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.

Low heat tolerance and high desiccation resistance in nocturnal bees and the implications for nocturnal pollination under climate change.

Predicting insect responses to climate change is essential for preserving ecosystem services and biodiversity. Due to high daytime temperatures and low humidity levels, nocturnal insects are expected to have lower heat and desiccation tolerance compared to diurnal species. We estimated the lower (CTMin) and upper (CTMax) thermal limits of Megalopta, a group of neotropical, forest-dwelling bees. We calculated warming tolerance (WT) as a metric to assess vulnerability to global warming and measured survival rates during simulated heatwaves and desiccation stress events. We also assessed the impact of body size and reproductive status (ovary area) on bees' thermal limits. Megalopta displayed lower CTMin, CTMax, and WTs than diurnal bees (stingless bees, orchid bees, and carpenter bees), but exhibited similar mortality during simulated heatwave and higher desiccation tolerance. CTMin increased with increasing body size across all bees but decreased with increasing body size and ovary area in Megalopta, suggesting a reproductive cost or differences in thermal environments. CTMax did not increase with increasing body size or ovary area. These results indicate a greater sensitivity of Megalopta to temperature than humidity and reinforce the idea that nocturnal insects are thermally constrained, which might threaten pollination services in nocturnal contexts during global warming.

A U.S. Isolate of Theileria orientalis Ikeda Is Not Transstadially Transmitted to Cattle by Rhipicephalus microplus.

Theileria orientalis Ikeda has caused an epidemic of bovine anemia and abortion across several U.S. states. This apicomplexan hemoparasite is transmitted by Haemaphysalis longicornis ticks; however, it is unknown if other North American ticks are competent vectors. Since the disease movement is largely determined by the host tick range(s), the prediction of the T. orientalis spread among U.S. cattle populations requires determination of additional competent tick vectors. Although Rhipicephalus microplus has mostly been eradicated from the U.S., outbreaks in populations occur frequently, and the U.S. remains at risk for reintroduction. Since R. microplus is a vector of Theileria equi and T. orientalis DNA has been detected in R. microplus, the goal of this study was to determine whether R. microplus is a competent vector of T. orientalis. Larval R. microplus were applied to a splenectomized, T. orientalis Ikeda-infected calf for parasite acquisition, removed as molted adults, and applied to two T. orientalis naïve, splenectomized calves for transmission. After 60 days, the naïve calves remained negative for T. orientalis by PCR and cytology. Additionally, T. orientalis was not detected in the salivary glands or larval progeny of acquisition-fed adults. These data suggest that R. microplus is not a competent vector of the U.S. T. orientalis Ikeda isolate.

Thermal limits of Africanized honey bees are influenced by temperature ramping rate but not by other experimental conditions.

Interest in assessing the critical thermal limits of bees is rapidly increasing, as these physiological traits are good predictors of bees' potential responses to extreme temperature changes, which is relevant in the context of global climate change. However, estimates of thermal limits may be influenced by several factors and published studies differ in experimental methods and conditions, such as the rate of temperature change (ramping rate) and feeding status, which might yield inaccurate predictions and limit comparisons across taxa and regions. Using Africanized honey bees as a model organism, we assessed the effect of ramping rate (0.25, 0.5, 0.75, 1.0 and 1.5 °C min-1) and length of starvation (recently fed vs. fasted for 6, 12, and 18 h) on foragers' lower (CTMin) and upper (CTMax) thermal limits, as well as the effect of cold stress on CTMax. In addition, we evaluated the two approaches currently used to assess CTMax with a water bath: floating or submerging the testing vials in the bath. We found that critical thermal limits were influenced by ramping rates but not by the other assessed experimental conditions. On average, at ramping rates faster than 0.5 °C min-1, bees displayed a CTMin 1.1-2.6 °C lower and a CTMax 5.3-6.9 °C higher than those of the slowest ramping rate. We discuss the implications of these results and provide suggestions for future thermal studies on bees.

High thermal tolerance in high-elevation species and laboratory-reared colonies of tropical bumble bees.

Bumble bees are key pollinators with some species reared in captivity at a commercial scale, but with significant evidence of population declines and with alarming predictions of substantial impacts under climate change scenarios. While studies on the thermal biology of temperate bumble bees are still limited, they are entirely absent from the tropics where the effects of climate change are expected to be greater. Herein, we test whether bees' thermal tolerance decreases with elevation and whether the stable optimal conditions used in laboratory-reared colonies reduces their thermal tolerance. We assessed changes in the lower (CTMin) and upper (CTMax) critical thermal limits of four species at two elevations (2600 and 3600 m) in the Colombian Andes, examined the effect of body size, and evaluated the thermal tolerance of wild-caught and laboratory-reared individuals of Bombus pauloensis. We also compiled information on bumble bees' thermal limits and assessed potential predictors for broadscale patterns of variation. We found that CTMin decreased with increasing elevation, while CTMax was similar between elevations. CTMax was slightly higher (0.84°C) in laboratory-reared than in wild-caught bees while CTMin was similar, and CTMin decreased with increasing body size while CTMax did not. Latitude is a good predictor for CTMin while annual mean temperature, maximum and minimum temperatures of the warmest and coldest months are good predictors for both CTMin and CTMax. The stronger response in CTMin with increasing elevation, and similar CTMax, supports Brett's heat-invariant hypothesis, which has been documented in other taxa. Andean bumble bees appear to be about as heat tolerant as those from temperate areas, suggesting that other aspects besides temperature (e.g., water balance) might be more determinant environmental factors for these species. Laboratory-reared colonies are adequate surrogates for addressing questions on thermal tolerance and global warming impacts.

Neotropical stingless bees display a strong response in cold tolerance with changes in elevation.

Tropical pollinators are expected to experience substantial effects due to climate change, but aspects of their thermal biology remain largely unknown. We investigated the thermal tolerance of stingless honey-making bees, the most ecologically, economically and culturally important group of tropical pollinators. We assessed changes in the lower (CTMin) and upper (CTMax) critical thermal limits of 17 species (12 genera) at two elevations (200 and 1500 m) in the Colombian Andes. In addition, we examined the influence of body size (intertegular distance, ITD), hairiness (thoracic hair length) and coloration (lightness value) on bees' thermal tolerance. Because stingless beekeepers often relocate their colonies across the altitudinal gradient, as an initial attempt to explore potential social responses to climatic variability, we also tracked for several weeks brood temperature and humidity in nests of three species at both elevations. We found that CTMin decreased with elevation while CTMax was similar between elevations. CTMin and CTMax increased (low cold tolerance and high heat tolerance) with increasing ITD, hair length and lightness value, but these relationships were weak and explained at most 10% of the variance. Neither CTMin nor CTMax displayed significant phylogenetic signal. Brood nest temperature tracked ambient diel variations more closely in the low-elevation site, but it was constant and higher at the high-elevation site. In contrast, brood nest humidity was uniform throughout the day regardless of elevation. The stronger response in CTMin, and a similar CTMax between elevations, follows a pattern of variation documented across a wide range of taxa that is commonly known as the Brett's heat-invariant hypothesis. Our results indicate differential thermal sensitivities and potential thermal adaptations to local climate, which support ongoing conservation policies to restrict the long-distance relocations of colonies. They also shed light on how malleable nest thermoregulation can be across elevations.

Warm Blood Meal Increases Digestion Rate and Milk Protein Production to Maximize Reproductive Output for the Tsetse Fly, Glossina morsitans.

The ingestion of blood represents a significant burden that immediately increases water, oxidative, and thermal stress, but provides a significant nutrient source to generate resources necessary for the development of progeny. Thermal stress has been assumed to solely be a negative byproduct that has to be alleviated to prevent stress. Here, we examined if the short thermal bouts incurred during a warm blood meal are beneficial to reproduction. To do so, we examined the duration of pregnancy and milk gland protein expression in the tsetse fly, Glossina morsitans, that consumed a warm or cool blood meal. We noted that an optimal temperature for blood ingestion yielded a reduction in the duration of pregnancy. This decline in the duration of pregnancy is due to increased rate of blood digestion when consuming warm blood. This increased digestion likely provided more energy that leads to increased expression of transcript for milk-associated proteins. The shorter duration of pregnancy is predicted to yield an increase in population growth compared to those that consume cool or above host temperatures. These studies provide evidence that consumption of a warm blood meal is likely beneficial for specific aspects of vector biology.

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.

Cold hardening improves larval tick questing under low temperatures at the expense of longevity.

Questing in ticks is essential for locating a host, and this behavioral response can occur at regionally specific low temperatures for most tick species. Little is known about the dynamics between tick questing behavior and temperature in ticks, specifically how this may impact other aspects of tick biology. Here, we examine whether cold hardening increases questing in three larval tick species (Ixodes uriae, Dermacentor variabilis, and Amblyomma americanum) at low temperatures and whether cold hardening impacts longevity. Rapid cold hardening and prolonged cold acclimation benefitted ticks by decreasing the temperature of chill coma onset, and increased survival, activity, and questing in ticks at low temperatures. Oxygen consumption increased at low temperatures following acclimation in larvae, suggesting this process has a distinct metabolic expense. This increased metabolism associated with hardening led to a substantial reduction in larval longevity as nutrient reserves are limited and cannot be replenished until a host is located. These studies suggest that tick larvae, and likely other developmental stages, require a delicate balance between the need for questing at low temperatures and survival until the first blood meal.

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.

Tonic Immobility Is Influenced by Starvation, Life Stage, and Body Mass in Ixodid Ticks.

The ability to escape predation modulates predator-prey interactions and represents a crucial aspect of organismal life history, influencing feeding, mating success, and survival. Thanatosis, also known as death feigning or tonic immobility (TI), is taxonomically widespread, but understudied in blood-feeding vectors. Hematophagous arthropods, such as ticks, are unique among animals as their predators (birds, mice, lizards, frogs, and other invertebrates) may also be their source of food. Therefore, the trade-off between predator avoidance and host-seeking may shift as the time since the last bloodmeal increases. Because ticks are slow-moving and unable to fly, or otherwise escape, we predicted that they may use TI to avoid predation, but that TI would be influenced by time since the last bloodmeal (starvation). We therefore aimed to quantify this relationship, examining the effect of starvation, body mass, and ontogeny on TI for two tick species: Dermacentor variabilis (Say) (Acari: Ixodidae) and Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae). As we predicted, the duration and use of TI decreased with time since feeding and emergence across species and life stages. Therefore, ticks may become more aggressive in their search for a bloodmeal as they continue to starve, opting to treat potential predators as hosts, rather than avoiding predation by feigning death. Antipredator behaviors such as TI may influence the intensity and amount of time ticks spend searching for hosts, driving patterns of tick-borne pathogen transmission. This identification and quantification of a novel antipredation strategy add a new component to our understanding of tick life history.

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.

Biogeographic parallels in thermal tolerance and gene expression variation under temperature stress in a widespread bumble bee.

Global temperature changes have emphasized the need to understand how species adapt to thermal stress across their ranges. Genetic mechanisms may contribute to variation in thermal tolerance, providing evidence for how organisms adapt to local environments. We determine physiological thermal limits and characterize genome-wide transcriptional changes at these limits in bumble bees using laboratory-reared Bombus vosnesenskii workers. We analyze bees reared from latitudinal (35.7-45.7°N) and altitudinal (7-2154 m) extremes of the species' range to correlate thermal tolerance and gene expression among populations from different climates. We find that critical thermal minima (CTMIN) exhibit strong associations with local minimums at the location of queen origin, while critical thermal maximum (CTMAX) was invariant among populations. Concordant patterns are apparent in gene expression data, with regional differentiation following cold exposure, and expression shifts invariant among populations under high temperatures. Furthermore, we identify several modules of co-expressed genes that tightly correlate with critical thermal limits and temperature at the region of origin. Our results reveal that local adaptation in thermal limits and gene expression may facilitate cold tolerance across a species range, whereas high temperature responses are likely constrained, both of which may have implications for climate change responses of bumble bees.

Local adaptation across a complex bioclimatic landscape in two montane bumble bee species.

Understanding evolutionary responses to variation in temperature and precipitation across species ranges is of fundamental interest given ongoing climate change. The importance of temperature and precipitation for multiple aspects of bumble bee (Bombus) biology, combined with large geographic ranges that expose populations to diverse environmental pressures, make these insects well-suited for studying local adaptation. Here, we analyzed genome-wide sequence data from two widespread bumble bees, Bombus vosnesenskii and Bombus vancouverensis, using multiple environmental association analysis methods to investigate climate adaptation across latitude and altitude. The strongest signatures of selection were observed in B. vancouverensis, but despite unique responses between species for most loci, we detected several shared responses. Genes relating to neural and neuromuscular function and ion transport were especially evident with respect to temperature variables, while genes relating to cuticle formation, tracheal and respiratory system development, and homeostasis were associated with precipitation variables. Our data thus suggest that adaptive responses for tolerating abiotic variation are likely to be complex, but that several parallels among species can emerge even for these complex traits and landscapes. Results provide the framework for future work into mechanisms of thermal and desiccation tolerance in bumble bees and a set of genomic targets that might be monitored for future conservation efforts.

Distance, elevation and environment as drivers of diversity and divergence in bumble bees across latitude and altitude.

Identifying drivers of dispersal limitation and genetic differentiation is a key goal in biogeography. We examine patterns of population connectivity and genetic diversity using restriction site-associated DNA sequencing (RADseq) in two bumble bee species, Bombus vosnesenskii and Bombus bifarius, across latitude and altitude in mountain ranges from California, Oregon and Washington, U.S.A. Bombus vosnesenskii, which occurs across a broader elevational range at most latitudes, exhibits little population structure while B. bifarius, which occupies a relatively narrow higher elevation niche across most latitudes, exhibits much stronger population differentiation, although gene flow in both species is best explained by isolation with environmental niche resistance. A relationship between elevational habitat breadth and genetic diversity is also apparent, with B. vosnesenskii exhibiting relatively consistent levels of genetic diversity across its range, while B. bifarius has reduced genetic diversity at low latitudes, where it is restricted to high-elevation habitat. The results of this study highlight the importance of the intersect between elevational range and habitat suitability in influencing population connectivity and suggest that future climate warming will have a fragmenting effect even on populations that are presently well connected, as they track their thermal niches upward in montane systems.