Latitudinal clines in the timing and temperature-sensitivity of photoperiodic reproductive diapause in Drosophila montana

Reproductive diapause is a primary mechanism used by arthropods to synchronize their life cycle with seasonal changes in temperate regions. Our study species, Drosophila montana, represents the northern insect species where flies enter reproductive diapause under short day conditions and where the precise timing of diapause is crucial for both survival and offspring production. We have studied clinal variation in the critical day length for female diapause induction (CDL) and their overall susceptibility to enter diapause (diapause incidence), as well as the temperature sensitivity of these traits. The study was performed using multiple strains from four latitudinal clines of the species – short clines in Finland and Alaska and long clines in the Rocky Mountains and the western coast of North America – and from one population in Kamchatka, Russia. CDL showed strong latitudinal clines on both continents, decreasing by one hour per five degrees decline in latitude, on average. CDL also decreased in all populations along with an increase in fly rearing temperature postponing the diapause to later calendar time, the effects of temperature being stronger in southern than in northern population. Female diapause incidence was close to 100% under short day/low temperature conditions in all populations, but decreased below 50% even under short days in 19°C in the southern North American western coast populations and in 22°C in most populations. Comparing a diversity of climatic data for the studied populations showed that while CDL is under a tight photoperiodic regulation linked with latitude, its length depends also on climatic factors determining the growing season length. Overall, the study deepens our understanding of how spatial and environmental parameters affect the seasonal timing of an important biological event, reproductive diapause and helps to estimate the evolutionary potential of insect populations to survive in changing climatic conditions.     

The influence of temperature and photoperiod on the reproductive diapause and cold tolerance of spotted‐wing drosophila,Drosophila suzukii

Knowledge regarding the reproductive status of spotted‐wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is of critical importance in predicting potential infestations of this invasive pest, as eggs are laid in ripe or ripening fruit of several commercially important small‐fruit crops. Token environmental stimuli for the induction of reproductive diapause and improved cold tolerance were identified for D. suzukii. Diapause induction was evaluated by assessing, via dissection, the number of mature eggs carried by field‐captured D. suzukii and laboratory‐reared D. suzukii held under various temperature and photoperiod regimes. Egg load decreased over time in females trapped from July to December at sites in Benton County, OR, and Ontario County, NY, both USA, and reached zero eggs by December at all sites. Photoperiods below 14 h of day length led to reduced egg maturation in laboratory‐reared flies held at moderate temperatures (15 or 20 °C). Whereas very few mature eggs were found in females held at 10 °C under short‐ or long‐day photoperiods for several weeks after eclosion, a spontaneous return to ovarian maturity was observed in short‐day‐entrained females after 7 weeks. Diapause termination was investigated by evaluating fecundity in diapausing females returned to optimal environmental conditions. Whereas long‐day‐entrained flies began producing offspring immediately upon return to optimal conditions, short‐day‐entrained flies returned after 1 and 6 weeks at 10 °C were slower to produce offspring than colony flies or short‐day‐entrained flies returned after 7 weeks. Cold tolerance was evaluated by observing chill coma recovery rates after 24 h exposure to ?1 °C. Cold‐acclimated (diapausing) females recovered from chill coma faster than cold‐hardened or unacclimated females.     

Photoperiodic regulation of cold tolerance and expression levels of regucalcin gene in Drosophila montana

Temperature-induced plasticity of cold tolerance has been reported in many insect species, but cold tolerance can also be affected by changes in day (or night) length. In the present study we elucidate the direct and indirect effects of photoperiod on the cold tolerance of females of two Drosophila montana strains--one which possesses a robust photoperiodic diapause and another which does not. In the diapause-strain the time needed for recovery from chill coma showed a positive correlation with day length, but diapause itself played only a minor role in photoperiodic acclimation. The strain that was not able to enter to diapause as a response to day length also lacked photoperiodic cold acclimation ability indicating that this strain has deficiencies in its photoperiodic time measurement system. In the diapause-strain, the expression level of regucalcin gene was more than two times higher in diapausing than in non-diapausing females maintained in a single photoperiod, but day length per se did not cause significant changes in expression levels of this gene in either of the strains. In the non-diapausing strain this gene showed no expression changes in any comparison. Overall, the study shows that a decrease in day length can induce cold acclimation in D. montana, while changes in regucalcin expression are linked with photoperiodic diapause.     

Circadian clock of Drosophila montana is adapted to high variation in summer day lengths and temperatures prevailing at high latitudes

Photoperiodic regulation of the circadian rhythms in insect locomotor activity has been studied in several species, but seasonal entrainment of these rhythms is still poorly understood. We have traced the entrainment of activity rhythm of northern Drosophila montana flies in a climate chamber mimicking the photoperiods and day and night temperatures that the flies encounter in northern Finland during the summer. The experiment was started by transferring freshly emerged females into the chamber in early and late summer conditions to obtain both non-diapausing and diapausing females for the studies. The locomotor activity of the females and daily changes in the expression levels of two core circadian clock genes, timeless and period, in their heads were measured at different times of summer. The study revealed several features in fly rhythmicity that are likely to help the flies to cope with high variation in the day length and temperature typical to northern summers. First, both the non-diapausing and the diapausing females showed evening activity, which decreased towards the short day length as observed in the autumn in nature. Second, timeless and period genes showed concordant daily oscillations and seasonal shifts in their expression level in both types of females. Contrary to Drosophila melanogaster, oscillation profiles of these genes were similar to each other in all conditions, including the extremely long days in early summer and the cool temperatures in late summer, and their peak expression levels were not locked to lights-off transition in any photoperiod. Third, the diapausing females were less active than the non-diapausing ones, in spite of their younger age. Overall, the study showed that D. montana clock functions well under long day conditions, and that both the photoperiod and the daily temperature cycles are important zeitgebers for seasonal changes in the circadian rhythm of this species.     

Localization of quantitative trait loci for diapause and other photoperiodically regulated life history traits important in adaptation to seasonally varying environments

Seasonally changing environments at high latitudes present great challenges for the reproduction and survival of insects, and photoperiodic cues play an important role in helping them to synchronize their life cycle with prevalent and forthcoming conditions. We have mapped quantitative trait loci (QTL) responsible for the photoperiodic regulation of four life history traits, female reproductive diapause, cold tolerance, egg‐to‐eclosion development time and juvenile body weight in Drosophila montana strains from different latitudes in Canada and Finland. The F2 progeny of the cross was reared under a single photoperiod (LD cycle 16:8), which the flies from the Canadian population interpret as early summer and the flies from the Finnish population as late summer. The analysis revealed a unique QTL for diapause induction on the X chromosome and several QTL for this and the other measured traits on the 4th chromosome. Flies’ cold tolerance, egg‐to‐eclosion development time and juvenile body weight had several QTL also on the 2nd, 3rd and 5th chromosome, some of the peaks overlapping with each other. These results suggest that while the downstream output of females’ photoperiodic diapause response is partly under a different genetic control from that of the other traits in the given day length, all traits also share some QTL, possibly involving genes with pleiotropic effects and/or multiple tightly linked genes. Nonoverlapping QTL detected for some of the traits also suggest that the traits are potentially capable of independent evolution, even though this may be restricted by epistatic interactions and/or correlations and trade‐offs between the traits.     

Adaptation to a seasonally varying environment: a strong latitudinal cline in reproductive diapause combined with high gene flow in Drosophila montana

Adaptation to seasonal changes in the northern hemisphere includes an ability to predict the forthcoming cold season from gradual changes in environmental cues early enough to prepare for the harsh winter conditions. The magnitude and speed of changes in these cues vary between the latitudes, which induces strong selection pressures for local adaptation.We studied adaptation to seasonal changes in Drosophila montana, a northern maltfly, by defining the photoperiodic conditions leading to adult reproductive diapause along a latitudinal cline in Finland and by measuring genetic differentiation and the amount of gene flow between the sampling sites with microsatellites. Our data revealed a clear correlation between the latitude and the critical day length (CDL), in which half of the females of different cline populations enter photoperiodic reproductive diapause. There was no sign of limited gene flow between the cline populations, even though these populations showed isolation by distance. Our results show that local adaptation may occur even in the presence of high gene flow, when selection for locally adaptive life-history traits is strong. A wide range of variation in the CDLs of the fly strains within and between the cline populations may be partly due to gene flow and partly due to the opposing selection pressures for fly reproduction and overwinter survival. This variation in the timing of diapause will enhance populations' survival over the years that differ in the severity of the winter and in the length of the warm period and may also help them respond to long-term changes in environmental conditions.     

Evolution of thermotolerance in seasonal environments: the effects of annual temperature variation and life-history timing in Wyeomyia smithii

In organisms with complex life cycles, the adaptive value of thermotolerance depends on life-history timing and seasonal temperature profiles. We illustrate this concept by examining variation in annual thermal environments and thermal acclimation among four geographic populations of the pitcher plant mosquito. Only diapausing larvae experience winter, whereas both postdiapause and nondiapause adults occur only during the growing season. Thus, adults experience transient cold stress primarily during the spring. We show that adult cold tolerance (chill coma recovery) is enhanced in spring-like conditions via thermal acclimation but is unaffected by diapause state. Moreover, adult mosquitoes from northern populations were more cold tolerant than those from southern populations largely because acclimation responses were steeper in the north. In contrast to cold tolerance, there was no significant acclimation of heat tolerance (heat knockdown), and no significant differences in heat tolerance between northern and southern populations. Field temperature data show that because of evolved differences in diapause timing, adult exposure to cold stress is remarkably consistent across geography. This suggests that geographic variation in cold tolerance may not be the result of direct selection on adults. Our results illustrate the importance of the interplay between phenological and thermal adaptation for understanding variation along climatic gradients.     

Evolution of pre- and post-copulatory traits in female Drosophila melanogaster as a correlated response to selection for resistance to cold stress

Exposure to low temperatures reduces gamete viability and fecundity in females of insect species like Drosophila. Hence, adaptation to cold stress can in principle involve modifications in reproductive traits in females. Studies on resistance to cold stress have mostly addressed the evolution of adult survivorship post cold shock. Very few studies have addressed the evolution of reproductive traits in females in response to cold stress. We have successfully selected replicate populations of Drosophila melanogaster for resistance to cold shock. After 50 generations of selection, we investigated pre- and post-copulatory traits i.e. mating latency, copulation duration, mating frequency and progeny production in female flies exposed to cold shock or control conditions. Post cold shock, females from the selected populations were better at recovery in terms of mating latency, mating success, and progeny production relative to females from the control populations. Performance of the two types of females was not different under control conditions. These findings clearly indicate that adaptation to cold stress involves rapid modification of the reproductive traits.     

Seasonal variation in basal and plastic cold tolerance: Adaptation is influenced by both long‐ and short‐term phenotypic plasticity

Understanding how thermal selection affects phenotypic distributions across different time scales will allow us to predict the effect of climate change on the fitness of ectotherms. We tested how seasonal temperature variation affects basal levels of cold tolerance and two types of phenotypic plasticity in Drosophila melanogaster. Developmental acclimation occurs as developmental stages of an organism are exposed to seasonal changes in temperature and its effect is irreversible, while reversible short‐term acclimation occurs daily in response to diurnal changes in temperature. We collected wild flies from a temperate population across seasons and measured two cold tolerance metrics (chill‐coma recovery and cold stress survival) and their responses to developmental and short‐term acclimation. Chill‐coma recovery responded to seasonal shifts in temperature, and phenotypic plasticity following both short‐term and developmental acclimation improved cold tolerance. This improvement indicated that both types of plasticity are adaptive, and that plasticity can compensate for genetic variation in basal cold tolerance during warmer parts of the season when flies tend to be less cold tolerant. We also observed a significantly stronger trade‐off between basal cold tolerance and short‐term acclimation during warmer months. For the longer‐term developmental acclimation, a trade‐off persisted regardless of season. A relationship between the two types of plasticity may provide additional insight into why some measures of thermal tolerance are more sensitive to seasonal variation than others.     

Geographic Variation of Photoperiodic Diapause but Not Adult Survival or Reproduction of the Invasive Mosquito Aedes albopictus (Diptera: Culicidae) in North America

Climate differences across latitude can result in seasonal constraints and selection on life-history characters. Because Aedes albopictus (Skuse) invaded North America in the mid-1980s, it has spread across a range of ≈14° latitude and populations in the north experience complete adult mortality because of cold winter temperatures that are absent in the south. Life-table experiments were conducted to test for differences in the adult survival and reproductive schedules of Ae. albopictus females from three populations from the northern (Salem, NJ; Springfield, IL; Eureka, MO; ≈39° N) and southern (Palm Beach, Palmetto, Tampa, FL; ≈27-28° N) extremes of the species distribution in North America. There were consistent differences between northern and southern populations in incidence of photoperiodically-induced egg diapause. Under short daylength, diapause eggs constituted twice the proportion of total viable eggs from northern females (81.9-92.1%) than southern females (35.9-42.7%). There were no consistent differences between northern and southern populations in resource allocation between reproduction and maintenance, reproduction over time, and reproductive investment among offspring, and no apparent trade-offs between diapause incidence with reproduction or longevity. Our results suggest that the main response of North American Ae. albopictus to unfavorable winter climates is via the life history strategy of producing diapausing eggs, rather than quantitative variation in reproduction, and that there are no detectable costs to adult survival. Inherent geographic variation in the expression of diapause, consistent with the latitudinal extremes of A. albopictus, indicates evolutionary loss of diapause response in southern populations because of the invasion of A. albopictus in North America.     

Seasonal trade-off between starvation resistance and cold resistance in temperate wild-caught Drosophila simulans

Abstract  Selection experiments with Drosophila have revealed a possible evolutionary trade-off between cold resistance and starvation resistance that may be controlled by lipid metabolism. To test this trade-off in naturally occurring Drosophila simulans populations, flies were simultaneously collected from two temperate locales experiencing contrasting seasons. Flies from a tropical locale served as a control. Cold coma recovery, starvation resistance and lipid proportion were assayed on adult males and females from each locale. Compared with the summer-collected flies from Canberra, the winter-collected flies from San Diego recovered from cold coma more quickly, were less starvation resistant and had lower lipid levels. These results support an evolutionary trade-off between cold resistance and starvation resistance. Combined, these data also suggest that differences in lipid metabolism may be an underlying mechanism for this trade-off.     

Population dependent effects of photoperiod on diapause related physiological traits in an invasive beetle (Leptinotarsa decemlineata)

Organisms undergoing latitudinal range expansion face a change in the photoperiod which can lead to a mismatch between the timing of seasonal changes in physiological and life history traits with seasonal environmental changes. This mismatch can lead to lowered survival, for example, due to unsynchronized diapause timing. Successful range expansion even in recent introductions requires that organisms which use the photoperiod for seasonal predictions should show interpopulational differences in photoperiodic responses at different latitudes, as the photoperiod is a function of latitude. We investigated among population differences in photoperiodic responses of life history and physiological traits linked to diapause in the invasive beetle Leptinotarsa decemlineata. Beetles from a northern marginal and a southern European population were reared under short day (12:12L:D) and long day (18:6L:D) photoperiods. Both populations reacted similarly to the short day photoperiod. Their abdominal total lipid content increased and water content decreased which suggests that the beetles prepared for diapause. This was also indicated by low mortality during diapause. In the long day photoperiod large interpopulational differences were found, the southern population ceased lipid accumulation after 5 days, while the northern population continued lipid accumulation as beetles in the short day photoperiod. This indicates that the northern population has a longer critical photoperiod than the southern one. Abdominal total lipid stores in 10 day old beetles were shown to be predominantly composed of neutral lipids (85%), most likely representing storage triacylglycerols. Fatty acid profiles of both the neutral lipids and the phospholipids showed large shifts during the first 10 day of adult life, predominantly in the fractions of 18:0, 18:1ω9, 18:2ω6 and 18:3ω3. Although the degree of unsaturation increased with age, it was not higher in diapausing than non-diapausing beetles. This indicates that this species does not increase diapause related cold tolerance via homeoviscous adaptation, and might have developed other means to cope with suboptimal temperatures, such as behavioral adaptations.     

Dissecting chill coma recovery as a measure of cold resistance: evidence for a biphasic response in Drosophila melanogaster

Cold resistance in insects has traditionally been measured in terms of survival following a stress, but alternative methods are increasingly being used because of their relevance to the ecology of organisms and their utility in characterizing variation among species, populations and individuals. One such method capable of discriminating among Drosophila species and conspecific Drosophila populations from different environments is adult chill coma recovery time, the time taken for adults to become active again after being knocked down by a cold stress. Here we characterized the chill coma response of D.melanogaster in detail. Adults were exposed to a range of temperatures and stressful periods prior to measuring recovery. Recovery from chill coma in D.melanogaster was biphasic; as flies were stressed under cooler temperatures, recovery times leveled off and then decreased before sharply increasing again as mortality starts to occur. This biphasic response has previously been observed in D.subobscura where it has a somewhat different shape. A second mechanism therefore acts at relatively lower temperatures to ameliorate the effects of the cold stress. When D.melanogaster were reared at 19 and 25 °C for two generations, the shape of the curve relating temperature to recovery time was similar, but flies from the warmer temperature had longer recovery times and showed responses that leveled off and then decreased at relatively higher temperatures. As exposure time to cold stress was increased, recovery times also increased except at mild stress levels. Chill coma recovery in D.melanogaster is a complex trait and likely to reflect multiple underlying components.     

The effect of selection for desiccation resistance on cold tolerance of Drosophila melanogaster

Abstract.  Low temperature and desiccation stress are thought to be mechanistically similar in insects, and several studies indicate that there is a degree of cross-tolerance between them, such that increased cold tolerance results in greater desiccation tolerance and vice versa . This assertion is tested at an evolutionary scale by examining basal cold tolerance, rapid cold-hardening (RCH) and chill coma recovery in replicate populations of Drosophila melanogaster selected for desiccation resistance (with controls for both selection and concomitant starvation) for over 50 generations. All of the populations display a RCH response, and there is no effect of selection regime on RCH or basal cold tolerance, although there are differences in basal cold tolerance between sampling dates, apparently related to inter-individual variation in development time. Flies selected for desiccation tolerance recover from chill coma slightly, but significantly, faster than control and starvation-control flies. These findings provide little support for cross-tolerance between survival of near-lethal cold and desiccation stress in D. melanogaster .     

Phenotypic plasticity,but not adaptive tracking,underlies seasonal variation in post‐cold hardening freeze tolerance of Drosophila melanogaster

In temperate regions, an organism's ability to rapidly adapt to seasonally varying environments is essential for its survival. In response to seasonal changes in selection pressure caused by variation in temperature, humidity, and food availability, some organisms exhibit plastic changes in phenotype. In other cases, seasonal variation in selection pressure can rapidly increase the frequency of genotypes that offer survival or reproductive advantages under the current conditions. Little is known about the relative influences of plastic and genetic changes in short‐lived organisms experiencing seasonal environmental fluctuations. Cold hardening is a seasonally relevant plastic response in which exposure to cool, but nonlethal, temperatures significantly increases the organism's ability to later survive at freezing temperatures. In the present study, we demonstrate seasonal variation in cold hardening in Drosophila melanogaster and test the extent to which plasticity and adaptive tracking underlie that seasonal variation. We measured the post‐cold hardening freeze tolerance of flies from outdoor mesocosms over the summer, fall, and winter. We bred outdoor mesocosm‐caught flies for two generations in the laboratory and matched each outdoor cohort to an indoor control cohort of similar genetic background. We cold hardened all flies under controlled laboratory conditions and then measured their post‐cold hardening freeze tolerance. Comparing indoor and field‐caught flies and their laboratory‐reared G1 and G2 progeny allowed us to determine the roles of seasonal environmental plasticity, parental effects, and genetic changes on cold hardening. We also tested the relationship between cold hardening and other factors, including age, developmental density, food substrate, presence of antimicrobials, and supplementation with live yeast. We found strong plastic responses to a variety of field‐ and laboratory‐based environmental effects, but no evidence of seasonally varying parental or genetic effects on cold hardening. We therefore conclude that seasonal variation in post‐cold hardening freeze tolerance results from environmental influences and not genetic changes.     

Diapause affects cuticular hydrocarbon composition and mating behavior of both sexes in Drosophila montana

Envipnmental cues,mainly photoperiod and temperature,are known to control female adult reproductive diapause in several insect species.Diapause enhances female survival during adverse conditions and postpones progeny production to the favorable season.Male diapause(a reversible inability to inseminate receptive females)has been studied much less than female diapause.However,if the males maximized their chances to fertilize females while minimizing their energy expenditure,they would be expected to be in diapause at the same time as females.We investigated Drosophila montana male mating bchavior under short-day conditions that induce diapause in females and found the males to be reproductively inactive.We also found that males reared under long-day conditions(reproducing individuals)court reproducing postdiapause fermales,but not diapausing ones.The diapausing fies of both sexes had more long-chain and less short-chain hydrocarbons on their cuticle than the reproducing ones,which presumably increase their survival under stressful conditions,but at the same time decrease their attractiveness.Our study shows that the mating behavior of females and males is well coordinated during and afier overwintering and it also gives support to the dual role of insect cuticular hydrocarbons in adaptation and mate choice.     

Are bottlenecks associated with colonization? Genetic diversity and diapause variation of native and introduced Rhagoletis completa populations

The success of invasive species appears to be a paradox: despite experiencing strong population bottlenecks, invasive species are able to successfully establish in new environments. We studied how the walnut husk fly, Rhagoletis completa, was able to successfully colonize California from the Midwestern United States, by examining genetic diversity and diapause variation of native and introduced fly populations. Climate plays an important role in the successful establishment of introduced insects, because insect diapause is highly dependent upon external climatic conditions. We examined if: (1) fly populations show signs of a population bottleneck, (2) native and introduced flies differ in diapause length when exposed to California and Midwestern climatic conditions, and (3) population genetic diversity is related to variation in diapause length. We assessed if fly diapause conformed more to a model of establishment by local adaptation or to a model of a highly plastic “general-purpose genotype”. Our results indicate that only two populations close to the original introduced location showed signs of a population bottleneck, and native and introduced populations did not differ in genetic diversity. Genetic diversity increased in the northern introduced populations, suggesting that multiple introductions have occurred. Flies emerged about 2 weeks earlier under the Midwestern treatment than the California treatment, and introduced flies emerged about a week earlier than native flies. All flies emerged when walnuts are typically available in California. Although variance in diapause length differed between populations, it did not vary between populations or regions. Furthermore, genetic diversity was not associated with diapause variation. Therefore, multiple introductions and a “general-purpose genotype” appear to have facilitated the fly’s invasion into California.     

Color polymorphism,development, and reproductive diapause of the multicolored Asian ladybird <Emphasis Type="Italic">Harmonia axyridis</Emphasis> (Pallas) (Coleoptera,Coccinellidae) females

Preimaginal development and maturation of females of two laboratory strains originated from two populations of the multicolored Asian ladybird Harmonia axyridis were investigated under laboratory conditions. Females of the autochthonous population from Irkutsk (Siberia) enter diapause under the short day conditions, whereas in females of the invasive population from Sochi (the Caucasus) photoperiodic induction of diapause is weak. The two populations also differ in the proportions of morphs: the population from Sochi is represented only by the morph succinea, whereas the population from Irkutsk is represented by the morphs succinea и axyridis with the predominance of the last one, which in our study was separated into two phenotypes, differed in the degree of melanization. Rearing of adults under the short day (12 h) conditions induced reproductive diapause in females of all the studied morphs and phenotypes of the Irkutsk population, whereas under the long day (18 h) conditions females of the morph succinea showed a slightly lower tendency to diapause. Evidently, the interpopulation differences in the responses regulating the seasonal cycle of the multicolored Asian ladybird are determined by genes that are not involved (or almost not involved) in the determination of the color polymorphism. The interpopulation differences in size and weight were quite substantial, whereas the differences between morphs and phenotypes were statistically significant in only some of our experiments. In combination with the literature data, these results suggest that the genes determining the color polymorphism in H. axyridis may have a pleiotropic effect on other traits including important eco-physiological parameters but this effect is relatively weak and manifests itself only against a particular genetic background and / or under particular environmental conditions.