Programmed Autophagy in the Fat Body of Aedes aegypti Is Required to Maintain Egg Maturation Cycles

Autophagy plays a pivotal role by allowing cells to recycle cellular components under conditions of stress, starvation, development and cancer. In this work, we have demonstrated that programmed autophagy in the mosquito fat body plays a critical role in maintaining of developmental switches required for normal progression of gonadotrophic cycles. Mosquitoes must feed on vertebrate blood for their egg development, with each gonadotrophic cycle being tightly coupled to a separate blood meal. As a consequence, some mosquito species are vectors of pathogens that cause devastating diseases in humans and domestic animals, most importantly malaria and Dengue fever. Hence, deciphering mechanisms to control egg developmental cycles is of paramount importance for devising novel approaches for mosquito control. Central to egg development is vitellogenesis, the production of yolk protein precursors in the fat body, the tissue analogous to a vertebrate liver, and their subsequent specific accumulation in developing oocytes. During each egg developmental cycle, the fat body undergoes a developmental program that includes previtellogenic build-up of biosynthetic machinery, intense production of yolk protein precursors, and termination of vitellogenesis. The importance of autophagy for termination of vitellogenesis was confirmed by RNA interference (RNAi) depletions of several autophagic genes (ATGs), which inhibited autophagy and resulted in untimely hyper activation of TOR and prolonged production of the major yolk protein precursor, vitellogenin (Vg). RNAi depletion of the ecdysone receptor (EcR) demonstrated its activating role of autophagy. Depletion of the autophagic genes and of EcR led to inhibition of the competence factor, betaFTZ-F1, which is required for ecdysone-mediated developmental transitions. Moreover, autophagy-incompetent female mosquitoes were unable to complete the second reproductive cycle and exhibited retardation and abnormalities in egg maturation. Thus, our study has revealed a novel function of programmed autophagy in maintaining egg maturation cycles in mosquitoes

Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics

Culex quinquefasciatus (the southern house mosquito) is an important mosquito vector of viruses such as West Nile virus and St. Louis encephalitis virus, as well as of nematodes that cause lymphatic filariasis. C. quinquefasciatus is one species within the Culex pipiens species complex and can be found throughout tropical and temperate climates of the world. The ability of C. quinquefasciatus to take blood meals from birds, livestock, and humans contributes to its ability to vector pathogens between species. Here, we describe the genomic sequence of C. quinquefasciatus: Its repertoire of 18,883 protein-coding genes is 22% larger than that of Aedes aegypti and 52% larger than that of Anopheles gambiae with multiple gene-family expansions, including olfactory and gustatory receptors, salivary gland genes, and genes associated with xenobiotic detoxification

Gut contents, digestive half-lives and feeding state prediction in the soil predatory mite Pergamasus longicornis (Mesostigmata: Parasitidae)

Mid- and hind-gut lumenal changes are described in the free-living predatory soil mite Pergamasus longicornis (Berlese) from a time series of histological sections scored during and after feeding on fly larval prey. Three distinct types of tangible material are found in the lumen. Bayesian estimation of the change points in the states of the gut lumenal contents over time is made using a time-homogenous first order Markov model. Exponential processes within the gut exhibit ’stiff’ dynamics. A lumen is present throughout the midgut from 5 min after the start of feeding as the gut rapidly expands. It peaks at about 21.5 h - 1.5 days and persists post-feeding (even when the gut is contracted) up until fasting/starvation commences 10 days post start of feeding. The disappearance of the lumen commences 144 h after the start of feeding. Complete disappearance of the gut lumen make take 5-9 weeks from feeding commencing. Clear watery prey material arrives up to 10 min from the start of feeding - driving gut lumen expansion. Intracellular digestion triggered by maximum gut expansion is indicated. Detectable granular prey material appears in the lumen during the concentrative phase of coxal droplet production and, despite a noticeable collapse around 12 h, lasts in part for 52.5 h. Posterior midgut regions differ slightly from anterior regions in their main prey food dynamics being somewhat faster in processing yet being slightly delayed. Posterior regions are confirmed as Last-In-Last-Out depots, anterior regions confirmed as First-In-First-Out conveyor belt processes. Evidence for differential lability of prey fractions is found. A scheme of granular imbibed prey material being first initially rapidly absorbed (t andfrac12; = 23 min), and also being quickly partly converted to globular material extra-corporeally/extracellularly (t andfrac12; = 36 min) - which then rapidly disappears (t andfrac12; =1.1 h, from a peak around 4 h) is presented. This is then followed by slow intracellular digestion (t andfrac12; = 6.9 h) of the resultant resistant prey residue matching the slow rate of appearance of opaque pre-excretory egestive refractive grains (overall t andfrac12; = 4.5 days). The latter confirmed latent ’catabolic fraction’ (along with Malpighian tubule produced guanine crystals) drives rectal vesicle expansion as ’faeces’ during the later phases of gut emptying/contraction. Catabolic half-lives are of the order of 6.3-7.8 h. Membraneous material is only present in the lumen of the gut in starving mites. No obvious peritrophic membrane was observed. The total feeding cycle time may be slightly over 52.5 h. Full clearance in the gut system of a single meal including egestive and excretory products may take up to 3 weeks. Independent corroborative photographs are included and with posterior predictive densities confirm the physiological sequence of:- ingestion/digestion; egestion; excretion; defecation; together with their timings. Visually dark midguts almost certainly indicate egestive refractive grains (?xanthine) production. Nomograms to diagnose the feeding state of P.longicornis in field samples are presented and show that the timing of these 4 phases in the wild could be inferred by scoring 10-12 mites out of a sample of 20. Suggestions to critically confirm or refute the conclusions are included

Improved reference genome of Aedes aegypti informs arbovirus vector control

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector

The cell biology of mosquito vitellogenesis

Insect vitellogenesis involves coordinated activities of the fat body and oocytes. We have studied these activities at the cellular level in the mosquito. During each vitellogenic cycle, the fat body undergoes three successive stages: 1) proliferation of biosynthetic organelles, 2) vitellogenin synthesis, 3) termination of vitellogenin synthesis and degradation of biosynthetic organelles by lysosomes. Analysis with monoclonal antibodies and radiolabelling demonstrated that the mosquito yolk protein consists of two subunits (200-kDa and 65-kDa). Both subunits are glycosylated, their carbohydrate moieties are composed of high-mannose oligosaccharides. The yolk protein subunits are derived from a single 220 kDa precursor detected by an in vitro translation. Oocytes become competent to internalize proteins as a result of juvenile hormone-mediated biogenesis of endocytotic organelles. The yolk protein is then accumulated by receptor-mediated endocytosis. A pathway of the yold protein and factors determining its routing in the oocyte have been studied

Juvenile hormone regulation of microRNAs is mediated by E75 in the Dengue vector mosquito Aedes aegypti

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in controlling posttranscriptional gene regulation and have a profound effect on mosquito reproduction and metabolism. Juvenile hormone (JH) is critical for achieving reproductive competence in the main vector of human arboviral diseases, Aedes aegypti We report a JH-mediated mechanism governing miRNA expression. Using a transcription factor screen with multiple primary miRNA (pri-miRNA) promoters, we identified that the Ecdysone-induced protein E75 (E75) isoform (E75-RD) induced miRNA gene promoter activity. E75 binding sites were determined in miRNA promoters by means of cell transfection assay. E75-RD was found to be up-regulated by JH, as shown by the JH application and RNA interference (RNAi) of the JH receptor Methoprene-tolerant (Met). Small RNA sequencing from RNAi of Met and E75 displayed an overlapping miRNA cohort, suggesting E75 to be an intermediate component within the JH hierarchical network controlling miRNAs. Further experiments confirmed that E75-RD positively regulates several miRNAs including miR-2940. Reducing miR-2940 resulted in the arrest of follicle development and number of eggs laid. Performing miRNA target predictions and RT-qPCR from antagomir Ant-2940-3p-treated fat body tissues identified the mRNA target Clumsy (AAEL002518) The molecular interaction between this gene target and miR-2940 was confirmed using an in vitro dual luciferase assay in Drosophila S2 cells and in Ae. aegypti Aag2 cell lines. Finally, we performed a phenotypic rescue experiment to demonstrate that miR-2940/Clumsy is responsible for the disruption in egg development. Collectively, these results established the role of JH-mediated E75-RD in regulation of miRNA gene expression during the mosquito reproductive cycle

Cross-talk of insulin-like peptides, juvenile hormone, and 20-hydroxyecdysone in regulation of metabolism in the mosquito Aedes aegypti

Female mosquitoes feed sequentially on carbohydrates (nectar) and proteins (blood) during each gonadotrophic cycle to become reproductively competent and effective disease vectors. Accordingly, metabolism is synchronized to support this reproductive cyclicity. However, regulatory pathways linking metabolism to reproductive cycles are not fully understood. Two key hormones, juvenile hormone (JH) and ecdysteroids (20-hydroxyecdysone, 20E, is the most active form) govern female mosquito reproduction. Aedes aegypti genome codes for eight insulin-like peptides (ILPs) that are critical for controlling metabolism. We examined the effects of the JH and 20E pathways on mosquito ILP expression to decipher regulation of metabolism in a reproducing female mosquito. Chromatin immunoprecipitation assays showed genomic interactions between ilp genes and the JH receptor, methoprene-tolerant, a transcription factor, Krüppel homolog 1 (Kr-h1), and two isoforms of the ecdysone response early gene, E74. The luciferase reporter assays showed that Kr-h1 activates ilps 2, 6, and 7, but represses ilps 4 and 5 The 20E pathway displayed the opposite effect in the regulation of ilps E74B repressed ilps 2 and 6, while E74A activated ilps 4 and 5 Combining RNA interference, CRISPR gene tagging and enzyme-linked immunosorbent assay, we have shown that the JH and 20E regulate protein levels of all eight Ae. aegypti ILPs. Thus, we have established a regulatory axis between ILPs, JH, and 20E in coordination of metabolism during gonadotrophic cycles of Ae. aegypti