Latitudinal clines in heat tolerance, protein synthesis rate and transcript level of a candidate gene in Drosophila melanogaster.

The occurrence of climatic adaptation in Drosophila melanogaster is highlighted by the presence of latitudinal clines in several quantitative traits, particularly clines in adult heat knockdown tolerance that is higher in tropical populations. However the presence of latitudinal patterns in physiological characteristics that may underlie these traits have rarely been assessed. Protein synthesis has been implicated as an important physiological process that influences thermal tolerance, and this has not been examined in a clinal context. Here, we characterise latitudinal variation in D. melanogaster from eastern Australia in both adult heat knockdown tolerance and rates of protein synthesis following rearing at both 25 °C, approximating summer conditions, and 18 °C approximating winter development. We also examined clinal variation in the predominant nuclear transcript of the heat-inducible RNA gene hsr-omega, which has been implicated in regulating protein synthesis. We find significant clines in heat-hardened tolerance when cultured at both 18 and 25 °C - tolerance increased towards the low latitude tropics. Rates of protein synthesis measured in ovarian tissue also associated negatively with latitude, however the presence of the clines depended on rearing temperature and heat stress conditions. Finally, omega-n levels measured without heat stress showed a positive linear cline. When measured after a mild heat stress higher levels of omega-n were detected and the clinal pattern became parabolic - mid-latitude populations had lower levels of the transcript. While congruent latitudinal trends were detected for these three traits, only a low level of positive association was detected between protein synthesis and thermal tolerance providing little evidence that these traits are related at the level of cellular physiology. However the new clinal patterns of protein synthesis and hsr-omega variation suggest that these variables exert important influences on traits involved with latitudinal climatic adaptation.

Transcripts from the Drosophila heat-shock gene hsr-omega influence rates of protein synthesis but hardly affect resistance to heat knockdown.

While hundreds of genes have recently been implicated in an organism's response to thermal stress, our insight into the cellular and physiological mechanisms affected by these genes has advanced to a lesser extent. We focus on an enigmatic Drosophila heat stress RNA gene, hsr-omega, which encodes two RNA transcripts that are constitutively expressed in almost all developing and adult tissues, omega-n in the nucleus and omega-c in the cytoplasm; both being readily induced to high levels by mild heat stress. We derived three hsr-omega mutant lines via imprecise P-element excision and characterised them for changes in expression, in both the presence and absence of heat stress. Viability estimates indicate that a low level of omega-n is required for normal development. Consistent with the model of omega-n as a negative regulator of intron-processed mRNA levels the mutants displayed a 1.5-fold increase in rates of protein synthesis measured in ovarian tissue in the absence of heat stress, a result suggesting that an important function of hsr-omega is the modulation of general protein synthesis. The mutants had little effect on two measures commonly used to assess heat tolerance, heat-knockdown time and heat hardening ability, suggesting that more subtle heat-related fitness components need to be examined for effects of these mutations.

Genetic structure, behaviour and invasion history of the Argentine ant supercolony in Australia.

Biological invasions have significant ecological, evolutionary and economic consequences. Ants are exemplary invaders and their invasion success is frequently attributed to a shift in social structure between native and introduced populations. Here, we use a multidisciplinary approach to determine the social structure, origin and expansion of the invasive Argentine ant, Linepithema humile, in Australia by linking behavioural and genetic studies with indicators of dispersal pathways and propagule pressure. Behavioural assays revealed a complete absence of aggression within and between three cities - Melbourne, Adelaide and Perth - spanning 2700 km across Australia. Microsatellite analyses showed intracity genetic homogeneity and limited but significant intercity genetic differentiation. Exceptions were two Perth nests that likely represent independent translocations from Adelaide. These patterns suggest efficient local gene flow with more limited jump dispersal via transport corridors between cities. Microsatellite analyses of L. humile from potential source regions, combined with data from port interceptions, trade pathways and the timeline of spread within Australia, implicate the main European supercolony as the source of L. humile in Melbourne. Such an introduction probably then redistributed across Australia and spread to New Zealand to form an expansive Australasian supercolony.

Protein synthesis rates in Drosophila associate with levels of the hsr-omega nuclear transcript.

Transcripts of the Drosophila hsr-omega gene are known to interact with RNA processing factors and ribosomes and are postulated to aid in co-ordinating nuclear and cytoplasmic activities particularly in stressed cells. However, the significance of these interactions for physiological processes and in turn for whole-organism fitness remains an open question. Because hsr-omega's cellular expression characteristics suggest it may influence protein synthesis, and because both genotypic and expression variation of hsr-omega have been associated with thermotolerance, we characterised 30 lines for variation in the rates of protein synthesis, measured in ovarian tissues, both before and after a mild heat shock, and for basal levels of the two main hsr-omega transcripts, omega-n and omega-c. As expected, the mild heat shock reduced protein synthesis rates. Large variation occurred among lines in levels of omega-n which was negatively associated with rates of basal protein synthesis--a result that supports the model for the cellular function of omega-n. Furthermore, omega-n levels were associated with hsr-omega genotype of the line parents. Little variation occurred among lines for omega-c levels and no associations were detected with protein synthesis or genotype. Since protein synthesis is a fundamental process for growth and development, we characterised the lines for several life-history traits; however, no associations with protein synthesis, omega-n or omega-c levels were detected. Our results are consistent with the idea that natural variation in hsr-omega expression influence rates of protein synthesis in this species.

Candidate genes and thermal phenotypes: identifying ecologically important genetic variation for thermotolerance in the Australian Drosophila melanogaster cline.

Clinal variation in traits often reflects climatic adaptation; in Drosophila melanogaster clinal variation provides an opportunity to link variation in chromosomal inversions, microsatellite loci and various candidate genes to adaptive variation in traits. We undertook association studies with crosses from a single population of D. melanogaster from eastern Australia to investigate the association between genetic markers and traits showing clinal variation. By genotyping parents and phenotyping offspring, we minimized genotyping costs but had the power to detect association between markers and quantitative traits. Consistent with prior studies, we found strong associations between the clinal chromosomal inversion In(3R)Payne and markers within it, as well as among these markers. We also found an association between In(3L)Payne and one marker located within this inversion. Of the five predicted associations between markers and traits, four were detected (increased heat, decreased cold resistance and body size with the heat shock gene hsr-omega S, increased cold resistance with the inversion In(3L)Payne), while one was not detected (heat resistance and the heat shock gene hsp68). In a set of eight exploratory tests, we detected one positive association (between hsp23a and heat resistance) but no associations of heat resistance with alleles at the hsp26, hsp83, Desat 2, alpha-Gpdh, hsp70 loci, while cold resistance was not associated with Frost and Dca loci. These results confirm interactions between hsr-omega and thermal resistance, as well as between In(3L)Payne and cold resistance, but do not provide evidence for associations between thermal responses and alleles at other clinically varying marker genes.

An independent non-linear latitudinal cline for the sn-glycerol-3-phosphate (alpha- Gpdh ) polymorphism of Drosophila melanogaster from eastern Australia.

Latitudinal variation of the polymorphic sn-glycerol-3-phosphate (alpha-Gpdh) locus in Drosophila melanogaster has been characterized on several continents; however, apparent clinal patterns are potentially confounded by linkage with an inversion, close associations with other genetic markers that vary clinally, and a tandem alpha-Gpdh pseudogene. Here we compare clinal patterns in alpha-Gpdh with those of other linked markers by testing field flies from eastern Australian locations collected in two separate years. The alpha-Gpdh variation exhibited a consistent non-linear cline reflecting an increase in the alpha-GpdhF allele at extreme latitudes. This pattern was not influenced by the In(2L)t inversion wherein this locus is located, nor was it influenced by the presence of the alpha-Gpdh pseudogene, whose presence was ubiquitous and highly variable among populations. The alpha-Gpdh pattern was also independent of a cline in allozyme frequencies at the alcohol dehydrogenase (Adh) locus, and two length polymorphisms in the Adh gene. These results suggest clinal selection at the alpha-Gpdh locus that is partially or wholly unrelated to linear climatic gradients along the eastern coast of Australia.

Response to selection for rapid chill-coma recovery in Drosophila melanogaster: physiology and life-history traits.

Resistance to low temperatures can vary markedly among invertebrate species and is directly related to their distribution. Despite the ecological importance of cold resistance this trait has rarely been studied genetically, mainly because low and variable fitness of offspring from cold-stressed mothers makes it difficult to undertake selection experiments and compare cold resistance of parents and offspring. One measure of cold resistance that varies geographically in Drosophila melanogaster and that is amenable to genetic analysis is chill-coma recovery. Three replicate lines of D. melanogaster were selected every second generation, for over 30 generations, for decreased recovery time following exposure to 0 degrees C. Correlated responses were scored to characterize underlying physiological traits and to investigate interactions with other traits. Lines responded rapidly to the intermittent selection regime with realized heritabilities varying from 33% to 46%. Selected lines showed decreased recovery time after exposure to a broad range of low temperatures and also had a lower mortality following a more severe cold shock, indicating that a general mechanism underlying cold resistance had been selected. The selection response was independent of plastic changes in cold resistance because the selected lines maintained their ability to harden (i.e. a short-term exposure to cool temperature resulted in decreased recovery time in subsequent chill-coma assays). Changes in cold resistance were not associated with changes in resistance to high temperature exposure, and selected lines showed no changes in wing size, development time or viability. However, there was a decrease in longevity in the selected lines due to an earlier onset of ageing. These results indicate that chill-coma recovery can be rapidly altered by selection, as long as selection is undertaken every second generation to avoid carry-over effects, and suggest that lower thermal limits can be shifted towards increased cold resistance independently of upper thermal limits and without tradeoffs in many life-history traits.

A cluster of diagnostic Hsp68 amino acid sites that are identified in Drosophila from the melanogaster species group are concentrated around beta-sheet residues involved with substrate binding.

The coding region of the hsp68 gene has been amplified, cloned, and sequenced from 10 Drosophila species, 5 from the melanogaster subgroup and 5 from the montium subgroup. When the predicted amino acid sequences are compared with available Hsp70 sequences, patterns of conservation suggest that the C-terminal region should be subdivided according to predominant secondary structure. Conservation levels between Hsp68 and Hsp70 proteins were high in the N-terminal ATPase and adjacent beta-sheet domains, medium in the alpha-helix domain, and low in the C-terminal mobile domain (78%, 72%, 41%, and 21% identity, respectively). A number of amino acid sites were found to be "diagnostic" for Hsp68 (28 of approximately 635 residues). A few of these occur in the ATPase domain (385 residues) but most (75%) are concentrated in the beta-sheet and alpha-helix domains (34% of the protein) with none in the short mobile domain. Five of the diagnostic sites in the beta-sheet domain are clustered around, but not coincident with, functional sites known to be involved in substrate binding. Nearly all of the Hsp70 family length variation occurs in the mobile domain. Within montium subgroup species, 2 nearly identical hsp68 PCR products that differed in length are either different alleles or products of an ancestral hsp68 duplication.

The latitudinal cline in the In(3R)Payne inversion polymorphism has shifted in the last 20 years in Australian Drosophila melanogaster populations.

Clinal variation has been described in a number of inversions in Drosophila but these clines are often characterized by cytological techniques using small sample sizes, and associations with specific genes are rarely considered. Here we have developed a molecular assay for In(3R)Payne in Drosophila melanogaster from eastern Australia populations. It shows in repeated samples that the inversion cline is very tightly associated with latitude and is almost fixed in tropical populations while relatively rare in temperate populations. This steep cline has shifted in position in the last 20 years. The heat shock gene, hsr-omega, located centrally inside the inversion sequence, shows a different clinal pattern to In(3R)Payne. These results suggest strong ongoing selection on In(3R)Payne over the last 100 years since the colonization of Australia that is partly independent of hsr-omega.

Immunocontraception for population control: will resistance evolve?

The prospect for successful biocontrol using immunocontraception is threatened if there is adaptation to the vaccine through natural selection of individuals that are genetically resistant to the contraceptive agent. To assess this possibility we examined the literature and found that little relevant data are available for any species on the appropriate trait, fertility variation among immunized individuals, or about appropriate population and genetic parameters influencing the likelihood of a selection response. Some data are available on variation in antibody response to immunocontraceptives, but the relationship between antibody response and fertility levels is poorly documented. The antibody response data indicate low heritability for this trait suggesting that fertility levels of contraceptive-resistant individuals will also have a low heritability. Slow evolution of contraception resistance might therefore be anticipated. The absence of information about relevant parameters makes the construction of quantitative models premature. We discuss factors in particular need of investigation if predictions about resistance evolution are to be made. These include: 1. the genetic basis of fertility retention, 2. the proportion of the population resistant to the contraceptive agent and how this is affected by gene flow from refuge populations, 3. the genetically-based fitness tradeoffs of resistant individuals that often accompany selection, 4. cross-generation effects that can thwart the effects of selection, and 5. the efficiency of delivery of the contraceptive agent. An understanding of the above for particular species, and the development of appropriate divergently acting multiple vaccines that can be used in temporal rotation or in mixtures, should facilitate the development of management options to minimize resistance evolution.

HERITABLE VARIATION IN RESOURCE UTILIZATION AND RESPONSE IN A WINERY POPULATION OF DROSOPHILA MELANOGASTER.

It has been found that Drosophila melanogaster lines from the "Chateau Tahbilk" winery cellar had higher larval ethanol resistance than lines originating from outside the cellar. Because the adaptive significance of this trait is unclear, we have reexamined genetic microdifferentiation at Tahbilk with other resources and different tests for ethanol adaptation. Cellar stocks tended to be more resistant to starvation after exposure to wine seepage than stocks originating from orchard traps outside the cellar. Lines from a grape residue pile were also more resistant to starvation after seepage exposure than orchard stocks even though these collection sites were a few meters apart. Cellar and orchard stocks did not differ in ethanol resistance as measured by larval viability tests on low sucrose medium. However, stocks from the grape residue pile showed an increase in adult longevity over ethanol vapor compared to those from the cellar or orchard stocks. These differences were not due to maternal effects. In laboratory tests of behavioral responses, cellar stocks were relatively more attracted to seepage than orchard stocks in one year but not in the other two years. The findings suggest some adaptive differentiation related to resource heterogeneity at Tahbilk.

HERITABLE VARIATION IN ETHANOL TOLERANCE AND ITS ASSOCIATION WITH BIOCHEMICAL TRAITS IN DROSOPHILA MELANOGASTER.

To help elucidate mechanisms of larval ethanol tolerance seven isochromosomal lines of Drosophila melanogaster with different second chromosomes were fed a growth-limiting concentration of ethanol (4.5% v/v) and examined for associations between growth traits and biochemical characteristics that had previously been implicated in the determination of tolerance variation. Repeated measures of survival and development time over four generations verified the inherited nature of these traits. Significant variation among the lines were evident for flux from ethanol into lipid, for activity levels of alcohol dehydrogenase and glycerol-3-phosphate oxidase (GPO), and for levels of long chain and unsaturated fatty acids. A high degree of positive association occurred among the variables. A partial correlation analysis controlling for performance of the lines on ethanol-free medium revealed a strong association between the degree of long chain fatty acid content and line survival when ethanol was fed. The correlation between GPO activity and survival in an ethanol environment appeared to depend on the association of GPO activity with long chain fatty acid content. The positive correlations of flux from ethanol into lipid with many of the other variables suggested that the ADH pathway influenced the level of ethanol tolerance. These associations are all consistent with the hypothesis that the lipid content of body tissues, especially the levels of long chain and unsaturated fatty acids in cell membranes, may have an important influence on both spatial and interspecific variation in the ethanol tolerance of larvae.