Characterization of pre-diapause phase in the northern Drosophila species D. ezoana.

Drosophila ezoana is a virilis group Drosophila species inhabiting northern latitudes. The flies enter adult reproductive diapause to survive winter upon exposure to short photoperiod conditions (short-day) over several consecutive days. Insect pre-diapause phase - the duration between the beginning of exposure to short days and expression of diapause is thought to be comprised of two distinct phases - (a) photoperiodic time measurement that detects short-days, followed by (b) physiological events leading to the expression of diapause phenotype. A short-day dependent segment of the pre-diapause phase thus approximates the process of photoperiodic time measurement. Continuous darkness has been found to be a neutral condition with respect to diapause regulation in many insect species. The effect of variable number of short-days followed by continuous darkness on diapause incidence thus allows identification of short-day dependent segment of pre-diapause phase thereby mapping the process of photo-periodic time measurement. Although, few weeks of exposure to short-days in adult stage is known to be sufficient for the expression of diapause in D. ezoana, the number of short days required for the completion of photo-periodic time measurement has never been systematically analysed. Our experiments show that continuous darkness is a neutral condition for diapause regulation also in D. ezoana. We utilized the neutral nature of continuous darkness to map the process of photoperiodic time measurement in the D. ezoana strain 124OJ8 which showed that integration of short-day photic cues over the first 10 days of pre-diapause phase is essential for diapause induction.

Differential expression of microRNAs in diapause and non-diapause gonads of Aspongopus chinensis Dallas (Hemiptera: Dinidoridae): implications for reproductive control.

Aspongopus chinensis Dallas, 1851 (Hemiptera: Dinidoridae), an edible and medicinal insect, usually found in China and Southeast Asia, offers substantial potential for various applications. The reproductive cycle of this particular insect occurs annually because of reproductive diapause, leading to inadequate utilization of available natural resources. Despite its considerable ecological importance, the precise mechanisms underlying diapause in A. chinensis are not yet well understood. In this study, we conducted an analysis of comparing the microRNA (miRNA) regulation in the diapause and non-diapause gonads of A. chinensis and identified 303 differentially expressed miRNAs, among which, compared with the diapause group, 76 miRNAs were upregulated and 227 miRNAs downregulated. The results, regarding the Enrichment analysis of miRNA-targeted genes, showed their involvement in several essential biological processes, such as lipid anabolism, energy metabolism, and gonadal growth. Interestingly, we observed that the ATP-binding cassette pathway is the only enriched pathway, demonstrating the capability of these targeted miRNAs to regulate the reproductive diapause of A. chinensis through the above essential pathway. The current study provided the role of gonadal miRNA expression in the control of reproductive diapause in A. chinensis, the specific regulatory mechanism behind this event remained unknown and needed more investigation.

Impact of photoperiod and functional clock on male diapause in cryptochrome and pdf mutants in the linden bug Pyrrhocoris apterus.

Numerous insect species living in temperate regions survive adverse conditions, such as winter, in a state of developmental arrest. The most reliable cue for anticipating seasonal changes is the day-to-night ratio, the photoperiod. The molecular mechanism of the photoperiodic timer in insects is mostly unclear. Multiple pieces of evidence suggest the involvement of circadian clock genes, however, their role might be independent of their well-established role in the daily oscillation of the circadian clock. Furthermore, reproductive diapause is preferentially studied in females, whereas males are usually used for circadian clock research. Given the idiosyncrasies of male and female physiology, we decided to test male reproductive diapause in a strongly photoperiodic species, the linden bug Pyrrhocoris apterus. The data indicate that reproduction is not under circadian control, whereas the photoperiod strongly determines males' mating capacity. Clock mutants in pigment dispersing factor and cryptochrome-m genes are reproductive even in short photoperiod. Thus, we provide additional evidence of the participation of circadian clock genes in the photoperiodic time measurement in insects.

Hormonal Regulation of Reproductive Diapause That Occurs in the Year-Round Mass Rearing of Bombus terrestris Queens.

Adult reproductive diapause is an adaptive strategy under adverse environments for insects and other arthropod species, including bumblebees, which enables queens to survive through a harsh winter and then build new colonies in the following spring. Little research has been done on the molecular regulatory mechanism of reproductive diapause in Bombus terrestris, which is an important pollinator of wild plants and crops. Our previous research identified the conditions that induced reproductive diapause during the year-round mass rearing of B. terrestris. Here, we performed combined transcriptomics and proteomics analyses of reproductive diapause in B. terrestris during and after diapause at three different ecophysiological phases, diapause, postdiapause, and founder postdiapause. The analyses showed that differentially expressed proteins/genes acted in the citrate cycle, insect hormone biosynthesis, insulin and mTOR signaling pathway. To further understand the mechanisms that regulated the reproductive diapause, genes involved in the regulation of JH synthesis, insulin/TOR signal pathway were determined. The BtRheb, BtTOR, BtVg, and BtJHAMT had lower expression levels in diapause queens. The JH III titer levels and the activities of the metabolic enzymes were significantly up-regulated in postdiapause queens. Also, after the microinjection of insulin-like peptides (ILPs) and JH analogue (JHA), hormones, cold-tolerance metabolites, metabolic enzymes, and reproduction showed significant changes. Together with results from other related research, a model of the regulation of reproductive diapause during the year-round mass rearing of B. terrestris was proposed. This study contributes to a comprehensive insight into the molecular regulatory mechanism of reproductive diapause in eusocial insects.

Dispersal and migration have contrasting effects on butterfly flight morphology and reproduction.

Movement may fundamentally alter morphology and reproductive states in insects. In long-distance migrants, reproductive diapause is associated with trade-offs between diverse life-history traits such as flight morphology and lifespan. However, many non-diapausing insects engage in shorter resource-driven dispersals. How diapause and other reproductive states alter flight morphology in migrating versus dispersing insects is poorly understood. To find out, we compared flight morphology in different reproductive states of multiple butterfly species. We found that dispersers consisted of ovulating females with higher egg loads compared with non-dispersing females. This trend was in stark contrast with that of migrating female butterflies in reproductive diapause, which made substantially higher investment in flight tissue compared with reproductively active, non-migrating females. Thus, long-distance migration and shorter resource-driven dispersals had contrasting effects on flight morphology and egg loads. By contrast, male flight morphology was not affected by dispersal, migration or associated reproductive states. Thus, dispersal and migration affected resource allocation in flight and reproductive tissue in a sex-specific manner across relatively mobile versus non-dispersing individuals of different species. These findings suggest that dispersals between fragmented habitats may put extra stress on egg-carrying females by increasing their flight burdens.

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.

[Effect of temperature on post-diapause reproductive development in Listronotus maculicollis (Coleoptera: curculionidae)].

The annual bluegrass weevil Listronotus maculicollis requires chilling exposure to terminate reproductive diapause during overwintering, but the effects of temperature on its post-diapause development in spring remain unclear. To explore this effect, overwintering adults were transferred from cold conditions (6°C/4°C, L:D 10:14) to different warm-up temperatures at L:D 12:12. When weevils were transferred to 7, 14 and 21°C in December and late January, the sizes of male and female reproductive organs were significantly smaller at 7°C than at 14 and 21°C. When weevils were transferred to 7, 9, 11, 13 and 15°C in late January, higher temperatures facilitated the post-diapause development. In both sexes, the sizes of reproductive organs and developmental rate increased with temperature. Reproductive organs did not grow significantly at 7°C in males and at 7-9°C in females, at which the percentage of developing weevils remained low. The time required for 50% of individuals to resume development was 44, 18, 13 and 8 days at 9, 11, 13 and 15°C, respectively, in males and 19, 14 and 8 days at 11, 13 and 15°C, respectively, in females. The threshold temperature for post-diapause development was 7.8°C in males, based on which 61.7 degree-days coincided with 50% of individuals developing. Under field conditions, the percentage of male and female maturity and insemination rate were low until early March, but all reached 100% by late March.

Migratory monarchs that encounter resident monarchs show life-history differences and higher rates of parasite infection.

Environmental change induces some wildlife populations to shift from migratory to resident behaviours. Newly formed resident populations could influence the health and behaviour of remaining migrants. We investigated migrant-resident interactions among monarch butterflies and consequences for life history and parasitism. Eastern North American monarchs migrate annually to Mexico, but some now breed year-round on exotic milkweed in the southern US and experience high infection prevalence of protozoan parasites. Using stable isotopes (δ2 H, δ13 C) and cardenolide profiles to estimate natal origins, we show that migrant and resident monarchs overlap during fall and spring migration. Migrants at sites with residents were 13 times more likely to have infections and three times more likely to be reproductive (outside normal breeding season) compared to other migrants. Exotic milkweed might either attract migrants that are already infected or reproductive, or alternatively, induce these states. Increased migrant-resident interactions could affect monarch parasitism, migratory success and long-term conservation.

State-Dependent Plasticity in Response to Host-Plant Volatiles in a Long-Lived Moth, Caloptilia fraxinella (Lepidoptera: Gracillariidae).

Volatile chemicals produced by plants mediate host location, mate-finding and oviposition behavior in insects. State-dependent response to plant cues allows for timing of foraging, mating and oviposition on ephemeral host plants or plant parts. Caloptilia fraxinella is a herbivorous specialist on the foliage of ash trees (Fraxinus). Adults are long-lived and undergo a nine-month reproductive diapause over the fall and winter. Mating and oviposition occur in the spring when volatile chemicals released by ash leaves mediate host location. This study tested the plasticity of olfactory response of C. fraxinella to host plant volatiles using both electroantennogram and behavioral bioassays. The effect of moth physiological state on olfactory response was tested on male and female moths in different nutritional, mating, and diapause states. Antennal responses to host plant volatiles were plastic and depended on moth physiological state, and were highest when moths were reproductively active and would be seeking oviposition hosts. Moth sex and nutritional status also impacted antennal response to host plant volatiles. Oriented flight of females to ash seedlings varied with physiological state and nutritional status, with fed, reproductively active females having the highest response. Physiological state impacted oriented flight of males to female-produced sex pheromone signals whether or not a host plant was present, and there was no increase in behavioral response to sex pheromone in the presence of an ash host.

Cell cycle features of C. elegans germline stem/progenitor cells vary temporally and spatially.

Many organisms accumulate a pool of germline stem cells during development that is maintained in later life. The dynamics of establishment, expansion and homeostatic maintenance of this pool are subject to both developmental and physiological influences including the availability of a suitable niche microenvironment, nutritional status, and age. Here, we investigated the dynamics of germline proliferation during stages of expansion and homeostasis, using the C. elegans germ line as a model. The vast majority of germ cells in the proliferative zone are in interphase stages of mitosis (G1, S, G2) rather than in the active mitotic (M) phase. We examined mitotic index and DNA content, comparing different life stages, mutants, and physiological conditions. We found that germ cells in larval stages cycle faster than in adult stages, but that this difference could not be attributed to sexual fate of the germ cells. We also found that larval germ cells exhibit a lower average DNA content compared to adult germ cells. We extended our analysis to consider the effects of distance from the niche and further found that the spatial pattern of DNA content differs between larval and adult stages in the wild type and among mutants in pathways that interfere with cell cycle progression, cell fate, or both. Finally, we characterized expansion of the proliferative pool of germ cells during adulthood, using a regeneration paradigm (ARD recovery) in which animals are starved and re-fed. We compared adult stage regeneration and larval stage expansion, and found that the adult germ line is capable of rapid accumulation but does not sustain a larval-level mitotic index nor does it recapitulate the larval pattern of DNA content. The regenerated germ line does not reach the number of proliferative zone nuclei seen in the continuously fed adult. Taken together, our results suggest that cell cycle dynamics are under multiple influences including distance from the niche, age and/or maturation of the germ line, nutrition and, possibly, latitude for physical expansion.

Knockdown of TOR causing ovarian diapause in a genetically stable brachypterous strain of Nilaparvata lugens.

Brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is one of the most damaging pests of rice crops. BPH is a migratory insect with a delayed ovarian development in migrants classified as reproductive diapause. The molecular mechanism of reproductive diapause remains unclear, although we suspect it might be regulated by one or more nutrient signaling pathways. The target of rapamycin (TOR) pathway regulates cell growth in response to nutritional information, which raised a hypothesis that TOR mediates BPH reproductive diapause. We used a pure brachypterous strain (BS) and a predominantly macropterous strain (MS) to investigate the roles of NlTOR in BPH reproductive diapause. We found that NlTOR is expressed from the nymphal to adult stages, with a higher expression level of NlTOR in BS adults at 1, 2, and 4 days posteclosion than in MS at the same time points. Injection of dsNlTOR into BS nymphs resulted in the termination of BPH female ovary development and the retardation of nymph development. We infer that TOR signaling functions in BPH reproductive diapause by regulating the expression of NlFoxA and NlVitellogenin.

MYST family histone acetyltransferases regulate reproductive diapause initiation.

Histone acetylation, a crucial epigenetic mechanism, has been suggested to play a role in diapause regulation, but this has not been confirmed through gene loss-of-function studies. In this work, we investigated the involvement of MYST family genes, which are key writers of histone acetylation, in initiating reproductive diapause using the cabbage beetle Colaphellus bowringi as a model. We identified C. bowringi orthologs of MYST, including Tip60, KAT6A, KAT7, and KAT8, from previous transcriptomes. Analyses of phylogenetic trees and protein domains indicated that these MYST proteins are structurally conserved across animal species. Expression of these MYST genes was found to be enriched in heads and ovaries of C. bowringi. Under reproductive photoperiod conditions, RNAi targeting MYST genes, especially KAT8, suppressed ovarian growth and yolk deposition, resembling the characteristics of diapausing ovaries. Additionally, KAT8 knockdown led to the upregulation of diapause-related genes, such as heat shock proteins and diapause protein 1, and the emergence of diapause-like guts. Moreover, KAT8 knockdown reduced the expression of a crucial enzyme involved in juvenile hormone (JH) biosynthesis, likely due to decreased H4K16ac levels. Consequently, our findings suggest that MYST family genes, specifically KAT8, influence the JH signal, thereby regulating the initiation of reproductive diapause.

Key roles of insulin receptor InR1 in initiating reproductive diapause in males of the cabbage beetle Colaphellus bowringi (Coleoptera: Chrysomelidae).

BACKGROUND: Reproductive diapause serves as a valuable strategy enabling insects to survive unfavorable seasonal conditions. However, forcing insects into diapause when the environment is conducive to their well-being can cause them to miss out on seasonal opportunities for reproduction. This outcome not only reduces insect populations but also minimizes crop losses caused by insect feeding. Therefore, altering the timing of diapause initiation presents a potential strategy for managing pests. In this study, we examined the possible role of the Insulin Receptor 1 (InR1) in controlling reproductive diapause entry in the male cabbage beetle, Colaphellus bowringi. RESULTS: Compared to short-day (SD) conditions, long-day (LD) conditions led to reproductive diapause of C. bowringi males, characterized by arrested gonad development, increased Triglyceride (TG) accumulation, and upregulated expression of diapause protein 1 and genes associated with lipogenesis and stress tolerance. Upon employing RNA interference to knock down InR1 under SD conditions, males destined for reproduction were compelled into diapause, evidenced by arrested gonadal development, accumulation of TG, and elevated expression of diapause-related genes. Intriguingly, despite the common association of the absence of juvenile hormone (JH) with reproductive diapause in females, the knockdown of InR1 in males did not significant affect the expression of JH biosynthesis and JH response gene. CONCLUSION: The study highlight InR1 is a key factor involved in regulating male reproductive diapause in C. bowringi. Consequently, targeting insulin signaling could be a viable approach to perturb diapause timing, offering a promising strategy for managing pests with reproductive diapause capabilities. © 2024 Society of Chemical Industry.

Functional analysis of adipokinetic hormone signaling in reproductive diapause of Coccinella septempunctata.

BACKGROUND: The ladybeetle, Coccinella septempunctata, an important predator, is widely used to control aphids, whiteflies, mites, thrips, and lepidopteran pests. Diapause control technology is key to extending C. septempunctata shelf-life and commercialization. Lipid accumulation is a major feature of reproductive diapause, but the function of AKH signaling as a regulator of lipid mobilization in reproductive diapause remains unclear. This study aimed to identify and characterize AKH and AKHR genes, and clarify their functions in reproductive diapause. RESULTS: The relative expression levels of CsAKH and CsAKHR were the highest in the head and fat body, respectively, and were significantly decreased under diapause conditions, both in developmental stages and tissues (head, midgut, fat body, and ovary). Furthermore, CsAKH and CsAKHR expression was increased significantly after juvenile hormone (JH) injection, but CsMet silencing significantly inhibited CsAKH and CsAKHR expression, whereas CsMet knockdown blocked the induction effect of JH. CsAKH and CsAKHR knockdown significantly reduced water content, increased lipid storage, and promoted the expression of genes related to lipid synthesis, but significantly blocked ovarian development, and induced forkhead box O (FOXO) gene expression in C. septempunctata under reproduction conditions. By contrast, injection of AKH peptide significantly inhibited FOXO expression, reduced lipid storage, and increased water content in C. septempunctata under diapause conditions. CONCLUSION: These results indicate that CsAKH and CsAKHR are involved in the regulation of lipid accumulation and ovarian development during diapause in C. septempunctata, and provide a promising target for manipulating C. septempunctata diapause. © 2024 Society of Chemical Industry.

Consuming royal jelly alters several phenotypes associated with overwintering dormancy in mosquitoes.

Introduction: Females of the Northern house mosquito, Culex pipiens, enter an overwintering dormancy, or diapause, in response to short day lengths and low environmental temperatures that is characterized by small egg follicles and high starvation resistance. During diapause, Culex pipiens Major Royal Jelly Protein 1 ortholog (CpMRJP1) is upregulated in females of Cx. pipiens. This protein is highly abundant in royal jelly, a substance produced by honey bees (Apis mellifera), that is fed to future queens throughout larval development and induces the queen phenotype (e.g., high reproductive activity and longer lifespan). However, the role of CpMRJP1 in Cx. pipiens is unknown. Methods: We first conducted a phylogenetic analysis to determine how the sequence of CpMRJP1 compares with other species. We then investigated how supplementing the diets of both diapausing and nondiapausing females of Cx. pipiens with royal jelly affects egg follicle length, fat content, protein content, starvation resistance, and metabolic profile. Results: We found that feeding royal jelly to females reared in long-day, diapause-averting conditions significantly reduced the egg follicle lengths and switched their metabolic profiles to be similar to diapausing females. In contrast, feeding royal jelly to females reared in short-day, diapause-inducing conditions significantly reduced lifespan and switched their metabolic profile to be similar nondiapausing mosquitoes. Moreover, RNAi directed against CpMRJPI significantly increased egg follicle length of short-day reared females, suggesting that these females averted diapause. Discussion: Taken together, our data show that consuming royal jelly reverses several key seasonal phenotypes of Cx. pipiens and that these responses are likely mediated in part by CpMRJP1.

Quantitative transcriptomic and proteomic analyses reveal the potential maintenance mechanism of female adult reproductive diapause in Chrysoperla nipponensis.

BACKGROUND: The green lacewing Chrysoperla nipponensis is an important natural enemy of many insect pests and exhibits reproductive diapause to overwinter. Our previous studies showed that adult C. nipponensis enters reproductive diapause under a short-day photoperiod. However, the molecular mechanism underlying diapause maintenance in C. nipponensis is still unknown. RESULTS: The total lipid and triglyceride content showed the reservation and degradation of energy during diapause in C. nipponensis. Thus, we performed combined transcriptomic and proteomic analyses of female reproductive diapause in C. nipponensis at three ecophysiological phases (initiation, maintenance and termination). A total of 64 388 unigenes and 5532 proteins were identified from the transcriptome and proteome. In-depth dissection of the gene-expression dynamics revealed that differentially expressed genes and proteins were predominately involved in the lipid and carbohydrate metabolic pathways, in particular fatty acid metabolism, metabolic pathways and the citrate cycle. Among of these genes, TIM, CLK, JHAMT2, PMK, HMGS, HMGR, FKBP39, Kr-h1, Phm, ECR, IR1, ILP3, ILP4, mTOR, ACC, LSD1 and LSD2 were differentially expressed in diapause and non-diapause female adults of C. nipponensis. The expression patterns of these genes were consistent with the occurrence of vitellogenesis and expression of either Vg or VgR. CONCLUSION: Our findings indicated that diapause adult C. nipponensis accumulate energy resources to overwinter. Transcriptomic and proteomic analyses suggested candidate key genes involved in the maintenance of C. nipponensis during adult reproductive diapause. Taken together, these results provide in-depth knowledge to understand the maintenance mechanism of C. nipponensis during adult reproductive diapause. © 2023 Society of Chemical Industry.

Morphology and Morphometry of the Reproductive Tract of the Cotton Boll Weevil after Prolonged Feeding on Alternative Diets.

Anthonomus grandis Boheman (Coleoptera: Curculionidae) survives on alternative diets; however, this induces reproductive diapause. The objective was to evaluate the morphology and morphometry of the reproductive tract of this weevil after feeding on alternative diets. The experimental design was completely randomized with 160 replications and treatments arranged in a factorial design 3 × 3, represented by A. grandis adults fed on 3 food types (fragments of banana (T1) or orange (T2) endocarp, or with cotton squares of cultivar BRS 286 (T3, control)) and three evaluation periods (30, 60, and 90 days) and after each of these periods they were fed with cotton squares for 10 days. The reproductive tract of 100% of A. grandis females fed banana endocarp, orange endocarp, and cotton squares for 30 and 60 days and then cotton squares were morphologically adequate for reproduction, and after 90 days, only 50% of those fed cotton squares were in this condition. The length of the ovarioles and the width of the mature oocyte were greater for A. grandis fed on cotton squares and smaller in those with banana and orange endocarps. Histological sections reveal that male testes even with strong degenerative signals are already producing spermatozoa. On the other hand, females displayed ovaries with nurse cells in the tropharium and some maturing oocytes in the vitellarium. The body length was longer and the testis area and diameter smaller in males fed on cotton squares than in those with banana and orange endocarp. Anthonomus grandis females fed for ≥90 days with alternative food sources do not recover the functionality of their reproductive tract, even after consuming, for 10 days, a diet that favors reproduction. On the other hand, the males remain with their reproductive organs functional with this condition.

Transcriptional Regulation of Reproductive Diapause in the Convergent Lady Beetle, Hippodamia convergens.

Diapause is an alternate development program that synchronizes an insect's life cycle with seasonally abundant resources and ensures survival in unfavorable conditions. The physiological basis of diapause has been well characterized, but the molecular mechanisms regulating it are still being elucidated. Here, we present a de novo transcriptome and quantify transcript expression during diapause in the convergent lady beetle Hippodamia convergens. H. convergens is used as an augmentative biocontrol agent, and adult females undergo reproductive diapause that is regulated by photoperiod. We sampled females at three stages (early, mid, and late diapause) and compared transcript expression to non-diapausing individuals. Based on principle component analysis, the transcriptomes of diapausing beetles were distinct from non-diapausing beetles, and the three diapausing points tended to cluster together. However, there were still classes of transcripts that differed in expression across distinct phases of diapause. In general, transcripts involved in muscle function and flight were upregulated during diapause, likely to support dispersal flights that occur during diapause, while transcripts involved in ovarian development were downregulated. This information could be used to improve biological control by manipulating diapause. Additionally, our data contribute to a growing understanding of the genetic regulation of diapause across diverse insects.

Plasticity in Photoperiodism: Drosophila montana Females Have a Life-Long Ability to Switch From Reproduction to Diapause.

Photoperiodic reproductive diapause is an essential part of female life cycle in several insect species living on high latitudes, where overwintering in reproductive stage involves high risks for survival and progeny production. The sensitive period (SP), during which photoperiodic cues can trigger the switch from direct development to diapause, can last from a few hours or days after emergence to the entire life span of females. Moreover, in some species, sexually mature females can enter post-reproductive diapause as a response to decreasing day length and/or temperature. We studied the duration of SP for diapause induction and the females' ability to enter post-reproductive diapause at short day lengths in Drosophila montana strains from different latitudes in Europe, North America, and Japan. Our study shows that the females of this species have a life-long SP and that they retain an ability to switch between reproduction and diapause as a response to back-and-forth changes in day length for at least 3 months. D. montana strains from different latitudes showed high variation in females' ability to enter post-reproductive diapause; females of the southern strains generally requiring longer time and/or lower temperature to enter this stage than those of the northern strains. Moreover, the proportion of females that switched to post-reproductive diapause in 3 weeks in short day conditions at 16 °C showed positive correlation with the critical day length (CDL) for diapause induction and the latitudinal and continental origin of the strains. Life-long SP increases females' flexibility to respond to short-term changes in environmental conditions and enables reproducing females to switch to post-reproductive diapause when the days get shorter and colder toward the autumn. This ability can play a major role in species phenology and should be taken into account in theoretical and empirical studies on insect adaptation to seasonal variation.

Juvenile hormone regulates photoperiod-mediated male reproductive diapause via the methoprene-tolerant gene in the ladybeetle Harmonia axyridis.

Juvenile hormone (JH) absence induces photoperiod-mediated reproductive diapause, which is characterized by reproductive cessation. Although the role of methoprene-tolerant (Met)-mediated JH signaling in photoperiod-mediated female reproduction has been well documented, its role in male reproduction remains unclear. In this study, we investigated the role of JH in regulating photoperiod-mediated development of the male internal reproductive system (IRS) in the predatory ladybeetle Harmonia axyridis (Pallas). In a previous study, we found that adult male H. axyridis reared under either a short-day (SD) or long-day (LD) photoperiod had obvious differences in IRS development, but we were unable to identify the regulators of male reproductive diapause. In this study, we found that beetles reared under an SD photoperiod had significantly lower JH titer and a relatively undeveloped male IRS compared with those reared under an LD photoperiod. Additionally, application of the JH analog (JHA) methoprene promoted IRS development. Furthermore, Met knockdown strongly blocked JH signaling in males reared under the LD photoperiod, thereby slowing IRS development. Moreover, exogenous JHA did not reverse the suppressed development of the male IRS caused by Met knockdown. These results indicate that photoperiod regulates male IRS development in H. axyridis through a conserved Met-dependent JH signaling pathway.