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Invasive species may impact pathogen transmission by altering the distributions and interactions among native vertebrate reservoir hosts and arthropod vectors. Here, we examined the direct and indirect effects of the red imported fire ant (Solenopsis invicta) on the native tick, small mammal and pathogen community in southeast Texas. Using a replicated large-scale field manipulation study, we show that small mammals were more abundant on treatment plots where S. invicta populations were experimentally reduced. Our analysis of ticks on small mammal hosts demonstrated a threefold increase in the ticks caught per unit effort on treatment relative to control plots, and elevated tick loads (a 27-fold increase) on one common rodent species. We detected only one known human pathogen (Rickettsia parkeri), present in 1.4% of larvae and 6.7% of nymph on-host Amblyomma maculatum samples but with no significant difference between treatment and control plots. Given that host and vector population dynamics are key drivers of pathogen transmission, the reduced small mammal and tick abundance associated with S. invicta may alter pathogen transmission dynamics over broader spatial scales., (© 2016 The Authors.)

Background: Marking wild mosquitoes is important for understanding their ecology, behaviours and role in disease transmission. Traditional insect marking techniques include using fluorescent dyes, protein labels, radioactive labels and tags, but such techniques have various limitations; notably low marker retention and inability to mark wild mosquitoes at source. Stable isotopes are gaining wide spread use for non-invasive marking of arthropods, permitting greater understanding of mosquito dispersal and responses to interventions. We describe here a simple technique for marking naturally-breeding malaria and dengue vectors using stable isotopes of nitrogen (15N) and carbon (13C), and describe potential field applications., Methods: We created man-made aquatic mosquito habitats and added either 15N-labelled potassium nitrate or 13C-labelled glucose, leaving non-adulterated habitats as controls. We then allowed wild mosquitoes to lay eggs in these habitats and monitored their development in situ. Pupae were collected promptly as they appeared and kept in netting cages. Emergent adults (in pools of ~4 mosquitoes/pool) and individually stored pupae were desiccated and analysed using Isotope Ratio Mass Spectrometry (IRMS)., Findings: Anopheles gambiae s.l and Aedes spp. from enriched 13C and enriched 15N larval habitats had significantly higher isotopic levels than controls (P = 0.005), and both isotopes produced sufficient distinction between marked and unmarked mosquitoes. Mean δ15N for enriched females and males were 275.6±65.1 and 248.0±54.6, while mean δ15N in controls were 2.1±0.1 and 3.9±1.7 respectively. Similarly, mean δ13C for enriched females and males were 36.08±5.28 and 38.5±6.86, compared to -4.3±0.2 and -7.9±3.6 in controls respectively. Mean δ15N and δ13C was significantly higher in any pool containing at least one enriched mosquito compared to pools with all unenriched mosquitoes, P<0.001. In all cases, there were variations in standardized isotopic ratios between mosquito species., Conclusion: Enrichment of semi-natural mosquito larval habitats with stable isotopes of nitrogen and carbon resulted in effective marking of Anopheles and Aedes mosquitoes colonizing these habitats. This approach can significantly enhance studies on mosquito eco-physiology, dispersal, pathogen transmission and responses to control measures.

Mosquito-based surveillance is a practical way to estimate the risk of transmission of West Nile virus (WNV) to people. Variations in temperature and precipitation play a role in driving mosquito infection rates and transmission of WNV, motivating efforts to predict infection rates based on prior weather conditions. Weather conditions and sequential patterns of meteorological events can have particularly important, but regionally distinctive, consequences for WNV transmission, with high temperatures and low precipitation often increasing WNV mosquito infection. Predictive models that incorporate weather can thus be used to provide early indications of the risk of WNV infection. The purpose of this study was first, to assess the ability of a previously published model of WNV mosquito infection to predict infection for an area within the region for which it was developed, and second, to improve the predictive ability of this model by incorporating new weather factors that may affect mosquito development. The legacy model captured the primary trends in mosquito infection, but it was improved considerably when calibrated with local mosquito infection rates. The use of interaction terms between precipitation and temperature improved model performance. Specifically, temperature had a stronger influence than rainfall, so that lower than average temperature greatly reduced the effect of low rainfall on increased infection rates. When rainfall was lower, high temperature had an even stronger positive impact on infection rates. The final model is practical, stable, and operationally valid for predicting West Nile virus infection rates in future weeks when calibrated with local data., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The local abundance of Culex mosquitoes is a central factor adding to the risk of West Nile virus transmission, and vector abundance data influence public health decisions. This study evaluated differences in abundance estimates from mosquitoes trapped using two common methods: CO2-baited CDC light traps and infusion-baited gravid traps in suburban, Chicago, Illinois. On a weekly basis, the two methods were modestly correlated (r = 0.219) across 71 weeks over 4 years. Lagged weather conditions of up to four weeks were associated with the number of mosquitoes collected in light and gravid traps. Collections in light traps were higher with higher temperature in the same week, higher precipitation one, two, and four weeks before the week of trapping, and lower maximum average wind speed. Collections in gravid traps were higher with higher temperature in the same week and one week earlier, lower temperature four weeks earlier, and with higher precipitation two and four weeks earlier. Culex abundance estimates from light traps were significantly higher in semi-natural areas compared to residential areas, but abundance estimates from gravid traps did not vary by the landscape type. These results highlight the importance of the surveillance methods used in the assessment of local Culex abundance estimates. Measures of risk of exposure to West Nile virus should assess carefully how mosquito abundance has been estimated and integrated into assessments of transmission risk.

Multiple factors lead to extensive variation in mosquito and mosquito-borne virus control programs throughout the United States. This variation is related to differences in budgets, number of personnel, operational activities targeting nuisance or vector species, integration of Geographical Information Systems, and the degree of research and development to improve management interventions through collaboration with academic institutions. To highlight this heterogeneity, the current study evaluates associations among the size of a mosquito control community, the research productivity, and the mosquito-borne virus human disease burden among states within the continental United States. I used the attendance at state mosquito and vector control meetings as a proxy for the size of the mosquito control community in each state. To judge research productivity, I used all peer-reviewed publications on mosquitoes and mosquito-borne viruses using data originating in each state over a 5- and 20-yr period. Total neuroinvasive human disease cases caused by mosquito-borne viruses were aggregated for each state. These data were compared directly and after adjusting for differences in human population size for each state. Results revealed that mean meeting attendance was positively correlated with the number of publications in each state, but not after correcting for the size of the population in each state. Additionally, human disease cases were positively correlated with the number of publications in each state. Finally, mean meeting attendance and human disease cases were only marginally positively associated, and no correlation existed after correcting for human population size. These analyses indicated that the mosquito control community size, research productivity, and mosquito-borne viral human disease burden varied greatly among states. The mechanisms resulting in this variation were discussed and the consequences of this variation are important given the constantly changing environment due to invasive mosquito species and arboviruses, urbanization, immigration, global travel, and climate change., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Chagas disease is a zoonotic parasitic disease well-documented throughout the Americas and transmitted primarily by triatomine 'kissing bug' vectors. In acknowledgment of the successful history of vector control programs based on community participation across Latin America, we used a citizen science approach to gain novel insight into the geographic distribution, seasonal activity, and Trypanosoma cruzi infection prevalence of kissing bugs in Texas while empowering the public with information about Chagas disease., Methodology/principal Findings: We accepted submissions of kissing bugs encountered by the public in Texas and other states from 2013-2014 while providing educational literature about Chagas disease. In the laboratory, kissing bugs were identified to species, dissected, and tested for T. cruzi infection. A total of 1,980 triatomines were submitted to the program comprised of at least seven species, of which T. gerstaeckeri and T. sanguisuga were the most abundant (85.7% of submissions). Triatomines were most commonly collected from dog kennels and outdoor patios; Overall, 10.5% of triatomines were collected from inside the home. Triatomines were submitted from across Texas, including many counties which were not previously known to harbor kissing bugs. Kissing bugs were captured primarily throughout April-October, and peak activity occurred in June-July. Emails to our dedicated account regarding kissing bugs were more frequent in the summer months (June-August) than the rest of the year. We detected T. cruzi in 63.3% of tested bugs., Conclusions/significance: Citizen science is an efficient approach for generating data on the distribution, phenology, and infection prevalence of kissing bugs-vectors of the Chagas disease parasite-while educating the public and medical community.

The prevalence of vector-borne parasites varies greatly across host species, and this heterogeneity has been used to relate infectious disease susceptibility to host species traits. However, a few empirical studies have directly associated vector-borne parasite prevalence with exposure to vectors across hosts. Here, we use DNA sequencing of blood meals to estimate utilization of different avian host species by Culex mosquitoes, and relate utilization by these malaria vectors to avian Plasmodium prevalence. We found that avian host species that are highly utilized as hosts by avian malaria vectors are significantly more likely to have Plasmodium infections. However, the effect was not consistent among individual Plasmodium taxa. Exposure to vector bites may therefore influence the relative number of all avian Plasmodium infections among host species, while other processes, such as parasite competition and host-parasite coevolution, delimit the host distributions of individual Plasmodium species. We demonstrate that links between avian malaria susceptibility and host traits can be conditioned by patterns of exposure to vectors. Linking vector utilization rates to host traits may be a key area of future research to understand mechanisms that produce variation in the prevalence of vector-borne pathogens among host species.

Identification of the vertebrate hosts upon which hematophagous arthropods feed provides key information for understanding the ecology and transmission of vector-borne diseases. Bloodmeal analysis of ticks presents unique challenges relative to other vectors, given the long interval between bloodmeal acquisition and host-seeking, during which DNA degradation occurs. This study evaluates DNA-based and stable isotope-based bloodmeal analysis methodologies for the lone star tick, Amblyomma americanum (Linneaus, 1758), in an experimental study with chicken as the known host. We subjected ticks of different ages and environmental rearing conditions to three DNA-based approaches and a stable isotopic analysis, which relies on the natural variation of nitrogen ((15)N/(14)N) and carbon ((13)C/(12)C) isotopes. While all three DNA-based approaches were successful in identifying the bloodmeal host of the engorged nymphs, only the probe-based RT-PCR was able to detect host DNA in aged ticks, the success of which was low and inconsistent across age and rearing treatments. In contrast, the stable isotope analysis showed utility in determining the host across all ages of ticks when isotopic values of ticks were compared with a panel of candidate vertebrate species. There was a positive shift in both δ(13)C and δ(15)N in adult A. americanum until 34 wk postnymphal bloodmeal. Through analyzing the isotopic signatures of eight potential vertebrate host species, we determined that the magnitude of this isotopic shift that occurred with tick age was minor compared with the heterogeneity in the δ(15)N and δ(13)C signatures among species. These results suggest that stable isotopes are a useful tool for understanding tick-host interactions., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Antibody duration, following a humoral immune response to West Nile virus (WNV) infection, is poorly understood in free-ranging avian hosts. Quantifying antibody decay rate is important for interpreting serologic results and for understanding the potential for birds to serorevert and become susceptible again. We sampled free-ranging birds in Chicago, Illinois, US, from 2005 to 2011 and Atlanta, Georgia, US, from 2010 to 2012 to examine the dynamics of antibody decay following natural WNV infection. Using serial dilutions in a blocking enzyme-linked immunosorbent assay, we quantified WNV antibody titer in repeated blood samples from individual birds over time. We quantified a rate of antibody decay for 23 Northern Cardinals (Cardinalis cardinalis) of 0.198 natural log units per month and 24 individuals of other bird species of 0.178 natural log units per month. Our results suggest that juveniles had a higher rate of antibody decay than adults, which is consistent with nonlinear antibody decay at different times postexposure. Overall, most birds had undetectable titers 2 yr postexposure. Nonuniform WNV antibody decay rates in free-ranging birds underscore the need for cautious interpretation of avian serology results in the context of arbovirus surveillance and epidemiology.

While the population of endangered whooping cranes (Grus americana) has grown from 15 individuals in 1941 to an estimated 304 birds today, the population growth is not sufficient to support a down-listing of the species to threatened status. The degree to which disease may be limiting the population growth of whooping cranes is unknown. One disease of potential concern is caused by two crane-associated Eimeria species: Eimeria gruis and E. reichenowi. Unlike most species of Eimeria, which are localized to the intestinal tract, these crane-associated species may multiply systemically and cause a potentially fatal disease. Using a non-invasive sampling approach, we assessed the prevalence and phenology of Eimeria oocysts in whooping crane fecal samples collected across two winter seasons (November 2012-April 2014) at the Aransas National Wildlife Refuge along the Texas Gulf coast. We also compared the ability of microscopy and PCR to detect Eimeria in fecal samples. Across both years, 26.5% (n = 328) of fecal samples were positive for Eimeria based on microscopy. Although the sensitivity of PCR for detecting Eimeria infections seemed to be less than that of microscopy in the first year of the study (8.9% vs. 29.3%, respectively), an improved DNA extraction protocol resulted in increased sensitivity of PCR relative to microscopy in the second year of the study (27.6% and 20.8%, respectively). The proportion of positive samples did not vary significantly between years or among sampling sites. The proportion of Eimeria positive fecal samples varied with date of collection, but there was no consistent pattern of parasite shedding between the two years. We demonstrate that non-invasive fecal collections combined with PCR and DNA sequencing techniques provides a useful tool for monitoring Eimeria infection in cranes. Understanding the epidemiology of coccidiosis is important for management efforts to increase population growth of the endangered whooping crane.

Multiple mosquito-borne parasites cocirculate in nature and potentially interact. To understand the community of parasites cocirculating with West Nile virus (WNV), we screened the bloodmeal content of Culex pipiens L. mosquitoes for three common types of hemoparasites. Blood-fed Cx. pipiens were collected from a WNV-epidemic area in suburban Chicago, IL, from May to September 2005 through 2010. DNA was extracted from dissected abdomens and subject to PCR and direct sequencing to identify the vertebrate host. RNA was extracted from the head or thorax and screened for WNV using quantitative reverse transcriptase PCR. Seventy-nine engorged females with avian host origin were screened using PCR and amplicon sequencing for filarioid nematodes, Haemosporida, and trypanosomatids. Filarioid nematodes were identified in 3.8% of the blooded abdomens, Plasmodium sp. in 8.9%, Haemoproteus in 31.6%, and Trypanosoma sp. in 6.3%. The sequences from these hemoparasite lineages were highly similar to sequences from birds in prior studies in suburban Chicago. Overall, 50.6% of blood-fed Culex pipiens contained hemoparasite DNA in their abdomen, presumably from current or prior bloodmeals. Additionally, we detected hemoparasite DNA in the blooded abdomen of three of 10 Cx. pipiens infected with WNV., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Animals can decrease their individual risk of predation by forming groups. The encounter-dilution hypothesis extends the potential benefits of gregariousness to biting insects and vector-borne disease by predicting that the per capita number of insect bites should decrease within larger host groups. Although vector-borne diseases are common and can exert strong selective pressures on hosts, there have been few tests of the encounter-dilution effect in natural systems. We conducted an experimental test of the encounter-dilution hypothesis using the American robin (Turdus migratorius), a common host species for the West Nile virus (WNV), a mosquito-borne pathogen. By using sentinel hosts (house sparrows, Passer domesticus) caged in naturally occurring communal roosts in the suburbs of Chicago, we assessed sentinel host risk of WNV exposure inside and outside of roosts. We also estimated per capita host exposure to infected vectors inside roosts and outside of roosts. Sentinel birds caged inside roosts seroconverted to WNV more slowly than those outside of roosts, suggesting that social groups decrease per capita exposure to infected mosquitoes. These results therefore support the encounter-dilution hypothesis in a vector-borne disease system. Our results suggest that disease-related selective pressures on sociality may depend on the mode of disease transmission., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Rift Valley fever virus (RVFV) is a mosquito-borne virus in the family Bunyaviridiae that has spread throughout continental Africa to Madagascar and the Arabian Peninsula. The establishment of RVFV in North America would have serious consequences for human and animal health in addition to a significant economic impact on the livestock industry. Published and unpublished data on RVFV vector competence, vertebrate host competence, and mosquito feeding patterns from the United States were combined to quantitatively implicate mosquito vectors and vertebrate hosts that may be important to RVFV transmission in the United States. A viremia-vector competence relationship based on published mosquito transmission studies was used to calculate a vertebrate host competence index which was then combined with mosquito blood feeding patterns to approximate the vector and vertebrate amplification fraction, defined as the relative contribution of the mosquito or vertebrate host to pathogen transmission. Results implicate several Aedes spp. mosquitoes and vertebrates in the order Artiodactyla as important hosts for RVFV transmission in the U.S. Moreover, this study identifies critical gaps in knowledge which would be necessary to complete a comprehensive analysis identifying the different contributions of mosquitoes and vertebrates to potential RVFV transmission in the U.S. Future research should focus on (1) the dose-dependent relationship between viremic exposure and the subsequent infectiousness of key mosquito species, (2) evaluation of vertebrate host competence for RVFV among North American mammal species, with particular emphasis on the order Artiodactyla, and (3) identification of areas with a high risk for RVFV introduction so data on local vector and host populations can help generate geographically appropriate amplification fraction estimates.

Background: Various ecological and physiological mechanisms might influence the probability that two or more pathogens may simultaneously or sequentially infect a host individual. Concurrent infections can have important consequences for host condition and fitness, including elevated mortality risks. In addition, interactions between coinfecting pathogens may have important implications for transmission dynamics., Methods: Here, we explore patterns of association between two common avian pathogens (West Nile virus and avian malaria parasites) among a suburban bird community in Chicago, IL, USA that share mosquito vectors. We surveyed 1714 individual birds across 13 species for both pathogens through established molecular protocols., Results: Field investigations of haemosporidian and West Nile virus (WNV) infections among sampled birds yielded an inverse association between WNV serostatus and Plasmodium infection status. This relationship occurred in adult birds but not in juveniles. There was no evidence for a relationship between Haemoproteus infection and WNV serostatus. We detected similar prevalence of Plasmodium among birds captured with active WNV infections and spatiotemporally paired WNV-naïve individuals of the same species, demonstrating that the two pathogens can co-infect hosts., Conclusions: Mechanisms explaining the negative association between WNV serostatus and Plasmodium infection status remain unclear and must be resolved through experimental infection procedures. However, our results highlight potential interactions between two common avian pathogens that may influence their transmission among hosts. This is especially relevant considering that West Nile virus is a common zoonotic pathogen with public health implications. Moreover, both pathogens are instructive models in infectious disease ecology, and infection with either has fitness consequences for their avian hosts.

Dispersal is a critical life history behavior for mosquitoes and is important for the spread of mosquito-borne disease. We implemented the first stable isotope mark-capture study to measure mosquito dispersal, focusing on Culex pipiens in southwest suburban Chicago, Illinois, a hotspot of West Nile virus (WNV) transmission. We enriched nine catch basins in 2010 and 2011 with 15N-potassium nitrate and detected dispersal of enriched adult females emerging from these catch basins using CDC light and gravid traps to distances as far as 3 km. We detected 12 isotopically enriched pools of mosquitoes out of 2,442 tested during the two years and calculated a mean dispersal distance of 1.15 km and maximum flight range of 2.48 km. According to a logistic distribution function, 90% of the female Culex mosquitoes stayed within 3 km of their larval habitat, which corresponds with the distance-limited genetic variation of WNV observed in this study region. This study provides new insights on the dispersal of the most important vector of WNV in the eastern United States and demonstrates the utility of stable isotope enrichment for studying the biology of mosquitoes in other disease systems.

Blood-feeding arthropod vectors are responsible for transmitting many parasites between vertebrate hosts. While arthropod vectors often feed on limited subsets of potential host species, little is known about the extent to which this influences the distribution of vector-borne parasites in some systems. Here, we test the hypothesis that different vector species structure parasite-host relationships by restricting access of certain parasites to a subset of available hosts. Specifically, we investigate how the feeding patterns of Culex mosquito vectors relate to distributions of avian malaria parasites among hosts in suburban Chicago, IL, USA. We show that Plasmodium lineages, defined by cytochrome b haplotypes, are heterogeneously distributed across avian hosts. However, the feeding patterns of the dominant vectors (Culex restuans and Culex pipiens) are similar across these hosts, and do not explain the distributions of Plasmodium parasites. Phylogenetic similarity of avian hosts predicts similarity in their Plasmodium parasites. This effect was driven primarily by the general association of Plasmodium parasites with particular host superfamilies. Our results suggest that a mosquito-imposed encounter rate does not limit the distribution of avian Plasmodium parasites across hosts. This implies that compatibility between parasites and their avian hosts structure Plasmodium host range.

Background: An important determinant of mosquito-borne pathogen transmission is the spatial distribution of vectors. The primary vectors of West Nile virus (WNV) in Illinois are Culex pipiens Linnaeus (Diptera: Culicidae) and Culex restuans Theobald. In urban environments, these mosquitoes commonly oviposit in roadside storm water catch basins. However, use of this habitat is inconsistent, with abundance of larvae varying significantly across catch basins at a fine spatial scale., Methods: We tested the hypothesis that attributes of the biotic and abiotic environment contribute to spatial and temporal variation in production of mosquito vectors, characterizing the relationship between terrestrial vegetation and aquatic chemistry and Culex abundance in Chicago, Illinois. Larvae were sampled from 60 catch basins from June 14 to October 3, 2009. Density of shrubs and 14 tree genera surrounding the basins were quantified, as well as aquatic chemistry content of each basin., Results: We demonstrate that the spatial pattern of Culex abundance in catch basins is strongly influenced by environmental characteristics, resulting in significant variation across the urban landscape. Using regression and machine learning techniques, we described landscape features and microhabitat characteristics of four Chicago neighborhoods and examined the implications of these measures for larval abundance in adjacent catch basins. The important positive predictors of high larval abundance were aquatic ammonia, nitrates, and area of shrubs of height <1 m surrounding the catch basins, whereas pH and area of flowering shrub were negatively correlated with larval abundance. Tree density, particularly of arborvitae, maple, and pear, also positively influenced the distribution of Culex during the fruit-bearing periods and early senescent periods in August and September., Conclusions: This study identifies environmental predictors of mosquito production in urban environments. Because an abundance of adult Culex is integral to efficient WNV transmission and mosquitoes are found in especially high densities near larval habitats, identifying aquatic sites for Culex and landscape features that promote larval production are important in predicting the spatial pattern of cases of human and veterinary illness. Thus, these data enable accurate assessment of regions at risk for exposure to WNV and aid in the prevention of vector-borne disease transmission.

Hosts are commonly infected with a suite of parasites, and interactions among these parasites can affect the size, structure, and behavior of host-parasite communities. As an important step to understanding the significance of co-circulating parasites, we describe prevalence of co-circulating hemoparasites in two important avian amplification hosts for West Nile virus (WNV), the American robin (Turdus migratorius) and house sparrow (Passer domesticus), during the 2010-2011 in Chicago, Illinois, USA. Rates of nematode microfilariemia were 1.5% of the robins (n = 70) and 4.2% of the house sparrows (n = 72) collected during the day and 11.1% of the roosting robins (n = 63) and 0% of the house sparrows (n = 11) collected at night. Phylogenetic analysis of nucleotide sequences of the 18S rRNA and cytochrome oxidase subunit I (COI) genes from these parasites resolved two clades of filarioid nematodes. Microscopy revealed that 18.0% of American robins (n = 133) and 16.9% of house sparrows (n = 83) hosted trypanosomes in the blood. Phylogenetic analysis of nucleotide sequences from the 18s rRNA gene revealed that the trypanosomes fall within previously described avian trypanosome clades. These results document hemoparasites in the blood of WNV hosts in a center of endemic WNV transmission, suggesting a potential for direct or indirect interactions with the virus.

Bird-facilitated introduction of ticks and associated pathogens is postulated to promote invasion of tick-borne zoonotic diseases into urban areas. Results of a longitudinal study conducted in suburban Chicago, Illinois, USA, during 2005-2010 show that 1.6% of 6,180 wild birds captured in mist nets harbored ticks. Tick species in order of abundance were Haemaphysalis leporispalustris, Ixodes dentatus, and I. scapularis, but 2 neotropical tick species of the genus Amblyomma were sampled during the spring migration. I. scapularis ticks were absent at the beginning of the study but constituted the majority of ticks by study end and were found predominantly on birds captured in areas designated as urban green spaces. Of 120 ticks, 5 were infected with Borrelia burgdorferi, spanning 3 ribotypes, but none were infected with Anaplasma phagocytophilum. Results allow inferences about propagule pressure for introduction of tick-borne diseases and emphasize the large sample sizes required to estimate this pressure.

Analysis of molecular genetic diversity in nine marker regions of five genes within the bacteriophage WO genomic region revealed high diversity of the Wolbachia pipentis strain wPip in a population of Culex pipiens L. sampled in metropolitan Chicago, IL. From 166 blood fed females, 50 distinct genetic profiles of wPip were identified. Rarefaction analysis suggested a maximum of 110 profiles out of a possible 512 predicted by combinations of the nine markers. A rank-abundance curve showed that few strains were common and most were rare. Multiple regression showed that markers associated with gene Gp2d, encoding a partial putative capsid protein, were significantly associated with ancestry of individuals either to form molestus or form pipiens, as determined by prior microsatellite allele frequency analysis. None of the other eight markers was associated with ancestry to either form, nor to ancestry to Cx. quinquefasciatus Say. Logistic regression of host choice (mammal vs. avian) as determined by bloodmeal analysis revealed that significantly fewer individuals that had fed on mammals had the Gp9a genetic marker (58.5%) compared with avian-fed individuals (88.1%). These data suggest that certain wPip molecular genetic types are associated with genetic admixturing in the Cx. pipiens complex of metropolitan Chicago, IL, and that the association extends to phenotypic variation related to host preference.

Culex pipiens L. (Diptera: Culicidae) and Culex restuans Theobald are the primary enzootic and bridge vectors of West Nile virus in the eastern United States north of 36 degrees latitude. Recent studies of the natural history of these species have implicated catch basins and underground storm drain systems as important larval development sites in urban and suburban locales. Although the presence of larvae in these habitats is well-documented, the influence of abiotic factors on the ecology of Culex larvae developing in them remains poorly understood. Therefore, we examined the effects of multiple abiotic factors and their interactions on abundance of Culex larvae in catch basins in the Chicago, IL, metropolitan area. Low precipitation and high mean daily temperature were associated with high larval abundance, whereas there was no correlation between catch basin depth or water depth and larval abundance. Rainfall was an especially strong predictor of presence or absence of larvae in the summer of 2010, a season with an unusually high precipitation. Regression tree methods were used to build a schematic decision tree model of the interactions among these factors. This practical, visual representation of key predictors of high larval production may be used by local mosquito abatement districts to target limited resources to treat catch basins when they are particularly likely to produce West Nile virus vectors.

Understanding mosquito dispersal is critically important for vector-borne disease control and prevention. Mark-release-recapture methods using various marking techniques have made substantial contributions to the study of mosquito biology. However, the ability to mark naturally breeding mosquitoes noninvasively and with life-long retention has remained problematic. Here, we describe a method to mark naturally breeding mosquitoes with stable isotopes. Culex pipiens f. molestus mosquitoes were provisioned as larvae in laboratory experiments with 15N-labeled potassium nitrate and 13C-labeled glucose. Larval enrichment was sufficient to differentiate marked adult mosquitoes from unmarked control mosquitoes and the natural source population from Chicago Illinois, using either delta 15N or delta 13C. Isotopic retention lasted for at least 55 d for adult male and females mosquitoes. There were no consistent effects of isotopic enrichment on immature mosquito survival or adult mosquito body size. We then applied this marking technique to naturally breeding Culex pipiens mosquitoes in suburban Chicago, IL, and for the first time, report successful isotopic enrichment of mosquitoes in the field. This stable isotope marking technique will facilitate studies of mosquito dispersal.

Culex flavivirus (CxFV) is an insect-specific flavivirus globally distributed in mosquitoes of the genus Culex. CxFV was positively associated with West Nile virus (WNV) infection in a case-control study of 268 mosquito pools from an endemic focus of WNV transmission in Chicago, United States. Specifically, WNV-positive Culex mosquito pools were four times more likely also to be infected with CxFV than were spatiotemporally matched WNV-negative pools. In addition, mosquito pools from residential sites characterized by dense housing and impermeable surfaces were more likely to be infected with CxFV than were pools from nearby urban green spaces. Further, 6/15 (40%) WNV-positive individual mosquitoes were also CxFV positive, demonstrating that both viruses can coinfect mosquitoes in nature. Phylogenetic analysis of CxFV from Chicago demonstrated a pattern similar to WNV, consisting of low global viral diversity and lack of geographic clustering. These results illustrate a positive ecological association between CxFV and WNV, and that coinfection of individual mosquitoes can occur naturally in areas of high flaviviral transmission. These conclusions represent a challenge to the hypothesis of super-infection exclusion in the CxFV/WNV system, whereby an established infection with one virus may interfere with secondary viral infection with a similar virus. This study suggests that infection with insect-specific flaviviruses such as CxFV may not exclude secondary infection with genetically distinct flaviviruses such as WNV, and that both viruses can naturally coinfect mosquitoes that are epidemic bridge vectors of WNV to humans.

The innate immune system is the first line of defense against pathogens, and it plays a fundamental role in coordinating a protective immune response in birds. Although many studies have evaluated avian immune responses in the laboratory, many fewer studies to date have done so in a field setting. To gain insight into interspecific differences in immune function in wild birds, we used a field-deployed in vitro microbicidal assay to measure constitutive innate immunity of whole blood collected from three common passerines in suburban Chicago, Illinois, in 2009. Data from one microbe, Escherichia coli 8739, revealed that American Robins (Turdus migratorius) had significantly lower bactericidal capacity than House Sparrows (Passer domesticus) or Gray Catbirds (Dumetella carolinensis). Bactericidal capacity for E. coli 8739 tended to be lower for birds infested with chewing lice than those without chewing lice, and male birds had lower microbicidal capacity than females in the case of Staphylococcus aureus. This study demonstrates the potential for field-deployable eco-immunologic tools to inform infectious disease ecology research.

Stormwater catch basins in urban areas provide important larval habitat for Culex mosquitoes. In this study we quantified adult Culex emergence using a newly designed emergence trap deployed in catch basins in suburban Chicago, IL. Traps were deployed from late June to mid-October, 2009-10, in 19 catch basins for a total of 461 trap-days. Based on laboratory trials, the percentage of adults emerging under the trap and reaching the collection cup ranged from 37.7 +/- 6.5% for closed-cup and 50.5 +/- 3.8% for open-cup configurations. In 2009, catch basins containing immature mosquitoes produced an estimated 58.9 +/- 30.8 female and 86.2 +/- 36.4 male Culex spp. per day. Most (84.4%) were Culex pipiens and the remainder were Cx. restuans. The trap was also effective in documenting reductions in adult emergence following intense precipitation events that caused "flushing" of larvae and pupae. In general, the new emergence trap was effective for studying Culex production in catch basins and should be broadly useful in studies of container-breeding mosquitoes.

The influence of host diversity on multi-host pathogen transmission and persistence can be confounded by the large number of species and biological interactions that can characterize many transmission systems. For vector-borne pathogens, the composition of host communities has been hypothesized to affect transmission; however, the specific characteristics of host communities that affect transmission remain largely unknown. We tested the hypothesis that vector host use and force of infection (i.e., the summed number of infectious mosquitoes resulting from feeding upon each vertebrate host within a community of hosts), and not simply host diversity or richness, determine local infection rates of West Nile virus (WNV) in mosquito vectors. In suburban Chicago, Illinois, USA, we estimated community force of infection for West Nile virus using data on Culex pipiens mosquito host selection and WNV vertebrate reservoir competence for each host species in multiple residential and semi-natural study sites. We found host community force of infection interacted with avian diversity to influence WNV infection in Culex mosquitoes across the study area. Two avian species, the American robin (Turdus migratorius) and the house sparrow (Passer domesticus), produced 95.8% of the infectious Cx. pipiens mosquitoes and showed a significant positive association with WNV infection in Culex spp. mosquitoes. Therefore, indices of community structure, such as species diversity or richness, may not be reliable indicators of transmission risk at fine spatial scales in vector-borne disease systems. Rather, robust assessment of local transmission risk should incorporate heterogeneity in vector host feeding and variation in vertebrate reservoir competence at the spatial scale of vector-host interaction.

West Nile virus has evolved in concert with its expansion across North America, but little is known about the evolutionary dynamics of the virus on local scales. We analysed viral nucleotide sequences from mosquitoes collected in 2005, 2006, and 2007 from a known transmission 'hot spot' in suburban Chicago, USA. Within this approximately 11 x 14 km area, the viral envelope gene has increased approximately 0.1% yr(-1) in nucleotide-level genetic diversity. In each year, viral diversity was higher in 'residential' sites characterized by dense housing than in more open 'urban green space' sites such as cemeteries and parks. Phylodynamic analyses showed an increase in incidence around 2005, consistent with a higher-than-average peak in mosquito and human infection rates that year. Analyses of times to most recent common ancestor suggest that WNV in 2005 and 2006 may have arisen predominantly from viruses present during 2004 and 2005, respectively, but that WNV in 2007 had an older common ancestor, perhaps indicating a predominantly mixed or exogenous origin. These results show that the population of WNV in suburban Chicago is an admixture of viruses that are both locally derived and introduced from elsewhere, containing evolutionary information aggregated across a breadth of spatial and temporal scales.

West Nile virus spread rapidly from east to west across North America, despite the north-south migratory flyways of its avian hosts. In this issue, Venkatesan & Rasgon (2010) present new data on the population genetics of Culex tarsalis, the dominant West Nile virus vector in the Western United States, suggesting that patterns of mosquito gene flow may better reflect the virus's expansion from the Midwest to the Pacific than patterns of bird movement. These findings suggest a more significant role for vector dispersal in arboviral range expansion than has previously been appreciated, and they highlight the value of molecular genetic studies of insect vector populations for understanding epidemiology and disease ecology.

Background: Models of the effects of environmental factors on West Nile virus disease risk have yielded conflicting outcomes. The role of precipitation has been especially difficult to discern from existing studies, due in part to habitat and behavior characteristics of specific vector species and because of differences in the temporal and spatial scales of the published studies. We used spatial and statistical modeling techniques to analyze and forecast fine scale spatial (2000 m grid) and temporal (weekly) patterns of West Nile virus mosquito infection relative to changing weather conditions in the urban landscape of the greater Chicago, Illinois, region for the years from 2004 to 2008., Results: Increased air temperature was the strongest temporal predictor of increased infection in Culex pipiens and Culex restuans mosquitoes, with cumulative high temperature differences being a key factor distinguishing years with higher mosquito infection and higher human illness rates from those with lower rates. Drier conditions in the spring followed by wetter conditions just prior to an increase in infection were factors in some but not all years. Overall, 80% of the weekly variation in mosquito infection was explained by prior weather conditions. Spatially, lower precipitation was the most important variable predicting stronger mosquito infection; precipitation and temperature alone could explain the pattern of spatial variability better than could other environmental variables (79% explained in the best model). Variables related to impervious surfaces and elevation differences were of modest importance in the spatial model., Conclusion: Finely grained temporal and spatial patterns of precipitation and air temperature have a consistent and significant impact on the timing and location of increased mosquito infection in the northeastern Illinois study area. The use of local weather data at multiple monitoring locations and the integration of mosquito infection data from numerous sources across several years are important to the strength of the models presented. The other spatial environmental factors that tended to be important, including impervious surfaces and elevation measures, would mediate the effect of rainfall on soils and in urban catch basins. Changes in weather patterns with global climate change make it especially important to improve our ability to predict how inter-related local weather and environmental factors affect vectors and vector-borne disease risk.Local impact of temperature and precipitation on West Nile virus infection in Culex species mosquitoes in northeast Illinois, USA.

Mosquitoes of the Culex pipiens complex are important vectors of West Nile virus in the United States. We examined the genetic variations of Cx. pipiens mosquitoes from Chicago, Illinois that were determined to be principally ornithophilic but exhibited a relatively higher inclination for mammalian hosts including humans. Microsatellite analysis of 10 polymorphic markers was performed on 346 engorged Cx. pipiens specimens with identified avian or mammalian blood meals. Our results indicated that there were no significant differences in allelic richness, the pattern of conformity to Hardy-Weinberg equilibrium, and linkage disequilibrium, nor was there overall genetic differentiation between specimens with avian- and mammalian-derived blood meals. However, Cx. pipiens form pipiens with mammalian- (including human-) derived blood meals had significantly higher ancestry (p < 0.001) and proportion of hybrids (p < 0.01) from the Cx. pipiens form molestus (population from New York City) than did those with avian-derived blood meals. By contrast, there were no significant differences in the ancestry (p > 0.05) and the proportion of hybrids (p > 0.05) from Cx. quinquefasciatus (population from Harris Country, Texas). No temporal genetic variation was detected in accordance with the observation that there was no shift in blood feeding from birds to mammals. The results of this study in conjunction with regional host-feeding behavior suggest that the probability of genetic ancestry from Cx. pipiens f. molestus may predispose mosquitoes to feed more readily on mammals; however, the genetic mechanisms are unknown.

Vertebrate host diversity has been postulated to mediate prevalence of zoonotic, vector-borne diseases, such that as diversity increases, transmission dampens. This "dilution effect" is thought to be caused by distribution of infective bites to incompetent reservoir hosts. We quantified avian species richness, avian seroprevalence for antibodies to West Nile virus (WNV), and infection of WNV in Culex mosquitoes, in the Chicago metropolitan area, Illinois, USA, a region of historically high WNV activity. Results indicated high overall avian seroprevalence and variation in seroprevalence across host species; however, there was no negative correlation between avian richness and Culex infection rate or between richness and infection status in individual birds. Bird species with high seroprevalence, especially northern cardinals and mourning doves, may be important sentinels for WNV in Chicago, since they were common and widespread among all study sites. Overall, our results suggest no net effect of increasing species richness to West Nile virus transmission in Chicago. Other intrinsic and extrinsic factors, such as variation in mosquito host preference, reservoir host competence, temperature, and precipitation, may be more important than host diversity for driving interannual variation in WNV transmission. These results from a fine-scale study call into question the generality of a dilution effect for WNV at coarser spatial scales.

Nestling birds have been hypothesized to be important hosts for mosquito-borne arboviruses, but the role of nestlings for West Nile virus (WNV) amplification remains unclear. We sampled open-cup and cavity-nesting passerines in Chicago, Illinois, an area of intense WNV transmission, to determine infection rates in nestlings and mosquitoes, and to test whether mosquitoes are attracted to nesting birds. Analysis of Culex pipiens mosquito populations demonstrated WNV amplification to high mosquito infection rates during both years of the study near the locations where nestlings were sampled. Nevertheless, of 194 nestlings representing 12 species, only one 8-day-old house wren was positive for WNV RNA, and only one 10-day-old mourning dove was seropositive for antibodies to WNV, but at a low titer (1:20). The number of mosquitoes captured in nest box traps and control traps was not significantly different. These combined results suggest that nestling passerines play no evident role in WNV amplification and transmission in the Chicago area.

Recent field studies have suggested that the dynamics of West Nile virus (WNV) transmission are influenced strongly by a few key super spreader bird species that function both as primary blood hosts of the vector mosquitoes (in particular Culex pipiens) and as reservoir-competent virus hosts. It has been hypothesized that human cases result from a shift in mosquito feeding from these key bird species to humans after abundance of the key birds species decreases. To test this paradigm, we performed a mosquito blood meal analysis integrating host-feeding patterns of Cx. pipiens, the principal vector of WNV in the eastern United States north of the latitude 36 degrees N and other mosquito species with robust measures of host availability, to determine host selection in a WNV-endemic area of suburban Chicago, Illinois, during 2005-2007. Results showed that Cx. pipiens fed predominantly (83%) on birds with a high diversity of species used as hosts (25 species). American robins (Turdus migratorius) were marginally overused and several species were underused on the basis of relative abundance measures, including the common grackle (Quiscalus quiscula), house sparrow (Passer domesticus), and European starling (Sturnus vulgaris). Culex pipiens also fed substantially on mammals (19%; 7 species with humans representing 16%). West Nile virus transmission intensified in July of both years at times when American robins were heavily fed upon, and then decreased when robin abundance decreased, after which other birds species were selected as hosts. There was no shift in feeding from birds to mammals coincident with emergence of human cases. Rather, bird feeding predominated when the onset of the human cases occurred. Measures of host abundance and competence and Cx. pipiens feeding preference were combined to estimate the amplification fractions of the different bird species. Predictions were that approximately 66% of WNV-infectious Cx. pipiens became infected from feeding on just a few species of birds, including American robins (35%), blue jays (17%, Cyanocitta cristata), and house finches (15%, Carpodacus mexicanus).

Mosquitoes and birds were sampled for West Nile virus (WNV) in suburban Chicago, USA, in a "hot spot" of arboviral transmission. Viral genetic diversity within this area was similar to that within Illinois and the United States. Diversity was higher among viruses from mosquitoes than from birds, higher among viruses from birds in urban "green spaces" than from birds in residential areas, but lower among viruses from mosquitoes in green spaces than from mosquitoes in residential areas. Viral transmission was distance-limited, as evidenced by decreasing autocorrelation of WNV sequences with increasing geographic separation. The evolutionary rate of WNV within the study area between 21 July and 4 October 2005 was ten times higher than that for WNV across North America between 2002 and 2005. These results indicate that WNV transmission and evolutionary dynamics can vary seasonally and in response to fine-scale environmental conditions and landscape characteristics related to urbanization.

Epizootic transmission of West Nile virus (WNV) often intensifies rapidly leading to increasing risk of human infection, but the processes underlying amplification remain poorly understood. We quantified epizootic WNV transmission in communities of mosquitoes and birds in the Chicago, Illinois (USA) region during 2005 and 2006. Using quantitative polymerase chain reaction (PCR) methods, we detected WNV in 227 of 1195 mosquito pools (19%) in 2005 and 205 of 1685 (12%) in 2006; nearly all were Culex pipiens. In both years, mosquito infection rates increased rapidly in the second half of July to a peak of 59/1000 mosquitoes in 2005 and 33/1000 in 2006, and then declined slowly. Viral RNA was detected in 11 of 998 bird sera (1.1%) in 2005 and 3 of 1285 bird sera (<1%) in 2006; 11 of the 14 virus-positive birds were hatch-year birds. Of 540 hatch-year birds, 100 (18.5%) were seropositive in 2005, but only 2.8% (14/493) tested seropositive in 2006 for WNV antibodies using inhibition enzyme-linked immunosorbent assay (ELISA). We observed significant time series cross-correlations between mosquito infection rate and proportion of virus-positive birds, proportion of hatch-year birds captured in mist nets (significant in 2006 only), seroprevalence of hatch-year birds, and number of human cases in both seasons. These associations, coupled with the predominance of WNV infection and seropositivity in hatch-year birds, indicate a key role for hatch-year birds in the amplification of epizootic transmission of WNV, and in increasing human infection risk by facilitating local viral amplification.

Host-feeding patterns of Culex pipiens L. collected in southwest suburban Chicago in 2005 were studied using polymerase chain reaction (PCR) and DNA sequencing techniques. Culex spp. mosquitoes, most identified to Cx. pipiens and the remainder to Cx. restuans by PCR, had fed on 18 avian species, most commonly American robin (Turdus migratorious), house sparrow (Passer domesticus), and mourning dove (Zenaida macroura). Additional blood meals were derived from four mammal species, primarily humans and raccoons (Procyon lotor). During a West Nile virus (WNV) epidemic in 2005, West Nile virus (WNV) RNA was detected in heads and thoraces of five Cx. pipiens (n = 335, 1.5%) using quantitative PCR. The hosts of these virus-infected, blood-fed mosquitoes included two American robins, one house sparrow, and one human. This is the first report of a WNV-infected Cx. pipiens mosquito collected during an epidemic of WNV that was found to have bitten a human. These results fulfill a criterion for incrimination of Cx. pipiens as a bridge vector.