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Supplementary Table S1. Representativeness of Study Participants Supplementary Table S2. Disease response as assessed by RECIST v1.1 by PD-L1 tumor cell expression (response-evaluable population). Supplementary Table S3. Disease response as assessed by RECIST v1.1 in prior line of therapy subgroups (response-evaluable population) and survival (as-treated population).

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

Supplementary Figure S1. Patient disposition. Supplementary Figure S2. Association of PD-L1 expression and HPV status with best antitumor response. Supplementary Figure S3. Kaplan-Meier distribution curves for (A) PFS and (B) OS in the as-treated population according to line of treatment and platinum-refractory status, including estimates of medians. Supplementary Figure S4. Peripheral HPV-18-specific T-cells on IFNγ ELISpot assay of PMBCs. Supplementary Figure S5. Peripheral HPV-16 (A, C) E6-specific and (B, D) E7-specific T-cell responses on IFNγ ELISpot assay of PMBCs. Supplementary Figure S6. Peripheral HPV-18 (A, C) E6-specific and (B, D) E7-specific T-cell responses on IFNγ ELISpot assay of PMBCs. Supplementary Figure S7. Baseline peripheral (A) HPV-16-specific and HPV-16 (B) E6-specific and (C) E7-specific T-cell counts on IFNγ ELISpot assay of PMBCs according to best response to treatment. Supplementary Figure S8. Baseline peripheral (A) HPV-18-specific and HPV-18 (B) E6-specific and (C) E7-specific T-cell counts on IFNγ ELISpot assay of PMBCs according to best response to treatment. Supplementary Figure S9. (A) HPV-16-specific and (B) HPV-18-specific T-cell count measured by IFNγ ELISpot assay of PMBCs over time per individual patient (n = 34), color-coded by best response, plotted on a log10 y-axis.

Purpose:Tumoral programmed cell death ligand-1 (PD-L1) expression is common in human papillomavirus (HPV)–associated head and neck squamous cell carcinoma (HNSCC). We assessed whether a DNA vaccine targeting HPV-16/18 E6/E7 with IL12 adjuvant (MEDI0457) combined with the PD-L1 inhibitor durvalumab could enhance HPV-specific T-cell response and improve outcomes in recurrent/metastatic HPV-16/18–associated HNSCC.Patients and Methods:In this phase Ib/IIa study, immunotherapy-naïve patients with ≥1 previous platinum-containing regimen (neoadjuvant/adjuvant therapy or for recurrent/metastatic disease) received MEDI0457 7 mg intramuscularly with electroporation on weeks 1, 3, 7, and 12, then every 8 weeks, plus durvalumab 1,500 mg intravenously on weeks 4, 8, and 12, then every 4 weeks, until confirmed progression and/or unacceptable toxicity. Coprimary objectives were safety and objective response rate (ORR; H0: ORR ≤ 15%); secondary objectives included 16-week disease control rate (DCR-16), overall survival (OS), and progression-free survival (PFS).Results:Of 35 treated patients, 29 were response evaluable (confirmed HPV-associated disease; received both agents). ORR was 27.6% [95% confidence interval (CI), 12.7–47.2; four complete responses, four partial responses]; responses were independent of PD-L1 tumor-cell expression (≥25% vs. 2-fold increase in tumor-infiltrating CD8+ T cells.Conclusions:MEDI0457 plus durvalumab was well tolerated. While the primary efficacy endpoint was not reached, clinical benefit was encouraging.

Purpose: Tumoral programmed cell death ligand-1 (PD-L1) expression is common in human papillomavirus (HPV)–associated head and neck squamous cell carcinoma (HNSCC). We assessed whether a DNA vaccine targeting HPV-16/18 E6/E7 with IL12 adjuvant (MEDI0457) combined with the PD-L1 inhibitor durvalumab could enhance HPV-specific T-cell response and improve outcomes in recurrent/metastatic HPV-16/18–associated HNSCC. Patients and Methods: In this phase Ib/IIa study, immunotherapy-naïve patients with ≥1 previous platinum-containing regimen (neoadjuvant/adjuvant therapy or for recurrent/metastatic disease) received MEDI0457 7 mg intramuscularly with electroporation on weeks 1, 3, 7, and 12, then every 8 weeks, plus durvalumab 1,500 mg intravenously on weeks 4, 8, and 12, then every 4 weeks, until confirmed progression and/or unacceptable toxicity. Coprimary objectives were safety and objective response rate (ORR; H0: ORR ≤ 15%); secondary objectives included 16-week disease control rate (DCR-16), overall survival (OS), and progression-free survival (PFS). Results: Of 35 treated patients, 29 were response evaluable (confirmed HPV-associated disease; received both agents). ORR was 27.6% [95% confidence interval (CI), 12.7–47.2; four complete responses, four partial responses]; responses were independent of PD-L1 tumor-cell expression (≥25% vs. 2-fold increase in tumor-infiltrating CD8+ T cells. Conclusions: MEDI0457 plus durvalumab was well tolerated. While the primary efficacy endpoint was not reached, clinical benefit was encouraging.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the devastating kinetoplastid parasites of the genusLeishmania, the causative agents of diseases collectively termed leishmaniasis. More than 40 pathogenicLeishmaniaspecies are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. As no approved efficacious vaccine exists, available drugs are expensive and/or toxic, and resistance is emerging, management of sand fly populations to break transmission is currently the most effective disease control strategy. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two of the most important sand fly species:Phlebotomus papatasi, a cutaneous leishmaniasis vector, (distributed in the Middle East and North Africa) andLutzomyia longipalpis,a visceral leishmaniasis vector (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission ofLeishmaniaparasites.Author SummaryThe leishmaniases are a group of neglected tropical diseases caused by protist parasites from the GenusLeishmania. DifferentLeishmaniaspecies present a wide clinical profile, ranging from mild, often self-resolving cutaneous lesions that can lead to protective immunity, to severe metastatic mucosal disease, to visceral disease that is ultimately fatal.Leishmaniaparasites are transmitted by the bites of sand flies, and as no approved vaccine exists, available drugs are toxic and/or expensive and resistance is emerging, new dual control strategies to combat these diseases must be developed, combining interventions on human infections and integrated sand fly population management. Effective vector control requires a good understanding of the biology of sand flies. To this end, we sequenced and annotated the genomes of two sand fly species that are important leishmaniasis vectors from the Old and New Worlds. These genomes allow us to better understand, at the genetic level, processes important in the vector biology of these species, such as finding hosts, blood-feeding, immunity, and detoxification. These genomic resources highlight the driving forces of evolution of two majorLeishmaniavectors and provide foundations for future research on how to better prevent leishmaniasis by control of the sand fly vectors.

Insects provide an unparalleled opportunity to link genomic changes with the rise of novel phenotypes, given tremendous variation in the numerous and complex adaptations displayed across the group. Among these numerous and complex adaptations, live-birth has arisen repeatedly and independently in insects and across the tree of life, suggesting this is one of the most common types of convergent evolution among animals. We sequenced the genome and transcriptome of the Pacific beetle-mimic cockroach, the only truly viviparous cockroach, and performed comparative analyses including two other viviparous insect lineages, the tsetse and aphids, to unravel the genomic basis underlying the transition to viviparity in insects. We identified pathways experiencing adaptive evolution, common in all viviparous insects surveyed, involved in uro-genital remodeling, maternal control of embryo development, tracheal system, and heart development. Our findings suggest the essential role of those pathways for the development of placenta-like structure enabling embryo development and nutrition. Viviparous transition seems also to be accompanied by the duplication of genes involved in eggshell formation. Our findings from the viviparous cockroach and tsetse reveal that genes involved in uterine remodeling are up-regulated and immune genes are down-regulated during the course of pregnancy. These changes may facilitate structural changes to accommodate developing young and protect them from the mothers immune system. Our results denote a convergent evolution of live-bearing in insects and suggest similar adaptive mechanisms occurred in vertebrates, targeting pathways involved in eggshell formation, uro-genital remodeling, enhanced tracheal and heart development, and reduced immunity.

The Antarctic mite (Alaskozetes antarcticus) is widely distributed on sub-Antarctic islands and throughout the Antarctic Peninsula, making it one of the most abundant terrestrial arthropods in the region. Despite the impressive ability of A. antarcticus to thrive in harsh Antarctic conditions, little is known about the biology of this species. In this study, we performed 16S rRNA gene sequencing to examine the microbiome of the final immature instar (tritonymph) and both male and female adults. The microbiome included a limited number of microbial classes and genera, with few differences in community membership noted among the different stages. However, the abundances of taxa that composed the microbial community differed between adults and tritonymphs. Five classes—Actinobacteria, Flavobacteriia, Sphingobacteriia, Gammaproteobacteria, and Betaproteobacteria—comprised ~ 82.0% of the microbial composition, and five (identified) genera—Dermacoccus, Pedobacter, Chryseobacterium, Pseudomonas, and Flavobacterium—accounted for ~ 68.0% of the total composition. The core microbiome present in all surveyed A. antarcticus was dominated by the families Flavobacteriaceae, Comamonadaceae, Sphingobacteriaceae, Chitinophagaceae and Cytophagaceae, but the majority of the core consisted of operational taxonomic units of low abundance. This comprehensive analysis reveals a diverse microbiome among individuals of different stages, with overlap likely due to their shared habitat and common feeding preferences as herbivores and detritivores. The microbiome of the Antarctic mite shows considerably more diversity than observed in mite species from lower latitudes.

The vertical transmission of microbes from mother to offspring is critical to the survival, development, and health of animals. Invertebrate systems offer unique opportunities to conduct studies on microbiome‐development‐reproduction dynamics since reproductive modes ranging from oviparity to multiple types of viviparity are found in these animals. One such invertebrate is the live‐bearing cockroach, Diploptera punctata. Females carry embryos in their brood sac, which acts as the functional equivalent of the uterus and placenta. In our study, 16S rRNA sequencing was used to characterize maternal and embryonic microbiomes as well as the development of the whole‐body microbiome across nymphal development. We identified 50 phyla and 121 classes overall and found that mothers and their developing embryos had significantly different microbial communities. Of particular interest is the notable lack of diversity in the embryonic microbiome, which is comprised exclusively of Blattabacteria, indicating microbial transmission of only this symbiont during gestation. Our analysis of postnatal development reveals that significant amounts of non‐Blattabacteria species are not able to colonize newborn D. punctata until melanization, after which the microbial community rapidly and dynamically diversifies. While the role of these microbes during development has not been characterized, Blattabacteria must serve a critical role providing specific micronutrients lacking in milk secretions to the embryos during gestation. This research provides insight into the microbiome development, specifically with relation to viviparity, provisioning of milk‐like secretions, and mother–offspring interactions during pregnancy.

Background The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. Results This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. Conclusions The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Introduction: Tertiary Lymphoid Structures (TLS) are highly organized ectopic lymphoid structures found in inflamed or tumor tissues, acting as sites of lymphoid recruitment and immune activation. A high TLS density within the tumor is commonly associated with an increased prognostic effect of TILs and with an improved disease free survival and overall survival for patients. However, the existence of conflicting studies suggest that multiple TLS features should be taken into account when assessing their prognostic value, such as their location, cellular composition, maturation stage and spatial organisation, as those may affect their functionalities. Methods: With the aim of gaining insights into TLS biology and evaluating the prognostic role of TLS in Non-Small Cell Lung Carcinoma (NSCLC) according to their multiple features, we developed a TLS multiplex immunofluorescent (mIF) panel that includes T cell (CD3, CD8), B cell (CD20), Follicular Dendritic cell (CD21, CD23) and mature dendritic cell (DC-LAMP) markers. We deployed this panel across a cohort of primary tumors from NSCLC patients (n=408) and established a mIF image analysis workstream to assess the status and spatial location of each cell within the tissue. A H&E staining of the same tissue section was performed to evaluate mIF spatial data in relation to the tumor context. Additional multi-omics assessments were conducted across the same cohort including; whole exome sequencing, NanoString transcriptomics, and immunohistochemistry (e.g. PD-L1, FOXP3). We have leveraged clinical metadata, including demographics (e.g. age, sex, smoking status) and clinical risk factors (e.g. stage, grade, Standard of Care treatment) with clinical follow up (e.g. OS, PFS) for prevalence analysis, novel biomarker identification, and survival association. Results: Assessment of the prevalence of each cell phenotype within the tumor tissue and TLS (tumor centre vs invasive margin; tumor epithelium vs stroma), the distance between each cell type, and the distance of non-TLS immune cells to the closest TLS will be described, demonstrating the different types of lymphoid aggregates and TLS and their functional status. An integrative analysis combining these spatial biology data with multi-omics and clinical data will be presented evaluating the prognostic value of TLS composition, maturation status and spatial organization, in correlation with additional biomarkers and clinical characteristics. Conclusion: This exploratory study using cutting-edge technologies enables us to better understand how TLS orchestrate an organised anti-tumour response, defining TLS spatial biomarker signatures, TLS gene signatures, and TLS features associated with NSCLC patient outcomes to evaluate in the clinic. Citation Format: Julie Berthe, Felix Segerer, Emily C. Jennings, Alma Andoni, Marco Testori, Megha Saraiya, Miljenka Vuko, Harald Hessel, Andreas Spitzmüller, Mari Heininen-Brown, Jorge Blando, Felicia Ng, Emma Jones, Sophie Willis, Michael Surace, Rieneke van de Ven, Tanja De Gruijl, Helen Angell. A multi-modal analysis approach leveraging multiplexed spatial phenotyping and multi-omics analysis to better understand the prognostic value of tertiary lymphoid structures in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1711.

Here, we report the genome sequence and characterization for a Blattabacterium strain isolated from the viviparous cockroach, Diploptera punctata, which provides amino acids critical for intrauterine embryo development. The genome was assembled by sequencing of the cockroach fat body, which is the location of this obligate symbiont.

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.

The oribatid mite Alaskozetes antarcticus, one of the most abundant terrestrial invertebrates in Antarctica, survives extreme temperature fluctuation and desiccation, and thrives in the short growing season characteristic of this polar environment. Several aspects of the mite’s ecology and physiology are well studied, but little is known about its reproduction. In this study, we utilize sex- and development-specific next-generation RNA-sequencing (RNA-seq) analyses to identify differentially regulated transcripts underlying reproduction of A. antarcticus. Pairwise comparisons between males, females, and tritonymphs revealed more than 4000 enriched transcripts based on different transcriptional levels among sexes and developmental stages. More than 500 of these enriched transcripts were differentially upregulated over 1000-fold. Many of the highly enriched and sex-specific transcripts were previously uncharacterized or have no known orthology. Of the transcripts identified, gene ontology-based analyses linked the transcriptional distinctions to differences in reproduction, chemosensation, and stress response. Our comparative approach allowed us to determine sexually dimorphic transcript expression in A. antarcticus. We anticipate that this study will provide a baseline to better understand the mechanisms that underlie reproduction in both polar and non-polar oribatid mites.

Current insights into the mosquito dehydration response rely on studies that examine specific responses but ultimately fail to provide an encompassing view of mosquito biology. Here, we examined underlying changes in the biology of mosquitoes associated with dehydration. Specifically, we show that dehydration increases blood feeding in the northern house mosquito, Culex pipiens, which was the result of both higher activity and a greater tendency to land on a host. Similar observations were noted for Aedes aegypti and Anopheles quadrimaculatus. RNA-seq and metabolome analyses in C. pipiens following dehydration revealed that factors associated with carbohydrate metabolism are altered, specifically the breakdown of trehalose. Suppression of trehalose breakdown in C. pipiens by RNA interference reduced phenotypes associated with lower hydration levels. Lastly, mesocosm studies for C. pipiens confirmed that dehydrated mosquitoes were more likely to host feed under ecologically relevant conditions. Disease modeling indicates dehydration bouts will likely enhance viral transmission. This dehydration-induced increase in blood feeding is therefore likely to occur regularly and intensify during periods when availability of water is low.

Viviparous reproduction is characterized by maternal retention of developing offspring within the reproductive tract during gestation, culminating in live birth. In some cases, a mother will provide nutrition beyond that present in the yolk; this is known as matrotrophic viviparity. While this phenomenon is best associated with mammals, it is observed in insects such as the viviparous cockroach, Diploptera punctata. Female D. punctata carry developing embryos in the brood sac, a reproductive organ that acts as both a uterus and a placenta by protecting and providing a nutritive secretion to the intrauterine developing progeny. While the basic physiology of D. punctata pregnancy has been characterized, little is known about the molecular mechanisms underlying this phenomenon. This study combined RNA-seq analysis, RNA interference, and other assays to characterize molecular and physiological changes associated with D. punctata reproduction. A comparison of four stages of the female reproductive cycle and males revealed unique gene expression profiles corresponding to each stage and between sexes. Differentially regulated transcripts of interest include the previously identified family of milk proteins and transcripts associated with juvenile hormone metabolism. RNA interference and methoprene application experiments established the potential impacts of bothbreakdown and synthesis reduction of juvenile hormone in maintaining pregnancy in D. punctata. These studies provide the comprehensive molecular mechanisms associated with cockroach viviparity, which will be a critical resource for comparative purposes among viviparity in insect systems.

Background Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. Results Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. Conclusions Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

BackgroundThe stable fly,Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the United States alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies.ResultsThis study examines stable fly biology by utilizing a combination of high-quality genome sequencing, microbiome analyses, and RNA-seq analyses targeting multiple developmental stages and tissues. In conjunction, manual curation of over 1600 genes was used to examine gene content related to stable fly reproduction, interactions with their host, host-microbe dynamics, and putative routes for control. Most notable was establishment of reproduction-associated genes and identification of expanded vision, chemosensation, immune repertoire, and metabolic detoxification pathway gene families.ConclusionsThe combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and knowledge will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship ofStomoxysto other blood-feeding (Glossina) and non-blood-feeding flies (medflies,Drosophila, house flies) will allow for understanding the evolution of blood feeding among Cyclorrhapha flies.

Background: Tsetse flies (Glossina sp.) are the sole vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood specific diet by both sexes and obligate bacterial symbiosis. This work describes comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans (G.m. morsitans), G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes) and Fusca (G. brevipalpis) which represent different habitats, host preferences and vectorial capacity. Results: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex linked scaffolds show increased rates of female specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse specific genes are enriched in protease, odorant binding and helicase activities. Lactation associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other characterized insects. Vision associated Rhodopsin genes show conservation of motion detection/tracking functions and significant variance in the Rhodopsin detecting colors in the blue wavelength ranges. Conclusions: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

One critical aspect of an insect's ability to overwinter successfully is the effective management of its water resources. Maintenance of adequate water levels during winter is challenging because of the prevailing low relative humidity at that time of year and the short supply of environmental water that is not in the form of ice. These issues are further exacerbated for insects overwintering as pupae, comprising an immobile stage that is unable to move to new microhabitats if conditions deteriorate. The present study compares the water balance attributes of diapausing and nondiapausing pupae of the corn earworm Helicoverpa zea Boddie, aiming to identify the mechanisms used by diapausing pupae to maintain water balance during winter. Diapausing pupae are 10% larger than nondiapausing individuals. Water loss rates for nondiapausing pupae are low (0.21 mg h−1) and are suppressed (0.01 mg h−1) in diapausing pupae. Cuticular lipids, which serve to waterproof the cuticle and thus suppress cuticular water loss, are more than two-fold more abundant on the surface of diapausing pupae, and oxygen consumption rates during diapause drop to almost one-third the rate observed in nondiapausing pupae. Water gain can be accomplished only when atmospheric water content is near saturation or during contact with free water. At moderate relative humidities (20–40%), water loss rates are very low for diapausing pupae, suggesting that these moth pupae have robust mechanisms for combating water loss. The exceptional ability of H. zea to suppress water loss during diapause is probably a result of the combined effects of increased size, more abundant cuticular lipids and decreased metabolic rates.

Mosquitoes are prone to dehydration and respond to this stress through multiple mechanisms, but previous studies have examined very specific responses and fail to provide an encompassing view of the role that dehydration has on mosquito biology. This study examined underlying changes in biology of the northern house mosquito,Culex pipiens, associated with short bouts of dehydration. We show that dehydration increased blood feeding propensity of mosquitoes, which was the result of both enhanced activity and a higher tendency to land on a host. Mosquitoes exposed to dehydrating conditions with access to water or rehydrated individuals experience no water loss and failed to display behavioral changes. RNA-seq and metabolome analyses following dehydration indicated that factors associated with energy metabolism are altered, specifically the breakdown of trehalose to yield glucose, which likely underlies changes in mosquito activity. Suppression of trehalose breakdown by RNA interference reduced phenotypes associated with dehydration. Comparable results were noted for two other mosquito species, suggesting this is a general response among mosquitoes. Lastly, field-based mesocosm studies usingC.pipiensrevealed that dehydrated mosquitoes were more likely to host feed, and disease modeling indicates dehydration bouts may increase transmission of West Nile virus. These results suggest that periods of dehydration prompt mosquitoes to utilize blood feeding as a mechanism to obtain water. This dehydration-induced increase in blood feeding is likely to intensify disease transmission during periods of low water availability.SignificanceDehydration stress has substantial impacts on the biology of terrestrial invertebrates. To date, no studies have elucidated the difference between dehydration exposure and realized water loss in relation to mosquito behavior and physiology. Our experiments show that direct dehydration stress increases mosquito activity and subsequent blood feeding, likely as a mechanism to locate and utilize a bloodmeal for rehydration. These dehydration-induced phenotypes were linked to altered carbohydrate metabolism that acts as a source of energy. This study provides important insight into the impact of mosquito-dehydration dynamics on disease transmission that is likely general among mosquitoes.

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite., The bed bug, Cimex lectularius, is a ubiquitous human ectoparasite with global distribution. Here, the authors sequence the genome of the bed bug and identify reductions in chemosensory genes, expansion of genes associated with blood digestion and genes linked to pesticide resistance.