Odorant and Gustatory Receptors in the Tsetse Fly Glossina morsitans morsitans

Tsetse flies use olfactory and gustatory responses, through odorant and gustatory receptors (ORs and GRs), to interact with their environment. Glossina morsitans morsitans genome ORs and GRs were annotated using homologs of these genes in Drosophila melanogaster and an ab initio approach based on OR and GR specific motifs in G. m. morsitans gene models coupled to gene ontology (GO). Phylogenetic relationships among the ORs or GRs and the homologs were determined using Maximum Likelihood estimates. Relative expression levels among the G. m. morsitans ORs or GRs were established using RNA-seq data derived from adult female fly. Overall, 46 and 14 putative G. m. morsitans ORs and GRs respectively were recovered. These were reduced by 12 and 59 ORs and GRs respectively compared to D. melanogaster. Six of the ORs were homologous to a single D. melanogaster OR (DmOr67d) associated with mating deterrence in females. Sweet taste GRs, present in all the other Diptera, were not recovered in G. m. morsitans. The GRs associated with detection of CO₂ were conserved in G. m. morsitans relative to D. melanogaster. RNA-sequence data analysis revealed expression of GmmOR15 locus represented over 90% of expression profiles for the ORs. The G. m. morsitans ORs or GRs were phylogenetically closer to those in D. melanogaster than to other insects assessed. We found the chemoreceptor repertoire in G. m. morsitans smaller than other Diptera, and we postulate that this may be related to the restricted diet of blood-meal for both sexes of tsetse flies. However, the clade of some specific receptors has been expanded, indicative of their potential importance in chemoreception in the tsetse.     

Bicluster pattern of codon context usages between flavivirus and vector mosquito Aedes aegypti: relevance to infection and transcriptional response of mosquito genes

The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in most of the subtropical and tropical countries. Besides DENV, yellow fever virus (YFV) is also transmitted by A. aegypti. Susceptibility of A. aegypti to West Nile virus (WNV) has also been confirmed. Although studies have indicated correlation of codon bias between flaviviridae and their animal/insect hosts, it is not clear if codon sequences have any relation to susceptibility of A. aegypti to DENV, YFV and WNV. In the current study, usages of codon context sequences (codon pairs for neighboring amino acids) of the vector (A. aegypti) genome as well as the flaviviral genomes are investigated. We used bioinformatics methods to quantify codon context bias in a genome-wide manner of A. aegypti as well as DENV, WNV and YFV sequences. Mutual information statistics was applied to perform bicluster analysis of codon context bias between vector and flaviviral sequences. Functional relevance of the bicluster pattern was inferred from published microarray data. Our study shows that codon context bias of DENV, WNV and YFV sequences varies in a bicluster manner with that of specific sets of genes of A. aegypti. Many of these mosquito genes are known to be differentially expressed in response to flaviviral infection suggesting that codon context sequences of A. aegypti and the flaviviruses may play a role in the susceptible interaction between flaviviruses and this mosquito. The bias in usages of codon context sequences likely has a functional association with susceptibility of A. aegypti to flaviviral infection. The results from this study will allow us to conduct hypothesis-driven tests to examine the role of codon context bias in evolution of vector–virus interactions at the molecular level.     

Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti

Molecular genetic analyses of biological properties characteristic of insect vectors of disease, such as hematophagy and competence for pathogens, require the ability to isolate and characterize genes involved in these processes. We have been working to develop molecular approaches for studying the promoter function of genes that are expressed specifically in the adult salivary glands of the yellow fever mosquito, Aedes aegypti. Genomic DNA fragments containing cis-acting promoter elements from the Maltase-like I (MalI) and Apyrase (Apy) genes were cloned so as to direct the expression of the reporter gene, luciferase (luc). The function of the promoters was assayed transiently in cultured insect cells and by germ-line transformation of Ae. aegypti. MalI and Apy DNA fragments consisting of at least 650 nucleotides (nt) of DNA immediately adjacent to the 5′-end of the initiation codon of the mosquito genes directed constitutive expression of the luc reporter gene in cultured cells. When introduced into Ae. aegypti chromosomes, approximately 1.5 kilobases (kb) of each promoter were able to direct the predicted developmental-, sex- and tissue-specific expression of the reporter gene in patterns identical to those determined for the respective endogenous genes.     

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites.     

Analysis of cycle Gene Expression in Aedes aegypti Brains by In Situ Hybridization

Even though the blood-sucking mosquito Aedes aegypti is one of the most important disease vectors, relatively little is known about the molecular mechanisms underlying processes involved in the temporal pattern of its activity and host seeking behavior. In this study, we analyzed the expression of the cycle (cyc) gene, one of the core components of the circadian clock, in Ae. aegypti brains by in situ hybridization at two different time points in light-dark conditions and compared the results with those obtained using a quantitative PCR assay (qPCR). Within the brain, differential labeling was detected according to distinct areas empirically pre-defined. Six out of seven of these areas showed significantly higher staining at ZT3 (three hours after light-on) compared to ZT11 (one before light-off), which is consistent with the qPCR data. Predominant staining was observed in three of those areas which correspond to positions of the optical and antennal lobes, as well as the region where the neurons controlling activity rhythms are presumably localized.     

Analysis of molecular markers for metamorphic competency and their response to starvation or feeding in the mosquito, Aedes aegypti (Diptera: Culicidae)

The nutritional condition of fourth instar larvae of the yellow fever mosquito, Aedes aegypti, governs female longevity and egg production, both are key determinants of pathogen transmission. As well, nutrition provisions larval growth and development and attains its greatest pace in the last larval instar in preparation for metamorphosis to an adult. These developmental processes are regulated by a complex endocrine interplay of juvenile hormone, neuropeptides, and ecdysteroids that is nutrition sensitive. We previously determined that feeding for only 24 h post-ecdysis was sufficient for fourth instar Ae. aegypti larvae to reach critical weight and accumulate sufficient nutritional stores to commit to metamorphosis. To understand the genetic basis of metamorphic commitment in Ae. aegypti, we profiled the expression of 16 genes known to be involved in the endocrine and nutritional regulation of insect metamorphosis in two ways. The first set is a developmental profile from the beginning of the fourth instar to early pupae, and the second set is for fourth instars starved or fed for up to 36 h. By comparing the two sets, we found that seven of the genes (AaegCYP302, AaegJHE43357, AaegBrCZ4, AaegCPF1-2, AaegCPR-7, AaegPpl, and AaegSlif) were expressed during metamorphic commitment in fourth instars and in fed but not starved larvae. Based on these results, the seven genes alone or in combination may serve as molecular indicators of nutritional and metamorphic status of fourth instar Ae. aegypti larvae and possibly other mosquito species in field and laboratory studies to gauge sub-lethal effects of novel and traditional cultural or chemical controls.     

Behavioral and molecular responses of Aedes aegypti to ultrasound

Mosquito-borne infectious diseases cause mortality and global infectious disease burden worldwide. There are several electronic mosquito repellents (EMRs) based on ultrasound have been developed and commercialized to reduce human-mosquito contacts. However, the efficacy of EMRs against mosquitoes is still unclear. In this study, we present experimental evidence that ultrasound of different frequency and sound pressure differentially affects the host-seeking behavior of Aedes aegypti females. Behavioral tests were accompanied by molecular experiments to check whether mosquitoes respond to ultrasound and are there any changes in specific mRNA expression. Experiments in bioassays revealed that the ultrasound of 100 kHz frequency and 90–110 dB pressure significantly disrupted CO?-oriented olfactory behaviors and blocked indoor invasion. Furthermore, a long time (>24 h) exposure to 100 kHz frequency/90 dB pressure of ultrasound decreased attractive behaviors to human skin. At the molecular level, there was no change in expression of odorant receptor co-receptor (AaOrco) in ultrasound treated animals, while one of the CO₂ receptor genes, AaGr3, and putative hearing-related gene, AAEL009258, were down-regulated and up-regulated, respectively. Our study indicates that high frequency (100 kHz) and pressure (90–110 dB) of the ultrasound has repellent effects to olfactory–driven behaviors of mosquitoes.     

TALEN-Based Gene Disruption in the Dengue Vector Aedes aegypti

In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20–40% of fertile survivors produced kmo alleles that failed to complement an existing kh^w mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1–7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G₁ progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector.     

The effects of various carbohydrate diets on Aedes aegypti infected with Dirofilaria immitis.

Aedes aegypti infected with Dirofilaria immitis and uninfected mosquitoes were maintained on various carbohydrate diets (glucose, galactose, fructose, sucrose, trehalose, maltose, and melibiose). The value of each of these sugars in supporting survival of adult A. aegypti, and in supporting egg production, viability of eggs, and development of third-stage larvae of D. immitis in A. aegypti was analyzed. Fructose, glucose, maltose, sucrose, and trehalose provided the strongest support for survival of adult male, and infected and uninfected adult female A. aegypti. Galactose and melibiose provided the least support for survival of all groups of mosquitoes. The mean number of eggs laid per uninfected adult female A. aegypti was greatest when mosquitoes were maintained on glucose, melibiose, maltose, fructose, sucrose, and trehalose. The same was true for female mosquitoes infected with D. immitis; except for melibiose which provided poor support for egg production. In both Dirofilaria-infected and in uninfected mosquitoes, galactose supported the production of low mean numbers of eggs per adult female A. aegypti. High percentages of eggs laid by uninfected and by infected female mosquitoes fed glucose, melibiose, maltose, sucrose, and trehalose hatched. While galactose supported a high percentage of hatching in eggs laid by uninfected A. aegypti, a much lower percentage of eggs laid by infected female mosquitoes maintained on this same carbohydrate hatched. The lowest percentages of eggs that hatched were from among those laid by infected and by uninfected females fed fructose. The highest mean number of D. immitis larvae (L₃) were recovered from adult A. aegypti fed glucose, maltose, fructose, and sucrose; the second best sugar in this regard was trehalose. The lowest mean number of D. immitis larvae were isolated from female A. aegypti fed galactose and melibiose.     

Natural arbovirus infection rate and detectability of indoor female Aedes aegypti from Mérida,Yucatán,Mexico

Arbovirus infection in Aedes aegypti has historically been quantified from a sample of the adult population by pooling collected mosquitoes to increase detectability. However, there is a significant knowledge gap about the magnitude of natural arbovirus infection within areas of active transmission, as well as the sensitivity of detection of such an approach. We used indoor Ae. aegypti sequential sampling with Prokopack aspirators to collect all mosquitoes inside 200 houses with suspected active ABV transmission from the city of Mérida, Mexico, and tested all collected specimens by RT-PCR to quantify: a) the absolute arbovirus infection rate in individually tested Ae. aegypti females; b) the sensitivity of using Prokopack aspirators in detecting ABV-infected mosquitoes; and c) the sensitivity of entomological inoculation rate (EIR) and vectorial capacity (VC), two measures ABV transmission potential, to different estimates of indoor Ae. aegypti abundance. The total number of Ae. aegypti (total catch, the sum of all Ae. aegypti across all collection intervals) as well as the number on the first 10-min of collection (sample, equivalent to a routine adult aspiration session) were calculated. We individually tested by RT-PCR 2,161 Aedes aegypti females and found that 7.7% of them were positive to any ABV. Most infections were CHIKV (77.7%), followed by DENV (11.4%) and ZIKV (9.0%). The distribution of infected Aedes aegypti was overdispersed; 33% houses contributed 81% of the infected mosquitoes. A significant association between ABV infection and Ae. aegypti total catch indoors was found (binomial GLMM, Odds Ratio > 1). A 10-min indoor Prokopack collection led to a low sensitivity of detecting ABV infection (16.3% for detecting infected mosquitoes and 23.4% for detecting infected houses). When averaged across all infested houses, mean EIR ranged between 0.04 and 0.06 infective bites per person per day, and mean VC was 0.6 infectious vectors generated from a population feeding on a single infected host per house/day. Both measures were significantly and positively associated with Ae. aegypti total catch indoors. Our findings provide evidence that the accurate estimation and quantification of arbovirus infection rate and transmission risk is a function of the sampling effort, the local abundance of Aedes aegypti and the intensity of arbovirus circulation.     

Wolbachia infection in Aedes aegypti mosquitoes alters blood meal excretion and delays oviposition without affecting trypsin activity

Blood feeding in Aedes aegypti is essential for reproduction, but also permits the mosquito to act as a vector for key human pathogens such as the Zika and dengue viruses. Wolbachia pipientis is an endosymbiotic bacterium that can manipulate the biology of Aedes aegypti mosquitoes, making them less competent hosts for many pathogens. Yet while Wolbachia affects other aspects of host physiology, it is unclear whether it influences physiological processes associated with blood meal digestion. To that end, we examined the effects of wMel Wolbachia infection in Ae. aegypti, on survival post-blood feeding, blood meal excretion, rate of oviposition, expression levels of key genes involved in oogenesis, and activity levels of trypsin blood digestion enzymes. We observed that wMel infection altered the rate and duration of blood meal excretion, delayed the onset of oviposition and was associated with a greater number of eggs being laid later. wMel-infected Ae. aegypti also had lower levels of key yolk protein precursor genes necessary for oogenesis. However, all of these effects occurred without a change in trypsin activity. These results suggest that Wolbachia infection may disrupt normal metabolic processes associated with blood feeding and reproduction in Ae. aegypti.