Diapausing Females Research Articles

RpedOBP1 plays key roles in aggregation pheromones reception of the Riptortus pedestris

Riptortus pedestris (Hemiptera: Alydidae) is a notable soybean pest, with diapause and non-diapause individuals showing different sensitivities to aggregation pheromones. This study aimed to investigate how R. pedestris detects aggregation pheromones through electroantennogram (EAG) and behavioral experiments, transcriptome sequencing and qRT-PCR, as well as competitive fluorescence-binding assay. Results indicated that diapausing females and males of R. pedestris exhibited a heightened EAG response and were more attracted to the aggregation pheromone components compared to their non-diapause counterparts. Transcriptome sequencing and qRT-PCR analyses revealed significantly higher expression of RpedOBP1 in the antennae of diapause females and males compared to non-diapausing R. pedestris. The competitive fluorescence-binding assay demonstrated that RpedOBP1 displayed the strongest binding affinity to E2HE2H, suggesting its crucial role in recognizing the aggregation pheromone. These findings have the potential to inform the development of integrated pest management strategies utilizing behavioral approaches for bean bug control.

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

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. 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. 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. 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.

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

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. 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. 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.

The behaviour of recently-hatched caddis larvae from temporary water bodies in the British Isles

Caddisflies use four basic methods to ensure that newly-hatched larvae become aquatic insects. One is to lay the eggs underwater so larvae can start a free life immediately after hatching. Another method is to lay the eggs above water and the hatched larvae fall or wriggle into the water. Caddisflies from waters that dry out in summer often have a female diapause gradually becoming sexually mature and often do not lay their clutches until late summer or autumn, when rising water levels flood the eggs. The hatched larvae develop into pupae by March-May, from which adult caddisflies hatch again emergence beginning in spring. Some caddisflies from waters that dry out lay in summer and those eggs hatch soon after laying so it is as a larva that they survive the dry period.

Suppression of the gene encoding PDZ domain-containing protein decreases cold tolerance and overwintering survival of the mosquito, Culex pipiens (Culicidae: Diptera).

In diapausing mosquitoes, cold tolerance and prolonged lifespan are important features that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that PDZ domain-containing protein (PDZ) (post synaptic density protein [PSD95], drosophila disc large tumor suppressor [Dlg1], and zonula occludens-1 protein [zo-1]) domain-containing protein is involved with these diapause features for overwintering survival in Culex mosquitoes. The expression level of pdz was significantly higher in diapausing adult females in the early stage in comparison to their nondiapausing counterparts. Suppression of the gene that encodes PDZ by RNA interference significantly decreased actin accumulation in the midgut of early-stage adult diapausing females. Inhibition of pdz also significantly reduced the survivability of diapausing females which indicates that this protein could play a key role in preserving the midgut tissues during early diapause.

On the Nature of Individual Variation in Diapause Induction among the Progeny of Trichogramma telengai Sor. (Hymenoptera, Trichogrammatidae)

In this communication, we analyze the variation in diapause incidence among isofemale substrains of the main laboratory strain of Trichogramma telengai Sor. (Hymenoptera, Trichogrammatidae) in order to clarify the nature of the previously discovered individual (intrastrain) variation in this trait. During the experiment, the maternal generation developed at a temperature of 20°C under a day length of either 12 or 18 h, and diapause in the progeny was induced by development at 14°C in darkness. In total, the experiment comprised eight replicates in which female diapause incidence was measured in 428 substrains in two consecutive generations. There was statistically significant individual (first-generation) and interfamilial (second-generation) variation in almost all of the replicates. However, the correlation between the diapause incidence values among the progeny of two consecutive generations of the same substrains was weak and largely nonsignificant. These results suggest that intrastrain variation in the percentage of diapausing progeny of T. telengai females is not primarily produced by genetic (hereditary) factors but is due to certain environmental (nonhereditary) effects.

Bacterial Communities of Lab and Field Northern House Mosquitoes (Diptera: Culicidae) Throughout Diapause.

Diapause is a hormonally driven response which is triggered by environmental cues that signal impending adverse conditions and prompts metabolic, developmental, and behavioral changes to allow survival until the return of favorable conditions. Microbial symbionts have been shown to influence the metabolism, development, and behavior of their host organisms, all of which are common diapause-associated characteristics. Surveys of bacterial components in relation to diapause have been examined in few systems, of which the species are usually inactive during dormancy, such as eggs or pupae. This is specifically intriguing as adult female diapause in Culex pipiens (Diptera: Culicidae) can last between 4 and 7 mo and females remain mobile within their hibernacula. Furthermore, it is unknown how microbiota changes associated with prolonged dormancy are different between the lab and field for insect systems. This study aims to characterize how the microbiota of C. pipiens changes throughout diapause under both field and lab settings when provided identical food and water resources. Based on these studies, C. pipiens microbiota shifts as diapause progresses and there are considerable differences between field and lab individuals even when provided the same carbohydrate and water sources. Specific bacterial communities have more association with different periods of diapause, field and lab rearing conditions, and nutritional reserve levels. These studies highlight that diapausing mosquito microbiota studies ideally should occur in field mesocosms and at multiple locations, to increase applicability to wild C. pipiens as prolonged exposure to artificial rearing conditions could impact metrics related to diapause-microbiome interactions. Additionally, these findings suggest that it would be worthwhile to establish if the microbiota shift during diapause impacts host physiology and whether this shift is critical to diapause success.

Effect of changing temperatures from the spawning and egg stages to the emerging adult stage on the induction of reproductive diapause in females of Pseudaulacaspis pentagona (Hemiptera: Diaspididae)

Effect of changing temperatures from the spawning and egg stages to the emerging adult stage on the induction of reproductive diapause in females of Pseudaulacaspis pentagona (Hemiptera: Diaspididae)

Juvenile hormone biosynthetic genes are critical for regulating reproductive diapause in the cabbage beetle

In insects, the juvenile hormone (JH) biosynthetic pathway regulates the in vivo JH titer. Thus, its downregulation potentially contributes to the lowering of JH titers typically observed in insects undergoing reproductive diapause, a developmental arrest at the adult stage. However, no systematic evidence has yet been presented to demonstrate the physiological and genetic roles of JH biosynthetic genes in reproductive diapause. In this work, we performed RNA interference (RNAi)-based reverse genetic analyses by targeting JH biosynthetic genes, followed by analysis of the reproductive diapause traits in Colaphellus bowringi, an economically important cabbage beetle. We identified a total of 22 genes encoding homologues of enzymes involved in the mevalonate pathway and the JH branch of JH biosynthesis in C. bowringi. Among these, 18 genes showed significant downregulation of their expression in the long day-induced diapausing females, compared to the short day-induced reproductive females. RNAi knockdown of almost any one of the 18 genes in reproductive females reduced the expression of the JH-responsive gene, Krüppel homolog1 (Kr-h1), indicating a lowered circulating JH. Most importantly, depleting transcripts of 3-hydroxy-3-methylglutaryl-CoA reductase 2 (HMGR2), farnesyl-pyrophosphate synthase 1 (FPPS1) and juvenile hormone acid methyltransferase 1 (JHAMT1) induced diapause-associated traits, including immature and inactive ovaries, large accumulations of lipids and adult burrowing behavior. Meanwhile, genes related to ovarian development, lipid accumulation and stress response showed expression patterns like those of diapausing females. RNAi-mediated diapause phenotypes could be reversed to reproductive phenotypes by application of methoprene, a JH receptor agonist. These results suggest that photoperiodic reproductive diapause in C. bowringi is triggered by transcriptional suppression of JH biosynthetic genes, with HMGR2, FPPS1 and JHAMT1 playing a critical role in this process. This work provides sufficient evidence to reveal the physiological roles of JH biosynthetic genes in reproductive diapause.

Diapause Termination in Invasive Populations of the Brown Marmorated Stink Bug (Hemiptera: Pentatomidae) in Response to Photoperiod.

Understanding cues for diapause termination in insects can be valuable in predicting phenological events in their lifecycles. Once identified, such cues can be utilized as a biofix, the point at which the majority of individuals within a population begin to accumulate degree days. We investigated the impact of photoperiod on completion of reproductive diapause in the invasive eastern North American population of the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), by exposing diapausing females to varying light regimes in otherwise identical environments. The critical photoperiod estimated to initiate reproductive development in at least 50% of the potential reproductive population was 13.0-13.5 h, with increasing photoperiods coinciding with increased probability of females reproducing, earlier time to first oviposition, and higher rates of fecundity. These data on the species' response to photoperiod are in agreement with previous modeling that predicted the twin constraints of photoperiod and temperature on H. halys reproduction prevents populations that undergo diapause from producing more than two generations annually anywhere within the continental U.S. However, the facultative nature of diapause in H. halys leaves open the possibility that sub-populations may not enter diapause in some conditions, potentially allowing for additional annual generations.

Microbiome reduction prevents lipid accumulation during early diapause in the northern house mosquito, Culex pipiens pipiens

The mosquito microbiome is critical to multiple facets of their biology, including larval development and disease transmission. For mosquitoes that reside in temperate regions, periods of diapause are critical to overwintering survival, but how the microbiome impacts this state is unknown. In this study, we compared the midgut microbial communities of diapausing and non-diapausing Culex pipiens and assessed how a reduced midgut microbiome influences diapause preparation. High community variability was found within and between non-diapausing and diapausing individuals, but no specific diapause-based microbiome was noted. Emergence of adult, diapausing mosquitoes under sterile conditions generated low bacterial load (LBL) lines with nearly a 1000-fold reduction in bacteria levels. This reduction in bacterial content resulted in significantly lower survival of diapausing females after two weeks, indicating acquisition of the microbiome in adult females is critical for survival throughout diapause. LBL diapausing females had high carbohydrate levels, but did not accumulate lipid reserves, suggesting an inability to process ingested sugars necessary for diapause-associated lipid accumulation. Expression patterns of select genes associated with mosquito lipid metabolism during diapause showed no significant differences between LBL and control lines, suggesting transcriptional changes may not underlie impaired lipid accumulation. Overall, a diverse, adult-acquired microbiome is critical for diapause in C. pipiens to process sugar reserves and accumulate lipids that are necessary to survive prolonged overwintering.

Krüppel homolog 1 regulates photoperiodic reproductive plasticity in the cabbage beetle Colaphellus bowringi

Many insects exhibit reproductive plasticity where the photoperiod determines whether the insect becomes reproductively active or enters diapause. Adult reproductive diapause is a strategy that allows insects to survive harsh environmental conditions. A deficiency in juvenile hormone (JH) leads to reproductive diapause. However, little is known about the molecular mechanisms by which JH signaling regulates reproductive diapause. In this study, we used the cabbage beetle Colaphellus bowringi, a serious pest, to investigate the role of Krüppel homolog 1 (Kr-h1) in controlling photoperiodic plasticity of female reproduction. We focused on Kr-h1, since it acts as a key mediator of JH signaling. We show here that JH-Methoprene-tolerant signaling upregulated the expression of Kr-h1 in reproductively active C. bowringi females when reared under short day conditions. In the long day-treated diapausing females, Kr-h1 transcripts decreased dramatically. Interfering with Kr-h1 function repressed reproductive development by blocking vitellogenesis and ovarian growth. Further, Kr-h1 depletion induced other diapause-like traits, including elevated lipid accumulation and high expression of diapause-related genes. RNA-Seq showed that Kr-h1 played both activating and repressive roles, depending on whether downstream genes were acting in reproduction- or diapause pathways, respectively. Finally, we identified the DNA replication gene mini-chromosome maintenance 4 and two triacylglycerol lipase genes as critical downstream factors of Kr-h1 that are critical for reproductive plasticity in C. bowringi. These results reveal that Kr-h1 is a key component of the regulatory pathway that coordinates reproduction and diapause in insects in response to photoperiodic input.

Suppressed expression of oxidoreductin-like protein, Oxidor, increases follicle degeneration and decreases survival during the overwintering diapause of the mosquito Culex pipiens

Throughout diapause in mosquitoes, stress resistance and subsequent prolonged lifespan are a few important features of diapause that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that oxidoreductin-like protein is involved with these diapause characteristics for overwintering survival. Expression of oxidor was more than two-fold higher in early stage diapausing females compared to their non-diapausing counterparts. Suppression of the gene that encodes oxidoreductin-like protein by RNAi significantly increased the proportion of degenerating follicles in early-stage adult diapausing females. Inhibition of oxidor also significantly reduced the survivability of diapausing females which indicates that this protein plays a key role in protecting multiple tissues during early diapause.

Nanda-Hamner Curves Show Huge Latitudinal Variation but No Circadian Components in Drosophila Montana Photoperiodism.

Insect species with a wide distribution offer a great opportunity to trace latitudinal variation in the photoperiodic regulation of traits important in reproduction and stress tolerances. We measured this variation in the photoperiodic time-measuring system underlying reproductive diapause in Drosophila montana, using a Nanda-Hamner (NH) protocol. None of the study strains showed diel rhythmicity in female diapause proportions under a constant day length (12 h) and varying night lengths in photoperiods ranging from 16 to 84 h at 16°C. In the northernmost strains (above 55°N), nearly all females entered diapause under all photoperiods and about half of them even in continuous darkness, while the females of the southern strains showed high diapause proportions only in the circadian 24 h photoperiod. Significant correlation between the strains’ mean diapause proportions in ≥ 24 h photoperiods and critical day length (CDL; half of the females enter diapause) suggests at least partial causal connection between the traits. Interestingly, females of the northern strains entered diapause even in ≤ 24 h photoperiods, where the night length was shorter than their critical night length (24 h - CDL), but where the females experienced a higher number of Light:Dark cycles than in 24 h photoperiods. NH experiments, performed on the control and selection lines in our previous selection experiment, and completed here, gave similar results and confirmed that selection for shorter, southern-type CDL decreases female diapausing rate in non-circadian photoperiods. Overall, our study shows that D. montana females measure night length quantitatively, that the photoperiodic counter may play a prominent but slightly different role in extra short and extra long photoperiods and that northern strains show high stability against perturbations in the photoperiod length and in the presence of LD cycles. These features are best explained by the quantitative versions of the damped external coincidence model.

Preimaginal caste-related bias in the paper wasp Polistes jokahamae is limited to the first brood

Whether the caste fate of social insects is determined before or after emergence is a key question for understanding the evolution of eusociality. Paper wasps are a suitable model for answering this question because there are no critical morphological differences between queens and workers in paper wasps, and these animals appear to represent an early stage of eusociality. We explored the above question by determining the effects of photoperiod during the adult stage on caste-fate determination in the paper wasp Polistes jokahamae. We collected colonies at different stages in the field and exposed emerging adults individually to long or short days. Under these isolated conditions, gyne-destined (diapausing) females were expected to exhibit large lipid stores without mature eggs, while the reverse was expected to be true for worker-destined (nondiapausing) females. The proportion of wasps with mature eggs was higher under long days in the second and subsequent broods, but not in the first brood. Lipid stores were larger among large females and under short days, and smaller for the first brood. These findings together suggest that the first brood emerges with a strong preimaginal bias toward workers (nondiapausing form), whereas the other broods emerge with no bias or an easily reversible bias. However, it is difficult to conclude whether the bias came from body size or the season of emergence. We discuss the possibility that the ancestor of paper wasps had workers with and without preimaginal bias toward becoming workers at emergence.

Late autumn occurrence of non-diapause male adults in various geographic populations of a pierid butterfly, Eurema mandarina

ABSTRACT Adult-overwintering insects show diversity in the occurrence of reproductive diapause of males. Mating can occur both before and after winter in different species, and pre-winter mating allows males to reproduce without overwintering. In a pierid butterfly, Eurema mandarina, both diapause and non-diapause male adults emerge in the late autumn generation. Diapause females mate with non-diapause males before winter and re-mate with diapause males the following spring. Although this species is distributed from sub-tropical regions to cool-temperate regions, the geographic variation of this characteristic mating system has not been examined. A rearing experiment and a field survey were conducted to determine the geographic patterns of the mating system of the late autumn generation of E. mandarina for various populations from sub-tropical to cool temperate regions. Non-diapause males were observed in the late autumn generation in all populations examined. The frequency of pre-winter mating of females was not correlated with latitude. These results show that the mating system of the late autumn generation is similar among various populations distributed over a wider range, suggesting that the characteristic mating system may be evolutionarily stable.

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.

Prolonged diapause has sex-specific fertility and fitness costs

Diapause in seasonal environments allows insects to survive adverse seasons. However, individuals can sometimes enter a prolonged diapause for more than a year, and also skip favourable seasons, which can bring additional costs through e.g. loss of metabolic resources. At the same time, prolonged diapause can be beneficial if it allows individuals to have a risk-spreading strategy to skip potentially suboptimal breeding seasons. We studied if prolonged diapause (2-year diapause) negatively affects the fertility and fitness of female and male Colorado potato beetles (Leptinotarsa decemlineata) compared to control (1-year diapause) beetles. We also tested the parental effects on the subsequent chemical stress tolerance of their offspring. We found that prolonged diapause carried fertility costs only for females who were less fertile than the control females. However, no differences in fertility were observed in males. Furthermore, prolonged diapause in females resulted in offspring with lower larvae-to-adult survival even though these offspring had accelerated development times. In contrast, paternal diapause duration had no effects on their offspring larvae-to adult survival, but prolonged diapause males sired offspring with slower development times than control males. Perhaps to compensate the costs related to prolonged diapause both older parents produced or sired offspring with higher body mass than control parents. Despite the differences in emergence mass, parental diapause duration did not affect offspring insecticide stress tolerance. The difference between females and males most likely results from the observed differences in prolonged diapause females’ capacity to fight against cellular oxidative damage which was poorer compared to the control females. Even though prolonged diapause allows individuals to have a risk-spreading strategy it carries sex-specific fertility and fitness costs indicating that selection could favour this in males but not in females.

Overwintering of Piezodorus guildinii (Heteroptera, Pentatomidae) Populations.

Piezodorus guildinii (Westwood) is a soybean pest that causes significant economic losses in the Americas. The variability of overwintering (diapause) traits was evaluated in populations of the Southwest (SW) (33°55'-34°17'S, 57°13'-57°46'W) during 2-year period (2011-2013) and of the Northwest (NW) (32°01'-33°02'S, 57°50'-57°24'W) during 1-year period (2014-2015) Regions of Uruguay. Samples were taken from different plant species (cultivated legumes, wild shrubs, and trees) and from overwintering sites (leaf litter and bark). Alfalfa, Medicago sativa L. was the main host, with a collection period of 10-11 months in the SW and 12 months in the NW. Cluster analysis for each sex was carried out to group the months according to the similarity in diapause traits of populations (body size, body lipid content, immature reproductive organs, and clear type of pronotum band and connexivum in females). Female diapause in the SW was longer (beginning of autumn to end of winter) than that in the NW (mid-autumn to mid-winter). Male diapause was longer (mid-autumn to mid-winter) in SW1 (1st year) than in SW2 (2nd year) and NW (late-autumn to mid-winter). In both regions, male diapause was shorter than female. Differences were associated with maximum temperature at daylight hours ≤ 12.1, being necessary maximum temperatures below 23.8 °C for females and 19.2 °C for males to initiate diapause.

Quantitative Proteomic and Transcriptomic Analyses of Metabolic Regulation of Adult Reproductive Diapause in Drosophila suzukii (Diptera: Drosophilidae) Females.

Diapause is a form of dormancy used by many insects to survive adverse environmental conditions, which can occur in specific developmental stages in different species. Drosophila suzukii is a serious economic pest and we determined the conditions for adult reproductive diapause by the females in our previous studies. In this study, we combined RNA-Seq transcriptomic and quantitative proteomic analyses to identify adult reproductive diapause-related genes and proteins. According to the transcriptomic analysis, among 242 annotated differentially expressed genes in non-diapause and diapause females, 129 and 113 genes were up- and down-regulated, respectively. In addition, among the 2,375 proteins quantified, 39 and 23 proteins were up- and down-regulated, respectively. The gene expression patterns in diapause- and non-diapause were confirmed by qRT-PCR or western blot analysis. The overall analysis of robustly regulated genes at the protein and mRNA levels found four genes that overlapped in the up-regulated group and six genes in the down-regulated group, and thus these proteins/genes may regulate adult reproductive diapause. These differentially expressed proteins/genes act in the citrate cycle, insulin signaling pathway, PI3K-Akt signaling pathway, and amino acid biosynthesis pathways. These results provide the basis for further studies of the molecular regulation of reproductive diapause in this species.