Human-Mosquito Contact: A Missing Link in Our Understanding of Mosquito-Borne Disease Transmission Dynamics.

Despite the critical role that contact between hosts and vectors, through vector bites, plays in driving vector-borne disease (VBD) transmission, transmission risk is primarily studied through the lens of vector density and overlooks host-vector contact dynamics. This review article synthesizes current knowledge of host-vector contact with an emphasis on mosquito bites. It provides a framework including biological and mathematical definitions of host-mosquito contact rate, blood-feeding rate, and per capita biting rates. We describe how contact rates vary and how this variation is influenced by mosquito and vertebrate factors. Our framework challenges a classic assumption that mosquitoes bite at a fixed rate determined by the duration of their gonotrophic cycle. We explore alternative ecological assumptions based on the functional response, blood index, forage ratio, and ideal free distribution within a mechanistic host-vector contact model. We highlight that host-vector contact is a critical parameter that integrates many factors driving disease transmission. A renewed focus on contact dynamics between hosts and vectors will contribute new insights into the mechanisms behind VBD spread and emergence that are sorely lacking. Given the framework for including contact rates as an explicit component of mathematical models of VBD, as well as different methods to study contact rates empirically to move the field forward, researchers should explicitly test contact rate models with empirical studies. Such integrative studies promise to enhance understanding of extrinsic and intrinsic factors affecting host-vector contact rates and thus are critical to understand both the mechanisms driving VBD emergence and guiding their prevention and control.

Metagenomic shotgun sequencing reveals host species as an important driver of virome composition in mosquitoes.

High-throughput nucleic acid sequencing has greatly accelerated the discovery of viruses in the environment. Mosquitoes, because of their public health importance, are among those organisms whose viromes are being intensively characterized. Despite the deluge of sequence information, our understanding of the major drivers influencing the ecology of mosquito viromes remains limited. Using methods to increase the relative proportion of microbial RNA coupled with RNA-seq we characterize RNA viruses and other symbionts of three mosquito species collected along a rural to urban habitat gradient in Thailand. The full factorial study design allows us to explicitly investigate the relative importance of host species and habitat in structuring viral communities. We found that the pattern of virus presence was defined primarily by host species rather than by geographic locations or habitats. Our result suggests that insect-associated viruses display relatively narrow host ranges but are capable of spreading through a mosquito population at the geographical scale of our study. We also detected various single-celled and multicellular microorganisms such as bacteria, alveolates, fungi, and nematodes. Our study emphasizes the importance of including ecological information in viromic studies in order to gain further insights into viral ecology in systems where host specificity is driving both viral ecology and evolution.

Patterns, Drivers, and Challenges of Vector-Borne Disease Emergence.

Vector-borne diseases are emerging at an increasing rate and comprise a disproportionate share of all emerging infectious diseases. Yet, the key ecological and evolutionary dimensions of vector-borne disease that facilitate their emergence have not been thoroughly explored. This study reviews and synthesizes the existing literature to explore global patterns of emerging vector-borne zoonotic diseases (VBZDs) under changing global conditions. We find that the vast majority of emerging VBZDs are transmitted by ticks (Ixodidae) and mosquitoes (Culicidae) and the pathogens transmitted are dominated by Rickettsiaceae bacteria and RNA viruses (Flaviviridae, Bunyaviridae, and Togaviridae). The most common potential driver of these emerging zoonoses is land use change, but for many diseases, the driver is unknown, revealing a critical research gap. While most reported VBZDs are emerging in the northern latitudes, after correcting for sampling bias, Africa is clearly a region with the greatest share of emerging VBZD. We highlight critical gaps in our understanding of VBZD emergence and emphasize the importance of interdisciplinary research and consideration of deeper evolutionary processes to improve our capacity for anticipating where and how such diseases have and will continue to emerge.

Estimates of introgression as a function of pairwise distances.

BACKGROUND: Research over the last 10 years highlights the increasing importance of hybridization between species as a major force structuring the evolution of genomes and potentially providing raw material for adaptation by natural and/or sexual selection. Fueled by research in a few model systems where phenotypic hybrids are easily identified, research into hybridization and introgression (the flow of genes between species) has exploded with the advent of whole-genome sequencing and emerging methods to detect the signature of hybridization at the whole-genome or chromosome level. Amongst these are a general class of methods that utilize patterns of single-nucleotide polymorphisms (SNPs) across a tree as markers of hybridization. These methods have been applied to a variety of genomic systems ranging from butterflies to Neanderthals to detect introgression, however, when employed at a fine genomic scale these methods do not perform well to quantify introgression in small sample windows. RESULTS: We introduce a novel method to detect introgression by combining two widely used statistics: pairwise nucleotide diversity dxy and Patterson's D. The resulting statistic, the distance fraction (df), accounts for genetic distance across possible topologies and is designed to simultaneously detect and quantify introgression. We also relate our new method to the recently published fd and incorporate these statistics into the powerful genomics R-package PopGenome, freely available on GitHub (pievos101/PopGenome) and the Comprehensive R Archive Network (CRAN). The supplemental material contains a wide range of simulation studies and a detailed manual how to perform the statistics within the PopGenome framework. CONCLUSION: We present a new distance based statistic df that avoids the pitfalls of Patterson's D when applied to small genomic regions and accurately quantifies the fraction of introgression (f) for a wide range of simulation scenarios.

Mosquito vector-associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod-borne diseases.

Vector-borne diseases are a major health burden, yet factors affecting their spread are only partially understood. For example, microbial symbionts can impact mosquito reproduction, survival, and vectorial capacity, and hence affect disease transmission. Nonetheless, current knowledge of mosquito-associated microbial communities is limited. To characterize the bacterial and eukaryotic microbial communities of multiple vector species collected from different habitat types in disease endemic areas, we employed next-generation 454 pyrosequencing of 16S and 18S rRNA amplicon libraries, also known as metabarcoding. We investigated pooled whole adult mosquitoes of three medically important vectors, Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus, collected from different habitats across central Thailand where we previously characterized mosquito diversity. Our results indicate that diversity within the mosquito microbiota is low, with the majority of microbes assigned to one or a few taxa. Two of the most common eukaryotic and bacterial genera recovered (Ascogregarina and Wolbachia, respectively) are known mosquito endosymbionts with potentially parasitic and long evolutionary relationships with their hosts. Patterns of microbial composition and diversity appeared to differ by both vector species and habitat for a given species, although high variability between samples suggests a strong stochastic element to microbiota assembly. In general, our findings suggest that multiple factors, such as habitat condition and mosquito species identity, may influence overall microbial community composition, and thus provide a basis for further investigations into the interactions between vectors, their microbial communities, and human-impacted landscapes that may ultimately affect vector-borne disease risk.

Metagenomic shotgun sequencing of a Bunyavirus in wild-caught Aedes aegypti from Thailand informs the evolutionary and genomic history of the Phleboviruses.

Arthropod-borne viruses significantly impact human health. They span multiple families, all of which include viruses not known to cause disease. Characterizing these representatives could provide insights into the origins of their disease-causing counterparts. Field-caught Aedes aegypti mosquitoes from Nakhon Nayok, Thailand, underwent metagenomic shotgun sequencing to reveal a Bunyavirus closely related to Phasi Charoen (PhaV) virus, isolated in 2009 from Ae. aegypti near Bangkok. Phylogenetic analysis of this virus suggests it is basal to the Phlebovirus genus thus making it ideally positioned phylogenetically for understanding the evolution of these clinically important viruses. Genomic analysis finds that a gene necessary for virulence in vertebrates, but not essential for viral replication in arthropods, is missing. The sequencing of this phylogenetically-notable and genomically-unique Phlebovirus from wild mosquitoes exemplifies the utility and efficacy of metagenomic shotgun sequencing for virus characterization in arthropod vectors of human diseases.

Mosquito vector diversity across habitats in central Thailand endemic for dengue and other arthropod-borne diseases.

Recent years have seen the greatest ecological disturbances of our times, with global human expansion, species and habitat loss, climate change, and the emergence of new and previously-known infectious diseases. Biodiversity loss affects infectious disease risk by disrupting normal relationships between hosts and pathogens. Mosquito-borne pathogens respond to changing dynamics on multiple transmission levels and appear to increase in disturbed systems, yet current knowledge of mosquito diversity and the relative abundance of vectors as a function of habitat change is limited. We characterize mosquito communities across habitats with differing levels of anthropogenic ecological disturbance in central Thailand. During the 2008 rainy season, adult mosquito collections from 24 sites, representing 6 habitat types ranging from forest to urban, yielded 62,126 intact female mosquitoes (83,325 total mosquitoes) that were assigned to 109 taxa. Female mosquito abundance was highest in rice fields and lowest in forests. Diversity indices and rarefied species richness estimates indicate the mosquito fauna was more diverse in rural and less diverse in rice field habitats, while extrapolated estimates of true richness (Chao1 and ACE) indicated higher diversity in the forest and fragmented forest habitats and lower diversity in the urban. Culex sp. (Vishnui subgroup) was the most common taxon found overall and the most frequent in fragmented forest, rice field, rural, and suburban habitats. The distributions of species of medical importance differed significantly across habitat types and were always lowest in the intact, forest habitat. The relative abundance of key vector species, Aedes aegypti and Culex quinquefasciatus, was negatively correlated with diversity, suggesting that direct species interactions and/or habitat-mediated factors differentially affecting invasive disease vectors may be important mechanisms linking biodiversity loss to human health. Our results are an important first step for understanding the dynamics of mosquito vector distributions under changing environmental features across landscapes of Thailand.

Hybridization reveals the evolving genomic architecture of speciation.

The rate at which genomes diverge during speciation is unknown, as are the physical dynamics of the process. Here, we compare full genome sequences of 32 butterflies, representing five species from a hybridizing Heliconius butterfly community, to examine genome-wide patterns of introgression and infer how divergence evolves during the speciation process. Our analyses reveal that initial divergence is restricted to a small fraction of the genome, largely clustered around known wing-patterning genes. Over time, divergence evolves rapidly, due primarily to the origin of new divergent regions. Furthermore, divergent genomic regions display signatures of both selection and adaptive introgression, demonstrating the link between microevolutionary processes acting within species and the origin of species across macroevolutionary timescales. Our results provide a uniquely comprehensive portrait of the evolving species boundary due to the role that hybridization plays in reducing the background accumulation of divergence at neutral sites.

History of Aedes mosquitoes in Hawaii.

As a geographically isolated island chain with no native mosquitoes, Hawaii is a model for examining the mechanisms behind insect vector invasions and their subsequent interactions with each other and with human populations. The yellow fever mosquito, Aedes aegypti, and the Asian tiger mosquito, Ae. albopictus, have been responsible for epidemics of dengue in Hawaii. As one of the world's earliest locations to be invaded by both species, Hawaii's history is particularly relevant because both species are currently invading new areas worldwide and are implicated in outbreaks of emergent or reemergent pathogens such as dengue, chikungunya, and yellow fever. Here we analyze the historical records of mosquito introductions in order to understand the factors that have led to the current distribution of these 2 mosquitoes in the Hawaiian Islands.

Multi-allelic major effect genes interact with minor effect QTLs to control adaptive color pattern variation in Heliconius erato.

Recent studies indicate that relatively few genomic regions are repeatedly involved in the evolution of Heliconius butterfly wing patterns. Although this work demonstrates a number of cases where homologous loci underlie both convergent and divergent wing pattern change among different Heliconius species, it is still unclear exactly how many loci underlie pattern variation across the genus. To address this question for Heliconius erato, we created fifteen independent crosses utilizing the four most distinct color pattern races and analyzed color pattern segregation across a total of 1271 F2 and backcross offspring. Additionally, we used the most variable brood, an F2 cross between H. himera and the east Ecuadorian H. erato notabilis, to perform a quantitative genetic analysis of color pattern variation and produce a detailed map of the loci likely involved in the H. erato color pattern radiation. Using AFLP and gene based markers, we show that fewer major genes than previously envisioned control the color pattern variation in H. erato. We describe for the first time the genetic architecture of H. erato wing color pattern by assessing quantitative variation in addition to traditional linkage mapping. In particular, our data suggest three genomic intervals modulate the bulk of the observed variation in color. Furthermore, we also identify several modifier loci of moderate effect size that contribute to the quantitative wing pattern variation. Our results are consistent with the two-step model for the evolution of mimetic wing patterns in Heliconius and support a growing body of empirical data demonstrating the importance of major effect loci in adaptive change.

Flavobacterium akiainvivens sp. nov., from decaying wood of Wikstroemia oahuensis, Hawai'i, and emended description of the genus Flavobacterium.

Strain IK-1(T) was isolated from decaying tissues of the shrub Wikstroemia oahuensis collected on O'ahu, Hawai'i. Cells were rods that stained Gram-negative. Gliding motility was not observed. The strain was oxidase-negative and catalase-positive. Zeaxanthin was the major carotenoid. Flexirubin-type pigments were not detected. The most abundant fatty acids in whole cells of IK-1(T) grown on R2A were iso-C(15:0) and one or both of C(16:1)ω7c and C(16:1)ω6c. Based on comparisons of the nucleotide sequence of the 16S rRNA gene, the closest neighbouring type strains were Flavobacterium rivuli WB 3.3-2(T) and Flavobacterium subsaxonicum WB 4.1-42(T), with which IK-1(T) shares 93.84 and 93.67% identity, respectively. The G+C content of the genomic DNA was 44.2 mol%. On the basis of distance from its nearest phylogenetic neighbours and phenotypic differences, the species Flavobacterium akiainvivens sp. nov. is proposed to accommodate strain IK-1(T) ( =ATCC BAA-2412(T) =CIP 110358(T)) as the type strain. The description of the genus Flavobacterium is emended to reflect the DNA G+C contents of Flavobacterium akiainvivens IK-1(T) and other species of the genus Flavobacterium described since the original description of the genus.

Spatially disaggregated disease transmission risk: land cover, land use and risk of dengue transmission on the island of Oahu.

Vector-borne diseases persist in transmission systems that usually comprise heterogeneously distributed vectors and hosts leading to a highly heterogeneous case distribution. In this study, we build on principles of classical mathematical epidemiology to investigate spatial heterogeneity of disease risk for vector-borne diseases. Land cover delineates habitat suitability for vectors, and land use determines the spatial distribution of humans. We focus on the risk of exposure for dengue transmission on the Hawaiian island of Oahu, where the vector Aedes albopictus is well established and areas of dense human population exist. In Hawai'i, dengue virus is generally absent, but occasionally flares up when introduced. It is therefore relevant to investigate risk, but difficult to do based on disease incidence data. Based on publicly available data (land cover, land use, census data, surveillance mosquito trapping), we map the spatial distribution of vectors and human hosts and finally overlay them to produce a vector-to-host ratio map. The resulting high-resolution maps indicate a high spatial variability in vector-to-host ratio suggesting that risk of exposure is spatially heterogeneous and varies according to land cover and land use.

Polymorphic butterfly reveals the missing link in ecological speciation.

Ecological speciation occurs when ecologically based, divergent selection causes the evolution of reproductive isolation. There are many empirical examples of this process; however, there exists a poorly characterized stage during which the traits that distinguish species ecologically and reproductively segregate in a single population. By using a combination of genetic mapping, mate-choice experiments, field observations, and population genetics, we studied a butterfly population with a mimetic wing color polymorphism and found that the butterflies exhibited partial, color-based, assortative mate preference. These traits represent the divergent, ecologically based signal and preference components of sexual isolation that usually distinguish incipient and sibling species. The association between behavior and recognition trait in a single population may enhance the probability of speciation and provides an example of the missing link between an interbreeding population and isolated species.

Man bites mosquito: understanding the contribution of human movement to vector-borne disease dynamics.

In metropolitan areas people travel frequently and extensively but often in highly structured commuting patterns. We investigate the role of this type of human movement in the epidemiology of vector-borne pathogens such as dengue. Analysis is based on a metapopulation model where mobile humans connect static mosquito subpopulations. We find that, due to frequency dependent biting, infection incidence in the human and mosquito populations is almost independent of the duration of contact. If the mosquito population is not uniformly distributed between patches the transmission potential of the pathogen at the metapopulation level, as summarized by the basic reproductive number, is determined by the size of the largest subpopulation and reduced by stronger connectivity. Global extinction of the pathogen is less likely when increased human movement enhances the rescue effect but, in contrast to classical theory, it is not minimized at an intermediate level of connectivity. We conclude that hubs and reservoirs of infection can be places people visit frequently but briefly and the relative importance of human and mosquito populations in maintaining the pathogen depends on the distribution of the mosquito population and the variability in human travel patterns. These results offer an insight in to the paradoxical observation of resurgent urban vector-borne disease despite increased investment in vector control and suggest that successful public health intervention may require a dual approach. Prospective studies can be used to identify areas with large mosquito populations that are also visited by a large fraction of the human population. Retrospective studies can be used to map recent movements of infected people, pinpointing the mosquito subpopulation from which they acquired the infection and others to which they may have transmitted it.

Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies.

BACKGROUND: With over 20 parapatric races differing in their warningly colored wing patterns, the butterfly Heliconius erato provides a fascinating example of an adaptive radiation. Together with matching races of its co-mimic Heliconius melpomene, H. erato also represents a textbook case of Müllerian mimicry, a phenomenon where common warning signals are shared amongst noxious organisms. It is of great interest to identify the specific genes that control the mimetic wing patterns of H. erato and H. melpomene. To this end we have undertaken comparative mapping and targeted genomic sequencing in both species. This paper reports on a comparative analysis of genomic sequences linked to color pattern mimicry genes in Heliconius. RESULTS: Scoring AFLP polymorphisms in H. erato broods allowed us to survey loci at approximately 362 kb intervals across the genome. With this strategy we were able to identify markers tightly linked to two color pattern genes: D and Cr, which were then used to screen H. erato BAC libraries in order to identify clones for sequencing. Gene density across 600 kb of BAC sequences appeared relatively low, although the number of predicted open reading frames was typical for an insect. We focused analyses on the D- and Cr-linked H. erato BAC sequences and on the Yb-linked H. melpomene BAC sequence. A comparative analysis between homologous regions of H. erato (Cr-linked BAC) and H. melpomene (Yb-linked BAC) revealed high levels of sequence conservation and microsynteny between the two species. We found that repeated elements constitute 26% and 20% of BAC sequences from H. erato and H. melpomene respectively. The majority of these repetitive sequences appear to be novel, as they showed no significant similarity to any other available insect sequences. We also observed signs of fine scale conservation of gene order between Heliconius and the moth Bombyx mori, suggesting that lepidopteran genome architecture may be conserved over very long evolutionary time scales. CONCLUSION: Here we have demonstrated the tractability of progressing from a genetic linkage map to genomic sequence data in Heliconius butterflies. We have also shown that fine-scale gene order is highly conserved between distantly related Heliconius species, and also between Heliconius and B. mori. Together, these findings suggest that genome structure in macrolepidoptera might be very conserved, and show that mapping and positional cloning efforts in different lepidopteran species can be reciprocally informative.

Parallel genetic architecture of parallel adaptive radiations in mimetic Heliconius butterflies.

It is unknown whether homologous loci underlie the independent and parallel wing pattern radiations of Heliconius butterflies. By comparing the locations of color patterning genes on linkage maps we show that three loci that act similarly in the two radiations are in similar positions on homologous chromosomes.

Linkage of butterfly mate preference and wing color preference cue at the genomic location of wingless.

Sexual isolation is a critical form of reproductive isolation in the early stages of animal speciation, yet little is known about the genetic basis of divergent mate preferences and preference cues in young species. Heliconius butterflies, well known for their diversity of wing color patterns, mate assortatively as a result of divergence in male preference for wing patterns. Here we show that the specific cue used by Heliconius cydno and Heliconius pachinus males to recognize conspecific females is the color of patches on the wings. In addition, male mate preference segregates with forewing color in hybrids, indicating a genetic association between the loci responsible for preference and preference cue. Quantitative trait locus mapping places a preference locus coincident with the locus that determines forewing color, which itself is perfectly linked to the wing patterning candidate gene, wingless. Furthermore, yellow-colored males of the polymorphic race H. cydno alithea prefer to court yellow females, indicating that wing color and color preference are controlled by loci that are located in an inversion or are pleiotropic effects of a single locus. Tight genetic associations between preference and preference cue, although rare, make divergence and speciation particularly likely because the effects of natural and sexual selection on one trait are transferred to the other, leading to the coordinated evolution of mate recognition. This effect of linkage on divergence is especially important in Heliconius because differentiation of wing color patterns in the genus has been driven and maintained by natural selection for Müllerian mimicry.

Localization of Müllerian mimicry genes on a dense linkage map of Heliconius erato.

We report a dense genetic linkage map of Heliconius erato, a neotropical butterfly that has undergone a remarkable adaptive radiation in warningly colored mimetic wing patterns. Our study exploited natural variation segregating in a cross between H. erato etylus and H. himera to localize wing color pattern loci on a dense linkage map containing amplified fragment length polymorphisms (AFLP), microsatellites, and single-copy nuclear loci. We unambiguously identified all 20 autosomal linkage groups and the sex chromosome (Z). The map spanned a total of 1430 Haldane cM and linkage groups varied in size from 26.3 to 97.8 cM. The average distance between markers was 5.1 cM. Within this framework, we localized two major color pattern loci to narrow regions of the genome. The first gene, D, responsible for red/orange elements, had a most likely placement in a 6.7-cM region flanked by two AFLP markers on the end of a large 87.5-cM linkage group. The second locus, Sd, affects the melanic pattern on the forewing and was found within a 6.3-cM interval between flanking AFLP loci. This study complements recent linkage analysis of H. erato's comimic, H. melpomene, and forms the basis for marker-assisted physical mapping and for studies into the comparative genetic architecture of wing-pattern mimicry in Heliconius.