Project description:Aedes aegypti mosquitoes infect hundreds of millions of people each year with dangerous viral pathogens including dengue, yellow fever, Zika, and chikungunya. Progress in understanding the biology of this insect, and developing tools to fight it, depends on the availablity of a high-quality genome assembly. Here we use DNA proximity ligaton (Hi-C) and Pacific Biosciences long reads to create AaegL5 - a highly contiguous A. aegypti reference.
Project description:The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24?32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.
Project description:Our previous findings [Hazelton & Lang (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37(6), 2378 (abstr.)] demonstrated a senescence-specific decrease in glutathione (GSH) concentration in the yellow-fever mosquito Aedes aegypti (Louisville)]. As a possible mechanism for this change, GSH biosynthesis was investigated in adult mosquitoes of different ages through the life-span. Biosynthesis was measured as the incorporation rate of [14C]glycine or [14C]cystine into glutathione. Essential information to validate the procedure was also obtained on the precursor-amino-acid pool sizes and kinetic parameters such as lag-time and time course of incorporation. Also, synthesis de novo rather than exchange was verified using buthionine sulphoximine, a specific inhibitor of GSH biosynthesis. The synthetic rates with either amino acid precursor varied throughout the adult life-span, but the patterns for both precursors were essentially identical. Biosynthesis was high in the newly emerged adult and decreased 62-70% (P less than 0.005) to a plateau during maturity. From the mature value there was a decrease of 36-41% (P less than 0.005) to a new plateau during senescence. Glutathione biosynthesis and concentration were correlated throughout maturity and senescence (r = 0.982) and thus biosynthesis was proportional to glutathione content. On this basis we concluded that impaired biosynthesis is the major and perhaps sole mechanism for the aging decrease in glutathione content.
Project description:Aedes aegypti utilizes blood for energy production, egg maturation and replenishment of maternal reserves. The principle midgut enzymes responsible for bloodmeal digestion are endoproteolytic serine-type proteases within the S1.A subfamily. While there are hundreds of serine protease-like genes in the A. aegypti genome, only five are known to be expressed in the midgut. We describe the cloning, sequencing and expression profiling of seven additional serine proteases and provide a genomic and phylogenetic assessment of these findings. Of the seven genes, four are constitutively expressed and three are transcriptionally induced upon blood feeding. The amount of transcriptional induction is strongly correlated among these genes. Alignments reveal that, in general, the conserved catalytic triad, active site and accessory catalytic residues are maintained in these genes and phylogenetic analysis shows that these genes fall within three distinct clades; trypsins, chymotrypsins and serine collagenases. Interestingly, a previously described trypsin consistently arose with other serine collagenases in phylogenetic analyses. These results suggest that multiple gene duplications have arisen within the S1.A subfamily of midgut serine proteases and/or that A. aegypti has evolved an array of proteases with a broad range of substrate specificities for rapid, efficient digestion of bloodmeals.
Project description:This study aimed at comparing gene transcription using microarrays and protein expression using 2D-DIGE between an Aedes aegypti insecticide-resistant strain (LiTOX) selected for 28 generations at the larval stage with field-collected leaf litter containing persistent Bacillus thuringiensis var. israelensis (Bti) toxins and the parental strain (Bora-Bora) susceptible to all insecticides. We focused on the tissue where the mode of action of the insecticide takes place: the midgut of the larvae.
Project description:Insects use their olfactory systems to obtain chemical information on mating partners, oviposition sites and food. The yellow fever mosquito Aedes aegypti, an important vector of human infectious diseases, shows strong preference for human blood meals. This study investigated the chemical basis of host detection by characterizing the neuronal responses of antennal olfactory sensilla of female Ae. aegypti to 103 compounds from human skin emanations. The effect of blood feeding on the responses of olfactory sensilla to these odorants was examined as well. Sensilla SBTII, GP, and three functional subtypes of SST (SST1, SST2, and SST3) responded to most of the compounds tested. Olfactory receptor neurons (ORNs) 'A' and 'B' in the trichoid sensilla, either activated or inhibited, were involved in the odour coding process. Compounds from different chemical classes elicited responses with different temporal structures and different response patterns across the olfactory sensilla. Except for their increased responses to several odorants, blood-fed mosquitoes generally evoked reduced responses to specific aldehydes, alcohols, aliphatics/aromatics, ketones, and amines through the SST1, SST2, SBTI, SBTII and GP sensilla. The odorants eliciting diminished responses in female mosquitoes after blood feeding may be important in Ae. aegypti host-seeking activity and thus can be candidates for mosquito attractants in the process of this disease vector management.
Project description:The mariner transposable element is capable of interplasmid transposition in the embryonic soma of the yellow fever mosquito, Aedes aegypti. To determine if this demonstrated mobility could be utilized to genetically transform the mosquito, a modified mariner element marked with a wild-type allele of the Drosophila melanogaster cinnabar gene was microinjected into embryos of a kynurenine hydroxylase-deficient, white-eyed recipient strain. Three of 69 fertile male founders resulting from the microinjected embryos produced families with colored-eyed progeny individuals, a transformation rate of 4%. The transgene-mediated complementation of eye color was observed to segregate in a Mendelian manner, although one insertion segregates with the recessive allele (female-determining) of the sex-determining locus, and a separate insertion is homozygous lethal. Molecular analysis of selected transformed families demonstrated that a single complete copy of the construct had integrated independently in each case and that it had done so in a transposase-mediated manner. The availability of a mariner transformation system greatly enhances our ability to study and manipulate this important vector species.
Project description:At the end of each developmental stage, the yellow fever mosquito Aedes aegypti performs the ecdysis behavioral sequence, a precisely timed series of behaviors that culminates in shedding of the old exoskeleton. Here we describe ecdysis triggering hormone-immunoreactive Inka cells located at branch points of major tracheal trunks and loss of staining coincident with ecdysis. Peptides (AeaETH1, AeaETH2) purified from extracts of pharate 4th instar larvae have--PRXamide C-terminal amino acid sequence motifs similar to ETHs previously identified in moths and flies. Injection of synthetic AeaETHs induced premature ecdysis behavior in pharate larvae, pupae and adults. Two functionally distinct subtypes of ETH receptors (AeaETHR-A, AeaETHR-B) of A. aegypti are identified and show high sensitivity and selectivity to ETHs. Increased ETHR transcript levels and behavioral sensitivity to AeaETHs arising in the hours preceding the 4th instar larva-to-pupa ecdysis are correlated with rising ecdysteroid levels, suggesting steroid regulation of receptor gene expression. Our description of natural and ETH-induced ecdysis in A. aegypti should facilitate future approaches directed toward hormone-based interference strategies for control of mosquitoes as human disease vectors.
Project description:BackgroundThe mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti.Methodology/principal findingsUsing a bioinformatics approach we screened genome databases and identified six putative AQPs in the genome of Ae. aegypti. Phylogenetic analysis showed that five of the six Ae. aegypti AQPs have high similarity to classical water-transporting AQPs of vertebrates. Using microarray, reverse transcription and real time PCR analysis we found that all six AQPs are expressed in distinct patterns in mosquito tissues/body parts. AaAQP1, 4, and 5 are strongly expressed in the adult female MT. RNAi-mediated knockdown of the MT-expressed mosquito AQPs resulted in significantly reduced diuresis.Conclusions/significanceOur results support the notion that AQP1, 4, and 5 function as water transporters in the MTs of adult female Ae. aegypti mosquitoes. Our results demonstrate the importance of these AQPs for mosquito diuresis after blood ingestion and highlight their potential as targets for the development of novel vector control strategies.
Project description:The yellow fever mosquito Aedes aegypti is one of the deadliest animals on the planet because it transmits several medically important arboviruses, including Zika, chikungunya, dengue, and yellow fever. Carbon-based nanoparticles (CNPs) derived from natural sources have previously been shown to have toxic effects on mosquito larvae and offer a potential alternative to chemical insecticides such as pyrethroids, for which mosquitoes have evolved resistance. However, CNPs derived from industrial sources, such as carbon black, have not previously been evaluated as larvicides. Here, we evaluate the effects of a commercially-available carbon black, EMPEROR® 1800 (E1800), on mortality and development of pyrethroid-susceptible (PS) and pyrethroid-resistant (PR) strains of Ae. aegypti. We found that E1800 exhibited concentration-dependent mortality against 1st instar larvae of both strains within the first 120 h after exposure, but after this period, surviving larvae did not show delays in their development to adults. Physical characterization of E1800 suspensions suggests that they form primary particles of ~30 nm in diameter that fuse into fundamental aggregates of ~170 nm in diameter. Notably, larvae treated with E1800 showed internal accumulation of E1800 in the gut and external accumulation on the respiratory siphon, anal papillae, and setae, suggesting a physical mode of toxic action. Taken together, our results suggest that E1800 has potential use as a larvicide with a novel mode of action for controlling PS and PR mosquitoes.