Egg hatching success is influenced by the time of thermal stress in four hard tick species.

Ticks are blood-feeding arthropods responsible for the transmission of disease-causing pathogens to a wide range of vertebrate hosts, including livestock and humans. Tick-borne diseases have been implicated in significant economic losses to livestock production, and this threat will increase as these obligate parasites widen their geographical ranges. Similar to other ectotherms, thermal stress due to changing global temperatures has been shown to influence tick survival and distribution. However, studies on the influence of extreme temperatures in ticks have focused on advanced, mobile stages, ignoring immobile stages that cannot move to more favorable microhabitats. In this study, low- and high-temperature regimens were assessed in relation to egg viability for hard tick species-Amblyomma maculatum (Gulf Coast tick), Ixodes scapularis (black-legged tick), Dermacentor variabilis (American dog tick), and Rhipicephalus sanguineus (Brown dog tick). Tick eggs exposed early in development (freshly laid during early embryo development) were significantly more susceptible to thermal stress when compared with those exposed later in development (late embryo development denoted by a fecal spot). Based on our studies, differences in egg hatching success among treatments were greater than in hatching success when comparing species. Lastly, there was evidence of extreme thermal exposure significantly altering the hatching times of tick eggs for specific treatments. These results provide insights into the critical period for tick egg viability in relation to thermal exposure and tick survival associated with stress and climate change.

Dual assessment of transcriptional and metabolomic responses in the American dog tick following exposure to different pesticides and repellents.

The American dog tick, Dermacentor variabilis, is a major pest to humans and animals, serving as a vector to Rickettsia rickettsii, a bacterium responsible for Rocky Mountain spotted fever, and Francisella tularensis, which is responsible for tularemia. Although several tactics for management have been deployed, very little is known about the molecular response following pesticidal treatments in ticks. In this study, we used a combined approach utilizing transcriptomics and metabolomics to understand the response of the American dog tick to five common pesticides (amitraz, chlorpyrifos, fipronil, permethrin, and propoxur), and analyzed previous experimental data utilizing DEET repellent. Exposure to different chemicals led to significant differential expression of a varying number of transcripts, where 42 were downregulated and only one was upregulated across all treatments. A metabolomic analysis identified significant changes in acetate and aspartate levels following exposure to chlorpyrifos and propoxur, which was attributed to reduced cholinesterase activity. Integrating the metabolomics study with RNA-seq analysis, we found the physiological manifestations of the combined metabolic and transcriptional differences, revealing several novel biomolecular pathways. In particular, we discovered the downregulation of amino sugar metabolism and methylhistidine metabolism after permethrin exposure, as well as an upregulation of glutamate metabolism in amitraz treated samples. Understanding these altered biochemical pathways following pesticide and repellent exposure can help us formulate more effective chemical treatments to reduce the burden of ticks.

Metabolomic and transcriptomic responses of ticks during recovery from cold shock reveal mechanisms of survival.

Ticks are blood-feeding ectoparasites but spend most of their life off-host where they may have to tolerate low winter temperatures. Rapid cold hardening (RCH) is a process commonly used by arthropods, including ticks, to improve survival of acute low temperature exposure. However, little is known about the underlying mechanisms in ticks associated with RCH, cold shock and recovery from these stresses. In the present study, we investigated the extent to which RCH influences gene expression and metabolism during recovery from cold stress in Dermacentor variabilis, the American dog tick, using a combined transcriptomics and metabolomics approach. Following recovery from RCH, 1860 genes were differentially expressed in ticks, whereas only 99 genes responded during recovery to direct cold shock. Recovery from RCH resulted in an upregulation of various pathways associated with ion binding, transport, metabolism and cellular structures seen in the response of other arthropods to cold. The accumulation of various metabolites, including several amino acids and betaine, corresponded to transcriptional shifts in the pathways associated with these molecules, suggesting congruent metabolome and transcriptome changes. Ticks, D. variabilis and Amblyomma maculatum, receiving exogenous betaine and valine demonstrated enhanced cold tolerance, suggesting cryoprotective effects of these metabolites. Overall, many of the responses during recovery from cold shock in ticks were similar to those observed in other arthropods, but several adjustments may be distinct from the responses in other currently examined taxa.

Larval thermal characteristics of multiple ixodid ticks.

Temperature limits the geographic ranges of several tick species. Little is known about the thermal characteristics of these pests outside of a few studies on survival related to thermal tolerance. In this study, thermal tolerance limits, thermal preference, and the impact of temperature on activity levels and metabolic rate were examined in larvae for six species of ixodid ticks. Tolerance of low temperatures ranged from -15 to -24 °C with Dermacentor andersoni surviving the lowest temperatures. High temperature survival ranged from 41 to 47 °C, with Rhipicephalus sanguineus sensu lato having the highest upper lethal limit. Ixodes scapularis showed the lowest survival at both low and high temperatures. Thermal preference temperatures were tested from 0 to 41 °C. The majority of species preferred temperatures between 17 and 22 °C, while Dermacentor variabilis preferred significantly lower temperatures, near 12 °C. Overall activity was measured across a range of temperatures from 10 to 60 °C, and most tick species had the greatest activity near 30 °C. Metabolic rate was the greatest between 30 and 40 °C for all tick species and was relatively stable from 5 to 20 °C. The optimal temperature for tick larvae is likely near the thermal preference for each species, where oxygen consumption is low and activity occurs that will balance questing and conservation of nutrient reserves. In summary, tick species vary greatly in their thermal characteristics, and our results will be critical to predict distribution of these ectoparasites with changing climates.

The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control.

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.

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

Multi-level analysis of reproduction in an Antarctic midge identifies female and male accessory gland products that are altered by larval stress and impact progeny viability.

The Antarctic midge, Belgica antarctica, is a wingless, non-biting midge endemic to Antarctica. Larval development requires at least 2 years, but adults live only 2 weeks. The nonfeeding adults mate in swarms and females die shortly after oviposition. Eggs are suspended in a gel of unknown composition that is expressed from the female accessory gland. This project characterizes molecular mechanisms underlying reproduction in this midge by examining differential gene expression in whole males, females, and larvae, as well as in male and female accessory glands. Functional studies were used to assess the role of the gel encasing the eggs, as well as the impact of stress on reproductive biology. RNA-seq analyses revealed sex- and development-specific gene sets along with those associated with the accessory glands. Proteomic analyses were used to define the composition of the egg-containing gel, which is generated during multiple developmental stages and derived from both the accessory gland and other female organs. Functional studies indicate the gel provides a larval food source as well as a buffer for thermal and dehydration stress. All of these function are critical to juvenile survival. Larval dehydration stress directly reduces production of storage proteins and key accessory gland components, a feature that impacts adult reproductive success. Modeling reveals that bouts of dehydration may have a significant impact on population growth. This work lays a foundation for further examination of reproduction in midges and provides new information related to general reproduction in dipterans. A key aspect of this work is that reproduction and stress dynamics, currently understudied in polar organisms, are likely to prove critical in determining how climate change will alter their survivability.

Genome-enabled insights into the biology of thrips as crop pests.

BACKGROUND: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. RESULTS: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. CONCLUSIONS: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest.

BACKGROUND: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies. RESULTS: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. CONCLUSIONS: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

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.

Progressive behavioural, physiological and transcriptomic shifts over the course of prolonged starvation in ticks.

Ticks are obligatorily hematophagous but spend the majority of their lives off host in an unfed state where they must resist starvation between bouts of blood feeding. Survival during these extended off-host periods is critical to the success of these arthropods as vectors of disease; however, little is known about the underlying physiological and molecular mechanisms of starvation tolerance in ticks. We examined the bioenergetic, transcriptomic and behavioural changes of female American dog ticks, Dermacentor variabilis, throughout starvation (up to nine months post-bloodmeal). As starvation progressed, ticks utilized glycogen and lipid, and later protein as energy reserves with proteolysis and autophagy facilitating the mobilization of endogenous nutrients. The metabolic rate of the ticks was expectedly low, but showed a slight increase as starvation progressed possibly reflecting the upregulation of several energetically costly processes such as transcription/translation and/or increases in host-seeking behaviours. Starved ticks had higher activity levels, increased questing behaviour and augmented expression of genes related to chemosensing, immunity and salivary gland proteins. The shifts in gene expression and associated behavioural and physiological processes are critical to allowing these parasites to exploit their ecological niche as extreme sit-and-wait parasites. The overall responses of ticks to starvation were similar to other blood-feeding arthropods, but we identified unique responses that could have epidemiological and ecological significance for ticks as ectoparasites that must be tolerant of sporadic feeding.

The genome of the water strider Gerris buenoi reveals expansions of gene repertoires associated with adaptations to life on the water.

BACKGROUND: Having conquered water surfaces worldwide, the semi-aquatic bugs occupy ponds, streams, lakes, mangroves, and even open oceans. The diversity of this group has inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders our ability to more thoroughly investigate the molecular mechanisms underlying the processes of adaptation and diversification within this group. RESULTS: Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome; the first water strider genome to be sequenced thus far. The size of the G. buenoi genome is approximately 1,000 Mb, and this sequencing effort has recovered 20,949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to a water surface lifestyle. These expansions may affect key processes associated with growth, vision, desiccation resistance, detoxification, olfaction and epigenetic regulation. Strikingly, the G. buenoi genome contains three insulin receptors, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes affecting with opsin genes may be associated with wavelength sensitivity shifts in opsins, which is likely to be key in facilitating specific adaptations in vision for diverse water habitats. CONCLUSIONS: Our findings suggest that local gene duplications might have played an important role during the evolution of water striders. Along with these findings, the sequencing of the G. buenoi genome now provides us the opportunity to pursue exciting research opportunities to further understand the genomic underpinnings of traits associated with the extreme body plan and life history of water striders.

Dehydration prompts increased activity and blood feeding by mosquitoes.

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.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Low and high thermal tolerance characteristics for unfed larvae of the winter tick Dermacentor albipictus (Acari: Ixodidae) with special reference to moose.

We report that larvae of the winter tick Dermacentor albipictus, the only stage that will quest for a host, can tolerate short-term cold shock down to -25°C and short-term heat shock as high as 46°C. Unlike a three host-tick, larvae of D. albipictus have no preconditioning response to low or high temperature exposure by rapid cold hardening (RCH) or heat hardening, and poor ability to acclimate to low and high temperature extremes. Thermal tolerance limits were not improved as the result of larval clustering, and there was only a minimal effect due to changes in photoperiod. These larvae are freeze intolerant and die at higher temperatures (-5 to -10°C) from contact with ice by inoculative freezing. In absence of cold-associated resistance mechanisms, winter survival requires that larvae procure a host before the first snow cover. Their low and high temperature tolerance, however, is a key survival element that adapts them for off-host periods during summer, which in the arctic could allow for northern expansion.

Dehydration and starvation yield energetic consequences that affect survival of the American dog tick.

Ticks are obligate hematophagous arthropods, but may have to endure extended time (1-2years) between feedings. During these off-host periods, ticks must contend with a multitude of environmental stresses including prolonged or repeated exposure to desiccating conditions. In this study, we measured the energetic consequences of single and repeated bouts of dehydration of American dog ticks, Dermacentor variabilis, and examined the impact of energy reserves on tick survival during dehydration. Recently molted ticks exposed to a single period at 0% relative humidity (RH) for 5d lost ∼26% of their body water and showed 1.3- and 1.7-fold reductions in protein and lipid, respectively. These reduced energy reserves coincided with increased O2 consumption in dehydrated ticks. Exposure to repeated cycles of dehydration (0% RH, 48h) and rehydration (100% RH, 24h) also reduced energy reserves; however, ticks were able to fully recover their body water after 12 cycles of dehydration/rehydration and endured >20 cycles. Starvation of ticks, in the absence of dehydration, for 18 or 36weeks resulted in the loss of ∼20-40% of protein and 60% of lipid reserves. When ticks were exposed to continuous dehydration at 0% RH, their survival after 18weeks of starvation was only minimally impacted; however, individuals starved for 36weeks succumbed to dehydration much more rapidly than recently fed ticks. Both single and repeated dehydration exposures resulted in substantial energetic costs and ticks with limited energy reserves were more susceptible to dehydration-induced mortality, indicating that adequate energy reserves are critical for tolerance to dehydration stress and long-term success of ticks.

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species.

BACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control. RESULTS: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A high-quality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT. CONCLUSIONS: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution.