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.

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.

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.

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.

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.

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.

Conserved molecular pathways underlying biting in two divergent mosquito genera.

Mosquitoes transmit a wide variety of devastating pathogens when they bite vertebrate hosts and feed on their blood. However, three entire mosquito genera and many individual species in other genera have evolved a nonbiting life history in which blood is not required to produce eggs. Our long-term goal is to develop novel interventions that reduce or eliminate the biting behavior in vector mosquitoes. A previous study used biting and nonbiting populations of a nonvector mosquito, Wyeomyia smithii, as a model to uncover the transcriptional basis of the evolutionary transition from a biting to a nonbiting life history. Herein, we ask whether the molecular pathways that were differentially expressed due to differences in biting behavior in W. smithii are also differentially expressed between subspecies of Culex pipiens that are obligate biting (Culex pipiens pipiens) and facultatively nonbiting (Culex pipiens molestus). Results from RNAseq of adult heads show dramatic upregulation of transcripts in the ribosomal protein pathway in biting C. pipiens, recapitulating the results in W. smithii, and implicating the ancient and highly conserved ribosome as the intersection to understanding the evolutionary and physiological basis of blood feeding in mosquitoes. Biting Culex also strongly upregulate energy production pathways, including oxidative phosphorylation and the citric acid (TCA) cycle relative to nonbiters, a distinction that was not observed in W. smithii. Amino acid metabolism pathways were enriched for differentially expressed genes in biting versus nonbiting Culex. Relative to biters, nonbiting Culex upregulated sugar metabolism and transcripts contributing to reproductive allocation (vitellogenin and cathepsins). These results provide a foundation for developing strategies to determine the natural evolutionary transition between a biting and nonbiting life history in vector mosquitoes.

A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation.

BACKGROUND: Many temperate insects survive the harsh conditions of winter by undergoing photoperiodic diapause, a pre-programmed developmental arrest initiated by short day lengths. Despite the well-established ecological significance of photoperiodic diapause, the molecular basis of this crucial adaptation remains largely unresolved. The Asian tiger mosquito, Aedes albopictus (Skuse), represents an outstanding emerging model to investigate the molecular basis of photoperiodic diapause in a well-defined ecological and evolutionary context. Ae. albopictus is a medically significant vector and is currently considered the most invasive mosquito in the world. Traits related to diapause appear to be important factors contributing to the rapid spread of this mosquito. To generate novel sequence information for this species, as well as to discover transcripts involved in diapause preparation, we sequenced the transcriptome of Ae. albopictus oocytes destined to become diapausing or non-diapausing pharate larvae. RESULTS: 454 GS-FLX transcriptome sequencing yielded >1.1 million quality-filtered reads, which we assembled into 69,474 contigs (N50 = 1,009 bp). Our contig filtering approach, where we took advantage of strong sequence similarity to the fully sequenced genome of Aedes aegypti, as well as other reference organisms, resulted in 11,561 high-quality, conservative ESTs. Differential expression estimates based on normalized read counts revealed 57 genes with higher expression, and 257 with lower expression under diapause-inducing conditions. Analysis of expression by qPCR for 47 of these genes indicated a high correlation of expression levels between 454 sequence data and qPCR, but congruence of statistically significant differential expression was low. Seven genes identified as differentially expressed based on qPCR have putative functions that are consistent with the insect diapause syndrome; three genes have unknown function and represent novel candidates for the transcriptional basis of diapause. CONCLUSIONS: Our transcriptome database provides a rich resource for the comparative genomics and functional genetics of Ae. albopictus, an invasive and medically important mosquito. Additionally, the identification of differentially expressed transcripts related to diapause enriches the limited knowledge base for the molecular basis of insect diapause, in particular for the preparatory stage. Finally, our analysis illustrates a useful approach that draws from a closely related reference genome to generate high-confidence ESTs in a non-model organism.

A Linkage-Based Genome Assembly for the Mosquito Aedes albopictus and Identification of Chromosomal Regions Affecting Diapause.

The Asian tiger mosquito, Aedes albopictus, is an invasive vector mosquito of substantial public health concern. The large genome size (~1.19-1.28 Gb by cytofluorometric estimates), comprised of ~68% repetitive DNA sequences, has made it difficult to produce a high-quality genome assembly for this species. We constructed a high-density linkage map for Ae. albopictus based on 111,328 informative SNPs obtained by RNAseq. We then performed a linkage-map anchored reassembly of AalbF2, the genome assembly produced by Palatini et al. (2020). Our reassembled genome sequence, AalbF3, represents several improvements relative to AalbF2. First, the size of the AalbF3 assembly is 1.45 Gb, almost half the size of AalbF2. Furthermore, relative to AalbF2, AalbF3 contains a higher proportion of complete and single-copy BUSCO genes (84.3%) and a higher proportion of aligned RNAseq reads that map concordantly to a single location of the genome (46%). We demonstrate the utility of AalbF3 by using it as a reference for a bulk-segregant-based comparative genomics analysis that identifies chromosomal regions with clusters of candidate SNPs putatively associated with photoperiodic diapause, a crucial ecological adaptation underpinning the rapid range expansion and climatic adaptation of A. albopictus.

Differences in Responses to Urbanization Between Invasive Mosquitoes, Aedes japonicus japonicus (Diptera: Culicidae) and Aedes albopictus, in Their Native Range, Japan.

The Asian mosquitoes, Aedes albopictus (Skuse) and Aedes japonicus japonicus (Theobald), have invaded North America, Europe, and other regions since the late 20th century. In invasive ranges, Ae. albopictus has well been recognized as urban, domestic species, whereas views about the macrohabitats of Ae. j. japonicus are inconsistent. Previous reports in Japan suggest the disappearance of Ae. j. japonicus from metropolises. However, container-mosquito larvae have not been inspected simultaneously for various macro and microhabitats in metropolises in Japan. The current study in Fukuoka City, a metropolis in southwest Japan, confirmed the absence of Ae. j. japonicus irrespective of macrohabitats (temples with graveyards, shrines, public graveyards, cultivated bamboo groves, and urban forests, all within the area densely inhabited by humans) and microhabitats (container types). In contrast, Ae. albopictus was dominant throughout the macro and microhabitats except forest tree holes rich with competitive species. Past records indicate the disappearance of Ae. j. japonicus from metropolitan Fukuoka within the last 70 yr. Based on careful examination of available evidence, we concluded that 1) both species benefit from human-made environments with artificial containers free from competitors, 2) Ae. j. japonicus disappeared due to hot, dry summer conditions facilitated by urban heat-island effects and a decrease in favored mammal hosts, and 3) Ae. albopictus has proliferated with higher tolerance to hot, dry climate and a wider blood-feeding host-range including humans. This difference is important for efficient control of each species as well as predicting and preventing the expansion into new distribution ranges.

Rapid adaptive evolution of the diapause program during range expansion of an invasive mosquito.

In temperate climates, the recurring seasonal exigencies of winter represent a fundamental physiological challenge for a wide range of organisms. In response, many temperate insects enter diapause, an alternative developmental program, including developmental arrest, that allows organisms to synchronize their life cycle with seasonal environmental variation. Geographic variation in diapause phenology contributing to local climatic adaptation is well documented. However, few studies have examined how the rapid evolution of a suite of traits expressed across the diapause program may contribute to climatic adaptation on a contemporary timescale. Here, we investigate the evolution of the diapause program over the past 35 years by leveraging a "natural experiment" presented by the recent invasion of the Asian tiger mosquito, Aedes albopictus, across the eastern United States. We sampled populations from two distinct climatic regions separated by 6° of latitude (∼700 km). Using common-garden experiments, we identified regional genetic divergence in diapause-associated cold tolerance, diapause duration, and postdiapause starvation tolerance. We also found regional divergence in nondiapause thermal performance. In contrast, we observed minimal regional divergence in nondiapause larval growth traits and at neutral molecular marker loci. Our results demonstrate rapid evolution of the diapause program and imply strong selection caused by differences in winter conditions.