Cheolho Sim

Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens

The short day lengths of late summer program the mosquito Culex pipiens to enter a reproductive diapause characterized by an arrest in ovarian development and the sequestration of huge fat reserves. We suggest that insulin signaling and FOXO (forkhead transcription factor), a downstream molecule in the insulin signaling pathway, mediate the diapause response. When we used RNAi to knock down expression of the insulin receptor in nondiapausing mosquitoes (those reared under long day lengths) the primary follicles were arrested in a stage comparable to diapause. The mosquitoes could be rescued from this developmental arrest with an application of juvenile hormone, an endocrine trigger known to terminate diapause in this species. When dsRNA directed against FOXO was injected into mosquitoes programmed for diapause (reared under short day lengths) fat storage was dramatically reduced and the mosquitos lifespan was shortened, results suggesting that a shutdown of insulin signaling prompts activation of the downstream gene FOXO, leading to the diapause phenotype. Thus, the results are consistent with a role for insulin signaling in the short-day response that ultimately leads to a cessation of juvenile hormone production. The similarity of this response to that observed in the diapause of Drosophila melanogaster and in dauer formation of Caenorhabditis elegans suggests a conserved mechanism regulating dormancy in insects and nematodes.

Insulin signaling and the regulation of insect diapause

A rich chapter in the history of insect endocrinology has focused on hormonal control of diapause, especially the major roles played by juvenile hormones (JHs), ecdysteroids, and the neuropeptides that govern JH and ecdysteroid synthesis. More recently, experiments with adult diapause in Drosophila melanogaster and the mosquito Culex pipiens, and pupal diapause in the flesh fly Sarcophaga crassipalpis provide strong evidence that insulin signaling is also an important component of the regulatory pathway leading to the diapause phenotype. Insects produce many different insulin-like peptides (ILPs), and not all are involved in the diapause response; ILP-1 appears to be the one most closely linked to diapause in C. pipiens. Many steps in the pathway leading from perception of daylength (the primary environmental cue used to program diapause) to generation of the diapause phenotype remain unknown, but the role for insulin signaling in mosquito diapause appears to be upstream of JH, as evidenced by the fact that application of exogenous JH can rescue the effects of knocking down expression of ILP-1 or the Insulin Receptor. Fat accumulation, enhancement of stress tolerance, and other features of the diapause phenotype are likely linked to the insulin pathway through the action of a key transcription factor, FOXO. This review highlights many parallels for the role of insulin signaling as a regulator in insect diapause and dauer formation in the nematode Caenorhabditis elegans.

Catalase and superoxide dismutase-2 enhance survival and protect ovaries during overwintering diapause in the mosquito Culex pipiens.

Lifespan extension and stress resistance are two important features of diapause that are essential for successful overwintering. We present several lines of evidence suggesting that genes encoding two antioxidant enzymes, catalase and superoxide dismutase-2, are critical in generating these characteristics during diapause in overwintering adults of the mosquito Culex pipiens. Expression of both catalase and sod-2 was dramatically higher in young diapausing females than in their nondiapausing counterparts at the same age. Suppression of catalase, but not sod-2, resulted in increased damage to the ovaries, as evidenced by signs of apoptosis in ovarian follicle cells. Adult survival time was shortened when levels of either catalase or sod-2 were suppressed using RNAi. Together these results imply that these two antioxidants are particularly important in promoting survival in diapausing females, while elevation of catalase also contributes to protection of the ovaries. In addition, RNAi directed against forkhead transcription factor (foxo), a gene thought to be upstream of the genes encoding these antioxidants, resulted in suppression of both catalase and sod-2. The linkage with FOXO suggests that the genes encoding these two antioxidants are components of an important gene network regulated by this transcription factor.

Transcription profiling and regulation of fat metabolism genes in diapausing adults of the mosquito Culex pipiens

Culex pipiens, the mosquito that vectors West Nile virus in North America, overwinters in an adult diapause (dormancy) that is programmed by the short day length and low temperatures of autumn. In response to these environmental signals, females cease feeding on blood and instead seek sources of nectar used to generate the huge lipid reserves required for winter survival. To identify regulatory networks that regulate fat accumulation and fat consumption during diapause, we compared expression of fat-related genes from nondiapausing females with expression of those same genes in early and late diapause and at diapause termination. Among the 31 genes we examined, 4 were expressed more highly in early diapause than in nondiapause, while 14 genes were downregulated in early diapause. In the transition from early to late diapause, 19 genes related to fat metabolism were upregulated. As reported previously, fatty acid synthase, identified as fas-1 in this study, was upregulated in early diapause. Numerous fat metabolism genes, including multiple kinetic classes and genes involved in beta-oxidation, an energy-generation step, were suppressed in early diapause but were highly expressed in late diapause and at diapause termination. RNA interference (RNAi) analysis revealed that the fas-1 gene and others (fas-3 and fabp) have important roles in fat storage during early diapause. When expression of these genes is suppressed, female mosquitoes fail to sequester the lipids needed for overwintering.

Comparative genomic analysis in the region of a major Plasmodium-refractoriness locus of Anopheles gambiae

We have sequenced six overlapping clones from a library of bacterial artificial chromosome (BAC) clones derived from a laboratory strain of the mosquito, Anopheles gambiae, the major vector of human malaria in Africa. The resulting uninterrupted 528-kb sequence is from the 8C region of the mosquito 2R chromosome, at or very near the major refractoriness locus associated with melanotic encapsulation of parasites. This sequence represents the first extensive view of the mosquito genome structure encompassing 48 genes. Genomic comparison reveals that the majority of the orthologues are found in six microsyntenic clusters in Drosophila melanogaster. A BAC clone that is wholly contained within this region demonstrates the existence of a remarkable degree of local polymorphism in this species, which may prove important for its population structure and vectorial capacity.

Identification of FOXO targets that generate diverse features of the diapause phenotype in the mosquito Culex pipiens

Significance Diapause is an alternative developmental pathway exploited by insects and other invertebrates to survive inimical seasons. Like many insects that enter an adult diapause, the mosquito Culex pipiens responds to the short day lengths of autumn by feeding extensively on sugar, stockpiling huge fat reserves, halting reproduction, suppressing metabolism, boosting defense responses, and migrating to protected sites where it can safely bridge the winter months. In the experiments presented here, we propose that many of the diverse features of the diapause phenotype are the consequence of activating forkhead transcription factor (FOXO), a transcription factor downstream of insulin and juvenile hormone signaling. Our experimental results reveal how these upstream hormonal signaling pathways act through FOXO to generate the complex phenotype known as diapause. Insulin and juvenile hormone signaling direct entry of the mosquito Culex pipiens into its overwintering adult diapause, and these two critical signaling pathways appear to do so by converging on the regulation of forkhead transcription factor (FOXO). Diapause is a complex phenotype, and FOXO emerges as a prime candidate for activating many of the diverse physiological pathways that generate the diapause phenotype. Here, we used ChIP sequencing to identify direct targets of FOXO. The nearest gene in a 10-kb region surrounding a predicted binding site was extracted for each binding site, resulting in a dataset containing genes potentially regulated by FOXO. By selecting candidate genes based on their functional relevance to diapause, we identified five gene categories of potential interest, including stress tolerance, metabolic pathways, lifespan extension, cell cycle and growth regulation, and circadian rhythms. Twelve targets were prioritized for further analysis, 10 of which were validated by ChIP-quantitative PCR and quantitative real-time PCR. These 10 genes activated by FOXO are highly up-regulated during diapause and are thus strong candidates for implementation of the diapause syndrome.

Juvenile hormone III suppresses forkhead of transcription factor in the fat body and reduces fat accumulation in the diapausing mosquito, Culex pipiens

Juvenile hormone (JH) controls diverse physiological and developmental events including diapause and nutrient metabolism. The focal point of endocrine regulation in adult reproductive diapause is initiated by a halt of JH synthesis. In diapausing females of the mosquito Culex pipiens, the other key molecular event is the signalling pathway from insulin to forkhead of transcription factor (FOXO). We hypothesized that a halt of JH synthesis is related to activation of FOXO, which results in increasing lipid reserves in the fat body at the onset of the diapause programme. In this study, the full‐length sequence of the foxo gene in C. pipiens was characterized, and the protein abundance pattern of the foxo gene product was analyzed by immunoblotting and immunohistochemistry. FOXO was much more abundant in the fat body of diapausing females than in the fat body of nondiapausing females; much lower levels were present in other adult tissues. When we topically applied JH III to diapause‐destined females, FOXO was suppressed, and fat accumulation was reduced, suggesting an interaction between JH synthesis and FOXO that is critical for expression of the diapause phenotype.

Suppression of allatotropin simulates reproductive diapause in the mosquito Culex pipiens

The cessation of juvenile hormone (JH) production is a key endocrine event that halts ovarian development and hence initiates diapause in females of the mosquito, Culex pipiens. The shutdown in endocrine activity of the corpora allata (CA), the source of JH, was manifested in the smaller size of CA in females reared under short daylengths (diapause) compared to those reared under long daylengths (nondiapause), as well as in low expression of the mRNA encoding allatotropin, the neuropeptide that promotes JH biosynthesis in the CA. Genes encoding both allatotropin and allatostatin were identified in C. pipiens, but only expression levels of allatotropin differed in the two types of females. Knockdown of allatotropin mRNA using RNA interference in females programmed for nondiapause resulted in a cessation of ovarian development akin to diapause. This arrest in development could be reversed with an application of JH. Our results thus suggest that suppression of allatotropin is a critical link in regulating the shutdown of the CA during diapause.

Genomic Resources Notes Accepted 1 August 2014–30 September 2014

This article documents the public availability of (i) transcriptome sequence data, assembly and annotation, and single nucleotide polymorphisms (SNPs) for the cone snail Conus miliaris; (ii) a set of SNP markers for two biotypes from the Culex pipiens mosquito complex; (iii) transcriptome sequence data, assembly and annotation for the mountain fly Drosophila nigrosparsa; (iv) transcriptome sequence data, assembly and annotation and SNPs for the Neotropical toads Rhinella marina and R. schneideri; and (v) partial genomic sequence assembly and annotation for 35 spiny lizard species (Genus Sceloporus).

Identification of Culex complex species using SNP markers based on high-resolution melting analysis

Mosquitoes belonging to the Culex pipiens complex are primary vectors for diseases such as West Nile encephalitis, Eastern equine encephalitis, many arboviruses, as well as lymphatic filariases. Despite sharing physiological characteristics, each mosquito species within the Culex complex has unique behavioural and reproductive traits that necessitate a proper method of identification. Unfortunately, morphometric methods of distinguishing members of this complex have failed to yield consistent results, giving rise to the need for molecular methods of identification. In this study, we propose a novel identification method using high‐resolution melting (HRM) analysis by examining single‐nucleotide polymorphisms in the acetylcholinesterase‐2 (ace‐2) locus. Our method provides a high confidence for species determination among the three Culex complex mosquitoes.