The Genetic Basis of Host Preference and Resting Behavior in the Major African Malaria Vector, Anopheles arabiensis.

Malaria transmission is dependent on the propensity of Anopheles mosquitoes to bite humans (anthropophily) instead of other dead end hosts. Recent increases in the usage of Long Lasting Insecticide Treated Nets (LLINs) in Africa have been associated with reductions in highly anthropophilic and endophilic vectors such as Anopheles gambiae s.s., leaving species with a broader host range, such as Anopheles arabiensis, as the most prominent remaining source of transmission in many settings. An. arabiensis appears to be more of a generalist in terms of its host choice and resting behavior, which may be due to phenotypic plasticity and/or segregating allelic variation. To investigate the genetic basis of host choice and resting behavior in An. arabiensis we sequenced the genomes of 23 human-fed and 25 cattle-fed mosquitoes collected both in-doors and out-doors in the Kilombero Valley, Tanzania. We identified a total of 4,820,851 SNPs, which were used to conduct the first genome-wide estimates of "SNP heritability" for host choice and resting behavior in this species. A genetic component was detected for host choice (human vs cow fed; permuted P = 0.002), but there was no evidence of a genetic component for resting behavior (indoors versus outside; permuted P = 0.465). A principal component analysis (PCA) segregated individuals based on genomic variation into three groups which were characterized by differences at the 2Rb and/or 3Ra paracentromeric chromosome inversions. There was a non-random distribution of cattle-fed mosquitoes between the PCA clusters, suggesting that alleles linked to the 2Rb and/or 3Ra inversions may influence host choice. Using a novel inversion genotyping assay, we detected a significant enrichment of the standard arrangement (non-inverted) of 3Ra among cattle-fed mosquitoes (N = 129) versus all non-cattle-fed individuals (N = 234; χ2, p = 0.007). Thus, tracking the frequency of the 3Ra in An. arabiensis populations may be of use to infer selection on host choice behavior within these vector populations; possibly in response to vector control. Controlled host-choice assays are needed to discern whether the observed genetic component has a direct relationship with innate host preference. A better understanding of the genetic basis for host feeding behavior in An. arabiensis may also open avenues for novel vector control strategies based on driving genes for zoophily into wild mosquito populations.

Differential Plasmodium falciparum infection of Anopheles gambiae s.s. molecular and chromosomal forms in Mali.

BACKGROUND: Anopheles gambiae sensu stricto (s.s.) is a primary vector of Plasmodium falciparum in sub-Saharan Africa. Although some physiological differences among molecular and chromosomal forms of this species have been demonstrated, the relative susceptibility to malaria parasite infection among them has not been unequivocally shown. The objective of this study was to investigate P. falciparum circumsporozoite protein infection (CSP) positivity among An. gambiae s.s. chromosomal and molecular forms. METHODS: Wild An. gambiae from two sites Kela (n=464) and Sidarebougou (n=266) in Mali were screened for the presence of P. falciparum CSP using an enzyme-linked immunosorbent assay (ELISA). Samples were then identified to molecular form using multiple PCR diagnostics (n=713) and chromosomal form using chromosomal karyotyping (n=419). RESULTS: Of 730 An. gambiae sensu lato (s.l.) mosquitoes, 89 (12.2%) were CSP ELISA positive. The percentage of positive mosquitoes varied by site: 52 (11.2%) in Kela and 37 (13.9%) in Sidarebougou. Eighty-seven of the positive mosquitoes were identified to molecular form and they consisted of nine Anopheles arabiensis (21.4%), 46 S (10.9%), 31 M (12.8%), and one MS hybrid (14.3%). Sixty of the positive mosquitoes were identified to chromosomal form and they consisted of five An. arabiensis (20.0%), 21 Savanna (15.1%), 21 Mopti (30.4%), 11 Bamako (9.2%), and two hybrids (20.0%). DISCUSSION: In this collection, the prevalence of P. falciparum infection in the M form was equivalent to infection in the S form (no molecular form differential infection). There was a significant differential infection by chromosomal form such that, P. falciparum infection was more prevalent in the Mopti chromosomal forms than in the Bamako or Savanna forms; the Mopti form was also the most underrepresented in the collection. Continued research on the differential P. falciparum infection of An. gambiae s.s. chromosomal and molecular forms may suggest that Plasmodium - An. gambiae interactions play a role in malaria transmission.

High degree of single nucleotide polymorphisms in California Culex pipiens (Diptera: Culicidae) sensu lato.

Resolution of systematic relationships among members of the Culex pipiens (L.) complex has important implications for public health as well as for studies on the evolution of sibling species. Currently held views contend that in California considerable genetic introgression occurs between Cx. pipiens and Cx. quinquefasciatus Say, and as such, these taxa behave as if they are a single species. Development of high throughput SNP genotyping tools for the analysis of Cx. pipiens complex population structure is therefore desirable. As a first step toward this goal, we sequenced 12 gene fragments from specimens collected in Marin and Fresno counties. On average, we found a higher single nucleotide polymorphism (SNP) density than any other mosquito species reported thus far. Coding regions contained significantly higher GC content (median 54.7%) than noncoding regions (42.4%; Wilcoxon rank sum test, P = 5.29 x 10(-5)). Differences in SNP allele frequencies observed between mosquitoes from Marin and Fresno counties indicated significant genetic divergence and suggest that SNP markers will be useful for future detailed population genetic studies of this group. The high density of SNPs highlights the difficulty in identifying species within the complex and may be associated with the large degree of phenotypic variation observed in this group of mosquitoes.

Asymmetric introgression between the M and S forms of the malaria vector, Anopheles gambiae, maintains divergence despite extensive hybridization.

The suggestion that genetic divergence can arise and/or be maintained in the face of gene flow has been contentious since first proposed. This controversy and a rarity of good examples have limited our understanding of this process. Partially reproductively isolated taxa have been highlighted as offering unique opportunities for identifying the mechanisms underlying divergence with gene flow. The African malaria vector, Anopheles gambiae s.s., is widely regarded as consisting of two sympatric forms, thought by many to represent incipient species, the M and S molecular forms. However, there has been much debate about the extent of reproductive isolation between M and S, with one view positing that divergence may have arisen and is being maintained in the presence of gene flow, and the other proposing a more advanced speciation process with little realized gene flow because of low hybrid fitness. These hypotheses have been difficult to address because hybrids are typically rare (<1%). Here, we assess samples from an area of high hybridization and demonstrate that hybrids are fit and responsible for extensive introgression. Nonetheless, we show that strong divergent selection at a subset of loci combined with highly asymmetric introgression has enabled M and S to remain genetically differentiated despite extensive gene flow. We propose that the extent of reproductive isolation between M and S varies across West Africa resulting in a 'geographic mosaic of reproductive isolation'; a finding which adds further complexity to our understanding of divergence in this taxon and which has considerable implications for transgenic control strategies.

Surveillance, insecticide resistance and control of an invasive Aedes aegypti (Diptera: Culicidae) population in California.

The invasion and subsequent establishment in California of Aedes aegypti in 2013 has created new challenges for local mosquito abatement and vector control districts. Studies were undertaken to identify effective and economical strategies to monitor the abundance and spread of this mosquito species as well as for its control. Overall, BG Sentinel (BGS) traps were found to be the most sensitive trap type to measure abundance and spread into new locations. Autocidal-Gravid-Ovitraps (AGO-B), when placed at a site for a week, performed equally to BGS in detecting the presence of female Ae. aegypti. Considering operational cost and our findings, we recommend use of BGS traps for surveillance in response to service requests especially in locations outside the known infestation area. We recommend AGO-Bs be placed at fixed sites, cleared and processed once a week to monitor mosquito abundance within a known infestation area. Long-term high density placements of AGO-Bs were found to show promise as an environmentally friendly trap-kill control strategy. California Ae. aegypti were found to be homozygous for the V1016I mutation in the voltage gated sodium channel gene, which is implicated to be involved in insecticide resistance. This strain originating from Clovis, California was resistant to some pyrethroids but not to deltamethrin in bottle bio-assays. Sentinel cage ultra-low-volume (ULV) trials using a new formulation of deltamethrin (DeltaGard®) demonstrated that it provided some control (average of 56% death in sentinel cages in a 91.4 m spray swath) after a single truck mounted aerial ULV application in residential areas.

A DNA extraction protocol for improved DNA yield from individual mosquitoes.

Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people's own DNA extraction protocols and not as an advertisement of a commercially available product.

A multi-detection assay for malaria transmitting mosquitoes.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Plasmodium falciparum infection rates for some Anopheles spp. from Guinea-Bissau, West Africa.

Presence of Plasmodium falciparum circumsporozoite protein (CSP) was detected by enzyme linked immunosorbent assay (ELISA) in a sample of Anopheles gambiae s.s., A. melas and A. pharoensis collected in Guinea-Bissau during October and November 2009. The percentage of P. falciparum infected samples (10.2% overall) was comparable to earlier studies from other sites in Guinea-Bissau (9.6-12.4%). The majority of the specimens collected were identified as A. gambiae which had an individual infection rate of 12.6 % across collection sites. A small number of specimens of A. coluzzii, A. coluzzii x A. gambiae hybrids, A. melas and A. pharoensis were collected and had infection rates of 4.3%, 4.1%, 11.1% and 33.3% respectively. Despite being present in low numbers in indoor collections, the exophilic feeding behaviors of A. melas (N=18) and A. pharoensis (N=6) and high infection rates observed in this survey suggest falciparum-malaria transmission potential outside of the protection of bed nets.

An analysis of two island groups as potential sites for trials of transgenic mosquitoes for malaria control.

Considerable technological advances have been made towards the generation of genetically modified mosquitoes for vector control. In contrast, less progress has been made towards field evaluations of transformed mosquitoes which are critical for evaluating the success of, and hazards associated with, genetic modification. Oceanic islands have been highlighted as potentially the best locations for such trials. However, population genetic studies are necessary to verify isolation. Here, we used a panel of genetic markers to assess for evidence of genetic isolation of two oceanic island populations of the African malaria vector, Anopheles gambiae s.s. We found no evidence of isolation between the Bijagós archipelago and mainland Guinea-Bissau, despite separation by distances beyond the known dispersal capabilities of this taxon. Conversely, the Comoros Islands appear to be genetically isolated from the East African mainland, and thus represent a location worthy of further investigation for field trials. Based on assessments of gene flow within and between the Comoros islands, the island of Grande Comore was found to be genetically isolated from adjacent islands and also exhibited local population structure, indicating that it may be the most suitable site for trials with existing genetic modification technologies.