Genome-wide divergence among invasive populations of Aedes aegypti in California.

BACKGROUND: In the summer of 2013, Aedes aegypti Linnaeus was first detected in three cities in central California (Clovis, Madera and Menlo Park). It has now been detected in multiple locations in central and southern CA as far south as San Diego and Imperial Counties. A number of published reports suggest that CA populations have been established from multiple independent introductions. RESULTS: Here we report the first population genomics analyses of Ae. aegypti based on individual, field collected whole genome sequences. We analyzed 46 Ae. aegypti genomes to establish genetic relationships among populations from sites in California, Florida and South Africa. Based on 4.65 million high quality biallelic SNPs, we identified 3 major genetic clusters within California; one that includes all sample sites in the southern part of the state (South of Tehachapi mountain range) plus the town of Exeter in central California and two additional clusters in central California. CONCLUSIONS: A lack of concordance between mitochondrial and nuclear genealogies suggests that the three founding populations were polymorphic for two main mitochondrial haplotypes prior to being introduced to California. One of these has been lost in the Clovis populations, possibly by a founder effect. Genome-wide comparisons indicate extensive differentiation between genetic clusters. Our observations support recent introductions of Ae. aegypti into California from multiple, genetically diverged source populations. Our data reveal signs of hybridization among diverged populations within CA. Genetic markers identified in this study will be of great value in pursuing classical population genetic studies which require larger sample sizes.

Temporal Variations of Microbiota Associated with the Immature Stages of Two Florida Culex Mosquito Vectors.

Microbiota associated with mosquito vector populations impact several traits of mosquitoes, including survival, reproduction, control, and immunity against pathogens. The influence of seasonal variations and mosquito species on mosquito gut microbiota is poorly understood. We sought to determine whether the mosquito microbiota associated with immature stages of two congeners (Culex coronator and Culex nigripalpus) differ temporally and between the two species. Using high throughput 16S rRNA gene sequence analysis, we characterized bacterial and archaeal communities found in the immature stages of the two Culex mosquito species sampled over three seasons to compare the diversity of bacteria between the two species. Beta diversity analyses of the larval microbiota sequences revealed that the two Culex species differed significantly, both temporally within each species and between the two species. Bacteria in Cx. coronator larvae were dominated by Alphaproteobacteria, mainly associated with Roseoccocus and unidentified species of Rhizobiales, and two unidentified species of Cyanobacteria. In contrast, Cx. nigripalpus was dominated by Thorsellia anophelis (Gammaproteobacteria), Clostridium, an unidentified species of Ruminococcacae (Clostridiales), and additional unidentified species associated with Erysipelotrichaceae (Erysipelotrichales), Bacteroidales, and Mollicutes. Results of our study revealed both seasonal and interspecies differences in bacterial community composition associated with the immature stages of Cx. coronator and Cx. nigripalpus vector populations in Florida. These results have important implications for our understanding of the underlying factors of variations in disease transmission among seasons, susceptibility to various pesticides, and other biotic factors, including the role of the microbiota on the spread of invasive species. In addition, our results suggest close associations of certain bacteria species with each of the two Culex species that will be further targeted for their potential in the development of microbial-based control strategies.

Dengue serotype-specific immune response in Aedes aegypti and Aedes albopictus.

BACKGROUND: Dengue viruses (DENV) are considered one of the most important emerging pathogens and dengue disease is a global health threat. The geographic expansion of dengue viruses has led to co-circulation of all four dengue serotypes making it imperative that new DENV control strategies be devised. OBJECTIVES: Here we characterize dengue serotype-specific innate immune responses in Aedes aegypti and Aedes albopictus using DENV from Puerto Rico (PR). METHODS: Ae. aegypti and Ae. albopictus were infected with dengue serotype 1 and 2 isolated from Puerto Rico. DENV infected mosquito samples were collected and temporal change in expression of selected innate immune response pathway genes analyzed by quantitative real time PCR. FINDINGS: The Toll pathway is involved in anti-dengue response in Ae. aegypti, and Ae. albopictus. Infections with PR DENV- 1 elicited a stronger response from genes of the Toll immune pathway than PR DENV-2 in Ae. aegypti but in infected Ae. albopictus expression of Toll pathway genes tended to be similar between the serotypes. Two genes (a ribosomal S5 protein gene and a nimrod-like gene) from Ae. albopictus were expressed in response to DENV. MAIN CONCLUSIONS: These studies revealed a role for antiviral genes in DENV serotype-specific interactions with DENV vectors, demonstrated that infections with DENV-2 can modulate the Toll immune response pathway in Ae. aegypti and elucidated candidate molecules that might be used to interfere with serotype specific vector-virus interactions.

Factors That Influence the Transmission of West Nile Virus in Florida.

West Nile virus (WNV) was first detected in North America in New York City during the late summer of 1999 and was first detected in Florida in 2001. Although WNV has been responsible for widespread and extensive epidemics in human populations and epizootics in domestic animals and wildlife throughout North America, comparable epidemics have never materialized in Florida. Here, we review some of the reasons why WNV has yet to cause an extensive outbreak in Florida. The primary vector of mosquito-borne encephalitis virus in Florida is Culex nigripalpus Theobald. Rainfall, drought, and temperature are the primary factors that regulate annual populations of this species. Cx. nigripalpus is a competent vector of WNV, St. Louis encephalitis virus, and eastern equine encephalitis virus in Florida, and populations of this species can support focal amplification and transmission of these arboviruses. We propose that a combination of environmental factors influencing Cx. nigripalpus oviposition, blood-feeding behavior, and vector competence have limited WNV transmission in Florida to relatively small focal outbreaks and kept the state free of a major epidemic. Florida must remain vigilant to the danger from WNV, because a change in these environmental factors could easily result in a substantial WNV epidemic rivaling those seen elsewhere in the United States.

Transcriptomics of differential vector competence: West Nile virus infection in two populations of Culex pipiens quinquefasciatus linked to ovary development.

BACKGROUND: Understanding mechanisms that contribute to viral dissemination in mosquito vectors will contribute to our ability to interfere with the transmission of viral pathogens that impact public health. The expression of genes in two Culex pipiens quinquefasciatus populations from Florida with known differences in vector competence to West Nile virus (WNV) were compared using high throughput sequencing. RESULTS: A total of 15,176 transcripts were combined for comparison of expression differences between the two populations and 118 transcripts were differentially expressed (p < 0.05). The fold change in expression of the differentially expressed genes ranged from -7.5 - 6.13. The more competent population for WNV (Gainesville) over expressed 77 genes and down regulated 44 genes, compared with the less competent population for WNV (Vero Beach). Also, splicing analysis identified 3 transcripts with significantly different splice forms between the two populations. The functional analysis showed that the largest proportion of transcripts was included in the catalytic activity and transporter activity groups except for those in the unknown group. Interestingly, the up- regulated gene set contained most of the catalytic activity function and the down- regulated gene set had a notable proportion of transcripts with transporter activity function. Immune response category was shown in only the down regulated gene set, although those represent a relatively small portion of the function. Several different vitellogenin genes were expressed differentially. Based on the RNAseq data analysis, ovary development was compared across the populations and following WNV infection. There were significant differences among the compared groups. CONCLUSIONS: This study suggests that ovary development is correlated to vector competence in two Culex populations in Florida. Both populations control energy allocations to reproduction as a response to WNV. This result provides novel insight into the defense mechanism used by Culex spp. mosquitoes against WNV.

Immune-related transcripts, microbiota and vector competence differ in dengue-2 virus-infected geographically distinct Aedes aegypti populations.

BACKGROUND: Vector competence in Aedes aegypti is influenced by various factors. Crucial new control methods can be developed by recognizing which factors affect virus and mosquito interactions. METHODS: In the present study we used three geographically distinct Ae. aegypti populations and compared their susceptibility to infection by dengue virus serotype 2 (DENV-2). To identify any differences among the three mosquito populations, we evaluated expression levels of immune-related genes and assessed the presence of microbiota that might contribute to the uniqueness in their vector competence. RESULTS: Based on the results from the DENV-2 competence study, we categorized the three geographically distinct Ae. aegypti populations into a refractory population (Vilas do Atlântico), a susceptible population (Vero) and a susceptible but low transmission population (California). The immune-related transcripts were highly expressed in the California population but not in the refractory population. However, the Rel-1 gene was upregulated in the Vilas do Atlântico population following ingestion of a non-infectious blood meal, suggesting the gene's involvement in non-viral responses, such as response to microbiota. Screening of the bacteria, fungi and flaviviruses revealed differences between populations, and any of these could be one of the factors that interfere with the vector competence. CONCLUSIONS: The results reveal potential factors that might impact the virus and mosquito interaction, as well as influence the Ae. aegypti refractory phenotype.

Relationships between infection, dissemination, and transmission of West Nile virus RNA in Culex pipiens quinquefasciatus (Diptera: Culicidae).

Culex pipiens quinquefasciatus Say fed blood containing 6.8 +/- 0.3 logs (mean +/- SE) plaque-forming units of West Nile virus (WNV)/ml were maintained at 28 degrees C for incubation periods (IP) of 7, 14, or 21 d. Several attributes of vector competence were determined at each IP using quantitative real-time reverse transcriptase polymerase chain reaction to estimate plaque forming unit equivalents including: infection rate (WNV-positive abdomens), dissemination rate (WNV-positive legs or thoraces), combined dissemination rate (WNV-positive legs and thoraces), transmission rate (WNV-positive saliva), and WNV titers in abdomens, legs, thoraces, and saliva. Each rate increased or was equivalent with increasing IP. Mosquitoes transmitting WNV in saliva also had significantly higher IP-dependent WNV titers in abdomens, legs, and thoraces. Titers of WNV in abdomens were significantly correlated with titers in legs and thoraces, but the degree of association changed with IP. However, titers of abdomens, legs, and thoraces were not correlated with WNV presence or titer in the saliva. The results show that WNV presence or titer in the saliva of infected Cx. p. quinquefasciatus was not directly influenced by processes involved in WNV replication in other tissues. The processes controlling midgut infection and escape are, in part, independent from the infection processes in other tissues. The relationship between infection, dissemination, and transmission varied over time. The infection and replication of WNV in different tissues is likely influenced by different barriers encountered during the extrinsic incubation period. The significance of these observations for understanding vector competence is discussed.

Permethrin Resistance in Aedes aegypti Affects Aspects of Vectorial Capacity.

Aedes aegypti, as one of the vectors transmitting several arboviruses, is the main target in mosquito control programs. Permethrin is used to control mosquitoes and Aedes aegypti get exposed due to its overuse and are now resistant. The increasing percentage of permethrin resistant Aedes aegypti has become an important issue around the world and the potential influence on vectorial capacity needs to be studied. Here we selected a permethrin resistant (p-s) Aedes aegypti population from a wild Florida population and confirmed the resistance ratio to its parental population. We used allele-specific PCR genotyping of the V1016I and F1534C sites in the sodium channel gene to map mutations responsible for the resistance. Two important factors, survival rate and vector competence, that impact vectorial capacity were checked. Results indicated the p-s population had 20 times more resistance to permethrin based on LD50 compared to the parental population. In the genotyping study, the p-s population had more homozygous mutations in both mutant sites of the sodium channel gene. The p-s adults survived longer and had a higher dissemination rate for dengue virus than the parental population. These results suggest that highly permethrin resistant Aedes aegypti populations might affect the vectorial capacity, moreover, resistance increased the survival time and vector competence, which should be of concern in areas where permethrin is applied.

Activation of the autophagy pathway decreases dengue virus infection in Aedes aegypti cells.

BACKGROUND: Mosquito-borne dengue virus (DENV) causes major disease worldwide, impacting 50-100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology, including antiviral mechanisms, and developing new control strategies have become an important step towards the elimination of DENV disease. In the study reported here, we focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential antiviral target in the mosquito. METHODS: To understand the role played by autophagy in Ae. aegypti, we examined the activation of this pathway in Aag-2 cells, an Ae. aegypti-derived cell line, infected with DENV. Rapamycin and 3-methyladenine, two small molecules that have been shown to affect the function of the autophagy pathway, were used to activate or suppress, respectively, the autophagy pathway. RESULTS: At 1-day post-DENV infection in Aag-2 cells, transcript levels of both the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) and autophagy-related protein 1 (ATG1) increased. Rapamycin treatment activated the autophagy pathway as early as 1-h post-treatment, and the virus titer had decreased in the Aag-2 cells at 2 days post-infection; in contrast, the 3-methyladenine treatment did not significantly affect the DENV titer. Treatment with these small molecules also impacted the ATG12 transcript levels in DENV-infected cells. CONCLUSIONS: Our studies revealed that activation of the autophagy pathway through rapamycin treatment altered DENV infection in the mosquito cells, suggesting that this pathway could be a possible antiviral mechanism in the mosquito system. Here we provide fundamental information needed to proceed with future experiments and to improve our understanding of the mosquito's immune response against DENV.

Resistance to permethrin alters the gut microbiota of Aedes aegypti.

Insecticide resistance has emerged as a persistent threat to the fight against vector-borne diseases. We compared the gut microbiota of permethrin-selected (PS) strain of Aedes aegypti relative to the parent (KW) strain from Key West, Florida. Bacterial richness but not diversity was significantly higher in PS strain compared to KW strain. The two mosquito strains also differed in their gut microbial composition. Cutibacterium spp., Corynebacterium spp., Citricoccus spp., Leucobacter spp., Acinetobacter spp., Dietzia spp., and Anaerococcus spp. were more abundant in PS strain than in KW strain. In contrast, Sphingomonas spp., Aquabacterium spp., Methylobacterium spp., Flavobacterium spp., Lactobacillus spp., unclassified Burkholderiaceae and unclassified Nostocaceae were more abundant in KW strain compared to PS strain. PS strain was enriched with propionate metabolizers, selenate reducers, and xylan, chitin, and chlorophenol degraders while KW strain was enriched with sulfur oxidizers, sulfur metabolizers, sulfate reducers and naphthalene and aromatic hydrocarbons degraders. These findings demonstrate an association between the gut microbiota and insecticide resistance in an important vector species and sets the foundation for future studies to investigate the contribution of gut microbiota to evolution of insecticide resistance in disease vectors.

A simple method for determining arbovirus transmission in mosquitoes.

We present a simplified method for the collection of mosquito saliva to determine Culex pipiens quinquefasciatus transmission of West Nile virus that can be used for experiments requiring large sample sizes.

Effects of West Nile virus dose and extrinsic incubation temperature on temporal progression of vector competence in Culex pipiens quinquefasciatus.

Culex pipiens quinquefasciatus were fed blood containing either 7.0 +/- 0.1 logs plaque-forming units (pfu)/ml (high dose) or 5.9 +/- 0.1 logs pfu/ml (low dose) of West Nile virus and held at extrinsic incubation temperatures (EIT) of 28 degrees C or 25 degrees C. Approximately 20 mosquitoes per dose were collected after incubation periods (IP) of 4, 6, 8, and 12 days postinfection (dpi). Infection rates were influenced by EIT and virus dose but not by IP. Body titer was significantly higher for mosquitoes fed the high dose and held at 28 degrees C at the later IPs (6, 8, and 12 dpi). However, leg titer was significantly higher for mosquitoes at the later IPs but did not differ between EITs or doses. Because infection rates varied with EIT and dose, there is likely a midgut infection barrier influenced by these factors that is not influenced by IP. Dissemination rates were influenced by all 3 factors consistent with the presence of a midgut escape barrier. Dissemination rate, body titer, and leg titer were dependent on IP, indicating the need to investigate multiple time points in vector competence studies to elucidate critical events in infection and dissemination.

Profiling Transcripts of Vector Competence between Two Different Aedes aegypti Populations in Florida.

A Chikungunya virus (CHIKV) outbreak in Italy in 2007 spread to include the islands of the Caribbean and most of the Americas and still circulates in Europe and Africa. Florida being close in distance to the Caribbean islands experienced a CHIKV outbreak in 2014 and continues to have a few travel-related cases each year. It is known that different environmental conditions in different regions can result in genetic variation that favor changes in competence to arbovirus. We evaluated the vector competence of Florida Aedes aegypti for CHIKV and determined if there is a geographic component that influences genes involved in CHIKV competence. We utilized a genomic approach to identify the candidate genes using RNA sequencing. The infection and dissemination results showed that field populations were more competent vectors for CHIKV than a lab population. The differentially expressed genes in the two field-collected CHIKV-infected populations, compared to the Rockefeller strain, were related to the Wnt/Notch signaling pathway, with similarity to genes scattered throughout the signaling pathway. This result suggested the possibility of identifying genes involved in the determination of vector competence in different gene pools of Ae. aegypti.

Expression of a novel member of the odorant-binding protein gene family in Culex nigripalpus (Diptera: Culicidae).

We previously showed that gene expression in the midgut of female Culex nigripalpus Theobald (Diptera: Culicidae) was altered after ingestion of a bloodmeal and some of the expressed cDNAs showed stage-, sex-, and tissue-specific expression. One of these expressed cDNAs, CN G11B, is here shown to be similar to members of a gene family that encodes odorant-binding proteins. CN G11B.1 is a cDNA fragment of 721 bp, is incomplete at the 5'-end, and contains a canonical polyadenylic acid tail signal sequence in the 103-bp 3'-untranslated region. Translation of CN G11B.1 provides a putative protein product of 207 amino acids. GenBank tblastx, VectorBase, and Pfam database searches showed identity to hypothetical proteins from Culex pipiens quinquefasciatus Say (86%) and Aedes aegypti (L.) (55%). These proteins have structural motifs similar to those found in the gene family that includes odorant-binding proteins. CN G11B.1 putative protein has two of the six cysteines that are highly conserved in most invertebrate odorant-binding proteins. CN G11B.1 expression increased in thoraces (6-12 h) and in abdomens (3-48 h) of blood fed female Cx. nigripalpus but was not detected in RNA from heads, indicating a possible role in both chemoreception and blood feeding.

Isolation and spatial expression analysis of a partial esterase gene of Culex nigripalpus (Diptera: Culicidae).

Mosquitoes play an important role as vectors for a variety of pathogens. Insecticide use for control means results in selection of tolerant/resistant mosquitoes. An esterase cDNA was isolated from Culex nigripalpus Theobald (Diptera: Culicidae), and its spatial expression was characterized. GenBank tblastx and VectorBase searches with the translation product of the longest cDNA fragment (397 bp, CN Temsha est-1) of the Cx. nigripalpus esterase revealed similarity (95%) to the estalpha2 esterase gene product of Culex pipiens quinquefasciatus Say, suggesting it is a member of the esterase family. Transcripts encoding Cx. nigripalpus esterase were detected in midgut tissue, thoraces, and abdomens of female Cx. nigripalpus, findings that may support possible roles in feeding, digestion, or reproduction. Gene expression studies to determine the role of Cx. nigripalpus esterase in the mosquito are being conducted.

A model to assess the accuracy of detecting arboviruses in mosquito pools.

Vigilant surveillance of virus prevalence in mosquitoes is essential for risk assessment and outbreak prediction. Accurate virus detection methods are essential for arbovirus surveillance. We have developed a model to estimate the probability of accurately detecting a virus-positive mosquito from pooled field collections using standard molecular techniques. We discuss several factors influencing the probability of virus detection, including the number of virions in the sample, the total sample volume, and the portion of the sample volume that is being tested. Our model determines the probability of obtaining at least 1 virion in the sample that is tested. The model also determines the optimal sample volume that is required in any test to ensure a desired probability of virus detection is achieved, and can be used to support the accuracy of current tests or to optimize existing techniques.

Microbiota variations in Culex nigripalpus disease vector mosquito of West Nile virus and Saint Louis Encephalitis from different geographic origins.

Although mosquito microbiota are known to influence reproduction, nutrition, disease transmission, and pesticide resistance, the relationship between host-associated microbial community composition and geographical location is poorly understood. To begin addressing this knowledge gap, we characterized microbiota associated with adult females of Culex nigripalpus mosquito vectors of Saint Louis Encephalitis and West Nile viruses sampled from three locations in Florida (Vero Beach, Palmetto Inland, and Palmetto Coast). High-throughput sequencing of PCR-amplified 16S rRNA genes demonstrated significant differences among microbial communities of mosquitoes sampled from the three locations. Mosquitoes from Vero Beach (east coast Florida) were dominated by uncultivated Asaia sp. (Alphaproteobacteria), whereas microbiota associated with mosquitoes collected from two mosquito populations at Palmetto (west coast Florida) sites were dominated by uncultured Spironema culicis (Spirochaetes), Salinisphaera hydrothermalis (Gammaproteobacteria), Spiroplasma (Mollicutes), uncultured Enterobacteriaceae, Candidatus Megaira (Alphaproteobacteria; Rickettsiae), and Zymobacter (Gammaproteobacteria). The variation in taxonomic profiles of Cx. nigripalpus gut microbial communities, especially with respect to dominating taxa, is a potentially critical factor in understanding disease transmission and mosquito susceptibility to insecticides among different mosquito populations.

CNAct-1 gene is differentially expressed in the subtropical mosquito Culex nigripalpus (Diptera: Culicidae), the primary West Nile Virus vector in Florida.

Analysis of differentially expressed genes is a common molecular biological tool to investigate changes in mosquito genes after a bloodmeal or parasite exposure. We report here the characterization of a differentially expressed actin gene, CNAct-1, from the subtropical mosquito, Culex nigripalpus Theobald (Diptera: Culicidae). The CNAct-1 genomic clone is 1.525 kb, includes one 66-bp intron, and a 328-bp 3'-untranslated region. The 376-amino acid putative translation product shares high similarity with muscle-specific actin proteins from other insects, including Culex pipiens pipiens L., Aedes aegypti (L.), Anopheles gambiae Giles and Drosophila melanogaster (Meigen). CNAct-1 is expressed in second and third instars, late pupae, and adult females and males. Interestingly, Cx. nigripalpus actin was highly expressed in female mosquito midgut tissue isolated 6-12 h after ingestion of a bloodmeal. This expression profile indicates a unique function for CNAct-1 in midgut processes that are initiated after blood ingestion.

Bloodmeal-induced differential gene expression in the disease vector Culex nigripalpus (Diptera: Culicidae).

We have characterized gene expression changes in the midgut tissue of Culex nigripalpus Theobold (Diptera: Culicidae) females, after they ingest a bloodmeal, by differential display of RNA isolated from pre- and postfeeding females. Seventy-two cDNA fragments exhibiting reproducible differences were observed. Of these, 22 showed an increase in gene expression and 50 showed a decrease in gene expression as a result of blood feeding. Eleven cDNA fragments exhibiting increased expression after a bloodmeal were cloned and sequenced. Seven of these were novel transcripts, and they did not match any sequences in GenBank. Three clones were similar to conserved proteins of unknown function from Aedes aegypti (L.) and Anopheles gambiae Giles. Here, we present the expression data on the first cDNA clones isolated from Cx. nigripalpus midgut tissue, including their molecular characterization, and we discuss their possible involvement in blood-feeding-associated processes and disease transmission.

Transcription Profiling for Defensins of Aedes aegypti (Diptera: Culicidae) During Development and in Response to Infection With Chikungunya and Zika Viruses.

Aedes aegypti (L.) is a vector of chikungunya, dengue, yellow fever and Zika viruses. These viruses encounter a variety of induced defense responses from the innate immune system of the mosquito. We cloned defensin A from Ae. aegypti using laboratory populations originating from Key West and Orlando, Florida. To characterize inducible immune defensin peptides, we examined the defensin A (DefA) and defensin C (DefC) expression through time course studies using quantitative real-time PCR. We observed that ingestion of chikungunya virus (CHIKV) and Zika virus (ZIKV) infected blood triggered early upregulated expression of DefA and DefC at 3 h after blood feeding. At 10-d post infection, there was significant downregulation of DefA and DefC in CHIKV-infected females and significant upregulation of DefA and DefC in ZIKV-infected females compared with control mosquitoes fed uninfected blood. Our studies demonstrate that the relative activity of DefA and DefC changed depending on whether Ae. aegypti was infected with CHIKV or ZIKV, suggesting differences in antiviral defense responses. In addition, we also examined DefA and DefC gene expression during the different developmental stages. Significant qualitative and quantitative differences were found in DefA and DefC transcripts between Key West and Orlando strains. We found that adult males consistently had higher expression than adult females of different ages. Together, these data show that members of the Ae. aegypti defensin gene family play a role in both Zika and chikungunya antiviral response.