Genome-wide analysis reveals transcriptional and translational changes during diapause of the Asian corn borer (Ostrinia furnacalis).

BACKGROUND: Diapause, a pivotal phase in the insect life cycle, enables survival during harsh environmental conditions. Unraveling the gene expression profiles of the diapause process helps uncover the molecular mechanisms that underlying diapause, which is crucial for understanding physiological adaptations. In this study, we utilize RNA-seq and Ribo-seq data to examine differentially expressed genes (DEGs) and translational efficiency during diapause of Asian corn borer (Ostrinia furnacalis, ACB). RESULTS: Our results unveil genes classified as "forwarded", "exclusive", "intensified", or "buffered" during diapause, shedding light on their transcription and translation regulation patterns. Furthermore, we explore the landscape of lncRNAs (long non-coding RNAs) during diapause and identify differentially expressed lncRNAs, suggesting their roles in diapause regulation. Comparative analysis of different types of diapause in insects uncovers shared and unique KEGG pathways. While shared pathways highlight energy balance, exclusive pathways in the ACB larvae indicate insect-specific adaptations related to nutrient utilization and stress response. Interestingly, our study also reveals dynamic changes in the HSP70 gene family and proteasome pathway during diapause. Manipulating HSP protein levels and proteasome pathway by HSP activator or inhibitor and proteasome inhibitor affects diapause, indicating their vital role in the process. CONCLUSIONS: In summary, these findings enhance our knowledge of how insects navigate challenging conditions through intricate molecular mechanisms.

Comparative Proteomics and Metabonomics Analysis of Different Diapause Stages Revealed a New Regulation Mechanism of Diapause in Loxostege sticticalis (Lepidoptera: Pyralidae).

Histone acetylation is an important epigenetic mechanism that has been shown to play a role in diapause regulation. To explore the physiological and molecular mechanisms of histone deacetylase in the diapause process, LC-MS/MS analysis was used to perform TMT proteomic and metabolomic analysis on non-diapause (ND), pre-diapause (PreD), diapause (D), cold treatment (CT), and post-diapause (RD) stages of the meadow moth. A total of 5367 proteins were identified by proteomics, including 1179 differentially expressed proteins. We found 975 (602 up-regulated and 373 down-regulated), 997 (608 up-regulated and 389 down-regulated), 1119 (726 up-regulated and 393 down-regulated), 1179 (630 up-regulated and 549 down-regulated), 94 (51 up-regulated and 43 down-regulated), 111 (63 up-regulated and 48 down-regulated), 533 (243 up-regulated and 290 down-regulated), 58 (31 up-regulated and 27 down-regulated), and 516 (228 up-regulated and 288 down-regulated) proteins in ND and PreD, ND and D, ND and CT, ND and RD, PreD and D, PreD and CT, PreD and RD, D and CT, D and RD, and CT and RD stages, respectively. A total of 1255 differentially expressed metabolites were annotated by metabolomics. Through KEGG analysis and time series analysis of differentially expressed metabolites, we found that phospholipids were annotated in significantly different modules, demonstrating their important role in the diapause process of the meadow moth. Using phospholipids as an indicator for weighted gene co-expression network analysis, we analyzed the most relevant differentially expressed proteins in the module and found that ribosomal 40s and 60s subunits were the most relevant proteins for diapause. Because there have been studies that have shown that histone deacetylase is associated with the diapause of meadow moths, we believe that histone deacetylase regulates the 40s and 60s subunits of ribosomes, which in turn affects the diapause of meadow moths. This finding expands our understanding of the regulation of meadow moth diapause and provides new insights into its control mechanism.

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.

HSP gene superfamily in Aspongopus chinensis Dallas: unravelling identification, characterisation and expression patterns during diapause and non-diapause stages.

Aspongopus chinensis Dallas 1851, an insect of important economic value, faces challenges in artificial breeding due to mandatory diapause and limited access to wild resources. Heat shock proteins (Hsps) are thought to influence diapause in insects, but little is known about their role in A. chinensis during diapause. This study used genomic methods to identify 25 Hsp genes in A. chinensis, including two Hsp90, 14 Hsp70, four Hsp60 and five small Hsp genes, were located on seven chromosomes, respectively. The gene structures among the same families are relatively conserved. Meanwhile, the motif compositions and secondary structures of A. chinensis Hsps (AcHsps) were predicted. RNA-seq data and fluorescence quantitative PCR analysis showed that there were differences in the expression patterns of AcHsps in diapause and non-diapause stages, and AcHsp70-5 was significantly differentially expressed in both analysis, which was enriched in the pathway of response to hormone. All the results showed that Hsps play an important role in the diapause mechanism of A. chinensis. Our observations highlight the molecular evolution of the Hsp gene and their effect on diapause in A. chinensis.

MYST family histone acetyltransferases regulate reproductive diapause initiation.

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

RNA N6-methyladenosine of DHAPAT and PAP involves in regulation of diapause of Bombyx mori via the lipid metabolism pathway.

Environment-induced epigenetics are involved in diapause regulation, but the molecular mechanism that epigenetically couples nutrient metabolism to diapause regulation remains unclear. In this study, we paid special attention to the significant differences in the level of N6-adenosine methylation (m6A) of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidate phosphatase (PAP) genes in the lipid metabolism pathway of the bivoltine silkworm (Bombyx mori) strain Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, non-diapause egg producer). We knocked down DHAPAT in the pupal stage of the QFLT group, resulting in the non-diapause destined eggs becoming diapausing eggs. In the PAP knockdown group, the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition, but they hatched as normal. Moreover, we validated that YTHDF3 binds to m6A-modified DHAPAT and PAP mRNAs to promote their stability and translation. These results suggest that RNA m6A methylation participates in the diapause regulation of silkworm by changing the expression levels of DHAPAT and PAP and reveal that m6A epigenetic modification can be combined with a lipid metabolism signal pathway to participate in the regulation of insect diapause traits, which provides a clearer image for exploring the physiological basis of insect diapause.

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

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

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.

Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica.

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.

Transcriptomic analysis of diapause-associated genes in Exorista civilis Rondani (Diptera:Tachinidae).

Exorista civilis Rondani (Diptera:Tachinidae) is an excellent dominant parasitic enemy all over the world. But there has been a lack of research on the molecular regulation of diapause in E. civilis. To investigate the important diapause-associated genes and metabolic pathways in E. civilis, we can provide a theoretical basis for clarifying the molecular mechanism of diapause at the transcriptome level. The Illumina HiSeq. 2000 platform was used to perform transcriptome sequencing and bioinformatics analysis of the non-diapause and diapause pupae of E. civilis. 58,050 unigenes were successfully assembled, in which 4355 upregulated and 3158 downregulated unigenes were differentially expressed. Moreover, by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments, 896 kinds of the differentially expressed genes were specifically analyzed and showed that diapause-associated genes were related to be involved in the pathways of cold resistance, amino acid metabolism, and energy metabolism. Furthermore, these upregulated five genes showed the same trends of expression patterns between quantitative real-time polymerase chain reaction and RNA-Seq. This study provides a theoretical basis for the further study of the diapausing molecular mechanisms of E. civilis.

Universal and differential transcriptional regulatory pathways involved in the preparation of summer and winter diapauses in Pieris melete.

Much progress has been made in understanding the environmental and hormonal systems regulating winter diapause. However, transcriptional regulation of summer diapause is still largely unknown, making it difficult to understand an all-around regulation profile of seasonal adaptation. To bridge this gap, comparison RNA-seq to profile the transcriptome and to examine differential gene expression profiles between non-diapause, summer diapause, and winter diapause groups were performed. A total number of 113 million reads were generated and assembled into 79,117 unigenes, with 37,492 unigenes categorized into 58 functional gene ontology groups, 25 clusters of orthologous group categories, and 256 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG analysis mapped 2108 differentially expressed genes to 48 and 67 pathways for summer and winter diapauses, respectively. Enrichment statistics showed that 11 identical pathways similarly overlapped in the top 20 enriched functional groups both related to summer and winter diapauses. We also identified 35 key candidate genes for universal and differential functions related to summer and winter diapause preparation. Furthermore, we identified some genes involved in the signaling and metabolic pathways that may be the key drivers to integrate environmental signals into the summer and winter diapause preparation. The current study provided valuable insights into global molecular mechanisms underpinning diapause preparation.

A region of the sex chromosome associated with population differences in diapause induction contains highly divergent alleles at clock genes.

Developmental plasticity describes the capacity of individuals with the same genotype to induce permanent change in a phenotype depending on a specific external input. One well-studied example of adaptive developmental plasticity is the induction of facultative diapause in insects. Studies investigating the inheritance of diapause induction have suggested diverse genetic origins. However, only few studies have performed genome-wide scans to identify genes affecting the induction decision. Here we compare two populations of the butterfly Pieris napi that differ in the propensity to enter diapause, and despite showing a low genome-wide divergence, we identify a few genomic regions that show high divergence between populations. We then identified a single genomic region associated with diapause induction by genotyping diapausing and directly developing siblings from backcrosses of these populations. This region is located on the Z chromosome and contained three circadian clock genes, cycle, clock, and period. Additionally, period harbored the largest number of SNPs showing complete fixation between populations. We conclude that the heritable basis of between-population variation in the plasticity that determines diapause induction resides on the Z chromosome, with the period gene being the prime candidate for the genetic basis of adaptive plasticity.

High-throughput profiling of diapause regulated genes from Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae).

BACKGROUND: The parasitoid wasp, Trichogramma dendrolimi, can enter diapause at the prepupal stage. Thus, diapause is an efficient preservation method during the mass production of T. dendrolimi. Previous studies on diapause have mainly focused on ecological characteristics, so the molecular basis of diapause in T. dendrolimi is unknown. We compared transcriptomes of diapause and non-diapause T. dendrolimi to identify key genes and pathways involved in diapause development. RESULTS: Transcriptome sequencing was performed on diapause prepupae, pupae after diapause, non-diapause prepupae, and pupae. Analysis yielded a total of 87,022 transcripts with an average length of 1604 bp. By removing redundant sequences and those without significant BLAST hits, a non-redundant dataset was generated, containing 7593 sequences with an average length of 3351 bp. Among them, 5702 genes were differentially expressed. The result of Gene Ontology (GO) enrichment analysis revealed that regulation of transcription, DNA-templated, oxidation-reduction process, and signal transduction were significantly affected. Ten genes were selected for validation using quantitative real-time PCR (qPCR). The changes showed the same trend as between the qPCR and RNA-Seq results. Several genes were identified as involved in diapause, including ribosomal proteins, zinc finger proteins, homeobox proteins, forkhead box proteins, UDP-glucuronosyltransferase, Glutathione-S-transferase, p53, and DNA damage-regulated gene 1 (pdrg1). Genes related to lipid metabolism were also included. CONCLUSIONS: We generated a large amount of transcriptome data from T. dendrolimi, providing a resource for future gene function research. The diapause-related genes identified help reveal the molecular mechanisms of diapause, in T. dendrolimi, and other insect species.

Unique genetic signatures of local adaptation over space and time for diapause, an ecologically relevant complex trait, in Drosophila melanogaster.

Organisms living in seasonally variable environments utilize cues such as light and temperature to induce plastic responses, enabling them to exploit favorable seasons and avoid unfavorable ones. Local adapation can result in variation in seasonal responses, but the genetic basis and evolutionary history of this variation remains elusive. Many insects, including Drosophila melanogaster, are able to undergo an arrest of reproductive development (diapause) in response to unfavorable conditions. In D. melanogaster, the ability to diapause is more common in high latitude populations, where flies endure harsher winters, and in the spring, reflecting differential survivorship of overwintering populations. Using a novel hybrid swarm-based genome wide association study, we examined the genetic basis and evolutionary history of ovarian diapause. We exposed outbred females to different temperatures and day lengths, characterized ovarian development for over 2800 flies, and reconstructed their complete, phased genomes. We found that diapause, scored at two different developmental cutoffs, has modest heritability, and we identified hundreds of SNPs associated with each of the two phenotypes. Alleles associated with one of the diapause phenotypes tend to be more common at higher latitudes, but these alleles do not show predictable seasonal variation. The collective signal of many small-effect, clinally varying SNPs can plausibly explain latitudinal variation in diapause seen in North America. Alleles associated with diapause are segregating in Zambia, suggesting that variation in diapause relies on ancestral polymorphisms, and both pro- and anti-diapause alleles have experienced selection in North America. Finally, we utilized outdoor mesocosms to track diapause under natural conditions. We found that hybrid swarms reared outdoors evolved increased propensity for diapause in late fall, whereas indoor control populations experienced no such change. Our results indicate that diapause is a complex, quantitative trait with different evolutionary patterns across time and space.

Selection of Reference Genes for Quantitative Real-Time PCR in Chrysoperla nipponensis (Neuroptera: Chrysopidae) Under Tissues in Reproduction and Diapause.

Chrysoperla nipponensis (Okamoto), which has the unique diapause phenotype distinguishable from nondiapause adult, is an ideal model organism for studying the mechanism of reproductive diapause. However, there is no reliable and effective reference genes used for the reproductive diapause study of C. nipponensis. Therefore, in this study, we evaluated the expression stability of 10 candidate reference genes (Tub1, Arpc5, EF1a, 128up, RpS5, RpS26e, GAPDH, Arp3, Actin, α-Tub) in adults under diapause and nondiapause induction conditions using four statistical algorithms including GeNorm, NormFinder, Bestkeeper, and ∆CT method. Results showed that Arp3 and Tub1 were the most stable reference genes in all samples and in the adult tissues group. Arp3 and RpS5 were the most stable reference genes in the development degree group. α-Tub and EF1a were unstable reference genes under the conditions of this study. Meanwhile, to verify the reliability of the reference genes, we evaluated the relative expression levels of Vg and VgR in different treatments. Significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was, respectively, observed when using Arp3 as the reference gene but not when using an unstable reference gene. The reference genes identified in this work provided not only the basis for future functional genomics research in diapause of C. nipponensis and will also identify reliable normalization factors for real-time quantitative real-time polymerase chain reaction data for other related insects.

Rapid adaptive evolution of the diapause program during range expansion of an invasive mosquito.

In temperate climates, the recurring seasonal exigencies of winter represent a fundamental physiological challenge for a wide range of organisms. In response, many temperate insects enter diapause, an alternative developmental program, including developmental arrest, that allows organisms to synchronize their life cycle with seasonal environmental variation. Geographic variation in diapause phenology contributing to local climatic adaptation is well documented. However, few studies have examined how the rapid evolution of a suite of traits expressed across the diapause program may contribute to climatic adaptation on a contemporary timescale. Here, we investigate the evolution of the diapause program over the past 35 years by leveraging a "natural experiment" presented by the recent invasion of the Asian tiger mosquito, Aedes albopictus, across the eastern United States. We sampled populations from two distinct climatic regions separated by 6° of latitude (∼700 km). Using common-garden experiments, we identified regional genetic divergence in diapause-associated cold tolerance, diapause duration, and postdiapause starvation tolerance. We also found regional divergence in nondiapause thermal performance. In contrast, we observed minimal regional divergence in nondiapause larval growth traits and at neutral molecular marker loci. Our results demonstrate rapid evolution of the diapause program and imply strong selection caused by differences in winter conditions.

Circadian transcription factors differentially regulate features of the adult overwintering diapause in the Northern house mosquito, Culex pipiens.

The short days of late summer and early fall are the environmental cues that most temperate insects and other animals use to predict winter's arrival. Although it is still unclear precisely how insects measure daylength, there is mounting evidence that the circadian clock regulates seasonal responses including photoperiodic diapause. Females of the Northern house mosquito, Culex pipiens, enter an adult reproductive diapause in response to short daylengths. While in this state, females divert their resources from reproduction to survival, arresting egg follicle development and increasing fat content. Here, we characterized the expression profile of two circadian transcription factors, vrille (vri) and Par domain protein 1 (Pdp1), as well as genes downstream of the clock, takeout (to) and Nocturnin (Noc) and under different seasonal conditions. We saw that while vri mRNA oscillated under both long day and short day conditions, Pdp1 expression oscillated only under long day conditions and was constitutively upregulated in diapausing females. We saw similar expression profiles for to and Noc, suggesting that PDP1 might regulate their expression or that Pdp1, to and Noc might be regulated by the same transcription factor. We suppressed vri and Pdp1 using RNA interference. dsRNA against vri provided inconsistent results, sometimes stimulating autogenous egg follicle development in both long and short day-reared females, and other times had no effect. In contrast, knocking down Pdp1 prevented short day-reared females from accumulating fat reserves, but increased expression of to and Noc. Taken together, these data suggest that the circadian transcription factors Vri and Pdp1 may independently regulate signaling pathways underlying arrested egg follicle development and fat accumulation in diapausing females of Cx. pipiens.

P-S6K is associated with insect diapause via the ROS/AKT/ S6K/CREB/HIF-1 pathway in the cotton bollworm, Helicoverpa armigera.

Diapause is a complex physiological response that allows insects to survive unfavorable environmental conditions, and many signaling pathways participate in regulating this process. However, little is known about TOR signaling in the regulation of diapause. In this study, we found that the TOR pathway-related proteins TOR and Raptor are expressed at low levels in the brains of diapause-destined pupae of Helicoverpa armigera, consistent with a previous report that TOR signaling is associated with development. Interestingly, another TOR signaling-related protein, p-S6K, was increased in the brains of diapause-destined pupae. Our results showed that p-S6K in the brains of diapause-destined pupae can respond to the upstream signals reactive oxygen species (ROS) and AKT and that S6K activates the level of CREB, which binds to the HIF-1α promoter and increases its expression. Previous study has shown that HIF-1α levels elevated by ROS in the brains of diapause-destined pupae cause low mitochondrial activity for insect diapause. Thus, p-S6K in response to ROS/AKT regulates HIF-1α via activating transcription factor CREB for diapause initiation.

DNA Methylation Is Correlated with Gene Expression during Diapause Termination of Early Embryonic Development in the Silkworm (Bombyx mori).

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.