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

Environmental 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 behavior 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 females, but not diapausing ones. The diapausing flies 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 after overwintering and it also gives support to the dual role of insect cuticular hydrocarbons in adaptation and mate choice.

Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence.

The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are inter-related is not well understood. We performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and/or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence from the control line in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled those detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, our study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity.

Strength of sexual and postmating prezygotic barriers varies between sympatric populations with different histories and species abundances.

The impact of different reproductive barriers on species or population isolation may vary in different stages of speciation depending on evolutionary forces acting within species and through species' interactions. Genetic incompatibilities between interacting species are expected to reinforce prezygotic barriers in sympatric populations and lead to cascade reinforcement between conspecific populations living within and outside the areas of sympatry. We tested these predictions and studied whether and how the strength and target of reinforcement between Drosophila montana and Drosophila flavomontana vary between sympatric populations with different histories and species abundances. All barriers between D. montana females and D. flavomontana males were nearly complete, while in the reciprocal cross strong postzygotic isolation was accompanied by prezygotic barriers whose strength varied according to population composition. Sexual isolation between D. flavomontana females and D. montana males was increased in long-established sympatric populations, where D. flavomontana is abundant, while postmating prezygotic (PMPZ) barriers were stronger in populations where this species is a new invader and still rare and where female discrimination against heterospecific males was lower. Strengthening of sexual and PMPZ barriers in this cross also induced cascade reinforcement of respective barriers between D. flavomontana populations, which is a classic signature of reinforcement process.

Direct and correlated responses to bi-directional selection on pre-adult development time in Drosophila montana.

Selection experiments offer an efficient way to study the evolvability of traits that play an important role in insects' reproduction and/or survival and to trace correlations and trade-offs between them. We have exercised bi-directional selection on Drosophila montana flies' pre-adult development time under constant light and temperature conditions for 10 generations and traced the indirect effects of this selection on females' diapause induction under different day lengths, as well as on the body weight and cold tolerance of both sexes. Overall, selection was successful towards slow, but not towards fast development. However, all fast selection line replicates showed at the end of selection increased variance in females' photoperiodic diapause response and about one hour increase in the critical day (CDL), where more than 50% of emerging females enter diapause. Indirect effects of selection on flies' body weight and cold-tolerance were less clear, as the flies of the slow selection line were significantly heavier and less cold-tolerant than the control line flies after five generations of selection, but lighter and more cold-tolerant at the end of selection. Changes in females' diapause induction resulting from selection for fast development could be due to common metabolic pathways underlying these traits, collaboration of circadian clock and photoperiodic timer and/or by the interaction between the endocrine and circadian systems.

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.

Involvement of circadian oscillation(s) in the photoperiodic time measurement and the induction of reproductive diapause in a northern Drosophila species.

An ability to predict forthcoming changes in environmental conditions and get prepared for them in advance is crucial for the survival and reproduction of organisms living in a seasonally changing environment. We have studied the possible involvement of circadian oscillator(s) in the photoperiodic timer controlling seasonal responses by tracing Drosophila montana females' diapause induction in constant darkness and in a classical Nanda-Hamner experiment. Nearly all females developed ovaries in continuous darkness, which shows the direct development to be their default developmental pathway in the absence of photoperiods. In Nanda-Hamner experiment the females' diapause incidence was close to zero in light:dark cycle 12:4 (photoperiod 16 h) and increased to nearly 100% in 12:8 and 12:12 (photoperiods 20 and 24 h). In longer photoperiods (28-72 h) the females' diapause percentages decreased gradually along with an increase in the length of the dark period, showing no peaks of high diapause incidence in the multiples of 24h. These findings suggest that the photoperiodic timer of D. montana is based on heavily damping circadian oscillator(s) or that it lacks strong oscillators. Damping of the photoperiodic timer under prolonged nights and constant darkness fits well with our earlier finding that these flies lose their locomotor activity rhythm in constant darkness, and suggests that the mechanisms underlying females' photoperiodic diapause response and their free-running locomotor activity rhythm may be partly based on same oscillators.

Flies in the north: locomotor behavior and clock neuron organization of Drosophila montana.

The circadian clock plays an important role in adaptation in time and space by synchronizing changes in physiological, developmental, and behavioral traits of organisms with daily and seasonal changes in their environment. We have studied some features of the circadian activity and clock organization in a northern Drosophila species, Drosophila montana, at both the phenotypic and the neuronal levels. In the first part of the study, we monitored the entrained and free-running locomotor activity rhythms of females in different light-dark and temperature regimes. These studies showed that D. montana flies completely lack the morning activity component typical to more southern Drosophila species in an entrained environment and that they are able to maintain their free-running locomotor activity rhythm better in constant light than in constant darkness. In the second part of the study, we traced the expression of the PDF neuropeptide and the CRY protein in the neurons of the brain in D. montana adults and found differences in the number and location of PDF- and CRY-expressing neurons compared with those described in Drosophila melanogaster. These differences could account, at least in part, for the lack of morning activity and the reduced circadian rhythmicity of D. montana flies in constant darkness, both of which are likely to be adaptive features during the long and dark winters occurring in nature.