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

Abstract

Questing in ticks is essential for locating a host, and this behavioral response can occur at fairly low temperatures. Little is known about the dynamics between questing 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 acclimation 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.

Introduction

Ticks are important parasites of humans and other animals and vectors of many pathogens (Sonenshine and Roe 2013; Dantas-Torres et al. 2012). The distribution of ticks is limited by a variety of factors ranging from host availability to environmental factors, such as dehydration and thermal stress. Low winter temperatures are a major factor that limits tick distributions as most, if not all, ticks are freeze intolerant (Minigan et al. 2018; Muñoz-Leal and González-Acuña 2015). Ticks appear to be capable of overwintering in any of the unfed stages, thus they are likely exposed to cold stress as larvae, nymphs and adults (Brunner, Killilea, and Ostfeld 2012; Rosendale et al. 2016; Sonenshine and Roe 2013; Semtner and Hair 1976). To resist cold exposure, ticks utilize a suite of mechanisms including synthesis of specific metabolites (Neelakanta et al. 2010; Rosendale et al. 2020; Z.-J. Yu et al. 2014), shifting gene expression to tolerate cold-induced stress (Rosendale et al. 2020; Z. Yu et al. 2020), induction of a diapause-like syndrome (Yoder et al. 2016; Belozerov, Fourie, and Kok 2002; Z Yu et al. 2020), and retreat into sheltered microhabitats (C. S. Burks et al. 1996; Burks et al. 1996; Rosendale et al. 2016). Recent studies have shown that rapid cold hardening (RCH) and more prolonged cold acclimation occurs in several tick species and are likely critical components of tick cold tolerance (Rosendale et al. 2016; Rosendale et al. 2020; Wang et al. 2017; Z.-J. Yu et al. 2014).

Rapid cold hardening and prolonged cold acclimation have been studied in many arthropod systems, where either short (minutes to hours) or long (days to weeks) periods of sub-lethal cold exposure improves cold hardiness (Denlinger and Lee 2010; Lee et al. 1987; Teets and Denlinger 2013; Teets et al. 2020). Diverse mechanisms underlying the processes of RCH and prolonged cold acclimation include synthesis and accumulation of specific cryoprotectants, changes in the composition of cell membranes, inhibition of apoptotic pathways, changes in stress protein abundance, protein phosphorylation shifts, and metabolic differences (reviewed in (Teets et al. 2020; Teets and Denlinger 2013). Importantly, RCH and cold acclimation can have beneficial effects where biological processes such as movement, feeding, and mating can occur at lower temperatures (Shreve et al. 2004; Teets et al. 2019; Kelty et al. 1996; Srithiphaphirom et al. 2019). A few studies have examined RCH and general cold acclimation in ticks. First, RCH is documented in some, but not all, tick species (Vandyk et al. 1996; Z.-J. Yu et al. 2014; Rosendale et al. 2020; Holmes et al. 2018; Wang et al. 2017). Short term cold acclimation in the bush tick corresponded to changes in water and protein content, but not glycerol, and developmental stages had differing responses (Z.-J. Yu et al. 2014). Recent studies in the American dog tick revealed specific transcriptional and metabolome changes associated with RCH, where betaine was identified as a critical component of tick RCH response (Rosendale et al. 2020). Beyond basic studies on survival, little is known about the ecological and behavioral impacts of RCH or prolonged cold acclimation on ticks.

Off-host periods are one of the most stressful periods in the life cycle of ticks, and this particularly critical for tick larvae (Leal et al. 2020). Tick larvae are highly susceptible to dehydration due to their small size (Benoit et al. 2007; Yoder et al. 2012), but have a relatively high resistance to cold (Rosendale et al. 2016; Wang et al. 2017; Fieler et al. 2020). Larvae, as well as other stages, rely on finite nutrient reserves that cannot be replenished until the next blood meal (Rosendale et al. 2019). Thus, tick longevity and starvation resistance are directly linked as long as individuals can remain hydrated (Rosendale et al. 2016, 2019, 2017). Importantly, periods of stress can increase the rate at which ticks consume nutrient reserves (McCue et al. 2017; Rosendale et al. 2017). Previous studies showed that metabolic rate increased following RCH (Teets et al. 2019), but despite increased metabolism the breakdown of nutrient reserves is lower compared to individuals directly exposed to cold stress without RCH. Ticks can quest at near freezing temperatures (Schulz et al. 2014), and there is significant variation in the temperature threshold at which ticks can remain active (Clark 1995). For ticks that reside in extreme regions, such as the Antarctic Peninsula, lower limits on activity fall below 0°C (Lee and Baust 1987), suggesting that questing is likely to occur at subzero temperatures, but higher temperatures likely increase the number of ticks feeding (Benoit et al. 2009). Previous observations that questing occurs at low temperatures and the dynamics between metabolism and activity during RCH and cold acclimation suggest that these processes may be important components of tick biology.

In this study, we examined the impact of RCH and prolonged cold acclimation on tick larvae, specifically how these forms of cold hardening impact survival, activity, and questing. Because increased questing and activity at low temperatures could have potential trade-offs, we examined how this could impact larval survival of three tick species, Ixodes uriae, Dermacentor variabilis, and Amblyomma americanum. The seabird tick, I. uriae, is distributed in the Northern and Southern hemispheres in polar and cold regions (Muñoz-Leal and González-Acuña 2015). The American dog tick, D. variabilis, and Gulf Coast tick, A. maculatum, are found in eastern North America while A. maculatum is present in more southern regions (Minigan et al. 2018; Teel et al. 2010). Based on our studies, RCH and cold acclimation increased questing at lower temperatures, which is likely due to activity occurring at lower temperatures. This increased activity was associated with a higher rate of oxygen consumption, which reduced starvation resistance and tick longevity. These results indicate that increased cold resistance following acclimation could allow ticks to locate a host at low temperatures and likely impact tick longevity.

Materials and Methods

Ticks

Fed females of I. uriae were collected under rocks by a penguin colony on Christine Island (64°47’S 64°01’W) near Palmer Station, Antarctica in Jan. 2017. Females were held at 15-20°C, 12:12 L:D and allowed to deposit eggs. Larvae were utilized four weeks after emergence. Fed females for D. variabilis and A. americanum were acquired from Ectoservices (Cary, NC). Both A. americanum and D. variabilis originated from Stillwater, OK. The seabird tick females most likely fed on Adélie penguin (Pygoscelis adeliae) based on the location of collection under rocks near penguin rookeries. Ticks from Ectoservices (Henderson, NC) were fed on sheep (Ovis aries). These were stored at 24-25°C and 12:12 L:D until eggs were deposited. Larvae were used for experiments two-three weeks after emergence. Relative humidities were held at 93% with a saturated salt solution of KNO₃ (Winston and Bates 1960)

Rapid cold hardening (RCH) and acclimation

RCH was accomplished based on methods previously used for tick larvae (Holmes et al. 2018; Rosendale et al. 2016; Rosendale et al. 2020). To induce RCH, larvae were exposed to 8-10°C for four hours. Prolonged acclimation was accomplished by exposing ticks to 14-16°C for one week. Relative humidity was maintained at 90-95% RH during the exposures. Controls were held under the same conditions as the fed females until use in the experiments. Survival was assessed following initial acclimation by confirming ticks could move legs and walk at least five body lengths.

Survival and chill coma onset assessment

Previous studies have shown that cold acclimation and RCH impact survival of ticks following subsequent cold tolerance in some, but not all, ticks (Holmes et al. 2018; Rosendale et al. 2016; Rosendale et al. 2020; Wang et al. 2017; Vandyk et al. 1996). To establish if RCH and cold acclimation occur in the larvae used in this study, a survival assay was performed. After RCH, chill coma onset and survival were assessed. The tick larvae were moved to −16 or −14 and −20 or −22°C based on the species for two hours. These discriminating temperatures were based on those established in previous studies on tick larval cold tolerance (Fieler et al. 2020). Following this cold treatment, survival was assessed after 48 h as described previously. Fifteen groups of 10 individual larvae were mixed from different gravid females.

Chill coma onset was determined as based on methods previously described for I. uriae (Lee and Baust 1987). Following RCH and cold acclimation, individual tick larvae were placed on a cooling stage (Bioquip) that was gradually lowered (~0.5°C/minute). The temperature at which individual larvae are unable to walk, move legs, and respond to probing is considered the chill coma onset temperature. Fifteen groups of 10 individual larvae were mixed from different gravid females. Survival and chill coma was assessed for A. americanum, D. variabilis, and I. uriae.

Assessment of activity

To measure activity, we used a Locomotor Activity Monitor (TriKinetics Inc., Waltham, MA, USA) in conjunction with DAMSystem3 Data Collection Software (TriKinetics). For these studies, we only used D. variabilis and A. americanum larvae. Individual tick larvae were placed in standard Drosophila vials (25mm diameter and 95 mm height). Tubes were placed horizontally in the monitor and the entire system was placed within a plastic container that was held at 93% RH using a saturated salt solution of potassium nitrate. This apparatus was placed within an incubator. After a two hour acclimation, general activity was measured for 6h at 5 and 10°C. A two-way ANOVA was used to examine the relationship between cold acclimation and activity at different temperatures. Twenty larvae were examined for each treatment and temperature that were the progeny of three different females. Monitoring of activity was conducted on A. americanum, D. variabilis, and I. uriae.

Larval questing assays

To determine the impact of RCH and prolonged acclimation, we performed an assay to evaluate questing based on previously developed methods that were scaled to examine larvae rather than adults (Rosendale et al. 2019). In the middle of each opening of an eight well plate, a wooden applicator stick (50 mm x 2.2 mm) was secured with hot glue. Moistened coconut fiber (5 mm in depth) was added to each well. Larvae in groups of five or individually were placed within each well. The walls of each well were coated with petroleum jelly to discourage ticks climbing the sides of each well. Following RCH, tick larvae were immediately moved to a questing apparatus and allowed to acclimate for 6 h before the start of each questing trial. Questing assays were conducted at 5 and 10°C. At the beginning of each trial, a pulse of breath (from the experimenter) was used to stimulate the tick larvae. The number that moved into the questing position within 10 min was recorded. After each experiment, ticks that did not quest were moved to 20°C and examined to ensure no mortality had occurred. For all treatments, 10 groups of six individuals were examined. Questing assay were conducted on A. americanum, D. variabilis, and I. uriae.

Oxygen consumption

Larval tick O₂ consumption was measured using microrespirometers used previously in tick systems (Rosendale et al. 2017) and modified for studies on tick larvae (Fieler et al. 2020). Groups of 15 larvae were positioned within the microrespirometers. Ticks were allowed space for movement ~ 1 cm³. Each of the units was placed within a temperature-controlled water bath at either 5 or 10°C. and allowed to equilibrate before measurements. CO₂ production, measured through the movement of KOH solution in a needle, was measured every hour for 6 hours. Assuming that one mole of O₂ is consumed for every mole of CO₂ that is released, oxygen consumption was calculated by using the distance traveled by the KOH and was expressed as nl O₂ mg⁻¹ mass tick⁻¹ h^-1. At the end of each assay, ticks were probed to ensure that individuals were capable of movement. For all treatments, three replicates were used from four different gravid females for a total of twelve treatments. Oxygen consumption studies were conducted on D. variabilis.

Longevity and starvation assay

Longevity is directly tied to starvation resistance in ticks at relative humidities that allow water balance to be maintained (Rosendale et al. 2017, 2019; Benoit and Oyen 2021). Briefly, tick larvae (two weeks after emergence) were exposed to RCH or prolonged acclimation, held at 5°C for 12 days, and then moved to 22-24°C and monitored for survival weekly or at specific timer intervals. Relative humidity was held at 93%. For all treatments, eight groups of ten ticks were mixed from four different gravid females. Longevity and starvation assay studies were conducted on I. uriae and D. variabilis.

Statistics

R was utilized for all statistical analyses (R Core Team 2013). All data were examined for normal distribution before analyses. ANOVA was used to compare the impact of RCH and cold acclimation of survival, chill come, activity, questing, and respiration. Differences between each treatment were established with Tukey’s posthoc test. To determine the effect of RCH and acclimation on larval survival, we performed a beta regression using treatments as fixed effects. Beta regressions are appropriate for continuous dependent variables that are restricted between 0 and 1, in this case the proportion of larvae alive each fortnight (Ferrari and Cribari-Neto 2004). Beta regressions were performed using the RStudio package ‘Betareg’ (Cribari-Neto and Zeileis 2009). Heatmaps were generated with the use of pheatmap. Other figures were generated with ggplot. All statistical results are in Table S1–S7.

Results

RCH and cold acclimation improves tick larval cold tolerance

Previous studies documented the process of RCH and cold acclimation in tick larvae, but other species, such as Ixodes scapularius, seem to lack RCH (Holmes et al. 2018; Rosendale et al. 2016; Burks et al. 1996; Z.-J. Yu et al. 2014). Cold acclimation and RCH did not impact larval tick survival (Fig. 1; Table S1). Survival was reduced during exposure to low temperature (Table S1). Survival was greater in all three tick species following RCH and exposure to temperatures near their thermal limits, with the exception of I. uriae at −16°C (Fig. 1; Table S1). A similar response was noted for cold acclimation (Fig. 1). Even when results were not significant, RCH and cold acclimation, in general, increased survival at all cold temperatures (Table S1).

Figure 1 -. Rapid cold hardening (RCH) and prolonged cold acclimation (Accl.) improve survival.

Left, survival of tick larvae following cold exposure (−14 and −20°C for Amblyomma americanum and −16 and −22°C for Ixodes uriae and Dermacentor variabilis). ✱, indicates significantly different compared to control at the respective temperature. Results and statistical analyses are included in Table S1. Fifteen replicates of ten individuals were conducted for each species. Right, chill coma onset of tick larvae. ✱, indicates significantly different compared to control at the respective temperature. Results and statistical analyses are included in Table S2. Twenty replicates were conducted for each species.

Chill coma onset varied significantly between species: I. uriae became unresponsive at temperatures below freezing, while the other two species became unresponsive at temperatures above freezing (Fig. 1; Table S2). Only D. variabilis had a significant decrease in chill coma onset at 5°C, which allowed the larvae to be more active 2°C lower than in the controls (Table S2). As with the survival analyses, RCH and cold acclimation tended to lower chill coma onset, but this response was not significant (Table S2).

RCH and cold acclimation increases tick activity and questing at low temperatures.

Previous studies have shown that RCH improves organismal performance at sublethal temperatures (Teets et al. 2019; Powell and Bale 2004). General larval activity was higher at 10°C when compared to 5°C (Fig. 2; Table S3). RCH had a significant impact on activity at 5°C for all three tick species and I. uriae at 10°C (Fig. 2; Table S3). A significant increase in activity compared to control was noted for A. americanum at 10°C (Fig. 2; Table S3). Similar to previous results, although insignificant, RCH and cold acclimation had a general trend of increased activity at lower temperatures.

Figure 2 -. Rapid cold hardening (RCH) and prolonged cold acclimation (Accl.) increase activity and questing at lower temperatures.

Top, activity of tick larvae following hardening and acclimation at 5°C and 10°C. ✱, indicates significantly different compared to control at the respective temperature. Results and statistical analyses are included in Table S3. Twenty replicates were conducted for each species and each treatment. Bottom, questing of tick larvae following hardening and acclimation at 5°C and 10°C. ✱, indicates significantly different compared to control at the respective temperature. Results and statistical analyses are included in Table S4. Ten replicates of six individuals were conducted for each species. Relative control levels based on comparison between treatments for each species.

The increased activity suggested that tick host-seeking (questing) could be shifted following RCH or acclimation. Significant increases in questing were noted at 5°C following RCH for A. americanum and D. variabilis when compared to those with no pre-treatment (Fig. 2; Table S4). No significant increases were noted for I. uriae following acclimation (Table S4). As with activity, most pre-treatment yielded showed the trend of increased, albeit not significant, in questing at both 5°C and 10°C.

RCH and cold acclimation increases oxygen consumption and reduces longevity

Trade-offs associated with RCH have been linked to increased heat tolerance (Overgaard and Sørensen 2008), survival (Overgaard et al. 2007; Basson et al. 2012), and fertility issues (Everman et al. 2018). Following RCH, oxygen consumption was significantly greater in cold-exposed larvae compared with control larvae at both 5°C and 10°C for D. variabilis (Fig. 3; Table S5). Cold acclimation did not significantly elevate oxygen consumption at 5°C and 10°C (Fig. 3, Table S5).

Figure 3 -. Rapid cold hardening (RCH) and prolonged cold acclimation (Accl.) increases oxygen consumption.

Left, oxygen consumption of tick larvae following hardening and acclimation at 5°C and 10°C. ✱, indicates significantly different compared to control at the respective temperature. Results and statistical analyses are included in Table S5. Ten replicates of fifteen individuals were conducted for each species and each treatment.

As metabolic output, starvation, and longevity are linked in ticks (Rosendale et al. 2019, 2017; Lighton and Fielden 1995), increased metabolic output suggests that tick longevity could be impacted. Although not statistically significant, the D. variabilis control larvae tended to survive longer than those exposed to RCH or acclimation, suggesting that both the process of RCH and cold acclimation could result in a decline in starvation resistance in D. variabilis (Fig. 4; Table S6). When examined for survival at 8, 20, and 32 weeks, RCH reduced longevity of I. uriae larvae (Fig. 4; Table S6). These results suggest a distinctive trade-off associated with cold acclimation in tick larvae is a reduction in longevity.

Figure 4 -. Rapid cold hardening (RCH) and prolonged cold acclimation (Accl.) decreases longevity.

Top, survival of Dermacentor variabilis larvae following hardening and acclimation at 5°C for two weeks and then at 22-24°C. Bottom, survival of I. uriae larvae following hardening and acclimation at 5°C for two weeks and then at 22-24°C. ✱, indicates significantly different compared to control at the respective temperature. Results and statistical analyses are included in Table S6. Ten replicates of ten individuals were conducted for each species.

Discussion

This study indicates that both RCH and prolonged cold acclimation directly impact the survival of three species of tick larvae. Chill coma onset temperatures declined following acclimation, suggesting the potential for increased activity under low temperatures. This was confirmed by measuring general activity, which was improved at 5 and 10°C. As with activity, questing occurred at lower temperatures when tick larvae experienced RCH and cold acclimation. Lastly, oxygen consumption increased at lower temperatures, which resulted in a reduction in longevity. These studies indicate that thermal acclimation can have substantial impacts on tick biology, both beneficial in allowing questing at lower temperatures but also detrimental as reflected in decreased longevity.

A key aspect of RCH and general cold acclimation is increased survival along with a lower chill coma onset as temperatures decline (Rosendale et al. 2016; Teets and Denlinger 2013; Lee et al. 1987; Chen et al. 1987). Initially, RCH and cold acclimation were thought to have only minor to no effects on tick cold tolerance (Burks et al. 1996). Importantly, these previous studies were performed on field-collected ticks, which can have drastically different physiological ages that can directly impact stress resistance (Pool et al. 2017; Uspensky et al. 2006; Uspensky 1995). Recent studies on adult D. variabilis concerning RCH have identified specific molecular mechanisms associated with this process. Metabolite changes include increases in several amino acids and betaine, which overlapped with the RNA-seq studies (Rosendale et al. 2020). Mechanisms used by larvae to accomplish cold acclimation are likely similar to those found in adults, with the major exception being that tick larvae which have not yet fed may have more limited nutrient reserves. Our studies confirm that RCH and more prolonged cold acclimation likely improve survival during cold exposure in most metastriate (Dermacentor and Amblyomma) and prostriate (Ixodes) ticks

Along with survival, chill coma tends to occur at a lower temperature following cold acclimation. Dynamics between RCH and chill coma have been studied in a few insect systems (Findsen et al. 2013; Larsen and Lee 1994; Kelty et al. 1996; Srithiphaphiron et al. 2019). The specific mechanism of chill coma is most likely due to acute neuromuscular dysfunction, followed by a subsequent loss of ion balance within the extracellular hemolymph (MacMillan et al. 2012; Findsen et al. 2013; Carrington et al. 2020). Ticks are commonly observed to quest at temperatures below 5°C (Schulz et al. 2014), an observation we have also made during our collections of ticks in southwestern Ohio. These temperatures are much lower than previous observations of the cold-induced arrestment of activity in ticks (Clark 1995; Lee and Baust 1987). As some tick larval species are likely to survive the winter or can be present during cooler winter or fall months (Davidson et al. 1994; Kollars et al. 2000; Ostfeld et al. 1996), a reduced chill coma onset may allow ticks to function at temperatures normally below their normal cold temperature threshold.

Given the slight decrease in the chill coma onset temperature, an increased activity level at lower temperatures following RCH and prolonged cold acclimation is unsurprising. We also noted that questing is increased at lower temperatures for all three tick species. These observations provide a direct ecological role for RCH and prolonged cold acclimation by decreasing the lower thermal limit of questing, one of the most critical periods in tick biology (Leal et al. 2020; Tomkins et al. 2014; Perret et al. 2004; Randolph 2004). The process of questing directly exposes ticks to environmental conditions and increases the risk of predation (Leal et al. 2020). Questing at lower temperatures could reduce threats to survival such as water loss and predator exposure (Leal et al. 2020; Benoit et al. 2007), allowing for prolonged periods of questing outside of protected microhabitats. Conversely, an absence of suitable hosts in colder temperatures, for example due to hibernation, negate the advantage of questing at low temperatures. Importantly, future studies modeling tick survival should account for host-seeking during the winter, specifically during short warm periods above the chill coma onset limits induced by RCH and prolonged cold acclimation.

Add polar paragraph.

Few trade-offs associated with RCH and cold acclimation have been identified (Teets et al. 2020; Overgaard and Sørensen 2008; Powell and Bale 2004; Basson et al. 2012). Importantly, this process has not been examined in invertebrates with limited nutrient reserves. Ticks are very different compared to many other invertebrates (Ogden and Lindsay 2016), including water replenishment by active uptake for the air and feeding once during each development stage. Here, we observed that the oxygen consumption increased at 5°C and 10°C following RCH when compared to larvae without a pre-treatment. This increased metabolism likely results in a reduction in longevity. Importantly, tick larvae are likely to be exposed to multiple bouts of RCH under natural conditions, which is likely to have even greater impacts on longevity based on previous studies of multiple bouts of stress in ticks and other invertebrate systems (Rosendale et al. 2017; Benoit et al. 2010; Marshall and Sinclair 2011). The importance of the trade-offs between low thermal tolerance and longevity is that the physiological age of tick larvae will likely be increased due to the process of cold acclimation, reducing longevity when more favorable conditions occur.

In summary, these studies indicate that cold acclimation likely occurs in most temperate and polar tick species and likely facilitates questing at lower temperatures. A significant trade-off is a decrease in longevity as the process of acclimation and RCH will likely increase oxygen consumption to remain active at a lower temperature. Of importance, this suggests that ticks could utilize two strategies to survive cold conditions. First, ticks could remain active at low temperatures, allowing a greater thermal range to find a host, but occurring at the cost of reduced longevity. Second, chill coma onset could occur at higher temperatures and allow ticks to retain nutrient reserves, enabling prolonged survival when environmental conditions are more favorable. Due to the extensive geographic range of most tick species, both strategies are possible because lower temperature thresholds are unlikely to be reached at southern areas of tick distributions, and lower thermal tolerance thresholds at northern range limits could facilitate increased questing and successful host seeking at lower temperatures.

Supplementary Material

Table S1 and S2

Table S1- Results and statistical comparison for survival studies.

Table S2 - Results and statistical comparison for chill coma onset.

Table S3 and S4

Table S3 - Results and statistical comparison for activity studies.

Table S4 - Results and statistical comparison for questing studies.

Table S5

Table S5 - Results and statistical comparison for oxygen consumption studies.

Table S6 and S7

Table S6 - Results and statistical comparison for starvation/longevity studies.