Revealing the profound influence of diapause on gene expression: Insights from the annual transcriptome of the copepod Calanus finmarchicus.

Annual rhythms are observed in living organisms with numerous ecological implications. In the zooplanktonic copepod Calanus finmarchicus, such rhythms are crucial regarding its phenology, body lipid accumulation, and global carbon storage. Climate change drives annual biological rhythms out of phase with the prevailing environmental conditions with yet unknown but potentially catastrophic consequences. However, the molecular dynamics underlying phenology are still poorly described. In a rhythmic analysis of C. finmarchicus annual gene expression, results reveal that more than 90% of the transcriptome shows significant annual rhythms, with abrupt and dramatic upheaval between the active and diapause life cycle states. This work explores the implication of the circadian clock in the annual timing, which may control epigenetic mechanisms to profoundly modulate gene expression in response to calendar time. Results also suggest an increased light sensitivity during diapause that would ensure the photoperiodic entrainment of the endogenous annual clock.

Genome-wide profiles of H3K9me3, H3K27me3 modifications, and DNA methylation during diapause of Asian corn borer (Ostrinia furnacalis).

Diapause represents a crucial adaptive strategy used by insects to cope with changing environmental conditions. In North China, the Asian corn borer (Ostrinia furnacalis) enters a winter larval diapause stage. Although there is growing evidence implicating epigenetic mechanisms in diapause regulation, it remains unclear whether dynamic genome-wide profiles of epigenetic modifications exist during this process. By investigating multiple histone modifications, we have discovered the essential roles of H3K9me3 and H3K27me3 during diapause of the Asian corn borer. Building upon previous findings in vertebrates highlighting the connection between DNA methylation and repressive histone methylations, we have examined changes in the genome-wide profile of H3K9me3, H3K27me3, and DNA methylation at the nondiapause, prediapause, and diapause stages. Data analysis reveals significant alterations in these three modifications during diapause. Moreover, we observe a correlation between the H3K9me3 and H3K27me3 modification sites during diapause, whereas DNA modifications show little association with either H3K9me3 or H3K27me3. Integrative analysis of epigenome and expression data unveils the relationship between these epigenetic modifications and gene expression levels at corresponding diapause stages. Furthermore, by studying the function of histone modifications on genes known to be important in diapause, especially those involved in the juvenile pathway, we discover that the juvenile hormone pathway lies downstream from H3K9me3 and H3K27me3 histone modifications. Finally, the analysis of gene loci with modified modifications unreported in diapause uncovers novel pathways potentially crucial in diapause regulation. This study provides a valuable resource for future investigations aiming to elucidate the underlying mechanisms of diapause.

Effects of overwintering on the transcriptome and fitness traits in a damselfly with variable voltinism across two latitudes.

Winter diapause consists of cessation of development that allows individuals to survive unfavourable conditions. Winter diapause may bear various costs and questions have been raised about the evolutionary mechanisms maintaining facultative diapause. Here, we explored to what extent a facultative winter diapause affects life-history traits and the transcriptome in the damselfly Ischnura elegans, and whether these effects were latitude-specific. We collected adult females at central and high latitudes and raised their larvae in growth chambers. Larvae were split into a non-diapausing and post-winter (diapausing) cohort, were phenotyped and collected for a gene expression analysis. At the phenotypic level, we found no difference in survival between the two cohorts, and the post-winter cohort was larger and heavier than the non-winter cohort. These effects were mostly independent of the latitude of origin. At the transcriptomic level, wintering affected gene expression with a small fraction of genes significantly overlapping across latitudes, especially those related to morphogenesis. In conclusion, we found clear effects of diapause on the phenotype but little evidence for latitudinal-specific effects of diapause. Our results showed a shared transcriptomic basis underpinning diapause demonstrated, here, at the intraspecific level and supported the idea of evolutionary convergence of the response to diapause across organisms.

Effect of cryopreservation on Odc1 and RhoA genes expression in diapausing mouse blastocysts.

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing in vivo-derived embryos continued their development in vitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system.

Telomere length is longer following diapause in two solitary bee species.

The mechanisms that underlie senescence are not well understood in insects. Telomeres are conserved repetitive sequences at chromosome ends that protect DNA during replication. In many vertebrates, telomeres shorten during cell division and in response to stress and are often used as a cellular marker of senescence. However, little is known about telomere dynamics across the lifespan in invertebrates. We measured telomere length in larvae, prepupae, pupae, and adults of two species of solitary bees, Osmia lignaria and Megachile rotundata. Contrary to our predictions, telomere length was longer in later developmental stages in both O. lignaria and M. rotundata. Longer telomeres occurred after emergence from diapause, which is a physiological state with increased tolerance to stress. In O. lignaria, telomeres were longer in adults when they emerged following diapause. In M. rotundata, telomeres were longer in the pupal stage and subsequent adult stage, which occurs after prepupal diapause. In both species, telomere length did not change during the 8 months of diapause. Telomere length did not differ by mass similarly across species or sex. We also did not see a difference in telomere length after adult O. lignaria were exposed to a nutritional stress, nor did length change during their adult lifespan. Taken together, these results suggest that telomere dynamics in solitary bees differ from what is commonly reported in vertebrates and suggest that insect diapause may influence telomere dynamics.

Evolution of diapause in the African turquoise killifish by remodeling the ancient gene regulatory landscape.

Suspended animation states allow organisms to survive extreme environments. The African turquoise killifish has evolved diapause as a form of suspended development to survive a complete drought. However, the mechanisms underlying the evolution of extreme survival states are unknown. To understand diapause evolution, we performed integrative multi-omics (gene expression, chromatin accessibility, and lipidomics) in the embryos of multiple killifish species. We find that diapause evolved by a recent remodeling of regulatory elements at very ancient gene duplicates (paralogs) present in all vertebrates. CRISPR-Cas9-based perturbations identify the transcription factors REST/NRSF and FOXOs as critical for the diapause gene expression program, including genes involved in lipid metabolism. Indeed, diapause shows a distinct lipid profile, with an increase in triglycerides with very-long-chain fatty acids. Our work suggests a mechanism for the evolution of complex adaptations and offers strategies to promote long-term survival by activating suspended animation programs in other species.

Symbiotic bacteria and fungi proliferate in diapause and may enhance overwintering survival in a solitary bee.

Host-microbe interactions underlie the development and fitness of many macroorganisms, including bees. Whereas many social bees benefit from vertically transmitted gut bacteria, current data suggests that solitary bees, which comprise the vast majority of species diversity within bees, lack a highly specialized gut microbiome. Here, we examine the composition and abundance of bacteria and fungi throughout the complete life cycle of the ground-nesting solitary bee Anthophora bomboides standfordiana. In contrast to expectations, immature bee stages maintain a distinct core microbiome consisting of Actinobacterial genera (Streptomyces, Nocardiodes) and the fungus Moniliella spathulata. Dormant (diapausing) larval bees hosted the most abundant and distinctive bacteria and fungi, attaining 33 and 52 times their initial copy number, respectively. We tested two adaptive hypotheses regarding microbial functions for diapausing bees. First, using isolated bacteria and fungi, we found that Streptomyces from brood cells inhibited the growth of multiple pathogenic filamentous fungi, suggesting a role in pathogen protection during overwintering, when bees face high pathogen pressure. Second, sugar alcohol composition changed in tandem with major changes in fungal abundance, suggesting links with bee cold tolerance or overwintering biology. We find that A. bomboides hosts a conserved core microbiome that may provide key fitness advantages through larval development and diapause, which raises the question of how this microbiome is maintained and faithfully transmitted between generations. Our results suggest that focus on microbiomes of mature or active insect developmental stages may overlook stage-specific symbionts and microbial fitness contributions during host dormancy.

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.

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.

Influence of abiotic factors on diapause termination and temperature requirements for postdiapause development in the European red mite, Panonychus ulmi (Acari: Tetranychidae).

The European red mite Panonychus ulmi (Koch) is widely distributed and it can severely affect pome fruit crops, particularly apple. Pest outbreaks are related to an overuse of non-selective pesticide treatments that lead to the development of resistance and the absence of natural enemies in the orchard. A key aspect to optimize the use of pesticide treatments in the context of IPM is to increase the knowledge on the biology and ecology of the pest to better predict population dynamics and outbreaks. For the European red mite, knowledge on the conditions that lead to diapause breaking by winter eggs is essential to model population dynamics. To increase this knowledge, winter eggs were collected during field surveys in northen Spain during three years and egg hatching was monitored under controlled temperature and photoperiod conditions in the laboratory. The "number of days exposed to cold temperatures" was the most significant factor that positively affected hatching of overwintering eggs. The time required for 50% of the egg population to hatch (T50%) was also negatively modulated by the duration of exposure to cold temperature. The temperature threshold for postdiapause eggs development collected from the field was estimated between 5 and 6 ºC in 2005 and 2007, respectively. Moreover, the degree-days required for post diapause development were estimated between 263.2 and 270.3, depending on the year of collection. Collectively, we provide additional information on the diapause termination and postdiapause development of the European red mite that may effectively contribute to optimize pest population models.

New insights into how to induce and maintain embryonic diapause in the blastocyst.

Embryonic diapause in mammals is a period of developmental pause of the embryo at the blastocyst stage. During diapause, the blastocyst has minimal cell proliferation, metabolic activity and gene expression. At reactivation, blastocyst development resumes, characterised by increases in cell number, biosynthesis and metabolism. Until recently, it has been unknown how diapause is maintained without any loss of blastocyst viability. This review focuses on recent progress in the identification of molecular pathways occurring in the blastocyst that can both cause and maintain the diapause state. A switch to lipid metabolism now appears essential to maintaining the diapause state and is induced by forkhead box protein O1. The forkhead box protein O transcription family is important for diapause in insects, nematodes and fish, but this is the first time a conclusive role has been established in mammals. Multiple epigenetic modifications are also essential to inducing and maintaining the diapause state, including both DNA and RNA methylation mechanisms. Finally, it now appears that diapause embryos, dormant stem cells and chemotherapeutic-resistant cancer cells may all share a universal system of quiescence.

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.

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.

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.

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.

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.

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.

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.

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.