Using haem concentration as a metric of physiological age to infer demographic structure in natural field and forest populations of host-seeking Amblyomma americanum adults

ABSTRACT Understanding how patterns of host-seeking behaviour differ across demographic classes in Amblyomma americanum (L.), the lone star tick, is essential in evaluating species potential as a vector of pathogens. Our objective was to characterize the relative contributions of two cryptic developmental cohorts, newly moulted and overwintered adults, to the A. americanum host-seeking population. To determine cohort identity, we used haem concentration as a metric of physiological age in ticks collected from field and forest habitats. Emergence of ticks displaying uncharacteristically high haem concentrations in late spring and early summer would indicate that newly moulted adults resume questing activity in the same season of their nymphal engorgement. Overall, haem concentration decreased significantly throughout the active season of adults from March to July of 2017 in northeast Missouri. Males displayed higher average haem concentrations than females when controlled for date of capture, but habitat-mediated differences were not significant. No subset of ticks with inflated haem concentrations was collected during the time that activization of newly moulted adults was feasible, suggesting that A. americanum adults undergo post-moulting behavioural diapause under natural conditions.


Introduction
Amblyomma americanum (L.), the lone star tick, is a three-host tick and vector of pathogens throughout the central and southeast United States. Recent range expansion (Springer et al. 2014) north and west from its historical distribution will likely expose more humans and domestic animals to the diseases caused by Amblyomma-associated pathogens, which include ehrlichiosis, southern tick-associated rash illness, Heartland virus disease, Bourbon virus disease, and tularaemia (Centers for Disease Control and Prevention 2018). Understanding pathogen dynamics in natural populations of A. americanum is therefore of rising importance to public health and agriculture.
To persist in the environment, tick-borne pathogens must optimally exploit phenology not only of their preferred host but also of physiologically distinct tick life stages. Maintenance of tick-borne encephalitis in some areas of Europe, for example, is dependent on horizontal pathogen transmission between larval and nymphal Ixodes ricinus co-feeding on the same rodent hosts (Randolph et al. 1999). Minor changes in tick phenology across life stages, therefore, can have drastic impacts on pathogen transmission cycles (Kurtenbach et al. 2006). For this reason, understanding the distinct patterns of activity across demographic classes in ticks is essential in evaluating their vector potential.
Host-seeking behaviour of larvae, nymphs, and adults is well documented in A. americanum (Lancaster 1955;Hair and Howell 1970;Mount et al. 1993;Jackson et al. 1996;Cilek and Olson 2000;Mangan et al. 2018), but behavioural variation within each life stage is difficult to interpret in natural populations. In part, this stems from our inability to determine the true chronological age of wild-caught ticks. However, physiological age, defined in this context as the gradual depletion of tick nutritional reserves along with any associated physical changes, serves as a tenable alternative that has been demonstrated across many ixodid species to have serious implications for tick activization, behaviour, susceptibility to acaricides, and suitability as a vector of pathogens (Uspensky 1995).
Quantification of physiological age in ixodid ticks is made possible by their unique life history characteristics. Ticks can survive long intervals without feeding, observed to span up to 2 years and 9 months in adult A. americanum (Semtner and Hair 1976). This ability results from having an exceptionally low metabolic rate relative to body mass (Lighton and Fielden 1995) and employing an energetically conservative sit-and-wait foraging strategy, where tick movement rarely occurs except in the presence of a host or in search of more hydrating microenvironments. Low variability in energy expenditure during extended fasting periods, therefore, allows tick nutritional reserves to serve as a reliable metric of physiological age.
Many histological, anatomical, and metabolic properties can be used to measure physiological age in ixodid ticks, though the precision of techniques varies across species (Uspensky 1995). In contrast to Ixodes ricinus (Randolph et al. 2002), for example, reductions in lipid content of A. americanum occur with too much variance to be a useful measure of ageing (Jaworski et al. 1984;Williams et al. 1986). Additionally, age-related changes in midgut ultrastructure of A. americanum are difficult to quantify accurately (Williams et al. 1985). The concentration of haemoglobin and its metabolites, however, was shown to be a reliable indicator of A. americanum physiological age in both laboratory (Jaworski et al. 1984) and field (Williams et al. 1986) conditions.
As ticks cannot synthesize haem (Perner et al. 2016), haemoglobin is absorbed from their bloodmeal and processed into nontoxic haematin crystals that are stored in haemosomes of digestive cells (Lara et al. 2003). From here, haem is either transported by carrier proteins (Maya-Monteiro et al. 2000;Gudderra et al. 2001) and vitellogenins (Thompson et al. 2007) to other tissues, or is expelled from the midgut epithelium into the gut lumen by exocytosis or cell detachment (Sonenshine and Anderson 2014 mechanisms cause haematin stores to be gradually depleted over time such that haem content can approximate physiological age. Estimations of physiological age may be especially useful in characterizing the demographic structure of host-seeking A. americanum adults. Adults and nymphs generally emerge from winter conditions to begin questing during March and April in northeast Missouri (Mangan et al. 2018). As nymphs in laboratory conditions only require 39 days to progress from host attachment to ecdysis (Troughton and Levin 2007), spring-feeding nymphs could feasibly moult into adults and resume host-seeking behaviour before May, expediting the A. americanum life cycle. Dynamic population modelling (Ludwig et al. 2015) and field observations of rabbit-fed nymphs (Semtner et al. 1973), however, do not support this hypothesis, instead suggesting that newly moulted adults undergo behavioural diapause in their first year rather than searching for hosts.
In this study, by using the concentration of haemoglobin equivalents to characterize relative contributions of aged and newly moulted adults to the host-seeking population, we determine whether adult-stage behavioural diapause is prevalent in A. americanum under natural conditions in northeast Missouri. If nymphs which fed and moulted into adults during the spring immediately join the active population, we would expect to observe adults with notably large haem concentrations during late spring and early summer relative to those earlier in the year. Additionally, we analyse how haem concentration differs in A. americanum adults according to sex and habitat. Discrepancies in haem concentration or rate of depletion across groups may indicate that differences in life history have consequential effects on tick energy utilization.

Tick collection
Amblyomma americanum adults were collected on dates from 8 March 2017 to 15 July 2017 in two habitats approximately 300 m apart in Adair County, Missouri, one representing an old field habitat of primarily non-native grasses and the other a second-growth forest dominated by hickory. Sampling effort was not standardized across days or between habitats, but all ticks were collected at times between 9:00 and 14:00 using a combination of drag sampling and dry ice baiting. In drag sampling, a 1 m 2 flannel cloth was pinned to a wooden dowel and dragged throughout the habitat, stopping approximately every 30 m to clear the cloth of ticks. In dry ice baiting, dry ice was placed on a flannel cloth and allowed to sublimate to attract ticks. Ticks found on clothes of researchers were also collected. To prevent cross-contamination, clothes and equipment were thoroughly checked for ticks prior to travel between sites. Ticks were gently handled by the legs using fine point tweezers, placed inside individually labelled 0.5 mL microcentrifuge tubes, and frozen in the lab at −20°C for later analysis. Collection was abandoned after 15 July 2017 due to lack of success in capturing adult A. americanum. Intervals between collection dates ranged from 2 to 18 days depending on weather and field worker availability.

Haem determination
The concentration of haem-containing compounds in each adult was determined using the cyanmethaemoglobin method (Cook 1973). Drabkin's reagent was created by adding 0.5 mL Brij L23 Solution (Sigma, Lot No. SLBT8516) to 1 g sodium bicarbonate, 200 mg potassium ferricyanide, and 50 mg potassium cyanide dissolved in 1 L of distilled water. Haemoglobin and its haemcontaining derivatives are oxidized in Drabkin's reagent by alkaline potassium ferricyanide to yield methaemoglobin. Methaemoglobin then reacts with potassium cyanide to form cyanmethaemoglobin, which is spectrophotometrically active at 540 nm. Lyophilized haemoglobin powder (Sigma Lot No. SLBR9638 V) was serially diluted in Drabkin's reagent to establish a standard curve of 10 points ranging from 15 to 220 μg/mL. The same haemoglobin standard curve and batch of Drabkin's reagent were used throughout the analysis.
Wet weights of frozen adults were recorded to the nearest microgram using a Cahn C-35 ultra-microbalance. Each adult was thoroughly homogenized in 0.5 mL Drabkin's reagent using finetipped scissors, after which an additional 1 mL Drabkin's reagent was added. Homogenates were centrifuged for three 10-min cycles at 5223 x g in an Eppendorf 5415 C centrifuge at room temperature, discarding the pellet each time. The absorbance of the final supernatant was read by a CARY 50 UV-Vis spectrophotometer, fitted to the standard curve, and adjusted for sample volume to determine haem contentthe total mass of haemoglobin and/or haemoglobin equivalents isolated from each sample. Haem content and tick wet weight were then used to calculate haem concentration for each tick, expressed as μg of haemoglobin equivalents per mg tick wet weight.

Statistical analyses
All statistical analyses were performed in R v3.6.2. Linear regression with indicator variables was used to determine how the concentration of haemoglobin equivalents was affected by the date of capture when controlled for tick sex and habitat. Square-roottransformation of tick haem concentration was used to meet the regression assumptions of equal variance and normality.

Results
Overall, 172 A. americanum adults were collected (59 field females, 14 forest females, 71 field males, 28 forest males; see Supplementary Table 1). Two field males and one forest male were excluded due to loss of tissue or apparent integumental damage. One outlier, a field male collected on 20 March 2017, displayed a concentration of haemoglobin equivalents over twice that of any other tick (224.6 μg/mg) and was therefore removed from subsequent analyses. Concentration of haemoglobin equivalents in remaining adult A. americanum collected between 8 March and 15 July 2017 ranged from 8.5 to 99.2 μg/mg. Of all ticks included in our analysis, only one adult collected on 18 April displayed a greater haem concentration (99.2 μg/mg) than the most haem-rich tick collected in March (89.0 μg/mg) (Figure 1).
Haem concentration significantly decreased with date of capture (t = −9.25, df = 164, p < 0.0001) and exhibited no interactions with habitat or sex (Figure 1). There was no significant difference (t = 1.84, df = 164, p = 0.0674) in tick haem concentration between forest and field habitats on average when controlled for date of capture, but males displayed a significantly higher concentration than females on average (t = 3.21, df = 164, p = 0.0016; Figure 1). Our final model (F 2,165 = 54.38, p < 0.0001, Radj 2 = 0.40) accounted for 40% of variation in haem concentration of collected ticks. Nine of the 168 ticks analysed (5.4%) displayed studentized residuals greater than two standard deviations above the mean predicted value of the model (Figure 2), which is more than the 2.5% expected due to chance. Only two ticks (1.2%) displayed studentized residuals greater than two standard deviations below the mean predicted value of the model.

Discussion
Our results indicate that newly moulted A. americanum adults do not become active in substantial numbers before winter inactivity in northeast Missouri. If fed nymphs joined the active adult population immediately after moulting, we would expect to observe a group of spring adults with inflated haem stores relative to those collected in March. No such group of ticks displaying high haem levels was apparent in our data. Instead, the concentration of haemoglobin equivalents decreased consistently throughout the adult active season. This lack of host-seeking activity in newly moulted adults, which we presume represents behavioural diapause, is consistent with observations of rabbit-fed ticks in Oklahoma (Semtner et al. 1973) and dynamic population modelling of the A. americanum life cycle (Ludwig et al. 2015).
Post-moult behavioural diapause in adults is likely an adaptive strategy to maximize offspring survivorship and minimize adult energy expenditure. Activization, feeding, and mating of newly moulted adults during early summer would result in egg incubation during the most desiccating months of the year, under which larval hatching success falls dramatically (Koch 1983). Delaying activization of newly moulted adults until late summer would also produce high offspring mortality, as the resulting autumn larvae would have little chance to find a host before winter temperatures prove fatal (Sonenshine and Levy 1971;Koch 1984). Unlike larvae, however, unfed adults exhibit high overwinter survivorship ranging between 50 (Semtner and Hair 1976) and 91% (Koch 1984). Inactivity of newly moulted adults until early spring, therefore, allows for the incubation of eggs during more mild spring and early summer temperatures, resulting in lower offspring mortality. As second-winter survivorship of unfed adults is also substantial (Koch 1984), early summer subsidence in activity of overwintered adults (Mangan et al. 2018) may occur for similar reasons.
Though the ticks in this study overwintered together from the previous year, there was substantial variation in haem concentration across individuals. As this variability suggests differences in physiological age, active adults may have obtained their nymphal bloodmeals across a wide range of time. Ticks with much higher  haem concentrations than our model predictions, for example, may represent adults which obtained a nymphal blood meal later in the previous year than the rest of their cohort. While nymphs are most active in spring, a small resurgence in nymphal activity is commonly observed during late summer and early autumn in northeast Missouri (Mangan et al. 2018). Adults arising from these second-wave nymphs would likely have higher haem reserves on average than those which fed and moulted several months earlier in the spring. In contrast, ticks with much lower haem concentrations than model predictions may represent adults that survived two consecutive winters without feeding, thereby depleting their nutritional reserves.
The outlier detected on 20 March may have found a host as a nymph during winter or late-autumn months, and therefore exhibits greater haem stores than other overwintered adults. Amblyomma americanum does not exhibit strict winter diapause (Stewart et al. 1998), and days with temperatures above the 12.3°C mean minimum coordinated activity threshold of nymphs (Clark 1995) occurred in Adair County during November, December, and January of 2016-2017 (National Centers for Environmental Information 2020). Additionally, replete nymphs under warmer April conditions required an average of 51 days to moult into adults, and moulting time consistently increased with decreasing temperature (Koch 1983). In aggregate, this indicates that the removed outlier likely acquired its nymphal bloodmeal during or before January 2017. In Oklahoma, Amblyomma americanum was documented feeding on white-tailed deer (Odocoileus virginianus) to some extent during all months of the year (Patrick and Hair 1977). The prevalence of late-autumn and winter feeding in A. americanum throughout its distribution, however, requires further documentation.
In concordance with our observation that haem concentration is higher in males than in females, Williams et al. (1986) found that male haem concentration decreased at a slower rate than females in field conditions. These observations may occur as a result of sexmediated differences in questing activity under cold temperatures. Females display an average minimum coordinated activity threshold 1.1°C lower than males (Clark 1995). If this allows females to emerge from winter quiescence earlier in the spring or quest for longer periods on a given day relative to males, females would expend more energy over time and deplete their haem reserves. Lower haem concentration in females could also, however, be an artefact of differential hydration status between sexes. Under desiccating laboratory conditions, male A. americanum aged 1 month and 8 months displayed significantly higher relative rates of water loss than similarly aged females (Sigal 1990). Therefore, if males in our study were more dehydrated than females, the concomitant decrease in wet weight would inflate male haem concentration. Total male per cent water content, however, was generally higher than females across age groups in both laboratory (Jaworski et al. 1984) and field conditions (Williams et al. 1986), which would result in dilution of apparent haem levels. Further research of sex-dependent differences in the life history of A. americanum may elucidate whether discrepancies in haem concentration and rate of utilization have consequential effects on patterns of tick activity.
While there were no significant differences in haem concentration between habitats, we analysed only a small number of forest ticks from a single site, none of which were captured before mid-April. Given the limits of our data, future research may reveal habitat-mediated differences in energy utilization that our study did not detect.
Overall, by using haem concentration as a metric of physiological age in wild-caught A. americanum, this study observed that adults arising from spring-fed nymphs in northeast Missouri do not comprise a significant portion of the questing population in the year of their moulting. Similar haem analysis of A. americanum nymphs may reveal whether a commonly observed late-summer and autumn resurgence in nymph activity (Lancaster 1955;Hair and Howell 1970;Mount et al. 1993;Jackson et al. 1996;Goddard 2007;Mangan et al. 2018) represents aged or newly moulted ticks. When employed in multiple regions, this method could provide insight into differences in demographic structure and life history across populations. If, for example, northern reaches of the expanding A. americanum distribution (Springer et al. 2014) have more mild summer temperatures which fail to trigger behavioural diapause in newly moulted adults, then life history and demographic structure could change in a manner which has significant implications for pathogen transmission. Furthermore, a shorter period of winter inactivity at southern latitudes (Lancaster 1955;Cilek and Olson 2000) suggests that warming due to climate change may allow ticks to resume questing earlier in the year. This could potentially allow for newly moulted adults to feed, mate, and oviposit well before summer temperatures reduce hatching success, ultimately shortening the A. americanum life cycle.