Mosquito population dynamics is shaped by the interaction among larval density, season, and land use.

Understanding how variation in key abiotic and biotic factors interact at spatial scales relevant for mosquito fitness and population dynamics is crucial for predicting current and future mosquito distributions and abundances, and the transmission potential for human pathogens. However, studies investigating the effects of environmental variation on mosquito traits have investigated environmental factors in isolation or in laboratory experiments that examine constant environmental conditions that often do not occur in the field. To address these limitations, we conducted a semi-field experiment in Athens, Georgia using the invasive Asian tiger mosquito (Aedes albopictus). We selected nine sites that spanned natural variation in impervious surface and vegetation cover to explore effects of the microclimate (temperature and humidity) on mosquitoes. On these sites, we manipulated conspecific larval density at each site. We repeated the experiment in the summer and fall. We then evaluated the effects of land cover, larval density, and time of season, as well as interactive effects, on the mean proportion of females emerging, juvenile development time, size upon emergence, and predicted per capita population growth (i.e., fitness). We found significant effects of larval density, land cover, and season on all response variables. Of most note, we saw strong interactive effects of season and intra-specific density on each response variable, including a non-intuitive decrease in development time with increasing intra-specific competition in the fall. Our study demonstrates that ignoring the interaction between variation in biotic and abiotic variables could reduce the accuracy and precision of models used to predict mosquito population and pathogen transmission dynamics, especially those inferring dynamics at finer-spatial scales across which transmission and control occur.

Para poder predecir la distribución y abundancia de las poblaciones de mosquitos y la transmisión potencial de patógenos a humanos, es crucial comprender cómo factores abióticos y bióticos clave para el éxito reproductivo y la dinámica poblacional de los mosquitos interactúan a escalas relevantes. Sin embargo, los estudios que han investigado los efectos de variables ambientales en las características demográficas de los mosquitos han considerado su efecto de forma aislada o en experimentos de laboratorio bajo condiciones ambientales constantes que, a menudo, no reflejan lo que ocurre en el campo. Para abordar estas limitaciones, llevamos a cabo un experimento de semi-campo en Athens, Georgia, utilizando el mosquito invasor tigre asiático (Aedes albopictus). Seleccionamos nueve sitios que abarcaban variaciones naturales en la superficie impermeable y cobertura vegetal para explorar los efectos del microclima (temperatura y humedad) en los mosquitos. También manipulamos la densidad de larvas de tigre asiático en dos experimentos que fueron realizados en el verano y otoño. Evaluamos los efectos de la cobertura vegetal, la densidad de larvas, la temporada climática, y la interacción entre estas variables en la proporción de hembras que emergieron, el tiempo de desarrollo de las larvas, el tamaño al momento de la emergencia, y el crecimiento demográfico per cápita previsto (éxito reproductivo). Encontramos efectos significativos de la densidad de larvas, la variación de la cobertura vegetal y la estación del año en todas las variables de respuesta. Más notablemente, observamos un fuerte efecto de la interacción entre la temporada climática y la densidad de larvas en todas las variables de respuesta, incluyendo una disminución no intuitiva en el tiempo de desarrollo con el aumento de la competencia intraespecífica en el otoño. Nuestro estudio evidencia que ignorar la interacción entre variables abióticas y bióticas podría reducir la exactitud y precisión de los modelos utilizados para predecir las dinámicas de las poblaciones de mosquitos, y por tanto, de la transmisión de patógenos. Esto, especialmente en modelos que infieren estas dinámicas a escalas espaciales más finas, en las cuales ocurre la transmisión y el control.

Leveraging insect-specific viruses to elucidate mosquito population structure and dynamics.

Several aspects of mosquito ecology that are important for vectored disease transmission and control have been difficult to measure at epidemiologically important scales in the field. In particular, the ability to describe mosquito population structure and movement rates has been hindered by difficulty in quantifying fine-scale genetic variation among populations. The mosquito virome represents a possible avenue for quantifying population structure and movement rates across multiple spatial scales. Mosquito viromes contain a diversity of viruses, including several insect-specific viruses (ISVs) and "core" viruses that have high prevalence across populations. To date, virome studies have focused on viral discovery and have only recently begun examining viral ecology. While nonpathogenic ISVs may be of little public health relevance themselves, they provide a possible route for quantifying mosquito population structure and dynamics. For example, vertically transmitted viruses could behave as a rapidly evolving extension of the host's genome. It should be possible to apply established analytical methods to appropriate viral phylogenies and incidence data to generate novel approaches for estimating mosquito population structure and dispersal over epidemiologically relevant timescales. By studying the virome through the lens of spatial and genomic epidemiology, it may be possible to investigate otherwise cryptic aspects of mosquito ecology. A better understanding of mosquito population structure and dynamics are key for understanding mosquito-borne disease ecology and methods based on ISVs could provide a powerful tool for informing mosquito control programs.

Humidity - The overlooked variable in the thermal biology of mosquito-borne disease.

Vector-borne diseases cause significant financial and human loss, with billions of dollars spent on control. Arthropod vectors experience a complex suite of environmental factors that affect fitness, population growth and species interactions across multiple spatial and temporal scales. Temperature and water availability are two of the most important abiotic variables influencing their distributions and abundances. While extensive research on temperature exists, the influence of humidity on vector and pathogen parameters affecting disease dynamics are less understood. Humidity is often underemphasized, and when considered, is often treated as independent of temperature even though desiccation likely contributes to declines in trait performance at warmer temperatures. This Perspectives explores how humidity shapes the thermal performance of mosquito-borne pathogen transmission. We summarize what is known about its effects and propose a conceptual model for how temperature and humidity interact to shape the range of temperatures across which mosquitoes persist and achieve high transmission potential. We discuss how failing to account for these interactions hinders efforts to forecast transmission dynamics and respond to epidemics of mosquito-borne infections. We outline future research areas that will ground the effects of humidity on the thermal biology of pathogen transmission in a theoretical and empirical framework to improve spatial and temporal prediction of vector-borne pathogen transmission.

Mapping current and future thermal limits to suitability for malaria transmission by the invasive mosquito Anopheles stephensi.

BACKGROUND: Anopheles stephensi is a malaria-transmitting mosquito that has recently expanded from its primary range in Asia and the Middle East, to locations in Africa. This species is a competent vector of both Plasmodium falciparum and Plasmodium vivax malaria. Perhaps most alarming, the characteristics of An. stephensi, such as container breeding and anthropophily, make it particularly adept at exploiting built environments in areas with no prior history of malaria risk. METHODS: In this paper, global maps of thermal transmission suitability and people at risk (PAR) for malaria transmission by An. stephensi were created, under current and future climate. Temperature-dependent transmission suitability thresholds derived from recently published species-specific thermal curves were used to threshold gridded, monthly mean temperatures under current and future climatic conditions. These temperature driven transmission models were coupled with gridded population data for 2020 and 2050, under climate-matched scenarios for future outcomes, to compare with baseline predictions for 2020 populations. RESULTS: Using the Global Burden of Disease regions approach revealed that heterogenous regional increases and decreases in risk did not mask the overall pattern of massive increases of PAR for malaria transmission suitability with An. stephensi presence. General patterns of poleward expansion for thermal suitability were seen for both P. falciparum and P. vivax transmission potential. CONCLUSIONS: Understanding the potential suitability for An. stephensi transmission in a changing climate provides a key tool for planning, given an ongoing invasion and expansion of the vector. Anticipating the potential impact of onward expansion to transmission suitable areas, and the size of population at risk under future climate scenarios, and where they occur, can serve as a large-scale call for attention, planning, and monitoring.

Streamlining sporozoite isolation from mosquitoes by leveraging the dynamics of migration to the salivary glands.

BACKGROUND: Sporozoites isolated from the salivary glands of Plasmodium-infected mosquitoes are a prerequisite for several basic and pre-clinical applications. Although salivary glands are pooled to maximize sporozoite recovery, insufficient yields pose logistical and analytical hurdles; thus, predicting yields prior to isolation would be valuable. Preceding oocyst densities in the midgut is an obvious candidate. However, it is unclear whether current understanding of its relationship with sporozoite densities can be used to maximize yields, or whether it can capture the potential density-dependence in rates of sporozoite invasion of the salivary glands. METHODS: This study presents a retrospective analysis of Anopheles stephensi mosquitoes infected with two strains of the rodent-specific Plasmodium berghei. Mean oocyst densities were estimated in the midguts earlier in the infection (11-15 days post-blood meal), with sporozoites pooled from the salivary glands later in the infection (17-29 days). Generalized linear mixed effects models were used to determine if (1) mean oocyst densities can predict sporozoite yields from pooled salivary glands, (2) whether these densities can capture differences in rates of sporozoite invasion of salivary glands, and (3), if the interaction between oocyst densities and time could be leveraged to boost overall yields. RESULTS: The non-linear effect of mean oocyst densities confirmed the role of density-dependent constraints in limiting yields beyond certain oocyst densities. Irrespective of oocyst densities however, the continued invasion of salivary glands by the sporozoites boosted recoveries over time (17-29 days post-blood meal) for either parasite strain. CONCLUSIONS: Sporozoite invasion of the salivary glands over time can be leveraged to maximize yields for P. berghei. In general, however, invasion of the salivary glands over time is a critical fitness determinant for all Plasmodium species (extrinsic incubation period, EIP). Thus, delaying sporozoite collection could, in principle, substantially reduce dissection effort for any parasite within the genus, with the results also alluding to the potential for changes in sporozoites densities over time to modify infectivity for the next host.

Correction: Detecting the impact of temperature on transmission of Zika, dengue, and chikungunya using mechanistic models.

[This corrects the article DOI: 10.1371/journal.pntd.0005568.].

Temperature impacts the environmental suitability for malaria transmission by Anopheles gambiae and Anopheles stephensi.

Extrinsic environmental factors influence the spatiotemporal dynamics of many organisms, including insects that transmit the pathogens responsible for vector-borne diseases (VBDs). Temperature is an especially important constraint on the fitness of a wide variety of ectothermic insects. A mechanistic understanding of how temperature impacts traits of ectotherms, and thus the distribution of ectotherms and vector-borne infections, is key to predicting the consequences of climate change on transmission of VBDs like malaria. However, the response of transmission to temperature and other drivers is complex, as thermal traits of ectotherms are typically nonlinear, and they interact to determine transmission constraints. In this study, we assess and compare the effect of temperature on the transmission of two malaria parasites, Plasmodium falciparum and Plasmodium vivax, by two malaria vector species, Anopheles gambiae and Anopheles stephensi. We model the nonlinear responses of temperature dependent mosquito and parasite traits (mosquito development rate, bite rate, fecundity, proportion of eggs surviving to adulthood, vector competence, mortality rate, and parasite development rate) and incorporate these traits into a suitability metric based on a model for the basic reproductive number across temperatures. Our model predicts that the optimum temperature for transmission suitability is similar for the four mosquito-parasite combinations assessed in this study, but may differ at the thermal limits. More specifically, we found significant differences in the upper thermal limit between parasites spread by the same mosquito (A. stephensi) and between mosquitoes carrying P. falciparum. In contrast, at the lower thermal limit the significant differences were primarily between the mosquito species that both carried the same pathogen (e.g., A. stephensi and A. gambiae both with P. falciparum). Using prevalence data, we show that the transmission suitability metric ST$$ S(T) $$ calculated from our mechanistic model is consistent with observed P. falciparum prevalence in Africa and Asia but is equivocal for P. vivax prevalence in Asia, and inconsistent with P. vivax prevalence in Africa. We mapped risk to illustrate the number of months various areas in Africa and Asia predicted to be suitable for malaria transmission based on this suitability metric. This mapping provides spatially explicit predictions for suitability and transmission risk.

Quantitative age grading of mosquitoes using surface-enhanced Raman spectroscopy.

Mosquito-borne pathogens, including malaria, Zika, dengue, and chikungunya continue to be a major public health concern globally. Based on the understanding that only older female mosquitoes are infectious and represent a risk to human health, scientists have sought to age-grade mosquitoes for decades. To date, however, no reliable, cost-effective and practical methods exist to age older mosquitoes despite the tremendous epidemiological value of this approach. This study is the first attempt to develop a surface-enhanced Raman spectroscopic (SERS) method to age mosquitoes. The water extracts of Aedes aegypti mosquitoes aged 0-22 days were mixed with silver nanoparticles. The SERS spectra, which were analysed by principal component analysis and partial least square (PLS), demonstrated the capability of this approach to predict the calendar age of mosquitoes between 0 and 22 days with the coefficient of correlation (R) = 0.994 and 0.990 for PLS model calibration and validation, respectively. Spectral analysis with both SERS and infrared spectroscopy revealed the key biological sources leading to changes in spectra allowing mosquito age-grading is adenine-containing compounds and proteins. In addition, we evaluated the impact of two arthropod-borne pathogen deactivating pre-treatments (bleach and ethanol) on the discrimination capability of the SERS approach. The result shows the ethanol treatment has the potential to enhance the discrimination capability and the safety of the approach. This study represents the first step towards developing the SERS approach as a quick, reliable and field-deployable method for mosquito age-grading, which would significantly improve the effectiveness of vector-borne disease monitoring and prevention.

Sexual selection theory meets disease vector control: Testing harmonic convergence as a "good genes" signal in Aedes aegypti mosquitoes.

BACKGROUND: The mosquito Aedes aegypti is a medically important, globally distributed vector of the viruses that cause dengue, yellow fever, chikungunya, and Zika. Although reproduction and mate choice are key components of vector population dynamics and control, our understanding of the mechanisms of sexual selection in mosquitoes remains poor. In "good genes" models of sexual selection, females use male cues as an indicator of both mate and offspring genetic quality. Recent studies in Ae. aegypti provide evidence that male wingbeats may signal aspects of offspring quality and performance during mate selection in a process known as harmonic convergence. However, the extent to which harmonic convergence may signal overall inherent quality of mates and their offspring remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To examine this, we measured the relationship between acoustic signaling and a broad panel of parent and offspring fitness traits in two generations of field-derived Ae. aegypti originating from dengue-endemic field sites in Thailand. Our data show that in this population of mosquitoes, harmonic convergence does not signal male fertility, female fecundity, or male flight performance traits, which despite displaying robust variability in both parents and their offspring were only weakly heritable. CONCLUSIONS/SIGNIFICANCE: Together, our findings suggest that vector reproductive control programs should treat harmonic convergence as an indicator of some, but not all aspects of inherent quality, and that sexual selection likely affects Ae. aegypti in a trait-, population-, and environment-dependent manner.

Sex, age, and parental harmonic convergence behavior affect the immune performance of Aedes aegypti offspring.

Harmonic convergence is a potential cue, female mosquitoes use to choose male mates. However, very little is known about the benefits this choice confers to offspring performance. Using Aedes aegypti (an important vector of human disease), we investigated whether offspring of converging parental pairs showed differences in immune competence compared to offspring derived from non-converging parental pairs. Here we show that harmonic convergence, along with several other interacting factors (sex, age, reproductive, and physiological status), significantly shaped offspring immune responses (melanization and response to a bacterial challenge). Harmonic convergence had a stronger effect on the immune response of male offspring than on female offspring. Further, female offspring from converging parental pairs disseminated dengue virus more quickly than offspring derived from non-converging parental pairs. Our results provide insight into a wide range of selective pressures shaping mosquito immune function and could have important implications for disease transmission and control.

Effects of changes in temperature on Zika dynamics and control.

When a rare pathogen emerges to cause a pandemic, it is critical to understand its dynamics and the impact of mitigation measures. We use experimental data to parametrize a temperature-dependent model of Zika virus (ZIKV) transmission dynamics and analyse the effects of temperature variability and control-related parameters on the basic reproduction number (R0) and the final epidemic size of ZIKV. Sensitivity analyses show that these two metrics are largely driven by different parameters, with the exception of temperature, which is the dominant driver of epidemic dynamics in the models. Our R0 estimate has a single optimum temperature (≈30°C), comparable to other published results (≈29°C). However, the final epidemic size is maximized across a wider temperature range, from 24 to 36°C. The models indicate that ZIKV is highly sensitive to seasonal temperature variation. For example, although the model predicts that ZIKV transmission cannot occur at a constant temperature below 23°C (≈ average annual temperature of Rio de Janeiro, Brazil), the model predicts substantial epidemics for areas with a mean temperature of 20°C if there is seasonal variation of 10°C (≈ average annual temperature of Tampa, Florida). This suggests that the geographical range of ZIKV is wider than indicated from static R0 models, underscoring the importance of climate dynamics and variation in the context of broader climate change on emerging infectious diseases.

Assessing temperature-dependent competition between two invasive mosquito species.

Invasive mosquitoes are expanding their ranges into new geographic areas and interacting with resident mosquito species. Understanding how novel interactions can affect mosquito population dynamics is necessary to predict transmission risk at invasion fronts. Mosquito life-history traits are extremely sensitive to temperature, and this can lead to temperature-dependent competition between competing invasive mosquito species. We explored temperature-dependent competition between Aedes aegypti and Anopheles stephensi, two invasive mosquito species whose distributions overlap in India, the Middle East, and North Africa, where An. stephensi is currently expanding into the endemic range of Ae. aegypti. We followed mosquito cohorts raised at different intraspecific and interspecific densities across five temperatures (16-32°C) to measure traits relevant for population growth and to estimate species' per capita growth rates. We then used these growth rates to derive each species' competitive ability at each temperature. We find strong evidence for asymmetric competition at all temperatures, with Ae. aegypti emerging as the dominant competitor. This was primarily because of differences in larval survival and development times across all temperatures that resulted in a higher estimated intrinsic growth rate and competitive tolerance estimate for Ae. aegypti compared to An. stephensi. The spread of An. stephensi into the African continent could lead to urban transmission of malaria, an otherwise rural disease, increasing the human population at risk and complicating malaria elimination efforts. Competition has resulted in habitat segregation of other invasive mosquito species, and our results suggest that it may play a role in determining the distribution of An. stephensi across its invasive range.

Socio-demographic, not environmental, risk factors explain fine-scale spatial patterns of diarrhoeal disease in Ifanadiana, rural Madagascar.

Precision health mapping is a technique that uses spatial relationships between socio-ecological variables and disease to map the spatial distribution of disease, particularly for diseases with strong environmental signatures, such as diarrhoeal disease (DD). While some studies use GPS-tagged location data, other precision health mapping efforts rely heavily on data collected at coarse-spatial scales and may not produce operationally relevant predictions at fine enough spatio-temporal scales to inform local health programmes. We use two fine-scale health datasets collected in a rural district of Madagascar to identify socio-ecological covariates associated with childhood DD. We constructed generalized linear mixed models including socio-demographic, climatic and landcover variables and estimated variable importance via multi-model inference. We find that socio-demographic variables, and not environmental variables, are strong predictors of the spatial distribution of disease risk at both individual and commune-level (cluster of villages) spatial scales. Climatic variables predicted strong seasonality in DD, with the highest incidence in colder, drier months, but did not explain spatial patterns. Interestingly, the occurrence of a national holiday was highly predictive of increased DD incidence, highlighting the need for including cultural factors in modelling efforts. Our findings suggest that precision health mapping efforts that do not include socio-demographic covariates may have reduced explanatory power at the local scale. More research is needed to better define the set of conditions under which the application of precision health mapping can be operationally useful to local public health professionals.

Warming temperatures could expose more than 1.3 billion new people to Zika virus risk by 2050.

In the aftermath of the 2015 pandemic of Zika virus (ZIKV), concerns over links between climate change and emerging arboviruses have become more pressing. Given the potential that much of the world might remain at risk from the virus, we used a previously established temperature-dependent transmission model for ZIKV to project climate change impacts on transmission suitability risk by mid-century (a generation into the future). Based on these model predictions, in the worst-case scenario, over 1.3 billion new people could face suitable transmission temperatures for ZIKV by 2050. The next generation will face substantially increased ZIKV transmission temperature suitability in North America and Europe, where naïve populations might be particularly vulnerable. Mitigating climate change even to moderate emissions scenarios could significantly reduce global expansion of climates suitable for ZIKV transmission, potentially protecting around 200 million people. Given these suitability risk projections, we suggest an increased priority on research establishing the immune history of vulnerable populations, modeling when and where the next ZIKV outbreak might occur, evaluating the efficacy of conventional and novel intervention measures, and increasing surveillance efforts to prevent further expansion of ZIKV.

Effects of seasonality and land use on the diversity, relative abundance, and distribution of mosquitoes on St. Kitts, West Indies.

BACKGROUND: Mosquito surveys that collect local data on mosquito species' abundances provide baseline data to help understand potential host-pathogen-mosquito relationships, predict disease transmission, and target mosquito control efforts. METHODS: We conducted an adult mosquito survey from November 2017 to March 2019 on St. Kitts, using Biogents Sentinel 2 traps, set monthly and run for 48-h intervals. We collected mosquitoes from a total of 30 sites distributed across agricultural, mangrove, rainforest, scrub and urban land covers. We investigated spatial variation in mosquito species richness across the island using a hierarchical Bayesian multi-species occupancy model. We developed a mixed effects negative binomial regression model to predict the effects of spatial variation in land cover, and seasonal variation in precipitation on observed counts of the most abundant mosquito species observed. RESULTS: There was high variation among sites in mosquito community structure, and variation in site level richness that correlated with scrub forest, agricultural, and urban land covers. The four most abundant species were Aedes taeniorhynchus, Culex quinquefasciatus, Aedes aegpyti and Deinocerites magnus, and their relative abundance varied with season and land cover. Aedes aegypti was the most commonly occurring mosquito on the island, with a 90% probability of occurring at between 24 and 30 (median = 26) sites. Mangroves yielded the most mosquitoes, with Ae. taeniorhynchus, Cx. quinquefasciatus and De. magnus predominating. Psorophora pygmaea and Toxorhynchites guadeloupensis were only captured in scrub habitat. Capture rates in rainforests were low. Our count models also suggested the extent to which monthly average precipitation influenced counts varied according to species. CONCLUSIONS: There is high seasonality in mosquito abundances, and land cover influences the diversity, distribution, and relative abundance of species on St. Kitts. Further, human-adapted mosquito species (e.g. Ae. aegypti and Cx. quinquefasciatus) that are known vectors for many human relevant pathogens (e.g. chikungunya, dengue and Zika viruses in the case of Ae. aegypti; West Nile, Spondweni, Oropouche virus, and equine encephalitic viruses in the case of Cx. quinqefasciatus) are the most wide-spread (across land covers) and the least responsive to seasonal variation in precipitation.

Land cover affects microclimate and temperature suitability for arbovirus transmission in an urban landscape.

The emergence of mosquito-transmitted viruses poses a global threat to human health. Combining mechanistic epidemiological models based on temperature-trait relationships with climatological data is a powerful technique for environmental risk assessment. However, a limitation of this approach is that the local microclimates experienced by mosquitoes can differ substantially from macroclimate measurements, particularly in heterogeneous urban environments. To address this scaling mismatch, we modeled spatial variation in microclimate temperatures and the thermal potential for dengue transmission by Aedes albopictus across an urban-to-rural gradient in Athens-Clarke County GA. Microclimate data were collected across gradients of tree cover and impervious surface cover. We developed statistical models to predict daily minimum and maximum microclimate temperatures using coarse-resolution gridded macroclimate data (4000 m) and high-resolution land cover data (30 m). The resulting high-resolution microclimate maps were integrated with temperature-dependent mosquito abundance and vectorial capacity models to generate monthly predictions for the summer and early fall of 2018. The highest vectorial capacities were predicted for patches of trees in urban areas with high cover of impervious surfaces. Vectorial capacity was most sensitive to tree cover during the summer and became more sensitive to impervious surfaces in the early fall. Predictions from the same models using temperature data from a local meteorological station consistently over-predicted vectorial capacity compared to the microclimate-based estimates. This work demonstrates that it is feasible to model variation in mosquito microenvironments across an urban-to-rural gradient using satellite Earth observations. Epidemiological models applied to the microclimate maps revealed localized patterns of temperature suitability for disease transmission that would not be detectable using macroclimate data. Incorporating microclimate data into disease transmission models has the potential to yield more spatially precise and ecologically interpretable metrics of mosquito-borne disease transmission risk in urban landscapes.

Field Relevant Variation in Ambient Temperature Modifies Density-Dependent Establishment of Plasmodium falciparum Gametocytes in Mosquitoes.

The relationship between Plasmodium falciparum gametocyte density and infections in mosquitoes is central to understanding the rates of transmission with important implications for control. Here, we determined whether field relevant variation in environmental temperature could also modulate this relationship. Anopheles stephensi were challenged with three densities of P. falciparum gametocytes spanning a ~10-fold gradient, and housed under diurnal/daily temperature range ("DTR") of 9°C (+5°C and -4°C) around means of 20, 24, and 28°C. Vector competence was quantified as the proportion of mosquitoes infected with oocysts in the midguts (oocyst rates) or infectious with sporozoites in the salivary glands (sporozoite rates) at peak periods of infection for each temperature to account for the differences in development rates. In addition, oocyst intensities were also recorded from infected midguts and the overall study replicated across three separate parasite cultures and mosquito cohorts. While vector competence was similar at 20 DTR 9°C and 24 DTR 9°C, oocyst and sporozoite rates were also comparable, with evidence, surprisingly, for higher vector competence in mosquitoes challenged with intermediate gametocyte densities. For the same gametocyte densities however, severe reductions in the sporozoite rates was accompanied by a significant decline in overall vector competence at 28 DTR 9°C, with gametocyte density per se showing a positive and linear effect at this temperature. Unlike vector competence, oocyst intensities decreased with increasing temperatures with a predominantly positive and linear association with gametocyte density, especially at 28 DTR 9°C. Oocyst intensities across individual infected midguts suggested temperature-specific differences in mosquito susceptibility/resistance: at 20 DTR 9°C and 24 DTR 9°C, dispersion (aggregation) increased in a density-dependent manner but not at 28 DTR 9°C where the distributions were consistently random. Limitations notwithstanding, our results suggest that variation in temperature could modify seasonal dynamics of infectious reservoirs with implications for the design and deployment of transmission-blocking vaccines/drugs.

Microclimate and Larval Habitat Density Predict Adult Aedes albopictus Abundance in Urban Areas.

The Asian tiger mosquito, Aedes albopictus, transmits several arboviruses of public health importance, including chikungunya and dengue. Since its introduction to the United States in 1985, the species has invaded more than 40 states, including temperate areas not previously at risk of Aedes-transmitted arboviruses. Mathematical models incorporate climatic variables in predictions of site-specific Ae. albopictus abundances to identify human populations at risk of disease. However, these models rely on coarse resolutions of environmental data that may not accurately represent the climatic profile experienced by mosquitoes in the field, particularly in climatically heterogeneous urban areas. In this study, we pair field surveys of larval and adult Ae. albopictus mosquitoes with site-specific microclimate data across a range of land use types to investigate the relationships between microclimate, density of larval habitat, and adult mosquito abundance and determine whether these relationships change across an urban gradient. We find no evidence for a difference in larval habitat density or adult abundance between rural, suburban, and urban land classes. Adult abundance increases with increasing larval habitat density, which itself is dependent on microclimate. Adult abundance is strongly explained by microclimate variables, demonstrating that theoretically derived, laboratory-parameterized relationships in ectotherm physiology apply to the field. Our results support the continued use of temperature-dependent models to predict Ae. albopictus abundance in urban areas.

Cryogenically preserved RBCs support gametocytogenesis of Plasmodium falciparum in vitro and gametogenesis in mosquitoes.

BACKGROUND: The malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for identifying and evaluating transmission in the laboratory is standard membrane feeding assays (SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to mosquitoes as part of a blood-meal. However, propagation of "infectious" gametocytes requires 10-12 days with considerable physico-chemical demands imposed on host RBCs and thus, "fresh" RBCs that are ≤ 1-week old post-collection are generally recommended. However, in addition to the costs, physico-chemical characteristics unique to RBC donors may confound reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs ("cryo-preserved RBCs") is accepted by European and US FDAs as an alternative to refrigeration (4 °C) for preserving RBC "quality" and while cryo-preserved RBCs have been used for in vitro cultures of other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to support parasite development for > 4 days. RESULTS: Using the standard laboratory strain, P. falciparum NF54, 11 SMFAs were performed with RBCs from four separate donors to demonstrate that RBCs cryo-preserved in the gaseous phase of liquid nitrogen (- 196 °C) supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensi mosquitoes. Overall levels of sporogony in the mosquito, as measured by oocyst and sporozoite prevalence, as well as oocyst burden, from each of the four donors thawed after varying intervals of cryopreservation (1, 4, 8, and 12 weeks) were comparable to using ≤ 1-week old refrigerated RBCs. Lastly, the potential for cryo-preserved RBCs to serve as a suitable alternative substrate is demonstrated for a Cambodian isolate of P. falciparum across two independent SMFAs. CONCLUSIONS: Basic guidelines are presented for integrating cryo-preserved RBCs into an existing laboratory/insectary framework for P. falciparum SMFAs with significant potential for reducing running costs while achieving greater reliability. Lastly, scenarios are discussed where cryo-preserved RBCs may be especially useful in enhancing the understanding and/or providing novel insights into the patterns and processes underlying human-to-mosquito transmission.

Estimating the effects of variation in viremia on mosquito susceptibility, infectiousness, and R0 of Zika in Aedes aegypti.

Zika virus (ZIKV) is an arbovirus primarily transmitted by Aedes mosquitoes. Like most viral infections, ZIKV viremia varies over several orders of magnitude, with unknown consequences for transmission. To determine the effect of viral concentration on ZIKV transmission risk, we exposed field-derived Ae. aegypti mosquitoes to four doses (10(3), 10(4), 10(5), 10(6) PFU/mL) representative of potential variation in the field. We demonstrate that increasing ZIKV dose in the blood-meal significantly increases the probability of mosquitoes becoming infected, and consequently disseminating virus and becoming infectious. Additionally, we observed significant interactions between dose and days post-infection on dissemination and overall transmission efficiency, suggesting that variation in ZIKV dose affects the rates of midgut escape and salivary gland invasion. We did not find significant effects of dose on mosquito mortality. We also demonstrate that detecting virus using RT-qPCR approaches rather than plaque assays potentially over-estimates key transmission parameters, including the time at which mosquitoes become infectious and viral burden. Finally, using these data to parameterize an R0 model, we showed that increasing viremia from 10(4) to 10(6) PFU/mL increased relative R0 3.8-fold, demonstrating that variation in viremia substantially affects transmission risk.