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

Genome Sequence Analysis of Dengue Virus 1 Isolated in Key West, Florida

Dengue virus (DENV) is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL), including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1) and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR) to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW), FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI) and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs

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

Permethrin Resistance in <i>Aedes aegypti</i> 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

The Effect of West Nile Virus Infection on the Midgut Gene Expression of Culex pipiens quinquefasciatus Say (Diptera: Culicidae)

The interaction of the mosquito and the invading virus is complex and can result in physiological and gene expression alterations in the insect. The association of West Nile virus (WNV) and Culex pipiens quinquefasciatus mosquitoes results in measurable changes in gene expression; 22 gene products were shown previously to have altered expression. Sequence analysis of one product, CQ G1A1, revealed 100% amino acid identity to gram negative bacteria binding proteins (CPQGBP) in Cx. p. quinquefasciatus, Aedes aegypti (70%) and Anopheles gambiae (63%) that function in pathogen recognition. CQ G1A1 also was differentially expressed following WNV infection in two populations of Cx. p. quinquefasciatus colonized from Florida with known differences in vector competence for WNV and showed spatial and temporal gene expression differences in midgut, thorax, and carcass tissues. These data suggest gene expression of CQ G1A1 is influenced by WNV infection and the WNV infection-controlled expression differs between populations and tissues

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

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

Abstract 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. Graphical Abstrac

Culex quinquefasciatus (Diptera: Culicidae) From Florida Transmitted Zika Virus

We report a laboratory colony of Culex quinquefasciatus mosquitoes were experimentally able to salivate Zika virus (ZIKV, Flaviviridae; Flavivirus) at 16 days post infection (dpi). ZIKV RNA was detected in bodies and in saliva deposited on filter paper cards with subsequent studies demonstrating the presence of live ZIKV in saliva