Nutrient deprivation induces mouse embryonic diapause mediated by Gator1 and Tsc2.

Embryonic diapause is a special reproductive phenomenon in mammals that helps embryos to survive various harsh stresses. However, the mechanisms of embryonic diapause induced by the maternal environment is still unclear. Here, we uncovered that nutrient deficiency in uterine fluid was essential for the induction of mouse embryonic diapause, shown by a decreased concentration of arginine, leucine, isoleucine, lysine, glucose and lactate in the uterine fluid of mice suffering from maternal starvation or ovariectomy. Moreover, mouse blastocysts cultured in a medium with reduced levels of these six components could mimic diapaused blastocysts. Our mechanistic study indicated that amino acid starvation-dependent Gator1 activation and carbohydrate starvation-dependent Tsc2 activation inhibited mTORC1, leading to induction of embryonic diapause. Our study elucidates the essential environmental factors in diapause induction.

A model for predicting the phenology of Philaenus spumarius.

The design and implementation of Philaenus spumarius control strategies can take advantage of properly calibrated models describing and predicting the phenology of vector populations in agroecosystems. We developed a temperature-driven physiological-based model based on the system of Kolmogorov partial differential equations to predict the phenological dynamics of P. spumarius. The model considers the initial physiological age distribution of eggs, the diapause termination process, and the development rate functions of post-diapausing eggs and nymphal stages, estimated from data collected in laboratory experiments and field surveys in Italy. The temperature threshold and cumulative degree days for egg diapause termination were estimated as 6.5 °C and 120 DD, respectively. Preimaginal development rate functions exhibited lower thresholds ranging between 2.1 and 5.0 °C, optimal temperatures between 26.6 and 28.3 °C, and upper threshold between 33.0 and 35 °C. The model correctly simulates the emergence of the 3rd, 4th, and 5th nymphal instars, key stages to target monitoring actions and control measures against P. spumarius. Precision in simulating the phenology of the 1st and 2nd nymphal stages was less satisfactory. The model is a useful rational decision tool to support scheduling monitoring and control actions against the late and most important nymphal stages of P. spumarius.

Photoperiodism of Diapause Induction in the Silkworm, Bombyx mori.

The silkworm Bombyx mori exhibits a photoperiodic response (PR) for embryonic diapause induction. This article provides a comprehensive review of literature on the silkworm PR, starting from early works on population to recent studies uncovering the molecular mechanism. Makita Kogure (1933) conducted extensive research on the PR, presenting a pioneering paper on insect photoperiodism. In the 1970s and 80s, artificial diets were developed, and the influence of nutrition on PR was well documented. The photoperiodic photoreceptor has been investigated from organ to molecular level in the silkworm. Culture experiments demonstrated that the photoperiodic induction can be programmed in an isolated brain (Br)-subesophageal ganglion (SG) complex with corpora cardiaca (CC)-corpora allata (CA). The requirement of dietary vitamin A for PR suggests the involvement of opsin pigment in the photoperiodic reception, and a cDNA encoding an opsin (Boceropsin) was cloned from the brain. The effector system concerning the production and secretion of diapause hormone (DH) has also been extensively investigated in the silkworm. DH is produced in a pair of posterior cells of SG, transported to CC by nervi corporis cardiaci, and ultimately released into the hemolymph. Possible involvement of GABAergic and corazonin (Crz) signal pathways was suggested in the control of DH secretion. Knockout (KO) experiments of GABA transporter (GAT) and circadian clock genes demonstrated that GAT plays a crucial role in PR through circadian control. A model outlining the PR mechanism, from maternal photoperiodic light reception to DH secretion, has been proposed.

GC-MS-based Metabolomics Unravels Metabolites across Larval Development and Diapause of a Specialist Insect.

Plant-insect interactions are a driving force into ecosystem evolution and community dynamics. Many insect herbivores enter diapause, a developmental arrest stage in anticipation of adverse conditions, to survive and thrive through seasonal changes. Herein, we investigated the roles of medium- to non-polar metabolites during larval development and diapause in a specialist insect herbivore, Chlosyne lacinia, reared on Aldama robusta leaves. Varying metabolites were determined using gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Sesquiterpenes and steroids were the main metabolites putatively identified in A. robusta leaves, whereas C. lacinia caterpillars were characterized by triterpenes, steroids, fatty acids, and long-chain alkanes. We found out that C. lacinia caterpillars biosynthesized most of the identified steroids and fatty acids from plant-derived ingested metabolites, as well as all triterpenes and long-chain alkanes. Steroids, fatty acids, and long-chain alkanes were detected across all C. lacinia instars and in diapausing caterpillars. Sesquiterpenes and triterpenes were also detected across larval development, yet they were not detected in diapausing caterpillars, which suggested that these metabolites were converted to other molecules prior to the diapause stage. Our findings shed light on the chemical content variation across C. lacinia development and diapause, providing insights into the roles of metabolites in plant-insect interactions.

The population genetics of partial diapause, with applications to the aestivating malaria mosquito Anopheles coluzzii.

Diapause, a form of dormancy to delay or halt the reproductive development during unfavourable seasons, has evolved in many insect species. One example is aestivation, an adult-stage diapause enhancing malaria vectors' survival during the dry season (DS) and their re-establishment in the next rainy season (RS). This work develops a novel genetic approach to estimate the number or proportion of individuals undergoing diapause, as well as the breeding sizes of the two seasons, using signals from temporal allele frequency dynamics. Our modelling shows the magnitude of drift is dampened at early RS when previously aestivating individuals reappear. Aestivation severely biases the temporal effective population size ( N e $$ {N}_e $$ ), leading to overestimation of the DS breeding size by 1 / 1 - α 2 $$ 1/{\left(1-\alpha \right)}^2 $$ across 1 year, where α $$ \alpha $$ is the aestivating proportion. We find sampling breeding individuals in three consecutive seasons starting from an RS is sufficient for parameter estimation, and perform extensive simulations to verify our derivations. This method does not require sampling individuals in the dormant state, the biggest challenge in most studies. We illustrate the method by applying it to a published data set for Anopheles coluzzii mosquitoes from Thierola, Mali. Our method and the expected evolutionary implications are applicable to any species in which a fraction of the population diapauses for more than one generation, and are difficult or impossible to sample during that stage.

The effect of artificial light at night (ALAN) on the characteristics of diapause of Aedes albopictus.

BACKGROUND: Recently, the effect of artificial light at night (ALAN) on the physiology and behavior of insects has gradually attracted the attention of researchers and has become a new research topic. Aedes albopictus is an important vector that poses a great public health risk. Further studies on the diapause of Ae. albopictus can provide a basis for new vector control, and it is also worth exploring whether the effect of ALAN on the diapause of Ae. albopictus will provide a reference for the prevention and control of infectious diseases mediated by Ae. albopictus. METHODS: In this study, we experimentally studied the diapause characteristics of different geographical strains of Ae. albopictus under the interference of ALAN, explored the effect of ALAN on the diapause of Ae. albopictus and explored the molecular mechanism of ALAN on the diapause process through RNA-seq. RESULTS: As seen from the diapause incidence, Ae. albopictus of the same geographic strain showed a lower diapause incidence when exposed to ALAN. The differentially expressed genes (DEGs) were mainly enriched in signaling and metabolism-related pathways in the parental females and diapause eggs of the ALAN group. CONCLUSIONS: ALAN inhibits Ae. albopictus diapause. In the short photoperiod induced diapause of Ae. albopictus in temperate strain Beijing and subtropical strain Guangzhou, the disturbance of ALAN reduced the egg diapause rate and increased the egg hatching rate of Ae. albopictus, and the disturbance of ALAN also shortened the life cycle of Ae. albopictus eggs after hatching.

Transcriptome-based analysis reveals a crucial role of the 20E/HR3 pathway in the diapause of Pieris rapae.

Pieris rapae is among the most damaging pests globally, and diapause makes it highly resistant to environmental stresses, playing a crucial role in the survival and reproduction of P. rapae while exacerbating the challenges of pest management and control. However, the mechanisms of its diapause regulation remain poorly understood. This research used RNA sequencing to profile the transcriptomes of three diapause phases (induction and preparation, initiation, maintenance) and synchronous nondiapause phases in P. rapae. During each comparison phase, 759, 1045, and 4721 genes were found to be differentially expressed. Among these, seven clock genes and seven pivotal hormone synthesis and metabolism genes were identified as having differential expression patterns in diapause type and nondiapause type. The weighted gene co-expression network analysis (WGCNA) revealed the red and blue modules as pivotal for diapause initiation, while the grey module was identified to be crucial to diapause maintenance. Meanwhile, the hub genes HDAC11, METLL16D, Dyw-like, GST, and so on, were identified within these hub modules. Moreover, an ecdysone downstream nuclear receptor gene, HR3, was found to be a shared transcription factor across all three phases. RNA interference of HR3 resulted in delayed pupal development, indicating its involvement in regulating pupal dipause in P. rapae. The further hormone assays revealed that the 20-hydroxyecdysone (20E) titer in diapause type pupae was lower than that in nondiapause type pupae, which exhibited a similar trend to HR3. When 20E was injected into diapause pupae, the HR3 expression levels were improved, and the pupal diapause were broken. These results indicate that the 20E/HR3 pathway is a critical pathway for the diapause regulation of P. rapae, and perturbing this pathway by ecdysone treatment or RNAi would result in the disruption of diapause. These findings provide initial insights into the molecular mechanisms of P. rapae diapause and suggest the potential use of ecdysone analogs and HR3 RNAi pesticides, which specifically target to diapause, as a means of pest control in P. rapae.

Impacts of winter warming events on spruce budworm: the importance of timing.

Winter climate change constitutes not only a shift in chronic conditions (i.e., shorter length and warmer average temperatures) but will also influence the dynamics of extreme warming events. The latter may be particularly important for the performance and survival of insects, given their susceptibility to temperature variation. However, metabolic sensitivity changes over the course of winter diapause, and thus, insect responses to warming may vary depending on when the event occurs. To determine the influence of warm-up timing, we exposed the spruce budworm, Choristoneura fumiferana ((Clem.), Lepidoptera: Tortricidae), to acute warming events in early-, mid-, and late-dormancy and measured impacts on survival, performance, and biochemistry. While we did not observe any impacts of warm-up timing on performance, survival to the adult stage was significantly reduced in response to earlier warming. Additionally, glycogen concentration was significantly higher in response to early and late warming exposure. Collectively, these results suggest that the timing of extreme winter warming events matters, with consequences for both lethal and sublethal responses.

Prolonged Heat Stress during Winter Diapause Alters the Expression of Stress-Response Genes in Ostrinia nubilalis (Hbn.).

During diapause, a state of temporarily arrested development, insects require low winter temperatures to suppress their metabolism, conserve energy stores and acquire cold hardiness. A warmer winter could, thus, reduce diapause incidence and duration in many species, prematurely deplete their energy reserves and compromise post-diapause fitness. In this study, we investigated the combined effects of thermal stress and the diapause program on the expression of selected genes involved in antioxidant defense and heat shock response in the European corn borer Ostrinia nubilalis. By using qRT-PCR, it has been shown that response to chronic heat stress is characterized by raised mRNA levels of grx and trx, two important genes of the antioxidant defense system, as well as of hsp70 and, somewhat, of hsp90, two major heat shock response proteins. On the other hand, the expression of hsc70, hsp20.4 and hsp20.1 was discontinuous in the latter part of diapause, or was strongly controlled by the diapause program and refractory to heat stress, as was the case for mtn and fer, genes encoding two metal storage proteins crucial for metal ion homeostasis. This is the first time that the effects of high winter temperatures have been assessed on cold-hardy diapausing larvae and pupae of this important corn pest.

Effects of photoperiod and temperature on the developmental duration and diapause in Dolycoris baccarum (Heteroptera: Pentatomidae) from Hohhot, Inner Mongolia, China.

The shield bug, Dolycoris baccarum (L.) (Heteroptera: Pentatomidae), is widely distributed across Asia and Europe. At high latitudes, it overwinters, as adult in diapause, which then becomes the insect source for the following year. To fully understand the developmental duration and diapause characteristics of D. baccarum, the effects of photoperiod and temperature were studied in a population from Hohhot, Inner Mongolia, China. The results indicated that the developmental duration was significantly prolonged at temperatures of 20 or 25 °C, with a prolonged light period; however, when the light period was prolonged to 16L:8D and 18L:6D, the developmental duration was shortened significantly. Furthermore, the developmental duration was also shortened significantly with increasing temperature, when the photoperiod was 12L:12D for short days and 16L:8D for long days. All individuals entered diapause under short-day conditions of 10L:14D and 12L:12D at a temperature of 20 °C; however, the diapause rate decreased significantly under 14L:10D and 16L:8D photoperiods, and the diapause rate decreased significantly at a temperature of 25 °C with prolonged photoperiod. Interestingly, when the photoperiod was fixed at 12L:12D, the diapause rates at different temperatures (20, 25, 28, and 30 °C) exceeded 95%; while the effect of temperature on diapauses was nonsignificant under this photoperiod, it was still sensitive to the photoperiod; at a photoperiod of 16L:8D, the effect of temperature on the diapause rate was noticeable, and the diapause rate decreased significantly with increasing temperature.

Developmental progression continues during embryonic diapause in the roe deer.

Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.

Different photoperiodic responses in diapause induction can promote the maintenance of genetic diversity via the storage effect in Daphnia pulex.

Understanding mechanisms that promote the maintenance of biodiversity (genetic and species diversity) has been a central topic in evolution and ecology. Previous studies have revealed that diapause can contribute to coexistence of competing genotypes or species in fluctuating environments via the storage effect. However, they tended to focus on differences in reproductive success (e.g. seed yield) and diapause termination (e.g. germination) timing. Here we tested whether different photoperiodic responses in diapause induction can promote coexistence of two parthenogenetic (asexual) genotypes of Daphnia pulex in Lake Fukami-ike, Japan. Through laboratory experiments, we confirmed that short day length and low food availability induced the production of diapausing eggs. Furthermore, we found that one genotype tended to produce diapausing eggs in broader environmental conditions than the other. Terminating parthenogenetic reproduction earlier decreases total clonal production, but the early diapausing genotype becomes advantageous by assuring reproduction in 'short' years where winter arrival is earlier than usual. Empirically parameterized theoretical analyses suggested that different photoperiodic responses can promote coexistence via the storage effect with fluctuations of the growing season length. Therefore, timing of diapause induction may be as important as diapause termination timing for promoting the maintenance of genetic diversity in fluctuating environments.

Diapause-like Drug-Tolerant Persister State: The Key to Nirvana Rebirth.

Cancer is one of the leading causes of death in the world. Various drugs have been developed to eliminate it but to no avail because a tumor can go into dormancy to avoid therapy. In the past few decades, tumor dormancy has become a popular topic in cancer therapy. Recently, there has been an important breakthrough in the study of tumor dormancy. That is, cancer cells can enter a reversible drug-tolerant persister (DTP) state to avoid therapy, but no exact mechanism has been found. The study of the link between the DTP state and diapause seems to provide an opportunity for a correct understanding of the mechanism of the DTP state. Completely treating cancer and avoiding dormancy by targeting the expression of key genes in diapause are possible. This review delves into the characteristics of the DTP state and its connection with embryonic diapause, and possible treatment strategies are summarized. The authors believe that this review will promote the development of cancer therapy.

Impact of diapause on the cuticular hydrocarbons and physiological status of Hippodamia variegata.

The spotted amber ladybird, Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae), is known to be a potent predator of aphids, psyllids, whiteflies, mealybugs, and some butterfly species. This ladybeetle overwinters in the diapausing adult stage. The current study aimed to evaluate the impact of diapause on the energy resources and cuticular hydrocarbons (CHCs) of the female ladybeetle, specifically comparing the changes in glycogen, lipid, and protein contents, and CHCs profile of diapausing and non-diapausing adults. In this study, gas chromatography-mass was used to analyze whole-body extracts of the beetles. Results showed no significant differences between the amount of glycogen, lipid, and protein contents of diapausing and non-diapausing ladybeetle. The CHCs profile of H. variegata consisted of 24 hydrocarbons categorized into 2 groups: linear aliphatic hydrocarbons (n-alkanes) and methyl-branched hydrocarbons (17 molecules), as well as unsaturated cyclic compounds (7 molecules). The n-alkanes, with 14 compounds, were identified as the primary constituents of the CHCs of the ladybeetle. Six molecules were common to non-diapausing and diapausing beetles, 5 were exclusive to non-diapausing beetles, and 13 were exclusive to diapausing beetles. Moreover, we noted a significant difference in the quantity and quality of CHCs between diapausing and non-diapausing beetles, with diapausing beetles synthesizing more CHCs with longer chains. This disparity in CHC profiles was concluded to be an adaptation of H. variegata to survive harsh environmental conditions during diapause.

Phenotypic plasticity of a winter-diapause mechanism copes with the effects of summer global warming in an ectothermic predator.

To adapt to changes in temperature, animals tend to invest more energy in thermal tolerance to enhance survival, which can have simultaneous costs on plastic traits. Would a decrease in genetic variability, due to global warming, affect the ability of populations with existing metabolic regulatory mechanisms to cope with extreme temperatures? To address this question, we conducted a series of experiments based on the A1B scenario of global warming, assessing within-population genetic variance in (a) morphological traits, (b) metabolic rate allometries, and (c) survival of a winter-diapausing predator ectotherm. Our study focused on the lacewing species Chrysoperla pallida, using both exogamic and endogamic artificial genetic lines. We discovered that both lines use their winter-diapausing phenotype to adapt to summer extreme temperatures caused by extreme heating conditions, but the exogamic line is prone to express phenotypic plasticity in metabolic scaling, with a trade-off between body size and mandible size, i.e. larger individuals tended to develop smaller mandibles to better survive. These findings highlight the significance of substantial phenotypic plasticity and pre-existing metabolic regulatory mechanisms in enabling ectotherms to cope with potential extreme heating occurring in global warming.

Significance of the clock gene period in photoperiodism in larval development and production of diapause eggs in the silkworm Bombyx mori.

Many insects living in seasonal environments sense seasonal changes from photoperiod and appropriately regulate their development and physiological activities. Genetic researches have indicated the importance of a circadian clock system in photoperiodic time-measurement for photoperiodic regulations. However, most previous studies have focused on the effects on a single photoperiodic phenotype, without elucidating whether the circadian clock is involved in the core photoperiodic mechanism or only in the production of one target phenotype, such as diapause. Here, we focused on two different phenotypes in a bivoltine Kosetsu strain of the silkworm Bombyx mori, namely, embryonic diapause and larval development, and examined their photoperiodic responses and relationship to the circadian clock gene period. Photoperiod during the larval stage clearly influenced the induction of embryonic diapause and duration of larval development in the Kosetsu strain; short-day exposure leaded to the production of diapause eggs and shortened the larval duration. Genetic knockout of period inhibited the short-day-induced embryonic diapause. Conversely, in the period-knockout silkworms, the larval duration was shortened, but the photoperiodic difference was maintained. In conclusion, our results indicate that the period gene is not causally involved in the photoperiodic response of larval development, while that is essential for the short-day-induced embryonic diapause.

Metabolic and transcriptomic characterization of summer and winter dormancy in the solitary bee, Osmia lignaria.

The solitary bee Osmia lignaria is a native pollinator in North America with growing economic importance. The life cycle of O. lignaria provides a unique opportunity to compare the physiological and molecular mechanisms underlying two ecologically contrasting dormancies within the same species. O. lignaria prepupae become dormant during the summer to avoid high temperatures. Shortly after adult eclosion, they enter a second dormancy and overwinter as diapausing adults. To compare these two dormancies, we measured metabolic rates and gene expression across development as bees initiate, maintain, and terminate both prepupal (summer) and adult (overwintering) dormancies. We observed a moderate temperature-independent decrease in gas exchange during both the prepupal dormancy after cocoon spinning (45 %) and during adult diapause after eclosion (60 %). We sequenced and assembled a high-quality reference genome from a single haploid male bee with a contiguous n50 of 5.5 Mbp to facilitate our transcriptomic analysis. The transcriptomes of dormant prepupae and diapausing adults clustered into distinct groups more closely associated with life stage than dormancy status. Membrane transport, membrane-bound cellular components, oxidoreductase activity, glutathione metabolism, and transcription factor activity increased during adult diapause, relative to prepupal dormancy. Further, the transcriptomes of adults in diapause clustered into two groups, supporting multiple phases of diapause during winter. Late adult diapause was associated with gene expression profiles supporting increased insulin/IGF, juvenile hormone, and ecdysone signaling.

Genetic constraints in genes exhibiting splicing plasticity in facultative diapause.

Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi. Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi.

The post-diapause vibrational behavior, motility, and survival of the brown marmorated stink bug Halyomorpha halys (Stål) adults at different temperatures.

Substrate-borne vibrational communication is common in pentatomids. Although several works exist on the vibrational communication of Halyomorpha halys, its vibrational behavior post diapause has not been investigated. In this study, we recorded H. halys overwintered adults using laser doppler vibrometers at three temperatures: 10 °C (inactivity), 18 °C (breaking of diapause), and 25 °C (peak of mating activity). The aim was to assess the effect of temperature on the signaling, motility, and survival of H. halys. The insects were sexed into different cages and recorded separately or joined with a cage of the opposite sex. We calculated the total time spent on signaling and walking per replica. The males predominantly emitted male signal 1 (MS1) throughout the four months of recordings. The females exclusively emitted female signal 2 (FS2) when joined with the opposite sex cage the first two months of recordings. Interestingly, they also started FS2 signaling when recorded separately, after two months. No signaling was recorded at 10 °C. At 25 °C, the signaling latency time before vibrational signaling was 24 h compared to 23 days at 18 °C. The short latency time at 25 °C correlated with a higher death rate in early stages of recording. Male walking activity was significantly higher in joined cages at 18 °C and 25 °C, suggesting the increased searching behavior near the opposite sex. Overwintered H. halys could adapt to different conditions whereas low temperatures maintain the diapause which is characterized by no signaling activity. Our results provide a foundation for bioclimatic modeling of climate change effects on H. halys and insights into the use of vibrational playbacks for mass trapping and monitoring as control techniques.

Regulation of forkhead box O transcription factor by insulin signaling pathway controls the reproductive diapause of the lady beetle, Coccinella septempunctata.

In biological control programs, knowledge about diapause regulation in natural enemy insects provides important insight for improving long-term storage, transportation, and field adoption of these biological control agents. As a natural predator of agricultural pests, the lady beetle Coccinella septempunctata has been commercially mass-cultured and widely employed in pest management. In some insects, insulin signaling, in conjunction with the downstream transcription factor Forkhead box O (FoxO), are master regulators of multiple physiological processes involved in diapause, but it is unclear whether insulin signaling and FoxO affect the diapause of C. septempunctata. In this study, we use a combination of approaches to demonstrate that insulin signaling and FoxO mediate the diapause response in C. septempunctata. In diapausing beetles, application of exogenous insulin and knocking down expression of CsFoxo with RNA interference (RNAi) both rescued beetles from developmental arrest. In non-diapausing beetles, knocking down expression of the insulin receptor (CsInR) with RNA interference (RNAi) arrested ovarian development and decreased juvenile hormone (JH) content to levels comparable to the diapause state. Taken together, these results suggest that a shutdown of insulin signaling prompts the activation of the downstream FoxO gene, leading to the diapause phenotype.