Thermal Ecology of the Western Tent Caterpillar

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2020

Heat for the Masses: Thermal Ecology of the Western Tent Caterpillar

Victoria Dahlhoff University of Montana, Missoula

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Recommended Citation Dahlhoff, Victoria, "Heat for the Masses: Thermal Ecology of the Western Tent Caterpillar" (2020). Graduate Student Theses, Dissertations, & Professional Papers. 11673. https://scholarworks.umt.edu/etd/11673

This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. HEAT FOR THE MASSES: THERMAL ECOLOGY OF THE WESTERN TENT CATERPILLAR

VICTORIA C. DAHLHOFF

B.S. Bates College, Lewison ME, 2015

Thesis

presented in partial fulfillment of the requirements for the degree of

Master of Science in Ecology and Evolution

The University of Montana Missoula, MT

September 2020

Submitted for approval by:

Scott Wittenburg, Graduate School Dean

H. Arthur Woods, Chair Division of Biological Sciences

Creagh Breuner Division of Biological Sciences

Bret Tobalske Division of Biological Sciences

Caroline Williams Department of Integrative Biology, University of California Berkeley

Abstract A unique feature of some gregarious, colonial insects is their ability to create external structures that alter environmental conditions for the entire (often family) group. A combination of physical alteration of local microhabitats and behavioral thermoregulation allows many of these animals to actively control their body temperatures, which allows them to regulate energy use and metabolism in variable thermal environments. Here I describe mechanisms of microhabitat modification and thermal regulation in the western tent caterpillar, Malacosoma californicum pluviale. Tent caterpillars build communal silk tents, whose temperatures can rise substantially above ambient air temperature. I experimentally manipulated colony sizes and examined effects on tent temperatures and on rates of larval development and mortality. I predicted that large colonies would construct warmer tents, allowing larvae to grow faster in cool spring conditions. I found that temperatures in tents of large colonies (100-150 individuals) were up to 22 °C higher than ambient air temperature, and on average 15 °C higher than the mean temperature of tent temperatures of smaller colonies (10 individuals). As a consequence, larvae from larger colonies reached the final instar 30% faster than those from smaller colonies, and only larvae from large colonies survived to pupation. I then tested three hypotheses regarding excess temperatures observed in for tents of larger caterpillar colonies: (1) The tent collects heat like a greenhouse, (2) aggregating caterpillars act biophysically as a larger organism, reducing heat loss and increasing insulation to trap heat, and (3) the caterpillars warm the tent with their own metabolic heat. Using infrared imaging, I recorded daytime body temperatures of caterpillars in naturally occurring colonies, alone or in groups, either on or off their tents. I found that grouped caterpillars located off their tent had slightly higher body temperatures than solitary ones (about 1-2 °C), a result that was replicated using operative temperature models, where no metabolic heat is generated. However, caterpillars grouped on their tent reached significantly higher temperatures than those off the tent (4-5 °C), suggesting that both a warm tent and behavioral thermal regulation by grouping contribute significantly to observed excesses in caterpillar habitat and body temperature. With the ability to both physically alter their local microhabitats and behaviorally thermoregulate, one might predict that this species may be less vulnerable to climate change than other temperate insects.

Keywords: insect physiology, climate change, extended phenotype, infrared imaging, colony size, Malacosoma, silk, body temperature, development rate, LT50

ii Introduction

A challenge facing climate biologists is the accuracy with which we are able to predict changes in the thermal environments that organisms experience. A significant body of literature demonstrates that changes in macroclimate cause complex and sometimes unpredictable changes in microclimate, and that organismal responses to these changes reflect their behavioral abilities to buffer or exploit local microclimates (Deutsch et al., 2008; Dillon et al., 2010; Huey et al., 2012; Pincebourde & Woods, 2012; Potter et al., 2013; Williams et al., 2014). (Woods et al., 2015). Insects are an excellent group with which to examine the importance of microclimate variation. As ectotherms, their body temperatures integrate macroclimatic conditions, local biophysical processes, and behavioral thermoregulation.

The degree of microhabitat modification, and the possibilities for behavioral thermoregulation, also depend on whether insects live singly or in colonies. There are different levels of sociality in organisms, with highly-organized, closely related groups with reproductive division of labor at the highest level, and loose aggregations of largely unrelated individuals at the lowest level (Wilson, 1971, 1975). Many social organisms fall somewhere between these extremes. A key characteristic influencing variation in groups is the number of individuals they contain (Wilson, 1971). The effects of colony size on group-living organisms have been studied in many systems. Group living has been shown to increase foraging success (Caraco & Wolf, 1975; Clark & Mangel, 1986) protection from predators (Alexander, 1974; Tyler, 1995), and success in competition (Buss, 1981). These studies, however, focus primarily on vertebrates, even though many species that Wilson would classify as “social” are arthropods, including arachnids (social spiders) and insects (bees, ants, etc.). Even in those systems, most studies focus only on effects of colony size in eusocial insects like social bees, which have hierarchical, genetically-based colonial social systems (Dornhaus et al., 2006; Herrmann et al., 2007).

To date, there has been little work examining effects of colony size on the fitness of moderately social arthropods. The most thorough work is by Avilés and colleagues, on social spiders (Avilés & Tufino, 1998; Hoffman & Avilés, 2017; Purcell & Avilés, 2008). They found that although offspring in larger colonies are more likely to survive to maturity, the probability of individual females reproducing decreases with colony size. Conversely, larger colonies do not survive better in the presence of parasitoids, as they are more likely to be parasitized. Avilés

1 concluded that there is an overall benefit to group living, with peak fitness reached in colonies of intermediate size. While this result is valuable for understanding how colony size affects fitness in moderately social arthropods, it still leaves open questions about how the thermal ecology of colonies changes with colony size, and whether colony members can modify their thermal environments in response to changing climatic conditions.

The extended phenotype An extended phenotype is an extension of an organism that modifies and interacts with the environment, ultimately influencing the physiology of that organism (Dawkins, 1999). For example, bird nests are extended phenotypes; they are phenotypes of the birds (objects that result from their nest-building behaviors) that buffer eggs and nestlings from variation in temperature and wind in the surrounding environment (e.g. weaver bird nests; Crook, 1963; White et al., 1975). Many insects and other arthropods also have extended phenotypes, most obviously those that engineer their environments to fit their physiological needs (Lill & Marquis, 2007). Web- building spiders use their webs to catch prey and as a shelter (Blamires, 2010). Extended phenotypes are observed in many insect species. For example, sawfly larvae mine leaves and develop inside galls (Wagner et al., 1993), some species of caterpillars construct shelters by rolling leaves or silking them together (Berenbaum, 1999), and weaver ants construct shelters out of leaves tied together with silk (Anderson & McShea, 2001). In some instances, especially in insects living on modified leaves, the extended phenotype emerges from complex interactions between insect and plant (Pincebourde & Casas, 2006). Pincebourde & Casas, 2019, for example, showed that six species of arthropods feeding on apple leaves could either warm up or cool down the leaves, depending on the arthropod’s effect on leaf transpiration. By altering the experience of organisms, extended phenotypes can modify the selection pressures these organisms experience, and can therefore change the course of evolution in these systems (Turner, 2004). Indeed, in Pincebourde and Casas’s study, species that raised leaf temperatures had higher upper thermal tolerances, whereas those that depressed leaf temperatures had lower upper thermal tolerances. By studying extended phenotypes and their role in modifying how an organism experiences its environment, researchers can understand better the selection pressures that shape populations and can more readily anticipate the effects of changing environments.

Tent caterpillars

2 In temperate insects, a key biophysical problem is inadequate heat in springtime (Fitzgerald, 1995; Joos et al., 1988; Knapp & Casey, 1986). Tent caterpillars (genus Malacosoma) solve this problem in part by building silk tents. Larvae orient the largest flat face of their tent toward the brightest incoming light, which is usually the noon sun (Fitzgerald & Willer, 1983; Moore et al., 1988). This face provides a platform on which larvae can bask, and creates a large boundary layer that lowers convective heat loss (Fitzgerald, 1995; Frid & Myers, 2002; Joos et al., 1988). On a sunny spring day, body temperatures of larvae on the surface of the tent may reach temperatures of 20°C or higher above ambient air temperature (Casey et al., 1988; Joos et al., 1988; Knapp & Casey, 1986). The tent is thus an extended phenotype by which larvae transform local macroenvironments into the microhabitats in which they operate most effectively (Bailey, 2012; Blamires, 2010; Casey et al., 1988; Dawkins, 1999; Fitzgerald, 1995; Laland & Sterelny, 2006; Odling-Smee et al., 2013). The ability to establish and maintain an intact tent may play a large role in enhancing survival to pupation for social, tent-building species (e.g., Ruf & Fiedler, 2005). Therefore, elucidating pathways of heat gained and lost by caterpillars on the tent is critical for understanding adaptive strategies and behaviors in these and other gregarious insect species.

Here I address three hypotheses regarding the role of tents in caterpillar thermal biology:

1. The tent collects heat like a greenhouse, and caterpillars gain heat by being in contact with it. Using operative temperature models of eastern tent caterpillars (Malacosoma americanum), (Joos et al., 1988) found that in the presence of solar radiation, tents with no live caterpillars were at least 4°C warmer than ambient air temperature, and under sunny conditions were up to 23°C higher than ambient. They proposed that the tent was acting as a greenhouse, absorbing and trapping incoming solar radiation. In the context of behavioral thermoregulation, basking caterpillars on the tent would therefore gain heat passively through conduction while sitting on the tent.

2. Caterpillars aggregate, conserving heat by functioning biophysically as a larger organism. Across different species of social caterpillars, aggregations of caterpillars consistently maintain body temperatures above those of single caterpillars of the same size, regardless of their orientation to the sun (Joos et al., 1988; Klok & Chown, 1999), and larger groups tend to achieve temperatures above those of smaller groups (Halperin, 1990). However, there is still a gap in

3 understanding how these aggregated caterpillars get so much warmer. Tent caterpillars, like many other species of caterpillars, are covered in hair-like setae. Setae increase the thickness of the boundary layer surrounding the caterpillar, thereby reducing the rate of convective heat loss that is caused by air moving over the surface of the caterpillar while still allowing for radiative heat gain. Heat loss savings provided by setae can be significant- for gypsy moth caterpillars, larvae with experimentally-removed setae lost heat 30-45% faster than those with intact setae (Casey & Hegel, 1981). Grouping behavior in social caterpillars with setae could further reduce convective heat loss, both by reducing passive heat loss among individuals via reduction of total surface area exposed to the environment, and because a larger surface of setae may create a more efficient insulating boundary layer in grouped caterpillars than solo ones.

3. Caterpillars warm their tents with metabolic heat Tent temperatures of the eggar moth, Eriogaster lanestris, rose ~2.5 to 3 °C above ambient air temperature when larvae aggregated on the tent, an increase that could be accounted for by metabolic heat production (Ruf & Fiedler, 2000). In a subsequent study of the same species, researchers found that the spectral characteristics of the silk did not allow for greenhouse conditions to develop, as the silk transmitted short wavelength radiation while blocking long wavelength radiation. Additionally, they found that tent temperatures dropped to match ambient air temperature once the source of radiation was gone, indicating that tents did not have the capacity to trap heat. The multiple layers of silk could reduce convective heat loss, but the main thermoregulatory function of the tent in this species seems to depend on caterpillars storing metabolic heat by grouping (Ruf & Fiedler, 2002b). This prediction is consistent with results of a study of M. americanum; body temperatures of grouped caterpillars exceed air temperatures by up to 13°C, but empty tents were not warmer than ambient air, even when exposed to solar radiation (Knapp & Casey, 1986).

These hypotheses are not mutually exclusive. Indeed, all three could contribute to elevating body temperatures above ambient air temperatures. Here I evaluate these three hypotheses in the western tent caterpillar Malacosoma californicum, in order to better understand the role of behavior and extended phenotype in thermal ecology and physiology, and how they may allow for survival in the face of a changing environment.

4 Materials and Methods

Study System Malacosoma californicum Packard, 1864 (Lasiocampidae) is a moth whose larvae – known as western tent caterpillars -- are voracious defoliators of trees and shrubs. It is common in forests, woodlands, grasslands and riparian habitats across the American and Canadian West and Great Plains, and its latitudinal distribution extends from southern Canada to northern Mexico (Furniss & Carolin, 1977). There are at least six known subspecies in the genus; populations of M. californicum pluviale, the focus of my work, are most prevalent in the Pacific Northwest and southern Canada, though isolated populations have been found as far east as western Quebec and northern New England (Ciesla & Ragenovich, 2008). Although western tent caterpillars are typically cosmopolitan, generalist herbivores, hostplant use varies among populations and subspecies across the geographic range (Fitzgerald, 1995) and may show some evidence of local adaptation (e.g. Barnes et al., 2016). The preferred hostplants for populations in western Montana (USA) are wild rose (Rosa spp.) and black hawthorn (Crataegus douglasii).

Western tent caterpillars are univoltine. Adults eclose in mid to late July to mate and lay eggs; they do not feed and only live for a few days. After mating with multiple males, females lay masses of 150 to 250 eggs, which encircle branches of the hostplant. Eggs develop through late summer and early fall; individuals overwinter inside their eggs as unhatched, first instar larvae (Furniss & Carolin, 1977). Larvae emerge from winter dormancy and hatch in spring, the timing of which is synchronized with hostplant development (Jones & Despland, 2006; Sarfraz et al., 2013). Larvae feed as a colony on the hostplant where they were oviposited, typically in family groups. They grow and develop from hatchling to pupae in 30 to 40 days, depending on local climatic conditions (Furniss & Carolin, 1977). Once larvae reach the pre-pupal stage, individuals construct a silk cocoon in which they pupate for 2-3 weeks.

In this genus, most species have gregarious larvae that form colonies and construct silk tents in the branches of their host plant. These tents alter the biotic and abiotic experience of the caterpillars, and therefore acts as an extended phenotype (Dawkins, 1999). When they are not feeding, caterpillars typically remain on or in their tent, which may also serve as a rain shelter, molting site, predator deterrent, and potential source of thermal mosaics that can be used for behavioral thermoregulation. It is this latter feature that is the focus of my study.

5 Field sites and environmental monitoring Experiments described here were conducted from late May to mid-June 2017 and 2019. In 2017, individuals were collected in Condon, Montana USA on the MPG North property (47°31'16.9" N, 113°40'01.5" W). In 2019, individuals were collected at MPG North and at Blue Mountain, near Missoula, MT (46°52'45.1" N, 114°09'19.9" W); after collection, Blue Mountain caterpillars were transported to MPG North for use in field experiments. In the spring of 2017, an RSR-100 weather station (Campbell Scientific Inc., Logan, UT) was launched at MPG North to measure air temperature, relative humidity, wind speed, wind direction, and solar radiation. Weather station data were used to compare field site macroclimate to microclimate data collected at the level of leaves, tents and larvae (described below).

Colony size manipulation and microhabitat temperature measurements To determine how colony size affects thermal ecology of tent caterpillars, natural colonies of second instar larvae were collected from wild rose host plants in May 2017 and randomly assigned to pseudo-colonies of 10, 50, 100, or 150 individuals. Tent caterpillars do not recognize kin (James T. Costa & Pierce, 1997), so it was assumed that mixing of individuals with different parents should not alter behavioral thermal regulation. Experimental colonies (N = 4 per treatment) were transplanted onto different wild rose plants in three plots established at MPG

North on 26 May 2017. Tent temperature (Tt) was measured by attaching a copper-constantan thermocouple at the center of the transplanted colony and allowing the caterpillars to build their tent around it. Newly transplanted colonies typically established tents within 24 h. Throughout the course of data collection, thermocouples stayed in the center of these tents. Additional thermocouples were attached to the underside of hostplant leaves to measure local air temperature (Ta). Both Ta and Tt were recorded every 5 min using HOBO 4-channel thermocouple loggers (Onset Computer Corp., Bourne, MA).

Statistical analyses for these and subsequent experiments were conducted in JMP (Version 15; SAS Institute, Cary NC USA) unless otherwise noted. For colony size manipulation experiment, temperature logger data were available for 11 of 16 colonies (N = 3 for all treatments except 10-individual colonies, where N = 2). Preliminary analysis showed that larvae from all colony size treatment groups were present from 28 May- 4 Jun. Hourly mean air and tent temperatures were calculated from logger output (N = 12 measurements per h for 24 h) for those 8 d and used to determine effects of colony size on tent temperature, using a mixed-models

6 ANOVA with day, hour, and colony size as fixed effects, colony nested in colony size as a random effect, and all interactions. Differences in relationship between air and tent temperature among colony-size treatment groups were determined using linear regression of average hourly mean air and tent temperatures of daylight hours (0800-1800).

Development rate To examine differences in development rate among caterpillars living in different-sized colonies, number of individuals of each developmental instar was recorded daily (N = 16 colonies). On each day, we calculated the average instar present on a colony as an index of colony development rate, following methods detailed in Rank, 1994. Effect of colony size on development rate was determined using mixed-model ANOVA with day and colony size as fixed effects, colony nested in colony size as a random effect, and all available interactions. A post-hoc Tukey multiple comparisons test assessed the probability that mean development rates were different from each other.

Caterpillar body temperature and behavior To test effects of aggregation on larval body temperature, 13 colonies were established at the MPG North site in late May 2019. For these experiments, hawthorn was used in place of wild rose as hostplant, as most natural colonies I observed at that time were feeding on hawthorn, not wild rose (which appeared unhealthy during that spring). For each experimental colony, tent and air temperature were measured as described above, with the exception that the thermocouple used to measure microhabitat air temperature was placed inside a white plastic radiation shield and mounted next to the experimental hostplant. On three days in late May and early June, individual body temperatures were measured using a T540sc infrared imaging camera (FLIR Systems, Inc., Wilsonville, OR). Thermal images of every experimental colony were recorded at each time point; all colony members were imaged. Images were captured approximately every 20 min from 0830-1600 hours, for a total of around 20 images per colony per day. Images were analyzed using FLIR Tools Thermal Analysis and Reporting software. The line tool was used to extract average body temperature of each caterpillar from each image; location on tent and grouping behavior were also noted. Typical images are shown in Fig. 1. Body temperatures were time-matched with thermocouple air temperatures and used to calculate the temperature excess

(TE), the degree to which caterpillar body temperature differed from air temperature (TE = Tb –

Ta). Effects of behavior on temperature excess was determined using mixed-models ANOVA

7 with date, position, and group as fixed effects, colony and trial as random effects, and all available interactions. A post-hoc Tukey multiple comparisons test assessed the probability that mean temperature excess differed among behavioral groups. Differences in relationship between air and body temperature among behavioral groups were determined using linear regression of average hourly mean air and body temperatures of daylight hours (0600-2100).

Operative Temperature Models To further test effects of grouping behavior on temperature excess, I made operative temperature models by freeze-drying individual caterpillars with thermocouples inserted into their body cavities. These caterpillars were then placed in 3 group sizes of 1, 5 or 15 individuals. Groups were glued to fine tulle mesh, mounted on embroidery hoops, and placed on a two-foot by four- foot frame and placed near site of experimental colonies at MPG North from June 13-19, 2019 (Fig. 2). Air and model temperatures were recorded with thermocouples attached to HOBO dataloggers as described above. To determine effects of grouping behavior on body temperature, air and model temperature data were sorted into Day (05:45-21:30) and Night (21:35-05:40) based on average time of sunrise and sunset during data collection and temperature excess calculated. Resulting data were analyzed using mixed-models ANOVA with time of day and colony size as fixed effects and hoop as a random effect. A post-hoc Tukey multiple comparisons test assessed the probability that mean temperature excess differed among sizes.

Thermal tolerance Upper lethal temperature was determined as the temperature at which 50% of the larvae died after treatment (LT50), following methods detailed in Mitchell et al., 1993. Third instar caterpillars (N =140) were placed individually into clear glass dram sample vials. A copper- constantan thermocouple was inserted into each vial to measure treatment temperature and was assumed to be representative of caterpillar body temperature. Vials were sealed and submerged into a water bath connected to a programmable temperature controller. Fourteen vials were kept at room temperature (~20°C) for the length of the exposure, to act as controls. Caterpillars were warmed at 0.1°C per min to 36°C. After 10 min, 14 vials were removed, and the temperature raised by 2°C. This procedure was repeated until 52°C was reached, a temperature well above upper critical temperatures found in the literature for related species. Upon removal, vials were opened, caterpillars prodded and immediately assessed for evidence of movement. Caterpillars were then allowed to recover for 24 h at room temperature (~20 C). During recovery, vials were

8 left partially open to allow air flow; larvae were fed after 12 h. After 24 h, caterpillars were scored as alive or dead by whether or not they moved in response to gentle, prodding stimuli.

The LT50 was determined using binomial logistic regression in R.

Results

Colony size manipulation Both air and tent temperatures varied significantly over the diurnal cycle during the 2017 colony manipulation experiment. During the day (between 08:00 – 18:00), mean tent temperatures varied among colony sizes, with larger colonies reaching higher temperatures (Figure 3; Table 1). However, as air temperatures cooled at night there was no significant difference in tent temperature between colony size treatments (Figure 3; Tukey HSD, P = 0.12). During the 8 days of data collection, large colonies reached tent temperatures up to 17°C higher than air temperatures, while tents of smaller colonies reached up to ~9°C higher than air temperatures. The larger two colony size treatments (100 & 150 individuals per colony) had higher daytime (8am – 6pm) hourly tent temperatures than the smaller colonies during the 8 days of data collection (Figure 4; Table 1). Development rate was lowest for smallest colony size and peaked at 100 individuals (Figure 5; Tukey HSD, P < 0.05; Table 2). Overall mortality was 86%. No caterpillars from small colonies survived to pupation, but 16% of those from large colonies did survive to pupate.

Caterpillar body temperature, behavior and climate The three days for which thermal images were recorded had significantly different climatic conditions. Days 1 and 2 (31 May and 3 Jun) were sunny and Day 3 (6 Jun) was cloudy and wet (Figure 6). In a comparison of macro- vs. microclimate data, air temperatures measured by the weather station were lower and much less variable than the measured body, tent, and local air temperatures across all 3 days of data collection (Figure 6A). Foraging bouts coincided with decreases in body and tent temperatures on Days 1 and 2, but not Day 3 (Figure 6A, C). I predict that larvae left the tent for a foraging bout before data collection started, but with the low air temperatures and wet conditions, they were unable to move very fast to return to the tent until the temperatures warmed up later in the afternoon. Caterpillars spent 57% of the sampling time resting on the tent. Caterpillars on the tent were significantly warmer than those off the tent, and grouped caterpillars were warmer than solitary ones (Figure 7), though this pattern was most

9 pronounced on sunny days. However, not all values coded as “on tent, grouped” are the same, as one might imagine a value where the entire colony is grouped on the tent might be different from a value where only some are grouped on the tent and others are off the tent. In order to do a proper comparison, only data points where all four position and behavior combinations were present were included. For this subset, caterpillars on the tent were always warmer than those off the tent and grouped caterpillars on the tent were even warmer than solitary caterpillars on the tent (Figure 8; Table 3). In the operative temperature models, all group sizes achieved temperatures above air temperature during the day, with group sizes of 5 and 15 reaching temperatures significantly above those of the solitary caterpillars (Figure 9). At night, all groups had temperatures similar to or below air temperature (Table 4).

Thermal tolerance There was no mortality in any treatment temperature below 46°C, and at 52°C there was 100% mortality (Table 5). LT50 for the third instar larvae was 49.3°C (Figure 10).

Discussion

Are large colonies better? In temperate regions, springtime insects often contend with temperatures cold enough to slow and even halt growth and development. Social, tent-building insects like those of the genus Malacosoma address this problem by building a silk tent that acts as a platform for behavioral thermoregulation. One question addressed here is how colony size affects tent building, body temperatures, and growth and survival of these insects. Compared to smaller colonies, larger colonies build larger tents more rapidly, reach higher temperatures, and develop faster and have higher rates of survival to pupation (Figs. 3- 5). Part of this can be attributed to increased temperatures in tents of larger colonies; higher temperatures (up to a point) allow caterpillars to eat, digest, and grow faster (Casey et al., 1988; Knapp & Casey, 1986; Ruf & Fiedler, 2002a). Having more individuals in a colony may provide other benefits as well. In other species of Malacosoma, mortality rates can range anywhere from 40% (Shiga, 1979) to more than 95% (Filip & Dirzo, 1985). In M. americanum, early instars have high rates of mortality, but mortality drops dramatically once caterpillars reach the third to fourth instar (Costa, 1993). Larger colonies may have higher overall fitness due to their ability to produce more surviving individuals even when mortality rates are high.

10 Egg masses of M. californicum typically contain 150 - 200 eggs. If larger colonies perform better, then why don’t natural colonies contain more larvae? First, experimental tents of larger colonies occasionally had regions with temperatures in excess of 52°C, well above the estimated larval LT50 of 49.3°C. On very hot days, larvae behaviorally thermoregulate by positioning themselves on the shady size of the tent, often hanging down from the tent to minimize conductive heat gains (Fitzgerald, 1995; Ruf & Fiedler, 2002b). However, there is still a chance of overheating in larger colonies, especially with increases in temperate spring temperatures (Seneviratne et al., 2012). Also, larger colonies may be more susceptible to predators and parasitoids (Chesson & Murdoch, 1986; Hoffman & Avilés, 2017; Lavalli & Spanier, 2001; Uetz & Hieber, 1994). I predict in addition that optimal colony sizes vary from year to year depending on spring climate and hostplant quality. Additional data would be needed to test these ideas.

Colony size and individual caterpillar size reflect the size and number of eggs in the overwintering egg mass, which may trade off against one another, particularly when one compares the needs of caterpillars in the spring versus the needs of the overwintering egg mass. Females could potentially produce more eggs (leading to larger maximum colony sizes) at a cost of producing smaller eggs. Smaller eggs may struggle to survive over winter, due to lack of energy reserves (Sinclair, 2015; Williams et al., 2012). Even though overwintering first instars have very low metabolic demands, temperature spikes in winter or early spring could cause larvae to burn through reserves too quickly before hatching (Abarca & Lill, 2015). Smaller hatchlings may also be less able to establish feeding sites and build tents successfully, contributing to high mortality rates in early instars (Hahn & Denlinger, 2007, 2011). In a study of the forest tent caterpillar M. disstria, researchers found that females laid fewer, larger eggs as latitude increased (Parry et al., 2001). Larger eggs may increase survival to spring hatching, and once those eggs hatch the resulting larger caterpillars can maintain more constant body temperatures, and may be more efficient at producing silk, both of which would mitigate the need for a larger number of individuals in a colony.

Effects of behavioral thermoregulation Social, tent-building insects use their tents for behavioral thermoregulation (Casey, 1981a; Casey et al., 1988; Joos et al., 1988; Knapp & Casey, 1986; Ruf & Fiedler, 2000, 2002b). Larger

11 colonies build warmer tents, but the tent temperature only matters insofar as it influences the body temperatures of the caterpillars living on it. My second experiment, in 2019, set out to address why the tents heat up, and how thermoregulatory behaviors using the tent influence individual body temperatures of caterpillars.

Hypothesis 1: The tent collects heat like a greenhouse, and caterpillars gain heat by being in contact with it. A study using operative temperature models of M. americanum found that when illuminated by daytime, temperatures of the models on tents with no living caterpillars were always at least 4°C warmer than ambient air temperature (Joos et al., 1988). In 2017, I measured tent temperatures even after all individuals in the colony had either died or pupated. Tents from the two larger colony size treatments still reached core temperatures up to 14°C above air temperatures in the middle of the day when solar radiation was strong. While the spectral characteristics of the silk itself may not be an effective greenhouse material (i.e. transmits short wavelength radiation like sunlight, but blocks longer wave radiation like heat from escaping)(Ruf & Fiedler, 2002b), these tents contain black molt casings and frass, both of which absorb solar radiation and increase the overall temperature of the tent. As the season progresses and the tent grows, and comes to contain more dark material, the magnitude of this effect likely also increases.

Hypothesis 2: Caterpillars aggregate, conserving heat by functioning biophysically as a larger organism. My experiments with operative temperature models support this idea, as the clusters of 5 and 15 individuals reached temperatures well above solitary models and ambient air temperature during the day. Likewise, in live caterpillars on sunny days (Figure 6, Days 1 & 2), especially in the mornings when direct solar radiation was elevated but air temperature still low, grouped caterpillars could maintain body temperatures well above air temperature. On cloudy days (Figure 6, Day 3), however, grouping was not enough to elevate body temperatures above ambient, as direct solar radiation was greatly reduced (Figure 6). In both the operative temperature models and the live caterpillars, grouping alone raised body temperatures only when solar radiation was high.

Hypothesis 3: Caterpillars warm their tents with metabolic heat.

12 While I did not directly measure metabolic heat production, my data still allow for some conclusions to be drawn regarding this hypothesis. Tent temperatures dropped when larvae left the tent and increased again when they returned from foraging bouts. This is highlighted in the Day 1 panel of Figure 6, in which tent temperatures declined when caterpillars left the tent. In addition, in tents of the small eggar moth and the eastern tent caterpillar, researchers attributed up to ~4°C of temperature excess to metabolic heat production (Knapp & Casey, 1986; Ruf & Fiedler, 2000, 2002b, 2002a). Most of my data, however, provide little support for this idea. If metabolic heat were a large contributor to body temperature excess, one would expect that live grouped larvae off the tent would have higher temperature excesses than the operative temperature models when the reverse was observed, likely because the grouped operative temperature models could not behaviorally thermoregulate as live caterpillars did. Additionally, on the cloudy day, when solar inputs were low, caterpillar temperatures were not significantly elevated above ambient (Figure 6). Even the lowering and raising of tent temperature as the caterpillars left and came back from foraging bouts could be attributed to conductive heat transfer of excess heat gained from solar radiation rather than metabolism. While there are instances in the literature of insects elevating or maintaining high body temperatures over a range of air temperatures, these are almost always in large, flying adults (Heinrich, 1971; Heinrich & Bartholomew, 1971; Heinrich, 1980). Even in a study of the eastern tent caterpillar, elevated thoracic temperatures were measured only in adult, flying moths (Casey, 1981b). Mathematical modeling also suggests that larval body masses and metabolic rates usually are not high enough to result in significant self-heating, even in relatively well-insulated environments like composted soil (Cooley et al., 2016).

In summary, my data support the first and second hypotheses – that western tent caterpillars warm significantly above ambient air temperatures by (Hypothesis 1) associating closely with their tents, which themselves warm to many degrees above ambient air temperature, especially when solar radiation is intense; and (Hypothesis 2) aggregating into large groups, which create large surface areas for absorbing incoming radiation. In large groups, caterpillar setae likely also more effectively increase the thickness of adherent boundary layers of air, which would depress rates of convective heat loss. Although I did not test the metabolic hypothesis (Hypothesis 3), the patterns of body temperature I observed suggest that metabolic heat plays a minor role in determining caterpillar body temperatures.

13 The complex behavior of the western tent caterpillar shows an organism combining both behavioral thermoregulation with an extended phenotype to create mosaics of microhabitats that vary both spatially and temporally. The novel approaches I took to measuring these factors in naturally occurring field environments combined with experimental manipulations provided new insights into these ecosystem engineers. We can only truly predict the adaptive strategies available to an organism in the face of changing environments by examining as many contributing factors to that organisms’ physiology as possible, whether it be the thermal mosaics created by ecosystem engineering and behavioral thermoregulation, or the plasticity of certain traits in the face of changing seasonal extremes.

14 Acknowledgements

These studies represent a thesis submitted as partial fulfillment of the requirements of a Masters of Science degree in the Department of Ecology and Evolution at the University of Montana. I thank my advisor Dr. Art Woods for his excellent recommendation that I study tent caterpillar thermal biology and his endless patience through this process. I thank my committee for their contributions in shaping my research questions and their helpful advice on my scientific career. I am especially grateful to Dr. Creagh Breuner for her unwavering emotional support. I am deeply appreciative of my “shadow committee members” Dr. Nathan Rank, who provided me with invaluable and timely assistance on statistical analyses, and Dr. Elizabeth Dahlhoff, for her help with data presentation. I also thank them both for their early encouragement of my curiosity about the natural world and for dragging me up mountains when I was a kid. I am indebted to current and former Woods Lab members, undergraduates, and friends who provided assistance with fieldwork, especially Hannah Beyl, Rachel Bingham, Markus Boyer, Dominic Cuellar, Kathleen Dahlhoff, Jameson Frakes, Matthew Jackson, Tylor Keeley, Steve Lane, and Brenna Shea. Finally, this work would not have been possible without the generosity of Beau Larkin and MPG, who provided funding for the field experiments and gave me access to an awesome place to conduct them.

I dedicate this thesis to my father, Geoffrey Dahlhoff. While he did not get to see the end of it, he was integral to getting me through it.

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20 Tables Table 1. Analysis of Variance (ANOVA) of tent temperatures for different-sized experimental caterpillar colonies. Data were analyzed using mixed models ANOVA, with main effects and interactions shown. Colony (nested in colony size) and all available interactions were included as random effects (model R2 = 0.99). Data shown in Figures 3 and 4.

Source DF Num DF Den Sum of Squares F P Fixed Effects Colony Size 3 7 358.23 2.19 0.18 Day of Year 7 49 7405.20 235.49 <0.0001 Hour 23 161 131656.00 357.11 <0.0001 Colony Size*Day 21 49 193.18 2.05 <0.0001 Colony Size*Hour 69 161 1612.81 1.46 0.0275 Day*Hour 161 1127 18905.80 80.69 <0.0001 Colony Size*Day*Hour 483 1127 840.12 1.2 0.0093 Random effects Colony [Colony Size] 7 181 381.23 2.86 0.0075 Colony [Colony Size]*Day 49 1127 220.12 3.09 <0.0001 Colony [Colony Size]*Hour 161 1127 2580.70 11.01 <0.0001 Error 1640.21

Table 2. ANOVA of effects of colony size on caterpillar development rate. Average larval instar was used to index development rate. Data were analyzed using mixed models ANOVA, with main effects and interactions shown. Colony (nested in colony size) was included as a random effect (model R2 = 0.95). Data shown in Figure 5.

Source DF Num DF Den Sum of Squares F P Fixed effects Colony Size 3 13 0.48 6.24 0.0075 Day of Year 3 33 12.81 181.06 <0.0001 Colony Size*Day 9 33 0.40 1.88 0.0906 Random effect Colony [Colony Size] 12 33 0.31 1.09 0.4024 Error 0.78

21 Table 3. ANOVA of effects of behavior on body temperature excess. Data were analyzed using mixed-models ANOVA, with main effects and interactions shown. Colony, trial (nested in date) and all available interactions were included as random effects. (model R2 = 0.97). Data shown in Figures 7 and 8.

Source DF Num DF Den Sum of Squares F P Fixed effects Date 2 30 158.85 1.06 0.36 Position 1 16 1413.82 27.95 <0.0001 Group 1 16 704.23 42.24 <0.0001 Date*Position 2 64 432.69 9.74 0.0002 Date*Group 2 86 87.33 2.83 0.06 Position*Group 1 67 656.46 42.48 <0.0001 Date*Position*Group 2 67 6.68 0.22 0.81 Random effects Trial[Date] 22 20 4533.89 6.15 <0.0001 Trial[Date]*Position 22 67 826.02 2.43 0.0029 Trial[Date]*Group 22 67 339.39 1.00 0.48 Colony 9 10 562.47 1.12 0.43 Colony*Position 9 67 634.44 4.56 0.0001 Colony*Group 9 67 154.88 1.11 0.37 Error 1035.41

Table 4. ANOVA of Operative Temperature Model experiment. Data were analyzed using mixed-models ANOVA, with main effects and interactions shown. Hoop was included as a random effect (model R2 = 0.27). Data shown in Figure 9.

Source DF Num DF Den Sum of Squares F P Fixed effects Time of Day 1 22167 86635.00 6475.19 <0.0001 Group Size 2 22167 6593.08 246.39 <0.0001 Time of Day*Group Size 2 22167 10131.30 378.61 <0.0001 Random effects Hoop 3 22167 2921.92 72.80 <0.0001 Error 296584.12

22 Table 5. Thermal tolerance raw data. Logistic regression shown in Figure 10 (LT50 = 49.3°C).

Maximum Temperature (°C) Alive Dead Proportion Alive 20 14 0 1 36 14 0 1 38 14 0 1 40 14 0 1 42 14 0 1 44 14 0 1 46 14 0 1 48 10 4 0.714 50 6 8 0.429 52 0 14 0

23 Figures

A B

C D

Figure 1. Thermal images of grouping behavior of larval Malacosoma californicum pluviale (western tent caterpillar). The four behavioral states examined are shown here – A: grouped, on tent; B: solitary, on tent; C: grouped, off tent; D: solitary, off tent.

Figure 2. Operative temperature model apparatus. A hoop represents a replicate for each group size (N = 6 per group size).

30 10 50 25 100 150 C) o 20

10 Tent temperature ( temperature Tent

0 0246810121416182022

Hour of day Figure 3. Average tent temperature of each colony size by hour of day. Data are least squares means ± SEM of hourly data for 8 days for 11 colonies, grouped by colony size. Colony size = 10, 50, 100, and 150 individuals (N = 16, 24, 24, 24). Shaded regions indicate nighttime. Tent temperature was measured with a copper-constantan thermocouple at the center of each tent, with 12 measurements per hour per colony (28 May – 4 Jun 2017). Analysis summary in Table 1.

35 10 50 100 150 30 Y = X C) o

20 Tent temperature ( temperature Tent

15 20 25 30 Air temperature (oC)

Figure 4. Average tent temperature by air temperature. Data are daytime hourly mean air and tent temperatures averaged for the same 8 day period as Figure 3. Linear regressions were calculated for each colony size treatment Colors and symbols of colony size treatment match those of Figure 3. Black dashed line is where Ta = Tt; any values above this line indicate where tent temperature exceeded air temperature (Colony Size 10: y = 0.851x + 3.230, N = 22 r2 = 0.86 F = 123.31 p < 0.0001; Colony Size 50: y = 0.925x + 2.961, N = 33 r2 = 0.70. F = 71.997 p < 0.0001; Colony Size 100: y = 0.877x + 5.523, N = 33 r2 = 0.64 F = 54.344 p < 0.0001; Colony Size 150: y = 1.078 + 0.560, r2 = 0.70 N = 33 F = 70.671 p < 0.0001).

3.0

2.8

2.6 instar (8 days) (8 instar

2.4

2.2 Mean average larval 2.0 10 50 100 150 COLONY SIZE

Figure 5. Development rates for each colony size treatment. Data are least squares means ± SEM of average instar calculated for each colony size for the same 8 day interval as Figures 3 and 4 (N = 4 per treatment). A post-hoc Tukey multiple comparisons showed the following for difference in mean development rate between treatments: 10, 50, 100, 150 = A, AB, B, AB. Symbols of colony size match those from Figures 3 and 4. Analysis summary in Table 2.

28 40 A: Temperature Tair T

C) 35 body o Ttent 30 TWS

15 Mean temperature ( 10

91113158101214169111315 ) 2 B: Light Direct Diffuse 3

Irradiance (W/m Irradiance 1 10

0 Mean log 91113158101214169111315

C: Caterpillar behavior

0.6

0.4

0.2 Proportion off tent off Proportion 0.0

91113158101214169111315 Time of day (h) Time of day (h) Time of day (h) 31 MAY 3 JUN 6 JUN

Figure 6. Macroclimate vs microclimate. Data are hourly means from 3 days of data collection. Day 1 (31 May 2019) had 17 time points, from 09:12 - 15:33; Day 2 (3 Jun 2019) had 22 time points, from 08:16 – 16:03; Day 3 (6 Jun 2019) had 18 time points, from 08:18 – 16:37. (A) Temperature - Colony 1 body temperature, Colony 1 tent temperature, local air temperature from HOBO data logger, and air temperature from Campbell RSR-100 weather station. (B) Irradiance – Direct and diffuse radiation as measured by the Campbell RSR-100 weather station. (C) Caterpillar Behavior – Proportion of caterpillars off the tent for Colony 1, where peaks indicate a foraging bout.

40 On tent, grouped On tent, solitary Off tent, grouped 35 Off tent, solitary Y = X C) o 30

20 Body temperature ( temperature Body 15

10 10 15 20 25 30 Air temperature (oC)

Figure 7. Average body temperature by air temperature. Data are daytime hourly mean air and body temperatures across the same 3 days of data collection as Figure 6. Data are coded by position and grouping, and linear regressions were calculated for each combination of position and grouping. (Off Tent, Solitary: y = 1.529x - 5.881, N = 22 r2 = 0.91 F = 224.8897 p < 0.0001; Off Tent, Grouped: y = 1.382x - 3.971, N = 22 r2 = 0.94 F = 299.7847 p < 0.0001; On Tent, Solitary: y = 1.503x - 5.167, N = 22 r2 = 0.98 F = 784.8605 p < 0.0001; On Tent, Grouped: y = 1.413x - 3.578, N = 22 r2 = 0.97 F = 656.7790 p < 0.0001). Black dashed line is where Ta = Tb; any values above this line indicates where body temperatures exceeded air temperatures.

ON TENT 6 OFF TENT

5 C) o

1 Temperature excess ( excess Temperature

SOLITARY GROUPED BEHAVIOR

Figure 8. Mid-day body temperature excesses of larvae as relates to position and grouping. Data are least squares means of average temperature excess (TE) ± SEM was calculated using body temperatures (Tb) measured with thermal imaging and local air temperature (Ta) from data loggers at given time points across the 3 days of data collection (TE = Tb - Ta). Least squares means of average TE were calculated only from time points where all four "behaviors" were present: On tent, grouped; On tent, solitary; Off tent, grouped; Off tent, solitary (N = 2582; Tukey = B, A, AC, C). Analysis summary in Table 3.

5 1 5 4 15 C) o 3

0 Temperature excess ( excess Temperature

-1

DAY NIGHT Time of Day

Figure 9. Temperature excesses of operative temperature models. Data are least squares means of temperature excess of freeze-dried caterpillars in 3 group sizes (1, 5, 15 individuals) during the day (05:45 – 21:30) and night (21:35 – 5:40) from June 13-19, 2019. Air and model temperatures were recorded every 5 minutes using HOBO dataloggers. Analysis summary in Table 4.

alive

LT50 = 49.3 Survival

dead

20 25 30 35 40 45 50

Maximum temperature (°C)

Figure 10. Upper thermal tolerance. In the plot, points are jittered slightly in both x and y directions for clarity. The response was modeled by logistic regression (using unjittered points) in R. Upper thermal tolerance was estimated as the temperature at which there was a 0.50 probability of survival (LT50 (± sem) = 49.3 ± 0.36 °C). Logistic regression created using data from Table 5.