Container-breeding mosquitoes and predator community dynamics along an urban-forest gradient: The effects of habitat type and isolation

Environmental disturbances such as deforestation, urbanization or pollution have been widely acknowledged to play a key role in the emergence of many infectious diseases, including mosquito-borne viruses. However, we have little understanding of how habitat isolation affects the communities containing disease vectors. Here, we test the effects of habitat type and isolation on the colonization rates, species richness and abundances of mosquitoes and their aquatic predators in water-filled containers in northwestern Thailand. For eight weeks water-filled containers were monitored in areas containing forest, urban and agricultural habitats and mixtures of these three. Mosquito larvae of the genera Aedes and Culex appeared to be differentially affected by the presence of the dominant predator; Toxorhynchites splendens (Culicidae). Therefore, a predation experiment was conducted to determine predator response to prey density and its relative effects on different mosquito prey populations. Colonization rates, species richness and abundances of mosquito predators were strongly related to forest habitat and to the distance from other aquatic habitats. Areas with more tree cover had higher predator species richness and abundance in containers. Containers that were close to surface water were more rapidly colonized than those further away. In all habitat types, including urban areas, when predators were present, the number of mosquito larvae was much lower. Containers in urban areas closer to water-bodies, or with more canopy cover, had higher predator colonization rates and species richness. T. splendens (Culicidae) preyed on the larvae of two mosquito genera at different rates, which appeared to be related to prey behaviour. This study shows that anthropogenic landscape modification has an important effect on the natural biological control of mosquitoes. Vector control programmes and urban planning should attempt to integrate ecological theory when developing strategies to reduce mosquito populations. This would result in management strategies that are beneficial for both public health and biodiversity.     

Blood-meal preferences and avian malaria detection in mosquitoes (Diptera: Culicidae) captured at different land use types within a neotropical montane cloud forest matrix

Human activities modify environmental conditions, altering ecological interactions that can contribute to the increasing number of vector-borne pathogens affecting both human and wildlife populations. There is a dearth of knowledge about mosquitoes feeding preferences and their role as potential vectors of haemosporidian parasites, particularly in modified habitats. During 2013–2014 we sampled mosquitoes in five different land use types within a cloud forest matrix. From a total of 4107 adult mosquitoes, 90 were engorged. We extracted DNA from mosquito blood-meals, abdomens, and thoraxes, which belonged to seven different species. Seventeen specimens were positive for avian Plasmodium parasites. We were able to identify the blood-meal source of 10 mosquitoes, the identified vertebrate species were: Homo sapiens (Human), Sturnira hondurensis (Bat), and Bos taurus (Cow). Our results show that Culex restuans is positive for avian malaria and it is feeding on both humans and domestic animals at urban and peri-urban habitat types, where it is also an abundant species throughout the year. Furthermore, Aedes quadrivittatus, also positive for avian malaria, is feeding on humans in the well-preserved cloud forest, where this mosquito species is highly abundant. This study is the first in Mexico to provide reference data showing generalist mosquito feeding preferences and presence of avian Plasmodium at locations with different land use types.     

Mosquito Vector Diversity across Habitats in Central Thailand Endemic for Dengue and Other Arthropod-Borne Diseases

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

The risk of a mosquito-borne infection in a heterogeneous environment

A common assumption about malaria, dengue, and other mosquito-borne infections is that the two main components of the risk of human infection—the rate at which people are bitten (human biting rate) and the proportion of mosquitoes that are infectious—are positively correlated. In fact, these two risk factors are generated by different processes and may be negatively correlated across space and time in heterogeneous environments. Uneven distribution of blood-meal hosts and larval habitat creates a spatial mosaic of demograPhic sources and sinks. Moreover, mosquito populations fluctuate temporally, forced by environmental variables such as rainfall, temperature, and humidity. These sources of spatial and temporal heterogeneity in the distribution of mosquito populations generate variability in the human biting rate, in the proportion of mosquitoes that are infectious, and in the risk of human infection. To understand how heterogeneity affects the epidemiology of mosquito-borne infections, we developed a set of simple models that incorporate heterogeneity in a stepwise fashion. These models predict that the human biting rate is highest shortly after the mosquito densities peak, near breeding sites where adult mosquitoes emerge, and around the edges of areas where humans are aggregated. In contrast, the proportion of mosquitoes that are infectious reflects the age structure of mosquito populations; it peaks where old mosquitoes are found, far from mosquito breeding habitat, and when mosquito population density is declining. Finally, we show that estimates for the average risk of infection that are based on the average entomological inoculation rate are strongly biased in heterogeneous environments.     

Immature mosquitoes in agricultural wetlands of the coastal plain of Georgia,U.S.A.: Effects of landscape and environmental habitat characteristics

This study is the first to report on the relationships between immature mosquitoes (larvae and pupae) and landscape and environmental habitat characteristics in wetlands associated with row crop agriculture. Indicator species analysis (ISA) was used to test for associations among mosquito species and groups of wetland sites with similar Landscape Development Intensity (LDI) values. Results indicated that Anopheles quadrimaculatus, Culex erraticus, and Psorophora columbiae were associated with agricultural wetlands (LDI > 2.0), whereas Anopheles crucians and Culex territans were associated with forested reference wetlands (LDI < 2.0) in both wet and dry years. The species fidelity to wetland type, regardless of the hydrologic regime, demonstrates these species are robust indicators of wetland condition. Data on immature mosquito assemblages were compared to selected landscape and environmental habitat variables using Akaike's Information Criterion (AIC_c) model selection. LDI indices, dissolved oxygen concentration, the proportion of emergent vegetation, and the proportion of bare ground in wetlands were important factors associated with the selected mosquito species. These results indicate that LDI indices are useful in predicting the distributions of disease vectors or other nuisance mosquito species across broad geographic areas. Additionally, these results suggest mosquitoes are valuable bioindicators of wetland condition that reflect land use and hydrologic variability.     

Impact of Land-use Change on Dengue and Malaria in Northern Thailand

Land-use change, a major constituent of global environmental change, potentially has significant consequences for human health in relation to mosquito-borne diseases. Land-use change can influence mosquito habitat, and therefore the distribution and abundance of vectors, and land use mediates human–mosquito interactions, including biting rate. Based on a conceptual model linking the landscape, people, and mosquitoes, this interdisciplinary study focused on the impacts of changes in land use on dengue and malaria vectors and dengue transmission in northern Thailand. Extensive data on mosquito presence and abundance, land-use change, and infection risk determinants were collected over 3 years. The results of the different components of the study were then integrated through a set of equations linking land use to disease via mosquito abundance. The impacts of a number of plausible scenarios for future land-use changes in the region, and of concomitant behavioral change were assessed. Results indicated that land-use changes have a detectable impact on mosquito populations and on infection. This impact varies according to the local environment but can be counteracted by adoption of preventive measures.     

Impacts of fungal entomopathogens on survival and immune responses of Aedes albopictus and Culex pipiens mosquitoes in the context of native Wolbachia infections

Microbial control of mosquitoes via the use of symbiotic or pathogenic microbes, such as Wolbachia and entomopathogenic fungi, are promising alternatives to synthetic insecticides to tackle the rapid increase in insecticide resistance and vector-borne disease outbreaks. This study evaluated the susceptibility and host responses of two important mosquito vectors, Ae. albopictus and Cx. pipiens, that naturally carry Wolbachia, to infections by entomopathogenic fungi. Our study indicated that while Wolbachia presence did not provide a protective advantage against entomopathogenic fungal infection, it nevertheless influenced the bacterial / fungal load and the expression of select anti-microbial effectors and phenoloxidase cascade genes in mosquitoes. Furthermore, although host responses from Ae. albopictus and Cx. pipiens were mostly similar, we observed contrasting phenotypes with regards to susceptibility and immune responses to fungal entomopathogenic infection in these two mosquitoes. This study provides new insights into the intricate multipartite interaction between the mosquito host, its native symbiont and pathogenic microbes that might be employed to control mosquito populations.     

Nestedness patterns of container-dwelling mosquitoes: effects of larval habitat within variable terrestrial matrices

Distributions of mosquito larvae likely are a consequence of multiple factors, although two commonly studied factors (quality of the larval environment and the terrestrial matrix in which these habitats reside) have rarely and simultaneously been varied in the field to understand distributions of larvae. We monitored aquatic containers of two leaf detritus levels within a forest, prairie, and industrial habitat across five months to understand the temporal and spatial colonization of aquatic invertebrates in Northcentral Illinois, USA. Data were collected monthly on mosquito populations and the composition of other invertebrates colonizing containers. Overall, six species of mosquitoes colonized containers, with Culex restuans and Aedes triseriatus having the highest relative abundances. There were strong seasonal abundance patterns for these two mosquito species, with the dominant species changing over time in the forest habitat. The responses of other mosquito taxa were more variable, with abundances reflective of either the terrestrial matrix or larval habitat quality. High detritus containers supported the highest abundances of most species encountered, regardless of habitat. Non-mosquito taxa were less common numerically, but analyses suggested that some taxa, such as syrphid larvae, often co-occurred with mosquitoes. Nested subset analysis indicated communities were strongly nested, and that both habitat type and detritus level were important in explaining nested patterns of aquatic invertebrates. Our data show that both the larval habitat and the surrounding terrestrial matrix shape patterns of container mosquitoes, and that other container invertebrates vary in similar ways as mosquitoes. Handling editor: K. Martens     

Horizontal distribution affects the vertical distribution of native and invasive container‐inhabiting Aedes mosquitoes within an urban landscape

The vertical dimension constitutes an important niche axis along which mosquitoes may adjust their distribution. Here, we evaluated whether the vertical distribution of container‐inhabiting Aedes mosquitoes differs along a gradient of anthropogenic land‐use intensity within an urban landscape. Using a pulley system, we hung oviposition cups at three heights (ground level, 4.5, and 9 m) and in three habitats: forest, park, and a built environment. We hypothesized that mosquito abundance and diversity would be highest in the least disturbed forest habitat, decrease in the park, and be lowest at the UNC‐Greensboro campus. We also expected Aedes albopictus (Skuse) and Ae. triseriatus (Say) to mainly oviposit at ground level and Ae. hendersoni (Cockerell) at canopy height. Aedes albopictus was the most common species (68.8%) collected in all three habitat types and was the only species found in the built environment. In that habitat, Ae. albopictus exhibited a bimodal distribution with the lowest activity at the intermediate height (4.5 m). Aedes triseriatus (28.9%) did not differ in egg abundance between the forest and park habitats but did exhibit diverse vertical habitat use while avoiding the canopy in the park habitat. Aedes hendersoni (2.3%) was the most sylvatic species and oviposited only at ground level. Our results indicate that the vertical distribution of mosquitoes is affected by the type of habitat in which they occur, and that this variation could be driven via local‐scale modification of microclimatic factors.     

Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.     

Role of Bunyamwera Orthobunyavirus NSs Protein in Infection of Mosquito Cells

Background

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line.

Methodology and Principal Findings

To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus.

Conclusions/Significance

Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.     

Landscape Effects on the Presence,Abundance and Diversity of Mosquitoes in Mediterranean Wetlands

Environment determines the distribution of mosquito-borne diseases in that it influences the vector-host-pathogen transmission cycle, including vector distribution, abundance and diversity. In this study, we analyse the relationship between environmental variables estimated by remote sensing and the spatial distribution (presence, abundance and diversity) of seven mosquito species vectors of West Nile and other pathogens (Usutu, avian malaria and dirofilariasis) in the Doñana Natural Park, Spain. Traps were distributed over an area of 54,984 ha divided into six ecological units: marshland, sand dunes, scrubland, ricefields, crops and fishponds. We collected mosquitoes once a month from up to 112 locations using BG-Sentinel traps baited with BG-lure and CO₂ during March-November 2010. Hydroperiod, NDVI and Inundation surface were estimated at several resolution scales (100, 250, 500, 1000 and 2000 metres) from corrected and normalized Landsat Images. We sampled 972,346 female mosquitoes, the most abundant species being Culex theileri, Ochlerotatus caspius, Culex modestus, Culex perexiguus, Culex pipiens, Anopheles atroparvus and Ochlerotatus detritus. Our results suggest that: (1) hydroperiod, inundation surface and NDVI are strongly related to the spatial distribution of mosquitoes; (2) the spatial scales used to measure these variables affected quantification of these relationships, the larger scale being more informative; (3) these relationships are species-specific; (4) hydroperiod is negatively related to mosquito presence and richness; (5) Culex abundance is positively related to hydroperiod; (6) NDVI is positively related to mosquito diversity, presence and abundance, except in the case of the two salt marsh species (Oc. caspius and Oc. detritus); and (7) inundation surfaces positively condition the abundance and richness of most species except the salt marsh mosquitoes. Remote sensing data provided reliable information for monitoring mosquito populations. Landscape significantly affected mosquito distribution and abundance, and as a result may alter disease risk. These results suggest that while environmental conditions affect the distribution and abundance of mosquitoes, other factors such as human modification of landscapes may give rise to significant changes in mosquito populations and consequently disease risk.     

A review of the role of fish as biological control agents of disease vector mosquitoes in mangrove forests: reducing human health risks while reducing environmental risk

The saltwater mosquito, Aedes vigilax, is prolific in coastal wetlands including mangroves and saltmarshes. Ae. vigilax is a vector for arboviruses such as Ross River and Barmah Forest viruses, with significant consequences for human health and economic productivity. In Australia the dominant form of mosquito control is chemicals. For mangroves, this is because there is a critical lack of knowledge supporting alternative approaches, such as environmental modification or biological control using larvivorous fish. This review examines the potential of fish as biological agents for the control of mosquito larvae in mangroves. We consider two key aspects: how larvivorous fish use mangroves; and can larvivorous fish reduce larval mosquito populations sufficiently to provide effective mosquito control? The link between fish and mangroves is reasonably well established, where mangroves act as refuge habitat for small and juvenile fish. Also, research has established that fish can be significant predators of mosquitoes, and therefore may be effective control agents. However, studies of fish activity within mangroves are limited to study of the fringe of the mangroves and not the internal structure of mangrove basins and as a result, fish populations within these areas remain unstudied. Also, until recently there was little appreciation of the mangrove-mosquito habitat relationship and, as a consequence, the importance of the mangrove basin as the key mosquito habitat has also been overlooked in the literature. Similarly, the predator/prey relationships between fish and mosquitoes within mangrove basin environments also remain unstudied, and therefore the importance of fish for mosquito management in mangrove basins is not known. There are substantial knowledge gaps regarding the potential of fish in controlling larval mosquitoes in mangroves. The gaps include: understanding of how larvivorous fish use mangrove basins; the nature of the fish-mosquito predator/prey relationship in mangrove basins; and whether larvivorous fish are effective as a mosquito control option in mangroves.     

Landscape Movements of Anopheles gambiae Malaria Vector Mosquitoes in Rural Gambia

Background

For malaria control in Africa it is crucial to characterise the dispersal of its most efficient vector, Anopheles gambiae, in order to target interventions and assess their impact spatially. Our study is, we believe, the first to present a statistical model of dispersal probability against distance from breeding habitat to human settlements for this important disease vector.

Methods/Principal Findings

We undertook post-hoc analyses of mosquito catches made in The Gambia to derive statistical dispersal functions for An. gambiae sensu lato collected in 48 villages at varying distances to alluvial larval habitat along the River Gambia. The proportion dispersing declined exponentially with distance, and we estimated that 90% of movements were within 1.7 km. Although a ‘heavy-tailed’ distribution is considered biologically more plausible due to active dispersal by mosquitoes seeking blood meals, there was no statistical basis for choosing it over a negative exponential distribution. Using a simple random walk model with daily survival and movements previously recorded in Burkina Faso, we were able to reproduce the dispersal probabilities observed in The Gambia.

Conclusions/Significance

Our results provide an important quantification of the probability of An. gambiae s.l. dispersal in a rural African setting typical of many parts of the continent. However, dispersal will be landscape specific and in order to generalise to other spatial configurations of habitat and hosts it will be necessary to produce tractable models of mosquito movements for operational use. We show that simple random walk models have potential. Consequently, there is a pressing need for new empirical studies of An. gambiae survival and movements in different settings to drive this development.     

Malaria's Missing Number: Calculating the Human Component of R0 by a Within-Host Mechanistic Model of Plasmodium falciparum Infection and Transmission

Human infection by malarial parasites of the genus Plasmodium begins with the bite of an infected Anopheles mosquito. Current estimates place malaria mortality at over 650,000 individuals each year, mostly in African children. Efforts to reduce disease burden can benefit from the development of mathematical models of disease transmission. To date, however, comprehensive modeling of the parameters defining human infectivity to mosquitoes has remained elusive. Here, we describe a mechanistic within-host model of Plasmodium falciparum infection in humans and pathogen transmission to the mosquito vector. Our model incorporates the entire parasite lifecycle, including the intra-erythrocytic asexual forms responsible for disease, the onset of symptoms, the development and maturation of intra-erythrocytic gametocytes that are transmissible to Anopheles mosquitoes, and human-to-mosquito infectivity. These model components were parameterized from malaria therapy data and other studies to simulate individual infections, and the ensemble of outputs was found to reproduce the full range of patient responses to infection. Using this model, we assessed human infectivity over the course of untreated infections and examined the effects in relation to transmission intensity, expressed by the basic reproduction number R₀ (defined as the number of secondary cases produced by a single typical infection in a completely susceptible population). Our studies predict that net human-to-mosquito infectivity from a single non-immune individual is on average equal to 32 fully infectious days. This estimate of mean infectivity is equivalent to calculating the human component of malarial R₀. We also predict that mean daily infectivity exceeds five percent for approximately 138 days. The mechanistic framework described herein, made available as stand-alone software, will enable investigators to conduct detailed studies into theories of malaria control, including the effects of drug treatment and drug resistance on transmission.     

miRNA Genes of an Invasive Vector Mosquito,Aedes albopictus

Aedes albopictus, a vector of Dengue and Chikungunya viruses, is a robust invasive species in both tropical and temperate environments. MicroRNAs (miRNAs) regulate gene expression and biological processes including embryonic development, innate immunity and infection. While a number of miRNAs have been discovered in some mosquitoes, no comprehensive effort has been made to characterize them from different developmental stages from a single species. Systematic analysis of miRNAs in Ae. albopictus will improve our understanding of its basic biology and inform novel strategies to prevent virus transmission. Between 10–14 million Illumina sequencing reads per sample were obtained from embryos, larvae, pupae, adult males, sugar-fed and blood-fed adult females. A total of 119 miRNA genes represented by 215 miRNA or miRNA star (miRNA*) sequences were identified, 15 of which are novel. Eleven, two, and two of the newly-discovered miRNA genes appear specific to Aedes, Culicinae, and Culicidae, respectively. A number of miRNAs accumulate predominantly in one or two developmental stages and the large number that showed differences in abundance following a blood meal likely are important in blood-induced mosquito biology. Gene Ontology (GO) analysis of the targets of all Ae. albopictus miRNAs provides a useful starting point for the study of their functions in mosquitoes. This study is the first systematic analysis of miRNAs based on deep-sequencing of small RNA samples of all developmental stages of a mosquito species. A number of miRNAs are related to specific physiological states, most notably, pre- and post-blood feeding. The distribution of lineage-specific miRNAs is consistent with mosquito phylogeny and the presence of a number of Aedes-specific miRNAs likely reflects the divergence between the Aedes and Culex genera.