Abstract
Parasites can be detrimental to the health, longevity, and reproduction of their hosts, but these costs are rarely quantified in nature. We removed ectoparasites and endoparasites from Cape ground squirrels (Xerus inauris), a highly social sciurid in southern Africa. Parasites were removed for 3 months during the dry winter season, when fewer resources are available and the impact of parasites may be greatest. We examined changes in female body mass, reproduction, grooming, and burrow use. Female body mass did not increase with parasite removal, but reproductive success (number of offspring raised to emergence) was nearly 4 times higher for treated females. Rates of allogrooming were lower for treated females, but we were unable to detect a significant change in burrow movements, perhaps because factors other than parasites (e.g., predator avoidance) affect such movements. Gestation and lactation are the most physiologically stressful processes that females undergo, and the dramatic increase in reproductive success in treated females suggests that parasites drain resources that would otherwise be allocated to reproduction. Our results suggest that nonparasitized females put the extra energy into reproduction rather than into their own body weight, which appears to enhance juvenile survival.
Life-history theory examines trade-offs in how organisms invest resources in growth, reproduction, and survival, such that increased investment in one component results in fewer resources available to the remaining components (Stearns 1992). These resource allocations can be altered by an organism’s interactions with its environment, particularly factors that reduce the overall resources available, such as parasites (Pagan et al. 2008). Parasites can have a profound effect on the fitness of a host. Direct fitness costs include loss of blood, nutrients, and energy allocated to immune response, whereas indirect fitness costs include increased chance of secondary infection and decreased time available for vigilance or foraging, which can translate into reduced survival or loss of lifetime reproductive success (Kollars et al. 1997; Rolff 2002; Neuhaus 2003; Johnson et al. 2004). Parasite infection may inhibit female reproduction by delaying reproduction or decreasing reproductive success (Arnold and Lichtenstein 1993; Hoogland 1995; Van Vuren 1996; Neuhaus 2003; Skorping and Jensen 2004). However, the relationship between parasitism and female fitness has not been well addressed in the literature, and few studies have experimentally examined the direct impact of parasites on female reproductive success because of the difficulties of manipulation (Neuhaus 2003; Brown et al. 2006).
In response to high parasite loads, individuals may use behavioral means to reduce parasites, such as grooming themselves (autogrooming; Hart 1992, 1994; Kollars et al. 1997) or conspecifics (allogrooming; Hart 1992, 1994; Johnson et al. 2004). High parasite loads may also prompt individuals to move away from infested areas (Hausfater and Meade 1982; Mooring and Hart 1992; Johnson et al. 2004).
In this study, we measured the costs of parasites to Cape ground squirrels (Xerus inauris) by removing parasites to reveal their impact on body mass, reproductive success, grooming, and burrow use of adult females. We removed both endoparasites and ectoparasites nonselectively because we had no prior information on which parasites might have the greatest impact, if any. We predicted that squirrels treated for parasites would increase in body mass and reproductive success because parasites are energetically taxing to the host (Scantlebury et al. 2007), decreasing the resources available for growth and reproduction. Treated squirrels should decrease time spent grooming because they will harbor fewer ectoparasites to stimulate grooming and should shift between sleeping burrows less frequently than untreated squirrels if parasite accumulation in burrows and nesting material triggers these shifts.
MATERIALS AND METHODS
Biology of the study animal
Cape ground squirrels are a highly social semi-fossorial species inhabiting the arid areas of southern Africa. Females live in matrilineal groups with other related adult females and their subadult and juvenile young (Waterman 1995). These groups of 4–13 squirrels share a communal sleeping burrow (Waterman 1995; Hillegass et al. 2008). Adult males usually live in all-male bands of up to 19 squirrels that travel and sleep together independent of female groups (Waterman 1997). Burrow clusters of the Cape ground squirrel can range in size from 120–1500 m2 and contain 20–149 burrow openings that house distinct burrow systems (series of connecting burrows) with multiple sleeping areas (Herzig-Straschil 1978; Waterman 1995; Waterman and Fenton 2000). Burrow clusters are composed of 1–3 burrow systems, and a typical burrow system spans around 10–15 m (Herzig-Straschil 1978). Periodically Cape ground squirrels shift from sleeping in one burrow system to another within their burrow cluster (Waterman JM, unpublished data).
Gestating females separate from their group prior to parturition and give birth in natal burrows that are located away from the burrow cluster and are difficult to detect (Waterman 1996). During the day, lactating females join their social group to forage, returning to the natal burrow only in the evening. When offspring first emerge from the natal burrow at the end of lactation, females return to their social group with their offspring (Waterman 1996). Subsequently, the interactions of offspring with their mothers and with other members of the social group do not differ, and they all sleep together (Waterman 1995). Gestation and lactation are approximately 50 and 52 days, respectively (Zumpt 1970; Waterman 1996). Maximum litter size is 2 (mean 1.45 ± 0.21; Waterman 1996); 40% of litters contain a single juvenile at emergence (Waterman JM, unpublished data). Litter loss is high; following estrus, two third of females lose their litters before offspring emergence (Waterman 1996). Thus, adult females can have up to 4 estrous cycles annually, with a maximum of 2 for those who successfully raise offspring to emergence (Waterman 1996; Pettitt et al. 2008). Age of first breeding in Cape ground squirrels appears to be influenced more by social factors than by body condition or other environmental factors (Waterman 2002; Pettitt 2006; Pettitt et al. 2008), and the first breeding attempt is rarely successful in rearing juveniles to emergence (Pettitt et al. 2008).
Adult male Cape ground squirrels carry 3 times as many ectoparasites (fleas, lice, and ticks) as females, probably because increased androgen levels reduce ectoparasite resistance (Hillegass et al. 2008). However, females carry nearly 3 times as many endoparasites (roundworms, hookworms, coccidia, and others) as males. Although some species may avoid foraging in areas contaminated with feces (Garnick et al. 2010), the small home range of female Cape ground squirrels restricts their foraging to areas around burrow sites where fecal matter is concentrated (Hillegass et al. 2008). Parasite loads are unaffected by group size (Hillegass et al. 2008).
Study site
This study was performed on the S.A. Lombard Nature Reserve near Bloemhof, South Africa (lat 27°35′S, long25°23′E) from May to September 2004. Cape ground squirrel social groups were located in burrow clusters on a floodplain, where the habitat is uniform short grass (van Zyl 1965). Austral winter is from May through September, coinciding with the dry season and time of lowest available food resources, yet when breeding peaks (Herzig-Straschil 1978; Waterman 1996). This reduction in resources when reproduction is high suggests that physiological stress on squirrels may be greatest during this season, and the impact of parasites on their hosts should be the most apparent (Soler et al. 1999; Villanua et al. 2007).
Trapping, handling, and antiparasite treatments
We captured and marked all squirrels in 18 social groups in May 2004 using Tomahawk live traps (15 × 15 × 50 cm; trapping and handling techniques are described in Waterman 1995, 2002). Squirrels were retrapped each month from June to September, and at each capture, we recorded body mass and reproductive condition. Adult females were identified by their long swollen nipples (swelling occurs after first estrus and remains over lifetime; Waterman 1996). Animals were tagged with small transponders (AVID Inc., Folsom, LA) under the skin for permanent identification. For identification at a distance, we dye-marked (Rodol D; Lowenstein & Sons Inc, New York, NY) and freeze-marked animals (Quik-freeze; Miller-Stephenson Product, Morton Grove, IL; Rood and Nellis 1980). All squirrels were handled in accordance with the animal care guidelines of the American Society of Mammalogists (Gannon et al. 2007) and the Animal Care and Use Committee of the University of Central Florida.
We quantified ectoparasites monthly for each squirrel by combing with a metal flea comb using 3 strokes on each plane of the back (left, middle, and right), from the shoulders to the base of the tail (Scantlebury et al. 2007; Hillegass et al. 2008). Ectoparasites were combed directly into 95% ethanol in a petri dish and then counted immediately. We estimated endoparasite loads using endoparasite egg counts from feces collected with plastic tarp sections placed under live traps in June and July (Pettitt et al. 2007). We collected feces with forceps dipped in 95% ethanol and sealed the samples in labeled plastic bags. Subsequently 0.5 g of fecal matter was weighed out and frozen. We later thawed these samples and prepared fecal flotations with a solution of magnesium sulfate (McCurin and Bassert 2002), which caused eggs released by adult endoparasites to float. Prepared samples were then observed under a compound microscope (×100) for endoparasite egg count.
We removed ectoparasites and endoparasites from squirrels in 9 female social groups and monitored 9 additional groups as controls. Treatment and control groups were selected across the floodplain, with 3 large areas containing treated groups alternated with 2 large groupings of controls. This arrangement allowed the control animals to be fairly isolated from treated groups and experience no effect of antiparasite treatments. These antiparasite treatments have been effective against multiple ectoparasites and endoparasites in this Cape ground squirrel population; parasite loads decreased significantly in treated animals (Scantlebury et al. 2007).
Antiparasite treatments were administered once a month for 3 months (June, July, and August), starting the beginning of June. Squirrels in treatment groups received a monthly subcutaneous injection of a systemic antiparasite treatment (0.1 ml, 0.1% solution ivermectin) to remove endoparasites (Campbell et al. 1983). This drug is absorbed into the blood stream and removes both adult and larval endoparasites from major phyla of nematodes found in the gut, as well as some arthropods, lasting approximately 4 weeks (Campbell et al. 1983; Heukelbach et al. 2004). To remove ectoparasites, we used the topical antiparasitic agent FRONTLINE (fipronil 0.29%; Merial, Duluth, GA), which kills fleas, ticks, and lice. As ectoparasites typically are concentrated behind the head, down the back, and around the hind regions (Nilsson 1981), we focused our treatment in this area. We sprayed 1.5 ml FRONTLINE topically over the back of each squirrel, protecting the entire body from parasites. FRONTLINE (fipronil) was tested for safety and effectiveness on rats with a 9.7% solution oral LD50 (97 mg/kg) and dermal LD50 (2000 mg/kg) (Merial Material Safety Data Sheet-FRONTLINE TOP SPOT 2001). The amount applied to Cape ground squirrels was thus 145 times lower than the oral LD50 and 1400 times lower than the dermal LD50. This drug is an external treatment only as it does not move past the dermis of the animal and is effective for over 30 days (Metzger and Rust 2002). Fipronil attaches to hair follicles in the dermal skin layer within 24 h (Kahn 2005). The remaining ingredients in FRONTLINE spray evaporate quickly; the main carrier is isopropyl alcohol, which evaporates in about 20 min. Thus, the impact on grooming and allogrooming behaviors is minimal. Even though our dosages were extremely low, to be extra cautious, no antiparasite treatments were given to juveniles (<6 months of age), which have very low parasite loads anyway (Hillegass et al. 2008). Treatment of some individuals in a group will reduce ectoparasite loads in all group members because they sleep together in close contact, allowing newly applied fipronil to pass between individuals and ectoparasites from untreated individuals to jump to treated individuals and die. Madden and Clutton-Brock (2009) found no differences in parasite loads in treated and untreated individuals in the same group using a similar antiparasite treatment; parasite loads declined significantly in both treated and untreated meerkats (Suricata suricatta). Thus, social group was our experimental unit.
Behavioral observations and analysis
Cape ground squirrels are diurnal and live in open habitats (Smithers 1971; Herzig-Straschil 1978), and the low vegetative cover on the floodplain allowed for relatively easy observation from hides on the roof of vehicles or observation towers. Observations focused on morning and evening hours (700–1000 and 1500–1800 h) when squirrels were nearest to burrows (Waterman 1996). We used all occurrences methods (Altmann 1974) to record any occurrence of autogrooming, allogrooming, and movement into and out of a burrow (Waterman 1995). Duration of autogrooming or allogrooming behavior was recorded in seconds. We recorded identity and sleeping burrow location for all squirrels in a burrow cluster and calculated linear distance from each emergence or immergence site to the next consecutive location. We considered a movement of ≥15 meters from one emergence location to the next immergence location as a shift to a new sleeping burrow. All colonies were closely watched for emergence of young. For several offspring, the mother’s identity was unknown, as more than a single mother returned to the social group with offspring at the same approximate time, and thus, mothers and litters could not be distinguished.
All data were checked for homogeneity and normality and transformed if necessary. Data that could not be normalized or homogenized were analyzed with nonparametric statistics (Fry 1993). We used repeated measures analysis of variance (ANOVA), controlling for social group identity, to determine if body mass changed during the study (June to August). Offspring born during the study (July to August) were used to calculate group reproductive output (number of offspring per adult female) for treated and control groups. We analyzed autogrooming and allogrooming data from July through September for squirrels with a minimum total observation time of 60 min. For each squirrel, we divided the cumulative amount of time (s) spent allogrooming (or autogrooming) by the total amount of time that animal was observed (h) to determine the proportion of time (s/h) that an individual spent allogrooming (or autogrooming). We also compared the minimum numbers of sleeping burrow shifts between treated and control groups. One treatment group disbanded in late June, decreasing the number of treated replicates from 9 to 8. Occasionally, not all individuals in a treated social group were captured within the same week for treatment, but parasites were still substantially reduced even in these groups (Scantlebury et al. 2007).
RESULTS
Body mass of nonpregnant adult females did not change from June to August in either control (repeated measures ANOVA, controlling for social group identity, F1,8 = 0.274, P = 0.62) or treated (F1,11 = 0.362, P = 0.56) groups (Figure 1a). However, reproductive success (offspring per adult female) was much higher in treated groups (Mann–Whitney U = 13.5, P = 0.025, n1 = 8, n2 = 9; Figure 1b). Of the litters we were able to distinguish, litter size did not differ between treatment (1.38 ± 0.18, n = 8) and control (1.60 ± 0.25, n = 5) groups (Mann–Whitney U = 15.5, P = 0.56). Likewise, sex ratio of offspring produced in control groups (4m:5f) and treatment groups (11m:11f) did not differ (chi squared = 0.079, P = 0.78; the sex of another 8 offspring in treatment groups was unknown). Treated groups (n = 8) spent less time autogrooming (square root transformation, nested ANOVA, controlling for social group, F1,23 = 5.59 P = 0.027) and less time allogrooming (using mean allogrooming rate for each group, Mann–Whitney U = 9.0, P = 0.02, n1 = 8, n2 = 9) than untreated groups (Figure 1c). The minimum number of burrow shifts made by control and treated groups did not differ (Mann–Whitney U = 29.5, P = 0.51, n1 = 8, n2 = 9; Figure 1d).
Effect parasite removal on adult female (a) body mass, (b) reproductive success (offspring surviving to emergence per adult female in the group), (c) grooming rates (seconds grooming per hour of observation), and (d) burrow shifts (≥15 m, from July to September). Only reproductive success and grooming differed between treated and untreated squirrels (P < 0.05). Data (mean ± standard error) are from the means of 8 treated groups and 9 control groups.
Effect parasite removal on adult female (a) body mass, (b) reproductive success (offspring surviving to emergence per adult female in the group), (c) grooming rates (seconds grooming per hour of observation), and (d) burrow shifts (≥15 m, from July to September). Only reproductive success and grooming differed between treated and untreated squirrels (P < 0.05). Data (mean ± standard error) are from the means of 8 treated groups and 9 control groups.
DISCUSSION
Animals must make trade-offs in investment in feeding, reproduction, and dealing with predators and parasites, and life-history theory suggests that females who are freed from energy-sapping parasites should invest more in reproduction. The 4-fold increase in reproductive success of treated females in our manipulation suggests that reproductive costs of parasites are substantial. Removal of parasites appears to free up energy that females can invest into the quality of their offspring (i.e., their survival), allowing them to survive through gestation and lactation (Hoste & Chartier 1993; Kristan 2004; Hinde 2007; Hayssen 2008). Thus, the increased reproductive success we observed in treated females was most likely due to higher juvenile survival.
Gestation length appears genetically fixed in most mammals (Racey 1981), but females in better condition can have shorter lactation (Lee et al. 1991; Arnold & Lichtenstein 1993). The energetic costs of lactation can be very high, requiring far more energy than gestation (Kenagy et al. 1990; Millesi et al. 1999; Kunkele 2000). Even for animals physiologically well adapted to low water availability, high aridity during the dry season can limit breeding success, especially when combined with other factors such as low food availability (Mutze et al. 1991; Degen 1997) and potentially parasite infestation.
Increased investment in reproduction could occur through an increase in litter size, but we found no indication that nonparasitized female Cape ground squirrels have larger litters than controls. In Columbian ground squirrels (Spermophilus columbianus), the removal of parasites resulted in significantly larger litters (Neuhaus 2003). However, Columbian ground squirrel litter sizes vary much more than those of Cape ground squirrels (Murie et al. 1980). Litter size in Cape ground squirrels is constrained to only 1 or 2 offspring (Waterman 1996; Pettitt et al. 2008), which would limit the ability of females to increase the production of juveniles in a single reproductive event. Thus, even with a longer parasite removal, we would not expect to see larger litters in this species.
Common sleeping burrows in social mammals have causally linked parasite transmission to burrow switching (Hoogland and Sherman 1976; Hausfater and Meade 1978, 1982; Hoogland 1995; Lewis 1996; Altizer et al. 2003). Studies in Brant’s whistling rats (Parotomys brantsii; Roper et al. 2002) and European badgers (Meles meles; Butler and Roper 1996) showed decreases in burrow and den shifts in response to parasite removal. Intense sociality and communal living of Cape ground squirrels create an environment ideal for parasite transmission, both in and out of the burrow system. However, the number of burrow shifts by Cape ground squirrels did not decrease significantly with parasite removal as expected (Figure 1d). Shifting burrows may be a learned or genetically programmed behavior that continues even after parasites are removed. Alternatively, because Cape ground squirrels (particularly young animals) are frequently preyed upon by snakes (Waterman 1997), they may continue to switch sleeping burrows even in the absence of parasites to avoid predators.
Autogrooming, an asocial activity, decreased in treated Cape ground squirrels. This result supports autogrooming as a stimulus-driven response to biting parasites (Hart 2000; Mooring et al. 2004; Hawlena et al. 2008). However, persistence in treated animals, at a lower rate, suggests that autogrooming may also improve dermal hygiene (e.g., removing dirt and other debris along hair shafts) in this semi-fossorial animal.
Allogrooming, a social activity that is an important component of dominance and other social interaction in many species (Spruijt et al. 1992; Stopka and Graciasova 2001; Kutsukake and Clutton-Brock 2006), almost completely stopped in treated female Cape ground squirrels. Because Cape ground squirrels females do not form dominance hierarchies (Waterman 1996), allogrooming may function solely to control parasites and may be why ectoparasite loads are not higher in larger groups in this species (Hillegass et al. 2008), even though parasite loads are expected to increase with group size (Brown and Brown 2004).
In conclusion, the impact of parasites on the reproductive success of this arid-living species is considerable. Female Cape ground squirrels are frequently unsuccessful in raising offspring, especially during periods of low rainfall, and this failure appears to be magnified by the physiological costs of bearing parasites. Behaviorally, females responded to the loss of parasites with less grooming, allowing more time for feeding, but we did not see the decline in sleeping burrow movements that we expected. Our treatments were purposely nonselective, and future parasite-specific removals are needed to determine whether ectoparasites or endoparasites have the greatest impact on squirrel reproductive success. Although we did not assess quality of offspring (e.g., size or condition), which may also affect offspring survival (Neuhaus 2003), future research should consider offspring quality, as well as metabolic costs of reproduction, in directly quantifying the costs of parasites to reproducing females.
FUNDING
National Science Foundation grant (IBN0130600 to J.M.W.).
Research protocols were approved by the animal care committee at the University of Central Florida and complied with their guidelines for animal research. We thank Northwest Parks and Tourism of South Africa and the staff of the S.A. Lombard Nature Reserve for their permission to conduct this research and their continued support. We also thank B. Pettitt, J. Calldo, M. Scantlebury, and T. Mulaundzi for their assistance in the field and C.J. Anderson, D. Jenkins, and M.B. Manjerovic for valuable suggestions and comments on the manuscript.
