Abstract: The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (-196 °C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.

Abstract: The Eurasian spruce bark beetle, Ips typographus, is one of the major forest insect pests in Europe, capable of mass-attacking and killing mature Norway spruce trees. The initiation and development of a new generation are strongly controlled by temperature and a warmer climate may affect the number of generations that is produced per year and hence the outbreak dynamics. Experimental knowledge regarding reproductive diapause adaptations is, however, too sparse for large-scale assessments of future trends. We developed a model description of diapause induction, and used gridded observational temperature data to evaluate multiple combinations of day length and temperature thresholds to find the model parameterisation most coherent with I. typographus monitoring data from Scandinavia. The selected model parameterisation is supported by European literature data, though further experimental studies are required to analyse population specific adaptations and capacity for adjustments to changing climate conditions. Implementing the model description of reproductive diapause in a temperature driven model of bark beetle phenology (swarming activity and development from egg to mature bark beetle), enabled us to assess the length of the late summer swarming period that is a critical determinant of the risk of forest damage. By using regional climate model data we show that higher temperatures can result in increased frequency and length of late summer swarming events, producing a second generation in southern Scandinavia and a third generation in lowland parts of central Europe. Reproductive diapause will not prevent the occurrence of an additional generation per year, but the day length cues may restrict the length of the late summer swarming period.

Abstract: Many temperate insects survive the harsh conditions of winter by undergoing photoperiodic diapause, a pre-programmed developmental arrest initiated by short day lengths. Despite the well-established ecological significance of photoperiodic diapause, the molecular basis of this crucial adaptation remains largely unresolved. The Asian tiger mosquito, Aedes albopictus (Skuse), represents an outstanding emerging model to investigate the molecular basis of photoperiodic diapause in a well-defined ecological and evolutionary context. Ae. albopictus is a medically significant vector and is currently considered the most invasive mosquito in the world. Traits related to diapause appear to be important factors contributing to the rapid spread of this mosquito. To generate novel sequence information for this species, as well as to discover transcripts involved in diapause preparation, we sequenced the transcriptome of Ae. albopictus oocytes destined to become diapausing or non-diapausing pharate larvae. 454 GS-FLX transcriptome sequencing yielded >1.1 million quality-filtered reads, which we assembled into 69,474 contigs (N50 = 1,009 bp). Our contig filtering approach, where we took advantage of strong sequence similarity to the fully sequenced genome of Aedes aegypti, as well as other reference organisms, resulted in 11,561 high-quality, conservative ESTs. Differential expression estimates based on normalized read counts revealed 57 genes with higher expression, and 257 with lower expression under diapause-inducing conditions. Analysis of expression by qPCR for 47 of these genes indicated a high correlation of expression levels between 454 sequence data and qPCR, but congruence of statistically significant differential expression was low. Seven genes identified as differentially expressed based on qPCR have putative functions that are consistent with the insect diapause syndrome; three genes have unknown function and represent novel candidates for the transcriptional basis of diapause. Our transcriptome database provides a rich resource for the comparative genomics and functional genetics of Ae. albopictus, an invasive and medically important mosquito. Additionally, the identification of differentially expressed transcripts related to diapause enriches the limited knowledge base for the molecular basis of insect diapause, in particular for the preparatory stage. Finally, our analysis illustrates a useful approach that draws from a closely related reference genome to generate high-confidence ESTs in a non-model organism.

Abstract: Environmental stresses and nutrition availability critically affect animal development. Numerous animal species across multiple phyla enter developmental arrest for long-term survival in unfavorable environments and resume development upon stress removal. Here we show that compromising overall microRNA (miRNA) functions or mutating certain individual miRNAs impairs the long-term survival of nematodes during starvation-induced L1 diapause. We provide evidence that miRNA miR-71 is not required for the animals’ entry into L1 diapause, but plays a critical role in long-term survival by repressing the expression of insulin receptor/PI3K pathway genes and genes acting downstream or in parallel to the pathway. Furthermore, miR-71 plays a prominent role in developmental recovery from L1 diapause partly through repressing the expression of certain heterochronic genes. The presented results indicate that interactions between multiple miRNAs and likely a large number of their mRNA targets in multiple pathways regulate the response to starvation-induced L1 diapause.

Abstract: The dormant state known as diapause is widely exploited by insects to circumvent winter and other adverse seasons For an insect to survive, feed, and reproduce at the appropriate time of year requires fine coordination of the timing of entry into and exit from diapause One of the hormones that regulates diapause in moths is the 24-aa neuropeptide, diapause hormone (DH) Among members of the Helicoverpa/Heliothis complex of agricultural pests, DH prompts the termination of pupal diapause Based on the structure of DH, we designed several agonists that are much more active than DH in breaking diapause One such agonist that we describe also prevents the entry into pupal diapause when administered to larvae that are environmentally programmed for diapause In addition, we used the unique antagonist development strategy of incorporating a dihydroimidazole (“Jones”) trans-Proline mimetic motif into one of our DH agonists, thereby converting the agonist into a DH antagonist that blocks the termination of diapause These results suggest potential for using such agents or next-generation derivatives for derailing the success of overwintering in pest species

Abstract: Background Diapause, a state of arrested development accompanied by a marked decrease of metabolic rate, helps insects to overcome unfavorable seasons. Helicoverpa armigera (Har) undergoes pupal diapause, but the molecular mechanism of diapause initiation is unclear. Using suppression subtractive hybridization (SSH), we investigated differentially expressed genes in diapause- and nondiapause-destined pupal brains at diapause initiation.

Abstract: 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.

Abstract: Summary 1. As organisms expand their range towards northern latitudes they will encounter selective factors like harsh winter conditions. The ability to cope with and adapt to harsh winters may depend on the variability and evolutionary potential of relevant traits. 2. One adaptation in insects is winter diapause. It is characterized by changes in physiology, behaviour or in both. Physiological changes include lowered metabolic rate that enhances survival by saving limited energy reserves during overwintering. Active behavioural changes like burrowing into the soil allow individuals to escape harsh conditions. 3. We examined variation in overwintering body mass, resting metabolic rate (CO2 production) and diapause behaviour (burrowing into the soil), and their effects on overwintering success in the adult Colorado potato beetles (Leptinotarsa decemlineata). We conducted a full-sib/half-sib rearing experiment to estimate the evolutionary potential (heritability) of these traits. 4. High overwintering body mass and low metabolic rate were phenotypically associated with high diapause propensity (i.e. burrowing), which was linked to high overwintering survival. We found that once beetles had entered the soil, only large body mass of males was associated with high overwintering survival. However, the heritability estimates in all traits examined were low. 5. Our results show that winter conditions impose selection on diapause behaviour, which was linked to lower metabolic rate and larger body mass. If range expansion to higher latitudes requires adaptive genetic changes in diapause behaviour, metabolism, or body mass, the insufficient genetic variation in these traits suggest that the Colorado potato beetle’s future potential to respond to selection due to harsher winters could be limited and thus, its range expansion could be hindered. Both physiological and behavioural adaptations are important to consider when assessing range expansion potential.

Abstract: Diapause is an adaptive response triggered by seasonal photoperiodicity to overcome unfavorable seasons. The photoperiodic clock is a system that controls seasonal physiological processes, but our knowledge about its physiological mechanisms and genetic architecture remains incomplete. The circadian clock is another system that controls daily rhythmic physiological phenomena. It has been argued that there is a connection between the two clocks. To examine the genetic connection between them, we analyzed the associations of five circadian clock genes (period, timeless, Clock, cycle and cryptochrome) with the occurrence of diapause in Drosophila triauraria, which shows a robust reproductive diapause with clear photoperiodicity. Non-diapause strains found in low latitudes were compared in genetic crosses with the diapause strain, in which the diapause trait is clearly dominant. Single nucleotide polymorphism and deletion analyses of the five circadian clock genes in backcross progeny revealed that allelic differences in timeless and cryptochrome between the strains were additively associated with the differences in the incidence of diapause. This suggests that there is a molecular link between certain circadian clock genes and the occurrence of diapause.

Abstract: Three populations of the copepod Acartia tonsa, two from the Baltic Sea and one from the U.S. East Coast, were compared for resting egg production at conditions of saturating and limiting food availability. All three populations produced eggs that hatched within 72 h when incubated at 17uC (subitaneous eggs), but the two Baltic populations in addition produced eggs that hatched at a much slower rate, in the course of a month (delayed hatching eggs [DHE]). Such eggs were not produced by the U.S. population. The fraction of DHE increased when food was limiting. Females from a Baltic population that were incubated individually all produced subitaneous eggs, but about half the females consistently also produced DHE. Cold storage that mimicked boreal winter conditions synchronized the hatching of DHE after extended storage, indicating that spring hatching of DHE might seed the water column with nauplii as an adaptation to match the timing of the spring bloom in boreal marine ecosystems. Low food availability promotes the production of resting eggs in marine copepods. Life history strategies may be adapted to unstable environments in various ways. Under stressful environmental conditions, mobile species can migrate to more suitable places, but migration in time by going into dormancy is also a suitable strategy to allow survival (Hairston and Bohonak 1998). Saving genes over time is the principle behind dormancy. Dormancy is a widespread life history adaptation in the plankton, both among phytoplankton and zooplankton. Phytoplankton, for example, produces resting spores that may survive unproductive seasons in the sediment (Hallegraeff and Bolch 1992). For zooplankton, dormancy is often more prevalent in freshwater as compared to marine environments, due to the typically larger fluctuations in limnic habitats. However, some marine crustaceans are known to rest (Alekseev et al. 2007); and, among marine copepods, diapause has been described for adult, copepodite, and egg stages (Mauchline et al. 1998). Diapausing in eggs has been described in close to 50 species of calanoid copepods (Engel and Hirche 2004) that all belong to the superfamily of Centropagoidea. Most if not all of these species are neritic and, thus, occur in habitats with rather large environmental variability.

Abstract: The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH2 (FXPRLa) neuropeptides: DH, PBAN, and α-, β-, and γ-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.

Abstract: Natural selection can generate parallel latitudinal clines in traits and gene frequencies across continents, but these have rarely been linked. An amino acid (isoleucine to lysine, or I462K) polymorphism of the couch potato (cpo) gene in Drosophila melanogaster is thought to control female reproductive diapause cline in North America (Schmidt et al. 2008, Proc Natl Acad Sci USA, 105, 16207–16211). Here, we show that under standard diapause-inducing conditions (12 °C and short photoperiod) (Saunders et al. 1989, Proc Natl Acad Sci USA, 86, 3748–3752), egg maturation in Australian flies is delayed, but not arrested at previtellogenic stages. At 12 °C, the phenotypic distribution in egg development was bimodal at stages 8 and 14 and showed a strong nonlinear pattern on the east coast of Australia, with incidence of egg maturation delay (ovarian dormancy) increasing both toward tropical and temperate climates. Furthermore, we found no evidence for an association between the cpo I462K polymorphism and ovarian dormancy at either 12 or 10 °C (when egg maturation was often delayed at stage 7). Owing to strong linkage disequilibrium, the latitudinal cline in cpo allele frequencies was no longer evident once variation in the In(3R)P inversion polymorphism was taken into account. Our results suggest that the standard diapause-inducing conditions (12 °C and short photoperiod) were not sufficient to cause the typical previtellogenic developmental arrest in Australian flies and that the cpo I462K polymorphism does not explain the observed delay in egg development. In conclusion, ovarian dormancy does not show a simple latitudinal cline, and the lack of cpo-dormancy association suggests a different genetic basis to reproductive dormancy in North America and Australia.

Abstract: Whether alien insects that are introduced into temperate regions adapt to seasonally changing environmental conditions is an important question in evolutionary biology. If rapid evolution has occurred in a non-native environment, a latitudinal cline in critical photoperiod for diapause induction (i.e., the photoperiod at which half of the individuals enter diapause) and in life cycle synchronization with host plant phenology should be evident among locations. The alien bruchid Acanthoscelides pallidipennis (Motschulsky) (Coleoptera: Bruchidae) is native to North America and introduced into Japan with the host plant Amorpha fruticosa L. (Fabaceae) in the late 1940s. To examine whether seasonal adaptation has occurred in A. pallidipennis, we conducted a laboratory experiment and phenological observations using three latitudinally different populations. We bred F1 eggs at 22 °C and five photoperiodic regimens – L:D = 10:14, 13:11, 14:10, 15:9, or 16:8 hours – and examined whether diapause was induced. The estimated critical photoperiod for diapause induction was longest in the most northern population and shortest in the most southern population. Life cycle was found to be synchronized with host phenology in each location. Also voltinism varied geographically, from univoltine in the northern population to bivoltine in the southern populations. These results showed that A. pallidipennis rapidly adapted to seasonal environmental conditions in Japan after its introduction.

Abstract: European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae), displays a larval diapause in response to short photoperiods and is adapted to a variety of local conditions throughout North America. Hence, the effective photoperiod inducing larval diapause will differ among geographic ecotypes. This study considers the inheritance of photoperiodic larval diapause induction by hybridization and backcrossing two latitudinally distinct ecotypes of the European corn borer collected between 41° N, 96° W and 48° N, 96° W and under a range of photoperiods representative of their respective locations: from 14:10 to16:8 (L:D) h. The ecotype adapted to a bivoltine habitat (southeastern Nebraska) exhibited a shorter critical photoperiod (1480 h) than the ecotype (1533 h), originating from a univoltine habitat (northwestern Minnesota). Reciprocal F1 crosses exhibited intermediate values with indication of sex-linked inheritance. In addition, the male parent had significantly more influence on diapause incidence of subsequent progeny than the female. The F2 and backcross progeny further supported the supposition that diapause response is a sex-linked inheritance. The minimum number of genes estimates, and the response from backcross progeny, suggest that diapause response of European corn borer larva may be controlled by only a few loci. The overall results indicated that both ecotypes had adopted unique diapause responses, which ultimately lead to seasonal synchrony in their ecosystems.

Abstract: At the south western border of its extensive distribution, the multivoltine large white butterfly, Pieris brassicae L., is exceptional in undergoing summer diapause or aestivation. In all other regions investigated, P. brassicae pupae only hibernate. The transitional zone from non-aestivating to aestivating populations is a geographically stable region south of the Pyrenees. The restric- tion of this response to this region cannot be accounted for in terms of genetics as aestivation is intermediately inherited, with the heritability (h 2 ) of aestivation in inbreeding lines between 0.35 and 0.77. Two hypotheses are presented to explain why this species does not aestivate in more northern regions. First, aestivation is a behaviour that serves to synchronize generations in areas where this species produces a high number of generations per year. Second, aestivation reduces the incidence of parasitism suffered by the butterfly by desynchronizing its life cycle from that of its main parasitoid, Cotesia glomerata. The two hypotheses are not mutually exclusive and both seem to be adaptive where the species is multivoltine.

Abstract: Organisms have evolved to optimally align the timing of diapause with the seasonal- ity of environmental conditions in order to maximize their fitness. Observations from the North- west Atlantic suggest that Calanus finmarchicus diapause initiation date varies significantly by geographic location, inconsistent with patterns of seasonal changes of any single environmental factor such as photoperiod, sea surface temperature, or food availability. In the present study, a life history-based model was used to reconcile the apparent inconsistency. The model results suggest that (1) the induction of diapause involves multiple environmental factors as a result of fitness maximization, and (2) in addition to temperature and food condition, predation pressure is another key factor that could affect diapause initiation process. It is further suggested that phenotypic plasticity is likely to be the major source of variation in the timing of diapause initiation for C. finmarchicus.

Abstract: Female Aedes albopictus, F2-F3 descendents from individuals collected as immatures at 6 geographic sites in the USA during 2008, exposed to short daylengths (10 h of light and 14 h of darkness at 21 degrees C) laid eggs in diapause, whose frequency depended upon population origin. Diapause responses in northern Florida and Illinois were strong, as had been reported approximately 10 years earlier for Ae. albopictus from these regions. For southern Florida, the diapause response was polymorphic, and its mean incidence decreased at 2 of 3 collection sites compared to 10 years earlier. Exposure in the field for 2- to 4-wk intervals in Vero Beach (lat 27 degrees 35'N) during January 2009 revealed that eggs laid by short-day females had significantly higher survivorship, even though <50% were estimated, from laboratory results, to be in diapause. Enhanced desiccation resistance may select for retention of diapause in southern Florida.

Abstract: For diapause eggs of the silkworm, Bombyx mori, diapause initiation is prevented with hydrochloric acid (HCl) at around 20 h post-oviposition while diapause status is terminated with chilling around 5°C. To investigate whether hydrogen peroxide (H(2)O(2)) and catalase expression are involved in diapause initiation and termination, the concentration of H(2)O(2), relatively higher levels of catalase mRNA and activity of catalase were compared between (1) 20-h-old diapause eggs and the HCl-treated diapause eggs, and (2) 10-day-old diapause eggs and the 5°C-chilled diapause eggs. Compared to diapause eggs, the HCl-treated eggs had significantly higher H(2)O(2) concentrations (up from approximately 1-3 µmol/g fresh mass to 5-8 µmol/g fresh mass), higher relative level of catalase mRNA (up from 0 to 35.2%) and higher catalase activity (up from 2.51 units/mg protein to 4.97 units/mg protein) at 96 h post-treatment. On the other hand, the 5°C chilling resulted in significant increases of H(2)O(2) concentration (up from 0.79 µmol/g fresh mass to 5.57 µmol/g fresh mass), relative level of catalase mRNA (up from 0 to 71.4%) and catalase activity (up from 0.88 units/mg protein to 3.42 units/mg protein) within 120 days. The results obtained in this work suggest that variations of H(2)O(2) and catalase expression in Bombyx eggs are involved in diapause initiation and termination.

Abstract: Larvae of the bean blister beetle Epicauta gorhami Marseul (Coleoptera: Meloidae) feed on grasshopper eggs in soil and undergo hypermetamorphosis. This beetle undergoes larval diapause in the fifth instar as a pseudopupa, a form characteristic of hypermetamorphosis in meloid beetles. The effects of temperature, photoperiod and soil humidity on larval development of E. gorhami are examined in a population in Miyazaki, Japan, using egg pods of Locusta migratoria L. as food. At lower temperatures (20 and 22.5 °C), all larvae become pseudopupae, regardless of the photoperiod. By contrast, at higher temperatures (27.5 and 30 °C), almost all larvae pupate at the end of the fourth instar, again regardless of the photoperiod. A long-day photoperiodic response occurs only at an intermediate temperature (25 °C): under an LD 12 : 12 h photocycle, all larvae enter diapause, although the diapause incidence tends to decrease as the day length becomes longer. Pseudopupae are immobile and remain in diapause for ≥120 days when they are kept under the same conditions, except that diapause terminates within a relatively short time at 30 °C. Although lower soil humidity retards post-feeding development, soil humidity has no effect on the diapause incidence. On the basis of the short developmental period and diapause avoidance under summer conditions, it is suggested that this beetle partially produces two generations a year in southwestern Japan.