DNA Barcoding Analysis of Trinidad Haemagogus Mosquitoes Reveals Evidence for Putative New Species.

Background: Haemagogus janthinomys is a primary sylvan vector of yellow fever virus and the emerging Mayaro virus. However, despite its medical importance, there is a dearth of data on the molecular taxonomy of this mosquito species. Methods: In this study, DNA barcoding analysis was performed on 64 adult female mosquitoes from Trinidad morphologically identified as Hg. janthinomys. The mitochondrial cytochrome c oxidase I (COI) gene and ribosomal DNA internal transcribed spacer 2 (ITS2) region of the mosquitoes were PCR amplified and sequenced, and molecular phylogenies inferred. Results: The BLASTN analysis showed that only 20% (n = 13/66) of COI sequences had high similarity (>99% identity) to Hg. janthinomys and the remaining sequences had low similarity (<90% identity) to reference GenBank sequences. Phylogenetic analysis of COI sequences revealed the presence of four strongly supported groups, with one distinct clade that did not align with any reference sequences. Corresponding ITS2 sequences for samples in this distinct COI group clustered into three clades. Conclusions: These molecular findings suggest the existence of a putative new Haemagogus mosquito species and underscore the need for further, more in-depth investigations into the taxonomy and classification of the Haemagogus genus.

Mating Competitiveness of Transgenic Aedes aegypti (Diptera: Culicidae) Males Against Wild-Type Males Reared Under Simulated Field Conditions.

Efforts directed at genetic modification of mosquitoes for population control or replacement are highly dependent on the initial mating success of transgenic male mosquitoes following their release into natural populations. Adult mosquito phenotypes are influenced by the environmental conditions experienced as larvae. Semifield studies conducted to date have not taken that under consideration when testing male mating fitness, and have compared mating success of males reared under identical environmental conditions. We performed pairwise mating challenges between males from a genetically modified laboratory strain (BF2) versus males from a recent Trinidad field isolate of Aedes aegypti (L.), a major vector of multiple arboviruses. We utilized larval density and nutrition to simulate environmental stress experienced by the Trinidad males and females. Our results indicated that environmental stress during larval development negatively influenced the competitiveness and reproductive success of males from the Trinidad population when paired with optimum reared BF2 males. Small (0.027 m3) and large (0.216 m3) trials were conducted wherein stressed or optimum Trinidad males competed with optimum BF2 males for mating with stressed Trinidad females. When competing with stress reared Trinidad males, optimum reared BF2 males were predominant in matings with stress reared Trinidad females, and large proportions of these females mated with males of both strains. When competing with optimum reared Trinidad males, no difference in mating success was observed between them and BF2 males, and frequencies of multiple matings were low. Our results indicate that future mating competition studies should incorporate appropriate environmental conditions when designing mating fitness trials of genetically modified males.

Environmental conditions for Jamestown Canyon virus correlated with population-level resource selection by white-tailed deer in a suburban landscape.

Suburban landscapes can alter spatial patterns by white-tailed deer (Odocoileus virginianus) and increase animal contact with vectors, pathogens, and humans. Close-contact relationships at a landscape level can have broad implications for disease epidemiology. From 1995-1999, we captured and radio-collared 41 deer in two suburban forest preserves in Chicago, Illinois. We collected blood to determine if animals were seronegative or seropositive for Jamestown Canyon virus and tracked deer movements within suburban habitats. We developed utilization distributions at the population-level and evaluated resource selection for seronegative and seropositive deer. We used maximum likelihood estimation for model selection via Akaike information criterion and then restricted maximum likelihood estimation to attain unbiased estimates of the parameters in the top-ranking models. The top-ranking model describing the resource selection of seronegative deer received almost the full weight of evidence (Akaike information criterion ωi = 0.93), and included the proportion of wetlands, precipitation in year t, and an interaction of the proportion of wetlands and precipitation in year t. The top-ranking model describing resource selection of seropositive deer received the full weight of evidence (Akaike information criterion ωi = 1.00). The model included distance to nearest populated place, distance to nearest river, length of road in each grid cell, precipitation in year t, and an interaction of the length of road in each grid cell and precipitation in year t. These results are valuable for mapping the spatial configuration of hotspots for Jamestown Canyon virus and could be used to educate local residents and recreationalists to reduce human exposure.

Identification of Host Blood Meals of Mosquitoes (Diptera: Culicidae) Collected at the Aripo Savannas Scientific Reserve in Trinidad, West Indies.

Surveillance for blood-fed female mosquitoes was performed between August 2015 and February 2016 at sites along the periphery of the Aripo Savannas Environmentally Reserve (ASSR) located in northeastern Trinidad, West Indies. We collected engorged female mosquitoes representing 13 species. DNA extractions from dissected abdomens were subjected to PCR amplification with three primer pairs targeting the mitochondrial cytochrome oxidase I and cytochrome b gene sequences. High-quality sequence information and host identification were obtained for 42 specimens representing eight mosquito species with at least one primer combination. A broad range of vertebrates including humans were identified, but the majority were nonhuman mammals, both domestic and wild. Domestic dogs were the most common host and may represent potential sentinel species for monitoring local enzootic arbovirus activity in Trinidad. Culex declarator Dyer and Knab and Culex nigripalpus Theobald were the most common blood-fed mosquito species comprising 79.1% of the total number identified. These species obtained blood meals from birds, nonhuman mammals, and human hosts, and therefore pose significant risks as potential bridge vectors for epizootic arbovirus transmission in the ASSR area as well as other sylvan areas in Trinidad. These data represent the first such results for Trinidad.

Changing patterns in the distribution of the Mayaro virus vector Haemagogus species in Trinidad, West Indies.

The Mayaro virus disease (MAYVD) is an emerging mosquito borne zoonosis that was first reported on the island of Trinidad in 1954. The viral agent for this disease is known to presently be endemic to Central and South America. The enzootic cycle of the Mayaro virus (MAYV) is not fully characterized, though primates are thought to be the main reservoir with Haemagogus species of mosquitoes as the primary vector. This virus has been responsible for several sporadic cases of infections and limited outbreaks, but it is postulated that the MAYVD will become a major epidemic in the future, following in the steps of the recent pandemics caused by Chikungunya and Zika viruses. Mitigating possible major outbreaks of MAYVD in the future would require effective strategies for vector control, for which knowledge on the ecology and distribution of the Haemagogus mosquitoes would be vitally important. In Trinidad, Haemagogus species have only been reported in the northwestern peninsula of the island based on studies up to 1995. However, no recent investigations have been completed to determine the status of this important vector on the island. The aim of this study was to investigate the current spatial distribution of Haemagogus species in the island of Trinidad, West Indies. Adult Haemagogus (Hag.) mosquitoes and larvae were surveyed during a twenty-month period using human bait trapping and ovitraps in major forested areas on the island. Mosquito species were identified using classical taxonomic keys. Haemagogus species were widespread and found in all forest types surveyed. Hag. janthinomys (85.7%) was the most widely distributed and dominant species on the island. Lower levels of Hag. leucocelaneus (7.3%), Hag. equinus (6.4%) and Hag. celeste (0.6%) were also collected. Overall, the proportion of mosquitoes collected in the wet season (June-December) was 3.5 times more than in the dry season (January-May). Mangroves, young secondary forests, semi-evergreen and evergreen forest types had relatively high mean abundance levels of Haemagogus species as compared to deciduous and montane forests. Proximity analysis suggests that population settlements within a 1 km buffer of the forest peripherals may be at risk for any emerging arboviral disease associated with these mosquito vectors. Haemagogus species showed a much wider distribution in Trinidad as compared to previous reports from up to 20 years ago and were prevalent in areas with no known presence of non-human primates. Since the MAYV has been previously implicated in causing infections in vertebrate hosts like rodents, birds and small mammals, the findings of this study suggest that there may be alternative hosts and reservoirs of this virus in the sylvatic cycle in Trinidad, other than primates. This has significant epidemiological implications for mosquito-borne viral infections in the region.

A transcriptomic survey of the impact of environmental stress on response to dengue virus in the mosquito, Aedes aegypti.

Populations of Aedes aegypti naturally exhibit variable susceptibility to dengue viruses. This natural variation can be impacted by nutritional stress resulting from larval-stage crowding, indicating the influence of environment components on the adult mosquito immune response. In particular, larval crowding was previously shown to reduce the susceptibility of adult females of a Trinidad field isolate of A. aegypti to the dengue serotype 2 (JAM1409) virus. Here, we present the first whole transcriptome study to address the impact of environmental stress on A. aegypti response to dengue virus. We examined expression profiles of adult females resulting from crowded and optimum reared larvae from the same Trinidad isolate at two critical early time points-3 and 18 hours post dengue virus infected blood meal. We exposed specimens to either a dengue or naïve blood meal, and then characterized the response in ten gene co-expression modules based on their transcriptional associations with environmental stress and time. We further analyzed the top 30 hub or master regulatory genes in each of the modules, and validated our results via qRT-PCR. These hub genes reveal which functions are critical to the mechanisms that confer dengue virus refractoriness or susceptibility to stress conditioned A. aegypti, as well as the time points at which they are most important.

Origin of a High-Latitude Population of Aedes aegypti in Washington, DC.

An overwintering population of Aedes aegypti has been documented in the Capitol Hill neighborhood of Washington, DC, since 2011. Mitochondrial cytochrome oxidase I (mtCOI) sequence data presented in a previous study traced the origin to the New World. Here, we use microsatellite and 14,071 single nucleotide polymorphisms along with mitochondrial DNA (mtDNA) sequences on Washington Ae. aegypti samples and samples from potential sources to further narrow the origin of this population. Genetically, Washington Ae. aegypti are closest to populations in Florida, meaning this is the most likely source. Florida experienced the first mosquito-borne transmission of dengue in the United States after decades of absence of this disease, as well as local transmission of chikungunya and Zika in recent years. This suggests that the Capitol Hill, Washington, DC population of Ae. aegypti is capable of transmitting viruses such as dengue, chikungunya, and Zika in modern US city environments.

Larval stress alters dengue virus susceptibility in Aedes aegypti (L.) adult females.

In addition to genetic history, environmental conditions during larval stages are critical to the development, success and phenotypic fate of the Aedes aegypti mosquito. In particular, previous studies have shown a strong genotype-by-environment component to adult mosquito body size in response to optimal vs stressed larval conditions. Here, we expand upon those results by investigating the effects of larval-stage crowding and nutritional limitation on the susceptibility of a recent field isolate of Aedes aegypti to dengue virus serotype-2. Interestingly, female mosquitoes from larvae subjected to a stressed regime exhibited significantly reduced susceptibility to disseminated dengue infection 14days post infection compared to those subjected to optimal regimes. Short term survivorship post-infected blood feeding was not significantly different. As with body size, dengue virus susceptibility of a mosquito population is determined by a combination of genetic and environmental factors and is likely maintained by balancing selection. Here, we provide evidence that under different environmental conditions, the innate immune response of field-reared mosquitoes exhibits a large range of phenotypic variability with regard to dengue virus susceptibility. Further, as with body size, our results suggest that mosquitoes reared under optimal laboratory conditions, as employed in all mosquito-pathogen studies to date, may not always be realistic proxies for natural populations.

Evidence for an Overwintering Population of Aedes aegypti in Capitol Hill Neighborhood, Washington, DC.

Aedes aegypti is an invasive, highly anthropophilic mosquito and a major vector for dengue and chikungunya. Population persistence in the continental United States is reportedly limited to southward of the average 10°C winter isotherm, which in the east, bisects Alabama, Mississippi, Georgia, and South Carolina. We report on summer collections and genotypic analyses of Ae. aegypti collected in the Capitol Hill neighborhood in Washington, DC (WDC). Analysis of a 441-bp fragment of the mitochondrial cytochrome oxidase I gene sequence identified the same two haplotype sequences during 2011-2014, and placed these within two discrete groups known to be derived from lineages resident in the Americas. Analysis of 10 microsatellite loci for specimens collected during 2011-2014 revealed no evidence for introgression of new alleles across years. Overall, our data support a conclusion that this represents a resident WDC population, likely maintained during winter months in a subterranean habitat that facilitates year-round survival.

Identification of a major Quantitative Trait Locus determining resistance to the organophosphate temephos in the dengue vector mosquito Aedes aegypti.

Organophosphate insecticides (OP) have extensively been used to control mosquitoes, such as the vector Aedes aegypti. Unfortunately, OP resistance has hampered control programs worldwide. We used Quantitative Trait Locus (QTL) mapping to evaluate temephos resistance in two F1 intercross populations derived from crosses between a resistant Ae. aegypti strain (RecR) and two susceptible strains (MoyoD and Red). A single major effect QTL was identified on chromosome 2 of both segregating populations, named rtt1 (resistance to temephos 1). Bioinformatics analyses identified a cluster of carboxylesterase genes (CCE) within the rtt1 interval. qRT-PCR demonstrated that different CCEs were up-regulated in F2 resistant individuals from both crosses. However, none exceeded the 2-fold expression. Primary mechanisms for temephos resistance may vary between Ae. aegypti populations, yet also appear to support previous findings suggesting that multiple linked esterase genes may contribute to temephos resistance in the RecR strain as well as other populations.

Mitotic-chromosome-based physical mapping of the Culex quinquefasciatus genome.

The genome assembly of southern house mosquito Cx. quinquefasciatus is represented by a high number of supercontigs with no order or orientation on the chromosomes. Although cytogenetic maps for the polytene chromosomes of this mosquito have been developed, their utilization for the genome mapping remains difficult because of the low number of high-quality spreads in chromosome preparations. Therefore, a simple and robust mitotic-chromosome-based approach for the genome mapping of Cx. quinquefasciatus still needs to be developed. In this study, we performed physical mapping of 37 genomic supercontigs using fluorescent in situ hybridization on mitotic chromosomes from imaginal discs of 4th instar larvae. The genetic linkage map nomenclature was adopted for the chromosome numbering based on the direct positioning of 58 markers that were previously genetically mapped. The smallest, largest, and intermediate chromosomes were numbered as 1, 2, and 3, respectively. For idiogram development, we analyzed and described in detail the morphology and proportions of the mitotic chromosomes. Chromosomes were subdivided into 19 divisions and 72 bands of four different intensities. These idiograms were used for mapping the genomic supercontigs/genetic markers. We also determined the presence of length polymorphism in the q arm of sex-determining chromosome 1 in Cx. quinquefasciatus related to the size of ribosomal locus. Our physical mapping and previous genetic linkage mapping resulted in the chromosomal assignment of 13% of the total genome assembly to the chromosome bands. We provided the first detailed description, nomenclature, and idiograms for the mitotic chromosomes of Cx. quinquefasciatus. Further application of the approach developed in this study will help to improve the quality of the southern house mosquito genome.

Influence of mosquito genotype on transcriptional response to dengue virus infection.

The mosquito Aedes aegypti is the principal vector that transmits dengue virus (DENV) to humans. The primary factors that trigger a susceptible or refractory interaction of A. aegypti with DENV are not well understood. In this study, our aim is to characterize the influence of vector genotype on differential gene expression of susceptible vs. refractory A. aegypti strains to DENV infection. To accomplish that, we identified differential expression of a set of complementary DNAs (cDNAs; n = 9,504) of the D2S3 (susceptible) and Moyo-D (refractory) strains of A. aegypti to DENV serotype 2 (JAM1409) and compared these results to the differential expression of cDNAs in a different susceptible vector genotype (Moyo-S) relative to the same refractory genotype (Moyo-D) identified from our previous study. We observed that, although the number of differentially expressed transcripts (DETs) was similar in both the studies, about ~95% of the DETs were distinct between Moyo-D/D2S3 vs. Moyo-D/Moyo-S. This suggested that A. aegypti response, to infection of a given genotype of dengue, is largely dependent upon the vector genotype. However, we observed a set of common DETs among the vector strains that were associated with predicted functions such as endocytosis, regulation of autophagy, peroxisome, and lipid metabolism that may be relatively universal in conferring mosquito response to DENV infection.

Stage-specific transcription during development of Aedes aegypti.

BACKGROUND: Aedes aegypti is the most important global vector of dengue virus infection in humans. Availability of the draft genome sequence of this mosquito provides unique opportunities to study different aspects of its biology, including identification of genes and pathways relevant to the developmental processes associated with transition across individual life stages. However, detailed knowledge of gene expression patterns pertaining to developmental stages of A. aegypti is largely lacking. RESULTS: We performed custom cDNA microarray analyses to examine the expression patterns among six developmental stages: early larvae, late larvae, early pupae, late pupae, and adult male and female mosquitoes. Results revealed 1,551 differentially expressed transcripts (DETs) showing significant differences in levels of expression between these life stages. The data suggests that most of the differential expression occurs in a stage specific manner in A. aegypti. Based on hierarchical clustering of expression levels, correlated expression patterns of DETs were also observed among developmental stages. Weighted gene correlation network analysis revealed modular patterns of expression among the DETs. We observed that hydrolase activity, membrane, integral to membrane, DNA binding, translation, ribosome, nucleoside-triphosphatase activity, structural constituent of ribosome, ribonucleoprotein complex and receptor activity were among the top ten ranked GO (Gene Ontology) terms associated with DETs. Significant associations of DETs were also observed with specific KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway modules. Finally, comparisons with the previously reported developmental transcriptome of the malaria vector, Anopheles gambiae, indicated that gene expression patterns during developmental processes reflect both species-specific as well as common components of the two mosquito species. CONCLUSIONS: Our study shows that genes involved in the developmental life cycle of A. aegypti are expressed in a highly stage-specific manner. This suggests that transcriptional events associated with transition through larval, pupal and adult stages are largely discrete.

Comparative expression profiles of midgut genes in dengue virus refractory and susceptible Aedes aegypti across critical period for virus infection.

BACKGROUND: Aedes aegypti is the primary mosquito vector for dengue virus (DENV) worldwide. Infectivity of dengue virus varies among natural populations of this mosquito. How A. aegypti responds to DENV infection relative to which genes and associated pathways contribute to its differential susceptibility as a vector is not well defined. METHODS/PRINCIPAL FINDINGS: Here, we used custom cDNA microarrays to identify groups of genes that were differentially expressed in midgut tissues between susceptible and refractory strains in a highly time specific manner. While genes involved in protein processing in the endoplasmic reticulum, mRNA surveillance, and the proteasome were significantly up-regulated in the susceptible strain, several metabolic processes including glycolysis, glycan biosynthesis and Wnt pathway were active in the refractory strain. In addition, several key signaling genes were expressed as common responsive genes in both susceptible and refractory mosquitoes that may be necessary for signal transduction to trigger the appropriate host response to the viral infection. These are coordinately expressed in the form of tight gene networks and expression clusters that may be necessary to differentially contribute to the progression of dengue infection between the two strains. CONCLUSIONS: Our data show that highly correlated differential expression of responsive genes throughout the post infection period in A. aegypti midgut tissues is necessary for a coordinated transcriptional response of the mosquito genes to host or defend the viral infection.

Global cross-talk of genes of the mosquito Aedes aegypti in response to dengue virus infection.

BACKGROUND: The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in humans, and DENV is the most important arbovirus across most of the subtropics and tropics worldwide. The early time periods after infection with DENV define critical cellular processes that determine ultimate success or failure of the virus to establish infection in the mosquito. METHODS AND RESULTS: To identify genes involved in these processes, we performed genome-wide transcriptome profiling between susceptible and refractory A. aegypti strains at two critical early periods after challenging them with DENV. Genes that responded coordinately to DENV infection in the susceptible strain were largely clustered in one specific expression module, whereas in the refractory strain they were distributed in four distinct modules. The susceptible response module in the global transcriptional network showed significant biased representation with genes related to energy metabolism and DNA replication, whereas the refractory response modules showed biased representation across different metabolism pathway genes including cytochrome P450 and DDT [1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane] degradation genes, and genes associated with cell growth and death. A common core set of coordinately expressed genes was observed in both the susceptible and refractory mosquitoes and included genes related to the Wnt (Wnt: wingless [wg] and integration 1 [int1] pathway), MAPK (Mitogen-activated protein kinase), mTOR (mammalian target of rapamycin) and JAK-STAT (Janus Kinase - Signal Transducer and Activator of Transcription) pathways. CONCLUSIONS: Our data revealed extensive transcriptional networks of mosquito genes that are expressed in modular manners in response to DENV infection, and indicated that successfully defending against viral infection requires more elaborate gene networks than hosting the virus. These likely play important roles in the global-cross talk among the mosquito host factors during the critical early DENV infection periods that trigger the appropriate host action in susceptible vs. refractory mosquitoes.

Enhancing genome investigations in the mosquito Culex quinquefasciatus via BAC library construction and characterization.

BACKGROUND: Culex quinquefasciatus (Say) is a major species in the Culex pipiens complex and an important vector for several human pathogens including West Nile virus and parasitic filarial nematodes causing lymphatic filariasis. It is common throughout tropical and subtropical regions and is among the most geographically widespread mosquito species. Although the complete genome sequence is now available, additional genomic tools are needed to improve the sequence assembly. FINDINGS: We constructed a bacterial artificial chromosome (BAC) library using the pIndigoBAC536 vector and HindIII partially digested DNA isolated from Cx. quinquefasciatus pupae, Johannesburg strain (NDJ). Insert size was estimated by NotI digestion and pulsed-field gel electrophoresis of 82 randomly selected clones. To estimate genome coverage, each 384-well plate was pooled for screening with 29 simple sequence repeat (SSR) and five gene markers. The NDJ library consists of 55,296 clones arrayed in 144 384-well microplates. Fragment insert size ranged from 50 to 190 kb in length (mean = 106 kb). Based on a mean insert size of 106 kb and a genome size of 579 Mbp, the BAC library provides ~10.1-fold coverage of the Cx. quinquefasciatus genome. PCR screening of BAC DNA plate pools for SSR loci from the genetic linkage map and for four genes associated with reproductive diapause in Culex pipiens resulted in a mean of 9.0 positive plate pools per locus. CONCLUSION: The NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes.

Complete sequences of mitochondria genomes of Aedes aegypti and Culex quinquefasciatus and comparative analysis of mitochondrial DNA fragments inserted in the nuclear genomes.

We present complete sequences of the mitochondrial genomes for two important mosquitoes, Aedes aegypti and Culex quinquefasciatus, that are major vectors of dengue virus and lymphatic filariasis, respectively. The A. aegypti mitochondrial genome is 16,655 bp in length and that of C. quinquefasciatus is 15,587 bp, yet both contain 13 protein coding genes, 22 transfer RNA (tRNA) genes, one 12S ribosomal RNA (rRNA) gene, one 16S rRNA gene and a control region (CR) in the same order. The difference in the genome size is due to the difference in the length of the control region. We also analyzed insertions of nuclear copies of mtDNA-like sequences (NUMTs) in a comparative manner between the two mosquitoes. The NUMT sequences occupy ~0.008% of the A. aegypti genome and ~0.001% of the C. quinquefasciatus genome. Several NUMTs were found localized in the introns of predicted protein coding genes in both genomes (32 genes in A. aegypti but only four in C. quinquefasciatus). None of these NUMT-containing genes had an ortholog between the two species or had paralogous copies within a genome that was also NUMT-containing. It was further observed that the NUMT-containing genes were relatively longer but had lower GC content compared to the NUMT-less paralogous copies. Moreover, stretches of homologies are present among the genic and non-genic NUMTs that may play important roles in genomic rearrangement of NUMTs in these genomes. Our study provides new insights on understanding the roles of nuclear mtDNA sequences in genome complexities of these mosquitoes.

Genome-based microsatellite development in the Culex pipiens complex and comparative microsatellite frequency with Aedes aegypti and Anopheles gambiae.

BACKGROUND: Mosquitoes in the Culex pipiens complex are among the most medically important vectors for human disease worldwide and include major vectors for lymphatic filariasis and West Nile virus transmission. However, detailed genetic studies in the complex are limited by the number of genetic markers available. Here, we describe methods for the rapid and efficient identification and development of single locus, highly polymorphic microsatellite markers for Cx. pipiens complex mosquitoes via in silico screening of the Cx. quinquefasciatus genome sequence. METHODOLOGY/PRINCIPAL FINDINGS: Six lab colonies representing four Cx. pipiens and two Cx. quinquefasciatus populations were utilized for preliminary assessment of 38 putative loci identified within 16 Cx. quinquefasciatus supercontig assemblies (CpipJ1) containing previously mapped genetic marker sequences. We identified and validated 12 new microsatellite markers distributed across all three linkage groups that amplify consistently among strains representing the complex. We also developed four groups of 3-5 microsatellite loci each for multiplex-ready PCR. Field collections from three cities in Indiana were used to assess the multiplex groups for their application to natural populations. All were highly polymorphic (Mean  = 13.0 alleles) per locus and reflected high polymorphism information content (PIC) (Mean  = 0.701). Pairwise F(ST) indicated population structuring between Terre Haute and Fort Wayne and between Terre Haute and Indianapolis, but not between Fort Wayne and Indianapolis. In addition, we performed whole genome comparisons of microsatellite motifs and abundance between Cx. quinquefasciatus and the primary vectors for dengue virus and malaria parasites, Aedes aegypti and Anopheles gambiae, respectively. CONCLUSIONS/SIGNIFICANCE: We demonstrate a systematic approach for isolation and validation of microsatellites for the Cx. pipiens complex by direct screen of the Cx. quinquefasciatus genome supercontig assemblies. The genome density of microsatellites is greater in Cx. quinquefasciatus (0.26%) than in Ae. aegypti (0.14%), but considerably lower than in An. gambiae (0.77%).

Genome-based polymorphic microsatellite development and validation in the mosquito Aedes aegypti and application to population genetics in Haiti.

BACKGROUND: Microsatellite markers have proven useful in genetic studies in many organisms, yet microsatellite-based studies of the dengue and yellow fever vector mosquito Aedes aegypti have been limited by the number of assayable and polymorphic loci available, despite multiple independent efforts to identify them. Here we present strategies for efficient identification and development of useful microsatellites with broad coverage across the Aedes aegypti genome, development of multiplex-ready PCR groups of microsatellite loci, and validation of their utility for population analysis with field collections from Haiti. RESULTS: From 79 putative microsatellite loci representing 31 motifs identified in 42 whole genome sequence supercontig assemblies in the Aedes aegypti genome, 33 microsatellites providing genome-wide coverage amplified as single copy sequences in four lab strains, with a range of 2-6 alleles per locus. The tri-nucleotide motifs represented the majority (51%) of the polymorphic single copy loci, and none of these was located within a putative open reading frame. Seven groups of 4-5 microsatellite loci each were developed for multiplex-ready PCR. Four multiplex-ready groups were used to investigate population genetics of Aedes aegypti populations sampled in Haiti. Of the 23 loci represented in these groups, 20 were polymorphic with a range of 3-24 alleles per locus (mean = 8.75). Allelic polymorphic information content varied from 0.171 to 0.867 (mean = 0.545). Most loci met Hardy-Weinberg expectations across populations and pairwise FST comparisons identified significant genetic differentiation between some populations. No evidence for genetic isolation by distance was observed. CONCLUSION: Despite limited success in previous reports, we demonstrate that the Aedes aegypti genome is well-populated with single copy, polymorphic microsatellite loci that can be uncovered using the strategy developed here for rapid and efficient screening of genome supercontig assemblies. These loci are suitable for genetic and population studies using multiplex-PCR.