Enzyme Activities and Alcohol Tolerance in Isofemale Lines of Drosophila melanogaster Originating from Different Habitats

Enzyme activity variation was studied in a Drosophila melanogaster population from two villages (Tiszafüred and Tiszaszolos) in Hungary. Two habitats (distillery and farmyard) were sampled in both villages and 8-9 isofemale lines were established from each sample with a total of 35 lines. The activities of ADH, alphaGPDH, IDH and 6PGDH were determined on starch gel after electrophoresis in 10 F1 females of each of the 35 isofemale lines. Three sublines were established from three selected isofemale lines of all four samples (altogether 36 sublines). Alcohol tolerance of the adult flies was assayed in these sublines. The activity of ADH was similar in the two habitats; so was the sensitivity to ethanol. Accordingly, no differences in adaptation to environmental ethanol were detected between the two habitats. The deviations between the two habitats in average activities and in the total variation of enzyme activities were not consistent in the two villages. These results suggest that founder effects and genetic drift are more pronounced in distilleries than selection. The association among enzyme activities varied greatly both between the two villages and between the two habitats. The two parameters of alcohol tolerance were not significantly different between the two habitats in any of the two villages.     

Structure of variation in enzyme activity in natural Drosophila melanogaster populations

Enzyme activity variation was assessed in several isofemale lines originating from two Hungarian Drosophila melanogaster populations. Samples from each population were taken from from two villages; 8-9 isofemale lines were established from each village. The activities of ADH, alphaGPDH, IDH and 6PGDH were determined in the adults (in the F1 generation) and in the larvae (in the F3 generation) as well. Enzyme activities were measured on starch gel after electrophoresis. The activity of the enzyme was detected in a single individual and it was also possible to determine its genotype. The results showed that most of the variation occurred within sites for all four enzymes. This within site variation was more or less equally partitioned into within and between isofemale line (family) components. A smaller portion of variation was attributable to the differences between the populations. Nevertheless, adult alphaGPDH, and larval IDH and 6PGDH activities exhibited significant differences between the two populations. Variation in larval activities of all enzymes was higher than that of the adults, but 6PGDH had considerably higher variation in the adults. The greater variation in larval activities probably reflected the greater environmental variation in the microhabitat of the larvae compared to that of the adults. Larval activities of the investigated enzymes showed much stronger correlation than adult activities. The correlation pattern in the adults differed greatly between the two populations.     

GENETIC VARIATION IN CACTOPHILIC DROSOPHILA FOR OVIPOSITION ON NATURAL YEAST SUBSTRATES

Theory predicts that environmental heterogeneity in space or in time can maintain genetic polymorphism. Stable polymorphisms are expected to be more readily maintained if there are genotype specific habitat preferences. Genotype specific preferences for oviposition sites in Drosophila could be a major factor promoting habitat selection, and thus the maintenance of genetic variation. This hypothesis is being tested using the cactophilic species, D. buzzatii and D. aldrichi, where available evidence indicates a potential for such habitat selection, the habitats (oviposition sites) being yeast species found in the natural environment of these flies (cactus rots). Genetic variation for oviposition preferences was tested using isofemale lines—for D. buzzatii, a total of 60 lines from seven localities widely distributed through the species range in Australia, and for D. aldrichi, 21 lines from three of these localities. Females were given a choice of five yeast species as oviposition sites. Genetic variation for oviposition preferences on these natural substrates was demonstrated. There was significant variation among isofemale lines within populations in their patterns of preferences for oviposition on the five yeast species. However, analyses of preferences for each yeast species separately showed that the genetic variation for preferences relates to only three of the five species. Heritabilities of individual female preferences for these three species were low, ranging up to 9%. Little geographic differentiation was apparent among populations, most likely due to similar selection regimes within each population. Within populations, this kind of habitat selection could act to maintain polymorphisms, both at loci determining the habitat preferences and at other loci in linkage disequilibrium with them.     

Allozyme markers to help define the South American origins of Microctonus hyperodae (Hymenoptera: Braconidae) established in New Zealand for biological control of Argentine stem weevil

The thelytokous parasitoid, Microctonus hyperodae Loan, was collected from eight South American locations and introduced to New Zealand in 1991 for biological control of Argentine stem weevil, Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). Parasitoids from each population were released in equal numbers at each New Zealand site to give them the same opportunities to establish. Population markers have been sought to identify the South American geographic populations that have become most successful in New Zealand. These would assist in determining the importance of concepts such as climate matching and host-parasitoid coevolution to the establishment of natural enemies in new regions for biological control. Vertical polyacrylamide electrophoresis was used to survey 16 enzymes and ten calcium binding proteins, and this paper reports variation at three putative loci. Malate dehydrogenase, a dihydrolipoamide dehydrogenase isozyme and a calcium binding protein exhibited clear genetic variation, each with two alleles. All M. hyperodae isofemale lines from east of the Andes mountains shared one genotype, all but one from west of the Andes shared another, while a population from within the Andes contained both genotypes. This variation was highly congruent with previously described morphometric variation. At two loci, the maintenance of heterozygotes, and the absence of homozygotes, within isofemale lines suggested M. hyperodae thelytoky is apomictic.     

Purging of inbreeding depression and fitness decline in bottlenecked populations of Drosophila melanogaster

Drastic reductions in population size, or bottlenecks, are thought to significantly erode genetic variability and reduce fitness. However, it has been suggested that a population can be purged of the genetic load responsible for reduced fitness when subjected to bottlenecks. To investigate this phenomenon, we put a number of Drosophila melanogaster isofemale lines known to differ in inbreeding depression through four ‘founder‐flush’ bottleneck cycles with flush sizes of 5 or 100 pairs and assayed for relative fitness (single‐pair productivity) after each cycle. Following the founder‐flush phase, the isofemale lines, with a large flush size and a history of inbreeding depression, recovered most of the fitness lost from early inbreeding, consistent with purging. The same isofemale lines, with a small flush size, did not regain fitness, consistent with the greater effect of genetic drift in these isofemale lines. On the other hand, the isofemale lines that did not show initial inbreeding depression declined in fitness after repeated bottlenecks, independent of the flush size. These results suggest that the nature of genetic variation in fitness may greatly influence the way in which populations respond to bottlenecks and that stochastic processes play an important role. Consequently, an attempt intentionally to purge a population of detrimental variation through inbreeding appears to be a risky strategy, particularly in the genetic management of endangered species.     

Enzyme polymorphism inDrosophila melanogaster populations collected in two different habitats in Hungary

The level of enzyme polymorphism was compared in tenDrosophila melanogaster populations collected in farmyards and distilleries in two regions of Hungary. The total genetic diversity was partitioned into between-and within-population components at each investigated locus using Wright's F-statistics. Population differentiation was studied in two different ways. Genetic distances between pairs of populations were calculated and a hierarchical analysis of gene diversity was performed. Based on the F values gene flow was estimated among the populations at different levels of the hierarchy. The results indicated that our farmyard populations collected within a region could be considered as parallel samples from a panmictic population rather than samples of distinct populations. In distilleries, the flies might be influenced by two different evolutionary forces: (i) selection due to the extremely high concentration of ethanol in the fermenting mash and (ii) genetic drift due to the combination of repeated founder effects and fluctuating population size. Our results suggested that distillery populations could not be regarded as real populations either. They could be considered as peculiar cases: founder individuals taken from the total population (region) established special populations which survived in the distilleries for many generations. Thus the dominating force acting on the distillery populations was genetic drift.     

Adaptive diversity in genetic control of egg-laying behavior in Drosophila melanogaster

Summary

In the absence of insemination or of a suitable oviposition site, Drosophila melanogaster females react with egg retention patterns which differ as to the position in the genital tract at which the mechanism operates (ovarioles for initial retention; uterus for oviposition blocking) and to physiological basis. A study of the first laboratory generation of isofemale lines, founded by using three different samples of wild flies collected the same year from a natural population, showed a significant genetic variability in initial egg retention by virgin females and in the capacity for oviposition blocking by mated flies reacting to an unfavorable oviposition site. Comparison of isofemale lines showed that these two adaptive behavior patterns also differ in their genetic determination. They are controlled, and therefore can react, independently to selection pressures resulting from seasonal variation in the natural environment.     

Seasonal fluctuation in susceptibility to insecticides within natural populations of Drosophila melanogaster: empirical observations of fitness costs of insecticide resistance

To investigate genetic variation and seasonal fluctuation in susceptibility to insecticides, natural populations of Drosophila melanogaster were collected from Katsunuma in mid summer and late fall for two consecutive years. After isofemale lines of each population collected in each season had been established in a laboratory, the susceptibility of each line to five insecticides, including permethrin, malathion, prothiophos, fenitrothion, and DDT, was examined. Lines of each population exhibited the broad ranges of variation in susceptibility to all chemicals. Comparison between populations in different seasons indicated that genetic variation in susceptibility to organophosphates fluctuated in consistency with the population size, in which the susceptibility increased in fall. In addition, highly significant correlations were observed among responses to organophosphates, and the correlations also fluctuated with seasons. On the other hand, genetic variation in susceptibility to permethrin and DDT was less fluctuated. These results suggest that not only a common resistance factor for organophosphate resistance but also different resistance factor(s) for each insecticide could be involved within a natural population, and that the fluctuation observed in the susceptibility to organophosphates could be associated with fitness costs of organophosphate resistance factor(s).     

Genotype by environment interactions in viability and developmental time in populations of cactophilic Drosophila

The genetic and ecological basis of viability and developmental time differences between Drosophila buzzatii and D. koepferae were analysed using the isofemale line technique. Several isofemale lines were sampled from pairs of allopatric/sympatric populations of each species. Flies were reared in media prepared with decaying tissues of two of the main natural cactus hosts of each species. This experimental design enabled us to evaluate the relative contribution of phenotypic plasticity, genetic variation and genotype by environment interaction (G x E) to total phenotypic variation for two fitness traits, viability and developmental time. Our results revealed significant G x E in both traits, suggesting that the maintenance of genetic variation can be explained, at least in part, by diversifying selection in different patches of a heterogeneous environment in both species. However, the relative importance of the factors involved in the G x E varied between traits and populations within species. For viability, the G x E can be mainly attributed to changes in the rank order of lines across cacti. However, the pattern was different for developmental time. In D. buzzatii the G x E can be mainly accounted for by changes in among line variance across cacti, whereas changes in the rank order of lines across cacti was the main component in D. koepferae. These dissimilar patterns of variation between traits and species suggest that the evolutionary forces shaping genetic variation for developmental time and viability vary between populations within species and between species.     

Testing for microevolution in body size in three blue tit populations

Quantifying the genetic variation and selection acting on phenotypes is a prerequisite for understanding microevolutionary processes. Surprisingly, long-term comparisons across conspecific populations exposed to different environments are still lacking, hampering evolutionary studies of population differentiation in natural conditions. Here, we present analyses of additive genetic variation and selection using two body-size traits in three blue tit (Parus caeruleus) populations from distinct habitats. Chick tarsus length and body mass at fledging showed substantial levels of genetic variation in the three populations. Estimated heritabilities of body mass increased with habitat quality. The poorer habitats showed weak positive selection on tarsus length, and strong positive selection on body mass, but there was no significant selection on either trait in the good habitat. However, there was no evidence of any microevolutionary response to selection in any population during the study periods. Potential explanations for this absence of a response to selection are discussed, including the effects of spatial heterogeneity associated with gene flow between habitats.     

PCR amplification of intergenic spacers in the ribosomal DNA of Drosophila melanogaster reveals high levels of turnover in length and copy-number of spacers in geographically separated populations

A recently described PCR-based method for the analysis of intergenic spacer (IGS) length variation in the ribosomal (r) DNA of Drosophila melanogaster was used to analyse the distribution of IGS length variants in the rDNA of a number of recently collected D. melanogaster populations. One group of populations, from Europe and North Africa, was shown to have low intrapopulation IGS length variation following maintenance of massed populations in the laboratory for an extended period. However, a greater degree of IGS profile variability was detected at a number of levels in the majority of laboratory-maintained isofemale lines from two of these populations plus a second group of populations which were collected more recently from the eastern coast of Australia; all of which were immediately divided into isofemale lines following collection. Interestingly, PCR analysis of pooled DNA extracts from 30 individuals of either sex showed almost identical PCR profiles from each of the Australian populations. These preliminary results are discussed with regard to the possible combinations of forces (natural selection, neutral drift and genomic molecular drive) on the patterns of IGS length variation.     

Quantitative variation of four morphological traits in Drosophila melanogaster under larval crowding

Effects of three different larval densities (low, intermediate and high) on phenotypic and genetic variation of four morphological traits (thorax and wing length, sternopleural and abdominal bristle number) were studied in Drosophila melanogaster using the isofemale line technique. Phenotypic variation was found to increase at high larval density in all traits examined. Environmental variance for three traits (exception was sternopleural bristle number) and fluctuating asymmetry for both bilateral traits were also increased under high density conditions. For estimates of genetic variability (among isofemale lines variance, heritability and evolvability), no statistically significant differences among density regimes were detected. However, the trends in changes of these estimates across densities indicated a possibility for enhanced genetic variation under larval crowding for all traits except abdominal bristle number. For the latter trait, genetic variation seemed not to be dependent on density regime. Generally, two metric traits (thorax and wing length) were more affected by larval crowding than two meristic ones (sternopleural and abdominal bristle number). The Results are in complete agreement with those previously obtained for D. melanogaster using extreme temperatures as stress-factors.     

Seasonal fluctuation in susceptibility to insecticides within natural populations of Drosophila melanogaster. II. Features of genetic variation in susceptibility to organophosphate insecticides within natural populations of D. melanogaster

To elucidate genetic variation in susceptibility to organophosphate insecticides within natural populations of Drosophila melanogaster, we conducted an analysis of variance for mortality data sets of isofemale lines (10-286 lines) used in the previous studies. Susceptibility of isofemale lines to the three organophosphate insecticides was continuously distributed within each natural population, ranging from susceptible to resistant. Analysis of variance showed highly significant variation among isofemale lines in susceptibility to each insecticide for each natural population. Significant genetic variances in susceptibility to the three chemicals were estimated for the Katsunuma population; 0.0529-0.2722 for malathion, 0.0492-0.1603 for prothiophos, and 0.0469-0.1696 for fenitrothion. Contrary to the consistent seasonal tendency towards an increase in mean susceptibility in the fall, reported in the previous study, genetic variances in susceptibility to the three organophosphates did not change significantly in 1997 but tended to increase by 2- to 5-times in 1998. We tested whether both the observed situations, maintenance and increase in genetic variance in organophosphate resistance, can be generated under circumstances in which the levels of resistance to the three organophosphates tended to decrease, by conducting a simulation analysis, based on the hypothesis that resistant genotypes have lower fitnesses than susceptible ones under the density-independent condition. The simulation analysis generally explained the pattern in the mean susceptibility and genetic variances in susceptibility to the three organophosphates, observed in the Katsunuma population of D. melanogaster. It was suggested that the differences in the frequencies of resistance genes in the summer population could affect the patterns in genetic variance in organophosphate resistance in the fall population.     

GENETIC COMPONENTS OF VARIATION IN ENERGY STORAGE IN DROSOPHILA MELANOGASTER

One approach to examining the underlying genetic structure of the variation in a continuous phenotype is to measure a set of possibly mechanistically related traits and determine the quantitative genetic aspects of their transmission. In this study the quantities of stored triacylglycerol and glycogen were measured along with the activities of 10 enzymes in related metabolic pathways in a set of 1,157 half-sib families of Drosophila melanogaster. The families were structured with each male being mated to 10 females and two offspring were scored from each female. Parents and offspring were scored for the phenotypes, and the components of variance (additive, dominance, and environmental) were estimated in three ways, including analysis of variance on offspring alone, parent-offspring regression, and maximum likelihood methods. While there were differences among the estimates made by the three methods, a consistent result was that substantial additive genetic variation was detected for all the traits. Consistent with models for the quantitative genetics of enzyme kinetics, the genetic variances of global properties were largely additive. Previous studies with extracted chromosome lines had indicated several significant genetic correlations among these characters, and much of the correlation was attributable to additive effects. The results imply that there is substantial opportunity for natural or artificial selection to act on quantities of stored lipid and carbohydrate, and that the response to selection is likely to be in part mediated by changes in the kinetics of the enzymes targeted in this study.     

Environmental heterogeneity and the maintenance of genetic variation for reproductive diapause in Drosophila melanogaster

Drosophila melanogaster has colonized temperate habitats on multiple continents over a historical time period, and many traits vary predictably with latitude. Despite considerable attention paid to clinal variation in Drosophila, the mechanisms generating such patterns in nature remain largely unidentified. In D. melanogaster, the expression of reproductive diapause can be induced by exposure to low temperatures and shortened photoperiods. Both diapause expression itself and the underlying genetic variance for diapause expression have widespread impacts on organismal fitness, and diapause incidence exhibits a 60% cline in frequency in the eastern United States. The major aim of this study was to evaluate whether the relative fitness of diapause and nondiapause genotypes varies predictably with environment. In experimental population cages in the laboratory, the frequency of genotypes that express diapause increased over time when flies were exposed to environmental stress, whereas the frequency of nondiapause genotypes increased when flies were cultured under benign control conditions. Other fitness traits correlated with the genetic variance for diapause expression (longevity, mortality rates, stress resistance, lipid content, preadult viability, fecundity profiles, and development time) also diverged between experimental treatments. Similarly, sampling of isofemale lines from natural populations revealed that the frequency of diapause incidence cycled over time in seasonal habitats: diapause expression was at high frequency following the winter season and subsequently declined throughout the summer months. In contrast, diapause expression was low and temporally homogeneous in isofemale line collections from human-associated urban habitats. These data suggest that genetic variation underlying the diapause-nondiapause dichotomy may be actively maintained by selection pressures that vary spatially and temporally in natural populations.     

Ancestral population reconstitution from isofemale lines as a tool for experimental evolution

Experimental evolution is a powerful tool to study adaptation under controlled conditions. Laboratory natural selection experiments mimic adaptation in the wild with better‐adapted genotypes having more offspring. Because the selected traits are frequently not known, adaptation is typically measured as fitness increase by comparing evolved populations against an unselected reference population maintained in a laboratory environment. With adaptation to the laboratory conditions and genetic drift, however, it is not clear to what extent such comparisons provide unbiased estimates of adaptation. Alternatively, ancestral variation could be preserved in isofemale lines that can be combined to reconstitute the ancestral population. Here, we assess the impact of selection on alleles segregating in newly established Drosophila isofemale lines. We reconstituted two populations from isofemale lines and compared them to two original ancestral populations (AP) founded from the same lines shortly after collection. No significant allele frequency changes could be detected between both AP and simulations showed that drift had a low impact compared to Pool‐Seq‐associated sampling effects. We conclude that laboratory selection on segregating variation in isofemale lines is too weak to have detectable effects, which validates ancestral population reconstitution from isofemale lines as an unbiased approach for measuring adaptation in evolved populations.     

Laboratory Performance Predicts the Success of Field Releases in Inbred Lines of the Egg Parasitoid Trichogramma pretiosum (Hymenoptera: Trichogrammatidae)

In this study we assessed the relationship between the laboratory and field performance of different isofemale lines of Trichogramma pretiosum Riley. In comparative assays, we used three rare mitochondrial haplotypes as genetic markers of the isofemale lines, and by introgressing these mitochondrial haplotypes into each of 15 genetically different nuclear lines, also tested the assumption that mitochondria are neutral markers. In a laboratory trial, 45 isofemale lines (15 nuclear genotypes x three mitochondrial haplotypes) were ranked in three categories (best, intermediate and worst) according to the mean offspring production and the proportion of female offspring. Subsequently, lines from each of the three categories were selected for field releases to quantify field parasitism on Ephestia kuehniella. Temporally separate releases were done in a transgenic Bt cornfield, with four plots, each with 50 points of recapture. The points of recapture consisted of trap cards with eggs of E. kuehniella collected daily. The trap cards were maintained in the laboratory at 25°C until the adult wasps emerged, and the maternal identity of the wasps was determined using qPCR and high-resolution melt curve analysis to determine the mitochondrial haplotype. The results showed that these measures of laboratory performance (fecundity and offspring sex ratio) were good predictors of field success in T. pretiosum. We also report strong evidence discrediting the assumption that mitochondria are neutral, in view of the correlation between performance and mitochondrial haplotype.     

Distribution and biomass of microphytes measured by benthic chlorophyll a in a tropical lagoon (New Caledonia,South Pacific)

Starch-gel electrophoretic techniques were applied to the investigation of molecular genetic variation in populations of the rough periwinkle Littorina saxatilis. The investigation comprised two phases: a) technique development to resolve as many as possible of the allozyme loci reported in the literature as having been screened in the genus Littorina and in Melarhaphe neritoides; b) the use of these loci to assess levels of genetic variation in and patterns of genetic differentiation among populations of L. saxatilis from a relatively isolated group of populations from Galway Bay, Ireland. More than 43 allozyme loci (of which four were screened for the first time here in this species), coding for 37 enzymes, were investigated and thirteen of these loci (including two loci screened for the first time here) were found to be variable and reliably scorable. Samples from five pairs of transects were collected from Inismór, Aran Islands, from sites with known exposure levels; one transect within each pair was collected from an exposed site and the other from a nearby, but relatively sheltered site. UPGMA for eleven loci, (ARK and PGDH were excluded from cluster and FST analysis as they were unscorable in a few samples), showed that the samples cluster mostly by pair, reflecting their geographic origin and is indicative of little gene flow between populations. Levels of population differentiation were high among samples from the top of the shore, but unusually so at AAT-1 which showed nearly three times the mean FST value for the eleven loci. There was also a significant regression of frequency of AAT-1100 against level of exposure. In addition, among midshore samples, there was a consistently higher frequency of AAT-1100 in sheltered habitats. These results support the findings of others, indicating that this locus may be subject to natural selection.     

High quantitative and no molecular differentiation of a freshwater snail (Galba truncatula) between temporary and permanent water habitats

We investigate the variation in quantitative and molecular traits in the freshwater snail Galba truncatula, from permanent and temporary water habitats. Using a common garden experiment, we measured 20 quantitative traits and molecular variation using seven microsatellites in 17 populations belonging to these two habitats. We estimated trait means in each habitat. We also estimated the distributions of overall genetic quantitative variation (QST), and of molecular variation (FST), within and between habitats. Overall, we observed a lack of association between molecular and quantitative variance. Among habitats, we found QST>FST, an indication of selection for different optima. Individuals from temporary water habitat matured older, at a larger size and were less fecund than individuals from permanent water habitat. We discuss these findings in the light of several theories for life-history traits evolution.     

Variation in the male pheromones and mating success of wild caught Drosophila melanogaster

Drosophila melanogaster males express two primary cuticular hydrocarbons (male-predominant hydrocarbons). These act as sex pheromones by influencing female receptivity to mating. The relative quantities of these hydrocarbons vary widely among natural populations and can contribute to variation in mating success. We tested four isofemale lines collected from a wild population to assess the effect of intrapopulation variation in male-predominant hydrocarbons on mating success. The receptivity of laboratory females to males of the four wild-caught lines varied significantly, but not consistently in the direction predicted by variation in male-predominant hydrocarbons. Receptivity of the wild-caught females to laboratory males also varied significantly, but females from lines with male-predominant hydrocarbon profiles closer to a more cosmopolitan one did not show a correspondingly strong mating bias toward a cosmopolitan male. Among wild-caught lines, the male-specific ejaculatory bulb lipid, cis-vaccenyl acetate, varied more than two-fold, but was not associated with variation in male mating success. We observed a strong inverse relationship between the receptivity of wild-caught females and the mating success of males from their own lines, when tested with laboratory flies of the opposite sex.