered equally from 3 weeks of heat stress in terms of shoot loss. In contrast, populations markedly differed in multivariate measures of gene expression. While the gene expression profiles converged to pre‐stress values directly after the heat wave, stress correlated genes were upregulated again 4 weeks later, in line with the observed delay in shoot loss. Target genes had to be selected based on functional knowledge in terrestrial plants, nevertheless, 10/12 genes were induced relative to the control treatment at least once during the heat wave in the fully marine plant Z. marina. This study underlines the importance of realistic stress and recovery scenarios in studying the impact of predicted climate change.     

Intraspecific variation in aerobic metabolism and glycolytic enzyme expression in heart ventricles

A previous phylogenetic analysis among 15 taxa of the teleost fish Fundulus suggested that there should be thermal-adaptive differences in heart metabolism among populations. To test this hypothesis, the rate of oxygen consumption and the activities of all 11 glycolytic enzymes were measured in isolated heart ventricle from two populations of Fundulus heteroclitus. Heart ventricular metabolism is greater in a northern population versus a southern population of these fish. Analysis of the amount of glycolytic enzymes indicates that 87% of the variation in cardiac metabolism within and between populations is explained by the variation in three enzymes (pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase). These enzymes are the same three enzymes that were predicted to be important based on previously determined phylogenetic patterns of expression. Our data indicate that near-equilibrium enzymes, as well as classically defined rate-limiting enzymes, can also influence metabolism.     

Measuring the immune system of the three‐spined stickleback – investigating natural variation by quantifying immune expression in the laboratory and the wild

Current understanding of the immune system comes primarily from laboratory‐based studies. There has been substantial interest in examining how it functions in the wild, but studies have been limited by a lack of appropriate assays and study species. The three‐spined stickleback (Gasterosteus aculeatus L.) provides an ideal system in which to advance the study of wild immunology, but requires the development of suitable immune assays. We demonstrate that meaningful variation in the immune response of stickleback can be measured using real‐time PCR to quantify the expression of eight genes, representing the innate response and Th1‐, Th2‐ and Treg‐type adaptive responses. Assays are validated by comparing the immune expression profiles of wild and laboratory‐raised stickleback, and by examining variation across populations on North Uist, Scotland. We also compare the immune response potential of laboratory‐raised individuals from two Icelandic populations by stimulating cells in culture. Immune profiles of wild fish differed from laboratory‐raised fish from the same parental population, with immune expression patterns in the wild converging relative to those in the laboratory. Innate measures differed between wild populations, whilst the adaptive response was associated with variation in age, relative size of fish, reproductive status and S. solidus infection levels. Laboratory‐raised individuals from different populations showed markedly different innate immune response potential. The ability to combine studies in the laboratory and in the wild underlines the potential of this toolkit to advance our understanding of the ecological and evolutionary relevance of immune system variation in a natural setting.     

Drosophila cdc2 homologs: a functional homolog is coexpressed with a cognate variant.

Using probes obtained by PCR amplification, we have cloned Drosophila cDNAs encoding structural homologs of the p34cdc2 cell cycle kinase. Southern blot experiments and in situ hybridization to polytene chromosomes demonstrated that the isolated cDNAs, were derived from two distinct genes, Dm cdc2 (31E) and Dm cdc2c (92F). Northern blot and in situ hybridization experiments revealed that these two genes are coexpressed during embryogenesis and that expression is correlated with cell proliferation. However, despite the similarity in structure and expression, the two gene products differed in functional assays in yeasts. Expression of Dm cdc2 in Schizosaccharomyces pombe and Saccharomyces cerevisiae rescued cell cycle arrest caused by mutations in cdc2+ and CDC28, the genes encoding the p34cdc2 kinase homologs of these yeasts. In contrast, the Dm cdc2c gene product did not restore cell cycle progression. Thus, in addition to the identification of a functional homolog in Drosophila, our results indicate the presence of a closely related cognate of the p34cdc2 cell cycle kinase.     

Gene expression remodelling and immune response during adaptive divergence in an African cichlid fish

Variation in gene expression contributes to ecological speciation by facilitating population persistence in novel environments. Likewise, immune responses can be of relevance in speciation driven by adaptation to different environments. Previous studies examining gene expression differences between recently diverged ecotypes have often relied on only one pair of populations, targeted the expression of only a subset of genes or used wild‐caught individuals. Here, we investigated the contribution of habitat‐specific parasites and symbionts and the underlying immunological abilities of ecotype hosts to adaptive divergence in lake–river population pairs of the cichlid fish Astatotilapia burtoni. To shed light on the role of phenotypic plasticity in adaptive divergence, we compared parasite and microbiota communities, immune response, and gene expression patterns of fish from natural habitats and a lake‐like pond set‐up. In all investigated population pairs, lake fish were more heavily parasitized than river fish, in terms of both parasite taxon composition and infection abundance. The innate immune response in the wild was higher in lake than in river populations and was elevated in a river population exposed to lake parasites in the pond set‐up. Environmental differences between lake and river habitat and their distinct parasite communities have shaped differential gene expression, involving genes functioning in osmoregulation and immune response. Most changes in gene expression between lake and river samples in the wild and in the pond set‐up were based on a plastic response. Finally, gene expression and bacterial communities of wild‐caught individuals and individuals acclimatized to lake‐like pond conditions showed shifts underlying adaptive phenotypic plasticity.     

Facing warm temperatures during migration: cardiac mRNA responses of two adult Oncorhynchus nerka populations to warming and swimming challenges

The main findings of the current study were that exposing adult sockeye salmon Onchorhynchus nerka to a warm temperature that they regularly encounter during their river migration induced a heat shock response at an mRNA level, and this response was exacerbated with forced swimming. Similar to the heat shock response, increased immune defence‐related responses were also observed after warm temperature treatment and with a swimming challenge in two different populations (Chilko and Nechako), but with some important differences. Microarray analyses revealed that 347 genes were differentially expressed between the cold (12–13° C) and warm (18–19° C) treated fish, with stress response (GO:0006950) and response to fungus (GO:0009620) elevated with warm treatment, while expression for genes involved in oxidative phosphorylation (GO:0006119) and electron transport chain (GO:0022900) elevated for cold‐treated fish. Analysis of single genes with real‐time quantitative PCR revealed that temperature had the most significant effect on mRNA expression levels, with swimming and population having secondary influences. Warm temperature treatment for the Chilko population induced expression of heat shock protein (hsp) 90α, hsp90β and hsp30 as well as interferon‐inducible protein. The Nechako population, which is known to have a narrower thermal tolerance window than the Chilko population, showed even more pronounced stress responses to the warm treatment and there was significant interaction between population and temperature treatment for hsp90β expression. Moreover, significant interactions were noted between temperature treatment and swimming challenge for hsp90α and hsp30, and while swimming challenge alone increased expression of these hsps, the expression levels were significantly elevated in warm‐treated fish swum to exhaustion. In conclusion, it seems that adult O. nerka currently encounter conditions that induce several cellular defence mechanisms during their once‐in‐the‐lifetime migration. As river temperatures continue to increase, it remains to be seen whether or not these cellular defences provide sufficient protection for all O. nerka populations.     

Cloning and characterization of cDNA clones encoding membrane-bound and potentially secreted major histocompatibility class I receptors from walleye (Stizostedion vitreum)

Major histocompatibility (MH) gene polymorphism has been used to type populations of humans, mice, and fish. Walleye ( Stizostedion vitreum) comprise an economically important fishery in Lake Erie, but whether those in the western basin form a single population or separate shoal- and river-breeding populations is not known. To develop MH gene markers for use in defining their population structure, we constructed a head kidney cDNA library from which five full-length class I heavy-chain clones were isolated and sequenced. Although they came in roughly three sizes, 1300, 1400, and over 2000 bp, the clones all exhibited a high degree of sequence similarity to each other and to known teleost MH class I cDNAs in the area encoding the extracellular domains, but showed dramatic differences in their transmembrane and cytoplasmic domains. One clone had an AG repeat that eliminated the hydrophobicity of the transmembrane domain, indicating that it may encode a secreted class I receptor. The other four clones encode three distinctly different cytoplasmic domains. The two clones that encode the same cytoplasmic domain resemble those of the known teleost MH class I sequences the most. Southern blotting indicated that there were four copies of the gene present in the walleye genome. Northern blotting showed that class I MH genes are expressed in most tissues and mRNAs of all three size classes can be detected. A preliminary survey of the polymorphism of these genes indicates that they will provide useful markers for differentiating fish stocks.     

Genes induced by WDS are differentially expressed in two populations of aleppo pine (<Emphasis Type="Italic">Pinus halepensis</Emphasis>)

Gene expression in response to water-deficit stress (WDS) is a critical factor determining the survival and growth of pine seedlings. To understand how gene expression varies with different water stress levels, we differentially screened a cDNA library from roots of aleppo pine (Pinus halepensis) seedlings subjected to WDS. We found 156 clones of genes up-regulated and 56 down-regulated in response to WDS. Of the 14 clones selected for further characterization, 8 resemble WDS-responsive genes previously identified in angiosperms. The genes showing similarities to known proteins included an aldehyde dehydrogenase, a late embryogenesis abundant (LEA) protein, a chitinase, a cyclophilin, an MYB factor, an inorganic pyrophosphatase, a sucrose synthase, and a 4-coumarate ligase. Six of the clones did not have any similarity to previously identified proteins of known function. Quantitative polymerase chain reaction (qPCR) was used to compare the differential expression of these genes during control (no stress), moderate, and severe WDS treatments of seedling progeny from two different geographic origins within Israel, Yirka, and Beit Jann. The difference in expression between the treatments for various genes ranged from 1.9 to 8.0 cycle threshold. Most of the genes were expressed at similar levels in trees from the two populations or at higher levels in trees from Beit Jann, Israel. Northern blot analyses done for four highly expressed genes verify the results of the qPCR.     

Swimming performance and energetics as a function of temperature in killifish Fundulus heteroclitus

Populations of the common killifish Fundulus heteroclitus are found along a latitudinal temperature gradient in habitats with high thermal variability. The objectives of this study were to assess the effects of temperature and population of origin on killifish swimming performance (assessed as critical swimming speed, U(crit)). Acclimated fish from northern and southern killifish populations demonstrated a wide zone (from 7 degrees to 33 degrees C) over which U(crit) showed little change with temperature, with performance declining significantly only at lower temperatures. Although we observed significant differences in swimming performance between a northern and a southern population of killifish in one experiment, with northern fish having an approximately 1.5-fold-greater U(crit) than southern fish across all acclimation temperatures, we were unable to replicate this finding in other populations or collection years, and performance was consistently high across all populations and at both low (7 degrees C) and high (23 degrees C) acclimation temperatures. The poor swimming performance of southern killifish from a single collection year was correlated with low muscle [glycogen] rather than with other indicators of fuel stores or body condition. Killifish acclimated to 18 degrees C and acutely challenged at temperatures of 5 degrees , 18 degrees , 25 degrees , or 34 degrees C showed modest thermal sensitivity of U(crit) between 18 degrees and 34 degrees C, with performance declining substantially at 5 degrees C. Thus, much of the zone of relative thermal insensitivity of swimming performance is intrinsic in this species rather than acquired as a result of acclimation. These data suggest that killifish are broadly tolerant of changing temperatures, whether acute or chronic, and demonstrate little evidence of local adaptation in endurance swimming performance in populations from different thermal habitats.