Improved piggyBac Transformation with Capped Transposase mRNA in Pest Insects.

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.

Ecological mechanism of climate-mediated selection in a rapidly evolving invasive species.

Recurring seasonal changes can lead to the evolution of phenological cues. For example, many arthropods undergo photoperiodic diapause, a programmed developmental arrest induced by short autumnal day length. The selective mechanisms that determine the timing of autumnal diapause initiation have not been empirically identified. We quantified latitudinal clines in genetically determined diapause timing of an invasive mosquito, Aedes albopictus, on two continents. We show that variation in diapause timing within and between continents is explained by a novel application of a growing degree day (GDD) model that delineates a location-specific deadline after which it is not possible to complete an additional full life cycle. GDD models are widely used to predict spring phenology by modelling growth and development as physiological responses to ambient temperatures. Our results show that the energy accumulation dynamics represented by GDD models have also led to the evolution of an anticipatory life-history cue in autumn.

Infection of Aedes albopictus Mosquito C6/36 Cells with the wMelpop Strain of Wolbachia Modulates Dengue Virus-Induced Host Cellular Transcripts and Induces Critical Sequence Alterations in the Dengue Viral Genome.

Dengue virus (DENV) causes frequent epidemics infecting ∼390 million people annually in over 100 countries. There are no approved vaccines or antiviral drugs for treatment of infected patients. However, there is a novel approach to control DENV transmission by the mosquito vectors, Aedes aegypti and Aedes albopictus, using the Wolbachia symbiont. The wMelPop strain of Wolbachia suppresses DENV transmission and shortens the mosquito life span. However, the underlying mechanism is poorly understood. To clarify this mechanism, either naive A. albopictus (C6/36) or wMelPop-C6/36 cells were infected with DENV serotype 2 (DENV2). Analysis of host transcript profiles by transcriptome sequencing (RNAseq) revealed that the presence of wMelPop dramatically altered the mosquito host cell transcription in response to DENV2 infection. The viral RNA evolved from wMelPop-C6/36 cells contained low-frequency mutations (∼25%) within the coding region of transmembrane domain 1 (TMD1) of E protein. Mutations with >97% frequencies were distributed within other regions of E, the NS5 RNA-dependent RNA polymerase (NS5POL) domain, and the TMDs of NS2A, NS2B, and NS4B. Moreover, while DENV2-infected naive C6/36 cells showed syncytium formation, DENV2-infected wMelPop-C6/36 cells did not. The Wolbachia-induced mutant DENV2 can readily infect and replicate in naive C6/36 cells, whereas in mutant DENV2-infected BHK-21 or Vero cells, virus replication was delayed. In LLC-MK2 cells, the mutant failed to produce plaques. Additionally, in BHK-21 cells, many mutations in the viral genome reverted to the wild type (WT) and compensatory mutations in NS3 gene appeared. Our results indicate that wMelPop impacts significantly the interactions of DENV2 with mosquito and mammalian host cells.IMPORTANCE Mosquito-borne diseases are of global significance causing considerable morbidity and mortality throughout the world. Dengue virus (DENV; serotypes 1 to 4), a member of the Flavivirus genus of the Flaviviridae family, causes millions of infections annually. Development of a safe vaccine is hampered due to absence of cross-protection and increased risk in secondary infections due to antibody-mediated immune enhancement. Infection of vector mosquitoes with Wolbachia bacteria offers a novel countermeasure to suppress DENV transmission, but the mechanisms are poorly understood. In this study, the host transcription profiles and viral RNA sequences were analyzed in naive A. albopictus (C6/36) and wMelPop-C6/36 cells by RNAseq. Our results showed that the wMelPop symbiont caused profound changes in host transcription profiles and morphology of DENV2-infected C6/36 cells. Accumulation of several mutations throughout DENV2 RNA resulted in loss of infectivity of progeny virions. Our findings offer new insights into the mechanism of Wolbachia-mediated suppression of DENV transmission.

Diapause-associated changes in the lipid and metabolite profiles of the Asian tiger mosquito, Aedes albopictus.

Diapause is an alternative life-history strategy that allows organisms to enter developmental arrest in anticipation of unfavorable conditions. Diapause is widespread among insects and plays a key role in enhancing overwinter survival as well as defining the seasonal and geographic distributions of populations. Next-generation sequencing has greatly advanced our understanding of the transcriptional basis for this crucial adaptation but less is known about the regulation of embryonic diapause physiology at the metabolite level. Here, we characterized the lipid and metabolite profiles of embryonic diapause in the Asian tiger mosquito, Aedes albopictus We used an untargeted approach to capture the relative abundance of 250 lipids and 241 metabolites. We observed adjustments associated with increased energy storage, including an accumulation of lipids, the formation of larger lipid droplets and increased lipogenesis, as well as metabolite shifts suggesting reduced energy utilization. We also found changes in neuroregulatory- and insulin-associated metabolites with potential roles in diapause regulation. Finally, we detected a group of unidentified, diapause-specific metabolites which have physical properties similar to those of steroids/steroid derivatives and may be associated with the ecdysteroidal regulation of embryonic diapause in A.albopictus Together, these results deepen our understanding of the metabolic regulation of embryonic diapause and identify key targets for future investigations.

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution.

The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

Comparative performance of transcriptome assembly methods for non-model organisms.

BACKGROUND: The technological revolution in next-generation sequencing has brought unprecedented opportunities to study any organism of interest at the genomic or transcriptomic level. Transcriptome assembly is a crucial first step for studying the molecular basis of phenotypes of interest using RNA-Sequencing (RNA-Seq). However, the optimal strategy for assembling vast amounts of short RNA-Seq reads remains unresolved, especially for organisms without a sequenced genome. This study compared four transcriptome assembly methods, including a widely used de novo assembler (Trinity), two transcriptome re-assembly strategies utilizing proteomic and genomic resources from closely related species (reference-based re-assembly and TransPS) and a genome-guided assembler (Cufflinks). RESULTS: These four assembly strategies were compared using a comprehensive transcriptomic database of Aedes albopictus, for which a genome sequence has recently been completed. The quality of the various assemblies was assessed by the number of contigs generated, contig length distribution, percent paired-end read mapping, and gene model representation via BLASTX. Our results reveal that de novo assembly generates a similar number of gene models relative to genome-guided assembly with a fragmented reference, but produces the highest level of redundancy and requires the most computational power. Using a closely related reference genome to guide transcriptome assembly can generate biased contig sequences. Increasing the number of reads used in the transcriptome assembly tends to increase the redundancy within the assembly and decrease both median contig length and percent identity between contigs and reference protein sequences. CONCLUSIONS: This study provides general guidance for transcriptome assembly of RNA-Seq data from organisms with or without a sequenced genome. The optimal transcriptome assembly strategy will depend upon the subsequent downstream analyses. However, our results emphasize the efficacy of de novo assembly, which can be as effective as genome-guided assembly when the reference genome assembly is fragmented. If a genome assembly and sufficient computational resources are available, it can be beneficial to combine de novo and genome-guided assemblies. Caution should be taken when using a closely related reference genome to guide transcriptome assembly. The quantity of read pairs used in the transcriptome assembly does not necessarily correlate with the quality of the assembly.

Simple Indices Provide Insight to Climate Attributes Delineating the Geographic Range of Aedes albopictus (Diptera: Culicidae) Prior to Worldwide Invasion.

Aedes albopictus (Skuse) has expanded its distribution worldwide during the past decades. Despite attempts to explain and predict its geographic occurrence, analyses of the distribution of Ae. albopictus in the context of broad climatic regions (biomes) has not been performed. We analyzed climate conditions at its distribution sites in the range before the worldwide invasions (from the easternmost Hawaii through westernmost Madagascar) by using thermal and aridity-humidity indices descriptive of major biomes. A significant advantage of this approach is that it uses simple indices clearly related to the population dynamics of Ae. albopictus. Although Ae. albopictus has been regarded as a forest species preferring humid climate, in areas with significant human habitation, the distribution sites extended from the perhumid, rain forest zone to the semiarid, steppe zone. This pattern was common from the tropics through the temperate zone. Across the distribution range, there was no seasonal discordance between temperature and precipitation; at sites where winter prevents Ae. albopictus reproduction (monthly means<10°C), precipitation was concentrated in warm months (>10°C) under the Asian summer monsoon. Absence of the species in northern and eastern coastal Australia and eastern coastal Africa was not attributable solely to climate conditions. However, Asia west of the summer monsoon range was climatically unsuitable because of low precipitation throughout the year or in warm months favorable to reproduction (concentration of precipitation in winter). We hypothesized that Ae. albopictus originated in continental Asia under the monsoon climate with distinct dry seasons and hot, wet summer, enabling rapid population growth.

Mosquito diapause.

Diapause, a dominant feature in the life history of many mosquito species, offers a mechanism for bridging unfavorable seasons in both temperate and tropical environments and serves to synchronize development within populations, thus directly affecting disease transmission cycles. The trait appears to have evolved independently numerous times within the Culicidae, as exemplified by the diverse developmental stages of diapause in closely related species. Its impact is pervasive, not only influencing the arrested stage, but also frequently altering physiological processes both before and after diapause. How the diapause response can be molded evolutionarily is critical for understanding potential range expansions of native and newly introduced species. The study of hormonal regulation of mosquito diapause has focused primarily on adult diapause, with little current information available on larval diapause or the intriguing maternal effects that regulate egg diapause. Recent quantitative trait locus, transcriptome, and RNA interference studies hold promise for interpreting the complex suite of genes that subserve the diapause phenotype.

Efficient RNAi knockdown at 20 °C in Aedes albopictus (Diptera: Culicidae).

RNA interference (RNAi) is a conserved cellular pathway found in nearly all eukaryotes that leads to the silencing of target mRNAs. Using RNAi as a mechanism to knockdown specific genes has enabled functional and reverse genetics studies in a wide range of eukaryotes. Previous work suggests that RNAi is inhibited at lower temperatures, potentially limiting the possibility to perform knockdown studies on ecologically relevant phenotypes that are only expressed at low temperatures. To determine whether RNAi is inhibited at low temperatures in Aedes albopictus (Skuse), we injected mosquitoes reared at 20 ±â€…1 °C, with dsRNA targeting yellow-g2 and compared knockdown efficacy to mosquitoes injected at 26.5 ±â€…1 °C. Our results demonstrate efficient knockdown at both temperatures, thereby establishing the feasibility of RNAi for functional genetic studies in A. albopictus at low temperatures.

How far do forest container mosquitoes (Diptera: Culicidae) invade rural and urban areas in Japan? - Simple landscape ecology with comparison of the invasive Aedes ecology between native and invasive ranges.

The distribution of container mosquitoes in relation to distances from forests was studied in temperate Japan. Mosquito larvae were collected between May and September in 4 years from tree holes, bamboo stumps, riverine rock pools, and artificial containers; sampling ranged spatially from the mountain forest across the deforested plain developed as agriculture and urban areas to the seacoast. Although tree holes, bamboo stumps, and artificial containers existed throughout the deforested plain area, 10 container species of 6 genera were found virtually only within 5 km from the nearest forest edge. Worldwide invasive Aedes albopictus (Skuse) and Aedes japonicus (Theobald) of Asian origin showed unique occurrence patterns different from other container species and from each other. Ae. japonicus was dominant in artificial containers in and near the forest but minor in forest natural containers and only occurred within 5 km from the forest. Ae. albopictus was minor in the forest irrespective of container types but not bound to the forest and dominant in natural and artificial containers throughout rural and urban areas. The 5-km range was designated as the circum-forest zone for container mosquitoes (except Ae. albopictus) in Japan, and an expanded concept, circum-boundary zone, is proposed. The widths of these zones primarily depend on the dispersal traits of mosquitoes. Whether the relation of Ae. albopictus and Ae. japonicus to forests we observed are common in the native and invasive ranges is discussed. The study of across-ecosystem dispersal is important for mosquito management under anthropogenically changing environments due to either deforestation or green restoration.

Phenotypic variation in biting behavior associated with differences in expression of olfactory genes in the vector mosquito, Aedes albopictus (Diptera: Culicidae).

We evaluated miRNA and mRNA expression differences in head tissues between avid-biting vs. reluctant-biting Aedes albopictus (Skuse) females from a single population over a 20-min timescale. We found no differences in miRNA expression between avid vs. reluctant biters, indicating that translational modulation of blood-feeding behavior occurs on a longer timescale than mRNA transcription. In contrast, we detected 19 differentially expressed mRNAs. Of the 19 differentially expressed genes at the mRNA level between avid-biting vs. reluctant-biting A. albopictus, 9 are implicated in olfaction, consistent with the well-documented role of olfaction in mosquito host-seeking. Additionally, several of the genes that we identified as differentially expressed in association with phenotypic variation in biting behavior share similar functions with or are inferred orthologues of, genes associated with evolutionary variation in biting behaviors of Wyeomyia smithii (Coq.) and Culex pipiens (Lin.). A future goal is to determine whether these genes are involved in the evolutionary transition from a biting to a non-biting life history.

Thermal tolerance of mosquito eggs is associated with urban adaptation and human interactions.

Climate change is expected to profoundly affect mosquito distributions and their ability to serve as vectors for disease, specifically with the anticipated increase in heat waves. The rising temperature and frequent heat waves can accelerate mosquito life cycles, facilitating higher disease transmission. Conversely, higher temperatures could increase mosquito mortality as a negative consequence. Warmer temperatures are associated with increased human density, suggesting a need for anthropophilic mosquitoes to adapt to be more hardy to heat stress. Mosquito eggs provide an opportunity to study the biological impact of climate warming as this stage is stationary and must tolerate temperatures at the site of female oviposition. As such, egg thermotolerance is critical for survival in a specific habitat. In nature, Aedes mosquitoes exhibit different behavioral phenotypes, where specific populations prefer depositing eggs in tree holes and prefer feeding non-human vertebrates. In contrast, others, particularly human-biting specialists, favor laying eggs in artificial containers near human dwellings. This study examined the thermotolerance of eggs, along with adult stages, for Aedes aegypti and Ae. albopictus lineages associated with known ancestry and shifts in their relationship with humans. Mosquitoes collected from areas with higher human population density, displaying increased human preference, and having a human-associated ancestry profile have increased egg viability following high-temperature stress. Unlike eggs, thermal tolerance among adults showed no significant correlation based on the area of collection or human-associated ancestry. This study highlights that the egg stage is likely critical to mosquito survival when associated with humans and needs to be accounted when predicting future mosquito distribution.

A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Deep sequencing reveals complex mechanisms of diapause preparation in the invasive mosquito, Aedes albopictus.

Seasonal environments present fundamental physiological challenges to a wide range of insects. Many temperate insects surmount the exigencies of winter by undergoing photoperiodic diapause, in which photoperiod provides a token cue that initiates an alternative developmental programme leading to dormancy. Pre-diapause is a crucial preparatory phase of this process, preceding developmental arrest. However, the regulatory and physiological mechanisms of diapause preparation are largely unknown. Using high-throughput gene expression profiling in the Asian tiger mosquito, Aedes albopictus, we reveal major shifts in endocrine signalling, cell proliferation, metabolism, energy production and cellular structure across pre-diapause development. While some hallmarks of diapause, such as insulin signalling and stress response, were not important at the transcriptional level, two genes, Pepck and PCNA, appear to show diapause-induced transcriptional changes across insect taxa. These processes demonstrate physiological commonalities between Ae. albopictus pre-diapause and diapause strategies across insects, and support the idea of a genetic 'toolkit' for diapause. Observations of gene expression trends from a comparative developmental perspective suggest that individual physiological processes are delayed against a background of a fixed morphological ontogeny. Our results demonstrate how deep sequencing can provide new insights into elusive molecular bases of complex ecological adaptations.

RNA-Seq reveals early distinctions and late convergence of gene expression between diapause and quiescence in the Asian tiger mosquito, Aedes albopictus.

Dormancy is a crucial adaptation allowing insects to withstand harsh environmental conditions. The pre-programmed developmental arrest of diapause is a form of dormancy that is distinct from quiescence, in which development arrests in immediate response to hardship. Much progress has been made in understanding the environmental and hormonal controls of diapause. However, studies identifying transcriptional changes unique to diapause, rather than quiescence, are lacking, making it difficult to disentangle the transcriptional profiles of diapause from dormancy in general. The Asian tiger mosquito, Aedes albopictus, presents an ideal model for such a study, as diapausing and quiescent eggs can be staged and collected for global gene expression profiling using a newly developed transcriptome. Here, we use RNA-Seq to contrast gene expression during diapause with quiescence to identify transcriptional changes specific to the diapause response. We identify global trends in gene expression that show gradual convergence of diapause gene expression upon gene expression during quiescence. Functionally, early diapause A. albopictus show strong expression differences of genes involved in metabolism, which diminish over time. Of these, only expression of lipid metabolism genes remained distinct in late diapause. We identify several genes putatively related to hormonal control of development that are persistently differentially expressed throughout diapause, suggesting these might be involved in the maintenance of diapause. Our results identify key biological differences between diapausing and quiescent pharate larvae, and suggest candidate pathways for studying metabolism and the hormonal control of development during diapause in other species.

Socioeconomic status affects mosquito (Diptera: Culicidae) larval habitat type availability and infestation level.

Mosquito populations are largely regulated by processes occurring at the larval stage. We sampled mosquito larval microhabitats (mostly water-holding containers) in six neighborhoods in the Washington, DC, area that varied in socioeconomic status (SES) and housing structure (row houses vs. stand-alone houses) to test associations among these neighborhood characteristics, microhabitat abundance and parameters, and mosquito occurrence and densities. Thirty-four percent (33.9%) of sampled microhabitats contained mosquito larvae, and 93.1% of larvae were Aedes albopictus Skuse or Culex pipiens L. Five specific container types (drains, corrugated flexible drainpipes, planters, garbage cans, and buckets) accounted for the majority of water-holding (56.0%) and mosquito-positive (50.6%) microhabitats sampled. We found no associations between SES or housing structure with total microhabitat abundance per yard, mosquito occurrence or mosquito densities per microhabitat. In contrast, container purpose varied with SES, with low SES neighborhoods having greater numbers of disused containers and lower numbers of functional containers than low and medium SES neighborhoods. Ae. albopictus were 83% more abundant in disused containers, whereas Cx. pipiens were more abundant in structural and functional containers, possibly owing to species-specific oviposition and development related to water quality. Ae. albopictus densities increased over the summer, whereas Cx. pipiens densities remained constant. Ae. albopictus is usually the dominant pest in urban areas in the eastern United States; therefore, integrated mosquito management programs should incorporate the elimination of disused containers to reduce its infestation and adult production, especially in low SES neighborhoods where they occur most frequently.

Contrasting effects of an extended fall period and winter heatwaves on the overwintering fitness of diapausing disease vector, Aedes albopictus.

Climate change is expected to dramatically alter autumnal and winter conditions in many temperate regions. However, limited data is available to accurately predict how these changes will impact species' overwinter survival and post-winter fitness. Here, we determine how a longer, warmer fall period and winter heatwaves affect overwintering fitness and post-winter performance of the invasive mosquito vector, Aedes albopictus. We found that a longer, warmer fall period representative of early entry into diapause did not affect overwinter survival but did lead to reduced post-winter performance for multiple traits. Specifically, larvae that experienced longer, warmer fall conditions as diapause embryos exhibited reduced post-diapause larval starvation tolerance, increased post-diapause larval mortality, and longer post-diapause larval development compared to individuals from the short-fall treatments. These negative post-diapause fitness effects likely resulted from the greater energetic demands and/or damage incurred during the warmer, longer fall period. In contrast, exposure to winter heatwaves increased overwinter survival, possibly by allowing diapausing embryos to escape or repair cold injury. Finally, fall treatment and winter heatwaves had an interactive effect on male development time, while neither treatment impacted pupal mass in either sex. Overall, our results highlight that experiments that fail to measure post-diapause fitness are likely to substantially under-estimate the impacts of climate change on post-winter performance. Additionally, our results emphasize that it is crucial to consider the potentially conflicting effects of different aspects of climate change on a species' overall overwintering success.

MicroRNA Expression Prior to Biting in a Vector Mosquito Anticipates Physiological Processes Related to Energy Utilization, Reproduction and Immunity.

Understanding the molecular and physiological processes underlying biting behavior in vector mosquitoes has important implications for developing novel strategies to suppress disease transmission. Here, we conduct small-RNA sequencing and qRT-PCR to identify differentially expressed microRNAs (miRNAs) in the head tissues of two subspecies of Culex pipiens that differ in biting behavior and the ability to produce eggs without blood feeding. We identified eight differentially expressed miRNAs between biting C. pipiens pipiens (Pipiens) and non-biting C. pipiens molestus (Molestus); six of these miRNAs have validated functions or predicted targets related to energy utilization (miR8-5-p, miR-283, miR-2952-3p, miR-1891), reproduction (miR-1891), and immunity (miR-2934-3p, miR-92a, miR8-5-p). Although miRNAs regulating physiological processes associated with blood feeding have previously been shown to be differentially expressed in response to a blood meal, our results are the first to demonstrate differential miRNA expression in anticipation of a blood meal before blood is actually imbibed. We compare our current miRNA results to three previous studies of differential messenger RNA expression in the head tissues of mosquitoes. Taken together, the combined results consistently show that biting mosquitoes commit to specific physiological processes in anticipation of a blood meal, while non-biting mosquitoes mitigate these anticipatory costs.