Rapid enzyme assays investigating the variation in the glycolytic pathway in field-caught populations ofFundulus heteroclitus

Variation in enzyme expression may be important in evolutionary adaptation, yet is seldom studied. Furthermore, no studies have examined the expression of all enzymes in a defined metabolic pathway. Enzyme concentration is a measure of enzyme expression and was ascertained by assaying maximal activity. Presented here is an analysis of variation of maximal enzyme activity for all the enzymes in a single metabolic pathway, glycolysis, from three clinically distributed populations of the fish,Fundulus heteroclitus. Techniques for rapidly analyzing maximal enzyme activity for all the enzymes of an entire metabolic pathway from many individuals are described. The high degree of repeatability (mean coefficient of variation for replicates, 4.4%) and sensitivity (less than 3 mg of tissue is required to measure all 10 enzymes) of these assays demonstrate the utility of such an approach for analyzing variation among populations for a large numbers of enzymes. Results from these studies indicate that (1) the average coefficient of variation for all enzyme determinations within a population is 45.3% and (2) between populations, the activity of 5 of the 10 glycolytic enzymes are significantly different. This considerable variation occurs even in populations where there is little allelic variation. These data demonstrating substantial variation in enzyme expression support the idea that changes in gene regulation may be as important as, or even more important than, changes in biochemical kinetic parameters in evolutionary processes.     

Variation in tissue-specific gene expression among natural populations

Background  

Variation in gene expression is extensive among tissues, individuals, strains, populations and species. The interactions among these sources of variation are relevant for physiological studies such as disease or toxic stress; for example, it is common for pathologies such as cancer, heart failure and metabolic disease to be associated with changes in tissue-specific gene expression or changes in metabolic gene expression. But how conserved these differences are among outbred individuals and among populations has not been well documented. To address this we examined the expression of a selected suite of 192 metabolic genes in brain, heart and liver in three populations of the teleost fish Fundulus heteroclitus using a highly replicated experimental design.     

Population proteomics: quantitative variation within and among populations in cardiac protein expression

Population analysis of gene expression is typically achieved by quantifying levels of mRNA; however, gene expression is also a function of protein translation and turnover. Therefore, a complete understanding of population variation in gene expression requires quantitative knowledge of protein expression within and among natural populations. We used two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) to quantitatively compare expression of heart ventricle proteins among 18 individuals in three populations of the teleost fish Fundulus. Among populations, expressions between orthologous proteins and mRNAs were generally positively correlated. Additionally, similar to the pattern of cardiac mRNA expression for the same populations, we found considerable variation in protein expression both within and among populations: Of 408 protein features in 2D gels, 34% are significantly different (P < 0.01) among individuals within a population, 9% differ between populations, and 12% have a pattern of expression that suggests they have evolved by natural selection. Although similar to mRNA expression, the frequency of significant differences among populations is larger for proteins. Similar to mRNA expressions, expressions of most proteins are correlated to the expressions of many other proteins. However, the correlations among proteins are more extensive than the correlation for similar RNAs. These correlations suggest a greater coordinate regulation of protein than mRNA expression. The larger frequency of significant differences among populations and the greater frequency of correlated expression among proteins versus among RNAs suggest that the molecular mechanisms affecting protein expression enhance the differences among populations, and these regulatory steps could be a source of variation for adaptation.     

Variation in tissue-specific gene expression among natural populations

Background

Variation in gene expression is extensive among tissues, individuals, strains, populations and species. The interactions among these sources of variation are relevant for physiological studies such as disease or toxic stress; for example, it is common for pathologies such as cancer, heart failure and metabolic disease to be associated with changes in tissue-specific gene expression or changes in metabolic gene expression. But how conserved these differences are among outbred individuals and among populations has not been well documented. To address this we examined the expression of a selected suite of 192 metabolic genes in brain, heart and liver in three populations of the teleost fish Fundulus heteroclitus using a highly replicated experimental design.

Results

Half of the genes (48%) were differentially expressed among individuals within a population-tissue group and 76% were differentially expressed among tissues. Differences among tissues reflected well established tissue-specific metabolic requirements, suggesting that these measures of gene expression accurately reflect changes in proteins and their phenotypic effects. Remarkably, only a small subset (31%) of tissue-specific differences was consistent in all three populations.

Conclusions

These data indicate that many tissue-specific differences in gene expression are unique to one population and thus are unlikely to contribute to fundamental differences between tissue types. We suggest that those subsets of treatment-specific gene expression patterns that are conserved between taxa are most likely to be functionally related to the physiological state in question.     

Isozyme Variation among Biological Species in the Gibberella fujikuroi Species Complex (Fusarium Section Liseola)

Isozyme phenotypes were determined for 101 strains of Gibberella fujikuroi and 2 strains of Gibberella nygamai that represent seven biological species (mating populations) isolated from a variety of plant hosts in dispersed geographic locations. Fourteen enzymes were resolved in one or more of three buffer systems. Two of the enzymes, arylesterase and acid phosphatase, were polymorphic within two or more biological species and are suitable for intraspecific studies of population variation. Six enzymes, alcohol dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphoglucomutase, and phosphogluconate dehydrogenase, were monomorphic in all of the isolates examined. The remaining six enzymes, fumarase, glucose phosphate isomerase, glutamate dehydrogenase (NADP), isocitrate dehydrogenase (NADP), malate dehydrogenase, and triose-phosphate isomerase, could potentially be used to distinguish the different biological species. Mating populations C and D are the most similar, since the mating population C isolates examined had the same isozyme phenotype as did a subset of the isolates in mating population D. Mating population E is the least similar to the other taxa examined. Unique isozyme phenotypes are present but are composed of banding patterns shared among the biological species. This finding supports the hypothesis that these biological species, with the possible exception of mating populations C and D, are reproductively isolated from one another and that no significant gene flow is occurring between them. Isozyme analysis is a useful method to distinguish these closely related biological species. Examination of isozyme phenotypes is more rapid than the present technique, which is based on sexual crosses; can be applied to strains that are not sexually fertile; and is more sensitive than traditional morphological characters, which cannot distinguish more than three or four morphological groups among the seven biological species. While emphasizing the discreteness of the mating populations as biological entities, our isozyme data also reaffirm the close genetic relationship among these groups.     

Morphological adaptation to thermal stress in a marine fish, Fundulus heteroclitus.

Populations of Fundulus heteroclitus (Cyprinodontidae), a coastal marine fish, were studied in control and artificially heated environments on the north shore of Long Island to determine patterns of variation in morphology and the extent to which this variation reflected adaptation to environmental characteristics. Principal components and discriminant function analyses were used to analyze variation in and among seventeen morphological characters. Fishes living in water artificially heated by a power plant exhibited marked divergence from control populations in head morphology, and convergence with a population sampled at more southern latitudes. Hence, these differences were interpreted as adaptations to warm environments. Greater morphological variation is detected at the heated locality than at control localities, and this may be partially due to a breakdown in developmental homeostasis, and partially due to selection favoring phenotypes that are rare in this environment.     

Phenotypic plasticity in gene expression contributes to divergence of locally adapted populations of Fundulus heteroclitus

We examine the interaction between phenotypic plasticity and evolutionary adaptation using muscle gene expression levels among populations of the fish Fundulus heteroclitus acclimated to three temperatures. Our analysis reveals shared patterns of phenotypic plasticity due to thermal acclimation as well as non‐neutral patterns of variation among populations adapted to different thermal environments. For the majority of significant differences in gene expression levels, phenotypic plasticity and adaptation operate on different suites of genes. The subset of genes that demonstrate both adaptive differences and phenotypic plasticity, however, exhibit countergradient variation of expression. Thus, expression differences among populations counteract environmental effects, reducing the phenotypic differentiation between populations. Finally, gene‐by‐environment interactions among genes with non‐neutral patterns of expression suggest that the penetrance of adaptive variation depends on the environmental conditions experienced by the individual.     

Genetic Organization and Adaptive Response of Allozymes to Ecological Variables in FUNDULUS HETEROCLITUS

Populations of Fundulus heteroclitus, (Cyprinodontidae) a widespread coastal marine fish, were studied in control and artificially heated environments on the North Shore of Long Island, New York to determine (1) patterns of variation in biochemical phenotypes and (2) the extent to which this variation reflected adaptation to environmental characteristics.—Variation at three of twelve polymorphic isoenzyme loci from the warm water population was beyond the range of variation among control populations, and resembled those determined for populations living at more southern latitudes. Hence, these differences were interpreted as adaptations to warm environments. Significant differences in allele frequencies and zygotic proportions at ten of twelve isoenzyme loci were found associated with differences in environments, sexes, and/or age classes. These data strongly support the view that protein polymorphisms are adaptive.—Several observations suggested that selection acts upon multilocus phenotypes rather than upon those of single loci. Several di-locus phenotypic distributions were demonstrated to be nonrandom, and those that exhibited similar patterns of dependence over years were postulated to be maintained by selection. Highly heterozygous fish exhibited superior viability when cohorts were compared over successive years.—The consequences of the polygynous mating system in this species for maintaining genetic variation and for allowing rapid evolutionary response to a variable environment are discussed.     

Variation in gene expression in response to stress in two populations of Fundulus heteroclitus

We used differential display PCR to identify hepatic genes responsive to handling stress and genes that differ in expression between populations of a fish, Fundulus heteroclitus, from different thermal environments. Despite substantial inter-individual variation, we cloned 20 putatively stress-regulated bands from Northern fish, 10 of which had high similarity to genes of known function. We selected five of these genes for further analysis based on their known roles in the stress response. Three of these genes (glucokinase, serine-threonine kinase 10 and cRAF) were confirmed as stress-responsive using real-time PCR. These genes increased in expression in response to a 7-day chronic stress protocol in fish from the Southern population of F. heteroclitus, but did not change significantly in fish from the Northern population. These three genes also differed in expression between populations in control fish, suggesting a link between the response to chronic stress and inter-population differences in gene expression in unstressed laboratory-acclimated fish. Two genes that did not respond to stress (glycogen synthase kinase and warm acclimation-related protein (WAP)) also differed between populations. Expression of WAP was eight-fold higher in Southern than in Northern fish, consistent with a previously suggested role for this gene in thermal acclimation or adaptation in fish.     

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.     

Fine quantitative trait loci mapping of carbon and nitrogen metabolism enzyme activities and seedling biomass in the maize IBM mapping population

Understanding the genetic basis of nitrogen and carbon metabolism will accelerate the development of plant varieties with high yield and improved nitrogen use efficiency. A robotized platform was used to measure the activities of 10 enzymes from carbon and nitrogen metabolism in the maize (Zea mays) intermated B73 × Mo17 mapping population, which provides almost a 4-fold increase in genetic map distance compared with conventional mapping populations. Seedling/juvenile biomass was included to identify its genetic factors and relationships with enzyme activities. All 10 enzymes showed heritable variation in activity. There were strong positive correlations between activities of different enzymes, indicating that they are coregulated. Negative correlations were detected between biomass and the activity of six enzymes. In total, 73 significant quantitative trait loci (QTL) were found that influence the activity of these 10 enzymes and eight QTL that influence biomass. While some QTL were shared by different enzymes or biomass, we critically evaluated the probability that this may be fortuitous. All enzyme activity QTL were in trans to the known genomic locations of structural genes, except for single cis-QTL for nitrate reductase, Glu dehydrogenase, and shikimate dehydrogenase; the low frequency and low additive magnitude compared with trans-QTL indicate that cis-regulation is relatively unimportant versus trans-regulation. Two-gene epistatic interactions were identified for eight enzymes and for biomass, with three epistatic QTL being shared by two other traits; however, epistasis explained on average only 2.8% of the genetic variance. Overall, this study identifies more QTL at a higher resolution than previous studies of genetic variation in metabolism.     

Relative influences of historical and contemporary forces shaping the distribution of genetic variation in the Atlantic killifish, Fundulus heteroclitus

A major goal of population genetics research is to identify the relative influences of historical and contemporary processes that serve to structure genetic variation. Most population genetic models assume that populations exist in a state of migration-drift equilibrium. However, in the past this assumption has rarely been verified, and is likely rarely achieved in natural populations. We assessed the equilibrium status at both local and regional scales of the Atlantic killifish, Fundulus heteroclitus . This species is a model organism for the study of adaptive clinal variation, but has also experienced a complicated history of range expansion and secondary contact following allopatric divergence, potentially obscuring the influence of contemporary evolutionary processes. Presumptively neutral genetic markers (microsatellites) demonstrated zones of secondary intergradation among coastal populations centred around northern New Jersey and the Chesapeake Bay region. Analysis of genetic variation indicated isolation by distance among some populations and provided supporting evidence that the Delaware Bay, but not the Chesapeake Bay, has acted as a barrier to dispersal among coastal populations. Bayesian estimates indicated large effective population sizes and low migration rates, and were in good agreement with empirically derived estimates of population and neighbourhood size from mark–recapture studies. These data indicate that populations are not in migration-drift equilibrium at a regional scale, and suggest that contributing factors include large population size combined with relatively low migration rates. These conditions should be considered when interpreting the evolutionary significance of the distribution of genetic variation among F. heteroclitus populations.     

The importance of sampling scale in the assessment of intraspecific life-history differences in plains killifish,Fundulus zebrinus,populations (Pisces: Cyprinodontidae)

Synopsis The purpose of this study was to determine the effects of unpredictable environmental fluctuations on the demographic and genetic structure of Fundulus zebrinus populations. Collections of F. zebrinus were taken from three rivers in the Arkansas River basin: the Arkansas, Chikaskia, and Ninnescah. Fish were sampled from three sites on each river on nine collection dates throughout 1984 and 1985. Totals of 2100 fish and 6000 fish were included in electrophoretic and demographic analyses, respectively. The results of the study indicate that within a limited geographic region (i.e. within rivers) spatial differences and temporal changes in both demographic and genetic population characteristics occur frequently and are primarily stochastic. However, on a larger spatial scale (i.e. across rivers), general trends emerge for demographic and especially for genetic population characteristics. These results illustrate the importance of sampling scale for conclusions of life-history evolution in fluctuating environments. In addition, it was found that regulation of Fundulus zebrinus populations includes an important density-independent component. Stochastic demographic differences across space and changes through time and spatially and temporally heterogeneous allele frequencies, are both indicative of density-independent regulation. Variation in population parameters, both demographic and genetic, was observed between populations sampled from each river. These population differences were attributed to differences between the rivers themselves.     

Shape variation in the least killifish: ecological associations of phenotypic variation and the effects of a common garden

Studies of the adaptive significance of variation among conspecific populations often focus on a single ecological factor. However, habitats rarely differ in only a single ecological factor, creating a challenge for identifying the relative importance of the various ecological factors that might be maintaining local adaptation. Here we investigate the ecological factors associated with male body shape variation among nine populations of the poeciliid fish, Heterandria formosa, from three distinct habitats and combine those results with a laboratory study of three of those populations to assess the contributions of genetic and environmental influences to shape variation. Field‐collected animals varied principally in three ways: the orientation of the gonopodium, the intromittent organ; the degree of body depth and streamlining; and the shape of the tail musculature. Fish collected in the spring season were larger and had a more anteriorly positioned gonopodium than fish collected in autumn. Fish collected from lotic springs were larger and more streamlined than those collected from lentic ponds or tidal marshes. Some of the variation in male shape among populations within habitats was associated with population‐level variation in species richness, adult density, vegetative cover, predation risk, and female standard length. Population‐level differences among males in body size, position of the gonopodium, and shape of the tail musculature were maintained among males reared in a common environment. In contrast, population variation in the degree of streamlining was eliminated when males were reared in a common environment. These results illustrate the complicated construction of multivariate phenotypic variation and suggest that different agents of selection have acted on different components of shape.     

Geographic Variation in Age Structure and Longevity in the Nine-Spined Stickleback (Pungitius pungitius)

Variation in age and size of mature nine-spined sticklebacks (Pungitius pungitius) within and among 16 Fennoscandian populations were assessed using skeletochronology. The average age of individuals in a given population varied from 1.7 to 4.7 years. Fish from pond populations were on average older than those from lake and marine populations, and females tended to be older than males. Reproduction in marine and lake populations commenced typically at an age of two years, whereas that in ponds at an age of three years. The maximum life span of the fish varied from 3 to 7 years. Mean body size within and among populations increased with increasing age, but the habitat and population differences in body size persisted even after accounting for variation in population age (and sex) structure. Hence, the population differences in mean body size are not explainable by age differences alone. As such, much of the pronounced intraspecific variation in population age structure can be attributed to delayed maturation and extended longevity of the pond fish. The results are contrasted and discussed in the context of similar data from the three-spined stickleback (Gasterosteus aculeatus) occupying the same geographic area.     

Genetic differentiation of piscivorous chub (genus Opsariichthys) in Japan,Korea and Russia

A comparison of allozyme variation, restriction fragment length polymorphisms in the mitochondrial DNA and partial sequences of the ND II gene (496 bp) was made among two lacustrine populations of the piscivorous chub (Opsariichthys uncirostris uncirostris) in Japan, four fluvial populations in Korea, one lacustrine population in Russia and one specimen from the Amur River (O. u. amurensis). All analyses indicate that the Japanese populations of piscivorous chub are separable from the Asian mainland populations of Korea and Russia. The latter populations were further divided into the Korean and Russian fish. Although opinions are divided on the phylogenetic position of the population in Lake Mikata, Japan, which shows unique morphological traits intermediate between those of the population in Lake Biwa and the mainland populations, the current analysis indicates a closer relationship to the population in Lake Biwa.     

Behavioral Plasticity in Response to Environmental Manipulation among Zebrafish (Danio rerio) Populations

Plastic responses can have adaptive significance for organisms occurring in unpredictable environments, migratory species and organisms occupying novel environments. Zebrafish (Danio rerio) occur in a wide range of habitats and environments that fluctuate frequently across seasons and habitats. We expect wild populations of fish to be behaviorally more flexible than fish reared in conventional laboratory and hatchery environments. We measured three behavioral traits among 2 wild (U and PN) and 1 laboratory bred (SH) zebrafish populations in four environments differing in water flow and vegetation regimes. We found that the degree of plasticity varied with the type of behavior and also among populations. In general, vegetation increased aggression and water flow decreased latency to feed after a disturbance, but the patterns were population dependent. For example, while wild U fish fed more readily after a disturbance in vegetated and/or flowing habitats, fish from the wild PN population and lab-reared SH strain showed little variation in foraging across different environmental conditions. Zebrafish from all the three populations were more aggressive when tested in an arena with vegetation. In contrast, while there was an inter- population difference in shoaling distances, variation in shoaling distance across environmental conditions within populations was not significant. These results suggest that both foraging and aggression in zebrafish are more plastic and influenced by immediate context than is shoaling distance, which may have a stronger genetic basis. Our findings point to different underlying mechanisms influencing the expression of these traits and warrants further investigations.     

A biochemical genetic analysis of populations of the northern pike, Esox lucius L., from Europe and North America

Genetic variation as identified by starch gel electrophoresis and enzyme staining was studied in seven populations of the northern pike, Esox lucius L. Samples derived from populations in northern U.S., Canada, Swedish fresh water, Swedish Baltic, the Netherlands, England and Ireland were analysed for variation at between 10 and 26 enzyme-coding loci. Polymorphism was found to be restricted to three loci: Sod-1 (mitochondrial superoxide dismutase), Ldh-2 (heart-type lactate dehydrogenase) and Got-1 (cytoplasmic glutamateoxalate transaminase). Mean heterozygosity ( H ) over all seven population samples was estimated to be 0·019. This extremely low variability, relative to reported data for other fish species, is discussed in terms of the structure and possible origins of pike populations.