Juvenile hormone, metabolic rate, body mass and longevity costs in parenting burying beetles

Animal Behaviour 92 (2014) 203e211

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Juvenile hormone, metabolic rate, body mass and longevity costs in parenting burying beetles Stephen T. Trumbo a, *, Claudia M. Rauter b a b

Department of Ecology and Evolutionary Biology, University of Connecticut, Waterbury, CT, U.S.A. Department of Biology, The University of Nebraska at Omaha, Omaha, NE, U.S.A.

a r t i c l e i n f o Article history: Received 15 January 2014 Initial acceptance 5 February 2014 Final acceptance 13 March 2014 Available online 10 May 2014 MS. number: A14-00040R Keywords: brood care burying beetle metabolism Nicrophorus parental effort parental investment reproductive cost reproductive stress

Levels of juvenile hormone (JH) are elevated during parental care in burying beetles (Nicrophorus) at a time when ovarian activity is suppressed, suggesting that JH plays an alternative role to its better known gonadotropic function. Because parental activity in burying beetles is time-intensive, it might be expected to be energetically stressful and to impart longevity costs. We predicted that the active (feeding) stage of care would be associated with elevated JH and greater energy expenditure (higher metabolic rate, lower body mass), and that this stress would result in shorter life span. Parents experimentally manipulated into providing more care for young had increased levels of JH. For females, there was a significant longevity cost associated with parental care but not with mating or egg production. During carcass preparation, females and males that would later regurgitate to larvae showed an initial increase in body mass followed by a significant decrease in mass during the intense period of parental provisioning of young (during and just after the JH peak). Males that provided little care (on small carcasses with a female partner) showed no such anticipatory weight gain. An indirect measure of metabolic rate (VCO2) was nearly twice as high in caregiving females compared to nonbreeding females. These results suggest that the energy demands and/or high JH levels during care extract a significant cost on longevity. We propose that JH has evolved to play a novel role in parental care in burying beetles associated with extreme energy demands during feeding of offspring. Ó 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

The costs of reproduction structure many fundamental life history parameters such as the timing of breeding, clutch size, interclutch intervals, parental care and life span (Calow, 1979; Snell & King, 1977; Trivers, 1972). Reproductive costs for females may result from mating, egg production and caregiving, but the importance of these components are quite variable across species and even within species. Mating costs, for example, can be substantial in some species (Kuijper, Stewart, & Rice, 2006), minimal in others (Reguera, Pomiankowski, Fowler, & Chapman, 2004), and apparently absent in still others (Kotiaho & Simmons, 2003; Reinhardt, Naylor, & Siva-Jothy, 2009). Within a species, the costs of a particular component of reproduction, such as provisioning young, can vary substantially with the availability of food resources for parents (Fletcher et al., 2012). Costs of parental care might be expected to vary with energetic stress. Effective parental care across a wide variety of taxa may require elevated metabolic rates that approach physiological limits, likely explaining weight gain

* Correspondence: S. T. Trumbo, Department of Ecology and Evolutionary Biology, University of Connecticut, Waterbury, CT 06702, U.S.A. E-mail address: [email protected] (S. T. Trumbo).

prior to the most active period of care (McNab, 2002; Moreno, 1989;  ski, & Konarzewski, Reardon & Chapman, 2010; Sadowska, Ge˛ bczyn 2013). Among birds, a high value and investment in the current brood may lead to a weaker response to stress and less investment in self-maintenance (Bokony et al., 2009), which might be reflected as a loss in body mass and decrease in life span. Correlates of reproductive stress such as hormone levels, rates of behaviour, metabolic activity, immune function and changes in body mass can provide insight (Harshman & Zera, 2007), first, into which components of reproduction are the most costly, and secondly, into how natural selection affects reproductive decisions. Among insects, juvenile hormone (JH) regulates allocation of resources to reproductive activities and to self-maintenance, including immunity (González-Tokman, González-Santoyo, Munguía-Steyer, & Córdoba-Aguilar, 2013). Because JH can be upregulated by stimulation of insulin-like receptors (Mutti et al., 2011) and JH increases susceptibility to oxidative stress (Salmon, Marx, & Harshman, 2001), it is not unexpected that JH can be an important mediator of reproductive costs (Tu, Flatt, & Tatar, 2006). In burying beetles (Nicrophorus spp.), much is known about changes in behaviour and immune function during reproduction. There is incomplete information, however, on changes in body

http://dx.doi.org/10.1016/j.anbehav.2014.04.004 0003-3472/Ó 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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mass and the role of JH, and nothing known about metabolic rates. The association of JH and metabolic state has received the most study in diapausing insects (Denlinger & Tanaka, 1989; Singtripop, Saeangsakda, Tatun, Kaneko, & Sakurai, 2007) where JH is related to mitochondrial function that may be independent of transcription  and translation (Farkas & Sut’áková, 2001; Stepien, Renaud, Savre, & Durand, 1988). JH can be associated with general metabolic activity, as, for example, cycles of O2 consumption in larval flesh flies (Denlinger & Tanaka, 1989). There has been little study of the JHe metabolism association in breeding insects, although Sullivan et al. (2003), working with adult worker honeybees found that removal of the source of JH lowered metabolic rates. The JHeenergy association may help us understand how JH relates to reproductive costs and why JH has such diverse behavioural and life history effects in adults. Insight might then be gained into why, nonintuitively, JH can have opposite associations with reproductive behaviour in different systems. For example, JH (1) increases social dominance in Polistes wasps (Tibbetts & Huang, 2010) but decreases it in a queenless ant (Brent, Peeters, Dietmann, Crewe, & Vargo, 2006), (2) decreases survival in young males of the damselfly Hetaerina americana but increases it in old males in the same population (González-Tokman et al., 2013) and (3) is found at low levels during caregiving in a viviparous cockroach and in earwigs (Rankin, McQuiston, & Jackson, 1999; Tobe et al., 1985) but is found at high levels in parenting burying beetles (Trumbo, 1997). Some of the variation in JH effects on the same category of behaviour across species might be explained by species differences in metabolic/ energy demands for those behaviours. Parental care among insects takes many forms, varying from passive guarding during a nonfeeding state to active provisioning and nest upkeep (Trumbo, 2012). The finding that JH is elevated during parental care of the burying beetle Nicrophorus orbicollis Say, when the ovaries are suppressed, was initially unexpected. In other parental insects, JH titres or JH synthesis is likely low during caregiving or brooding, and application of JH analogues can terminate care (Tallamy, Monaco, & Pesek, 2002; Trumbo, 2002 and references therein). Trumbo and Robinson (2008) suggested that a key difference is that parental care is very active in burying beetles. The female parent works close to maximal capacity except with the very smallest broods (Fetherston, Scott, & Traniello, 1990; Rauter & Moore, 2004), where paired N. orbicollis females spend more than 80% of their time processing carrion, regurgitating to young, maintaining the carcass and controlling the microbial environment in the nest. While some species can offset reproductive costs with less investment in immunity (Rolff & Siva-Jothy, 2003; Zuk & Stoehr, 2002), caregiving in burying beetles occurs at a time of elevated individual and social immunity in adults, probably because of the microbe-rich resource being utilized and the need to offset the lower level of immunity in young larvae (Cotter, Littlefair, Grantham, & Kilner, 2013; Cotter, Topham, Price, & Kilner, 2010; Steiger, Gershman, Pettinger, Eggert, & Sakaluk, 2011, 2012;  ski, Czarniewska, Baraniak, & Rosin  ski, 2014). Some species Urban can also reduce the costs of reproduction by withholding care, but this offset is limited in our study species because the first two instars of N. orbicollis will not develop without regurgitations (Trumbo, 1992). The combination of elevated behavioural activity, immune investment and JH levels during regurgitation to young suggests that caregiving might be expected to impose significant costs. In the present study, we predicted that (1) mating, egg production and parental care would result in longevity costs, with the greatest cost associated with care; (2) the regurgitation stage of care would also be associated with a loss in body mass and with elevated JH levels; and (3) the regurgitation stage of parental care in N. orbicollis would be a time of elevated resting metabolic

rate, comparable to the high levels of energy expenditure and stress in caregiving mammals and birds (McNab, 2002). We investigated the relationships between activity, JH, metabolism and costs of reproduction in N. orbicollis in four ways. (1) We partitioned the longevity costs of reproduction into mating, egg production/carcass preparation and posthatching care by terminating reproductive bouts at different stages, and assessing the effects on life span. (2) We developed the most complete profile in a subsocial invertebrate of changes in body mass prior, during and after breeding in males and females on small and large carcasses, to reflect the energetics of breeding. (3) We compared metabolic rates of breeding age N. orbicollis females that were providing active care versus females that were not breeding. (4) We assessed JH levels of beetles manipulated into providing more care, first in single males (N. orbicollis) forced to provide all care versus paired males, which provide less care, and second in males and females providing care on large versus small carcasses. This last comparison utilized Nicrophorus pustulatus Herschel because this species will produce the largest broods of any Nicrophorus (Trumbo, 1992), a capacity related to its ability to exploit a large amount of resource in nests of snake eggs (Blouin-Demers & Weatherhead, 2000; Smith, Trumbo, Sikes, Scott, & Smith, 2007). The results demonstrate strong correlations between regurgitating to young, JH level, metabolic rate and body mass, and provide evidence that posthatching care of young is the most costly component of reproduction. Our results also suggest that elevated JH in burying beetles has been co-opted for a novel role in parental care. Study Animals A single female burying beetle or a maleefemale pair will bury a small vertebrate carcass, strip it of hair or feathers and apply secretions to control the microbial environment (Rozen, Engelmoer, & Smiseth, 2008) during carcass preparation (the first 3e4 days after discovery of the resource). Oviposition begins less than 24 h after discovery and is usually complete by day 3. Both males and females are reported to increase their body mass on the carcass (day 1, Nicrophorus vespilloides: Steiger, Gershman, et al., 2012; day 3, N. orbicollis: Panaitof, Scott, & Borst, 2004; by the time larvae hatch, day 3 or 4, N. vespilloides: Jenkins, Morris, & Blackman, 2000). JH in N. orbicollis spikes in both males and females immediately after a carcass is discovered (Trumbo, Borst, & Robinson, 1995), although this may not be related to subsequent ovarian development and deposition of vitellogenin (Panaitof & Scott, 2006; Scott & Panaitof, 2004). JH levels then decline but will later reach a higher peak during the most active period of parental care, that is, during the first 48 h that larvae are on the carcass (days 4e6) when most of the processing and regurgitation of carrion occur (Panaitof et al., 2004; Scott, Trumbo, Neese, Bailey, & Roe, 2001; Trumbo, 1997). During the late nesting period, larvae feed almost exclusively from the carcass and the parent(s) becomes less active but will defend against intruders. Male care is much more variable than female care in both duration and intensity. Males feed larvae less than females and they leave the nest sooner, especially on a smaller carcass (Fetherston et al., 1990; Trumbo, 1991). Males increase their activity and duration of care substantially if the female is removed, but female behaviour in the reverse manipulation changes little, probably because females may be working close to their maximum potential, except on the very smallest carcasses (N. orbicollis: Fetherston, Scott, & Traniello, 1994; Rauter & Moore, 2004; N. vespilloides: Smiseth & Moore, 2004; Smiseth, Dawson, Varley, & Moore, 2005; Nicrophorus quadripunctatus: Suzuki & Nagano, 2009). Reproduction has clear longevity and fecundity costs in N. orbicollis (Creighton, Heflin, & Belk, 2009) but mixed effects in

S. T. Trumbo, C. M. Rauter / Animal Behaviour 92 (2014) 203e211

205

N. vespilloides (Cotter et al., 2010; Steiger, Meier, & Müller, 2012; Ward, Cotter, & Kilner, 2009) and N. quadripunctatus Kraatz (Satou, Nisimura, & Numata, 2001). The relative longevity costs of mating, egg production and parental care are poorly understood.

15. On one small and one large carcass, no larvae were produced and the adult pairs were excluded from the analysis.

METHODS

To test the prediction that resting metabolic rate is elevated during parental care, we measured resting metabolic rate of each female N. orbicollis twice, once when the females were sexually mature but not reproducing and once when each female had been providing parental care to second-instar larvae, when feeding rates are highest (Fetherston et al., 1990). To measure metabolic rate, we placed virgin females (age 36e42 days) into an animal chamber (50 ml) of a flow-through respiratory system for 114e180 min. We took metabolic measurements during the day (when nonreproducing beetles are inactive) under red light to simulate the conditions in the ground, since the beetles are buried in the ground during the day. After the measurements, we placed the beetles back into their containers. The same evening, or up to 6 evenings later, we added a male to the container with the female. One to three days later, we removed the male and placed the female into a new container (15  10  5 cm) filled with 5 cm of moist peat and containing a previously frozen mouse (range 19.6e 26.7 g). These new containers were kept in a dark room at 20.0e 24.0
C and were checked twice daily for newly hatched larvae. The day after the first larvae had hatched and most of the larvae had moulted into the second instar, we removed the female to measure metabolic rate a second time. The metabolic measurements of beetles in the parental phase lasted 110e180 min, after which the female was returned to the larvae. We measured metabolic rate of 16 sexually mature, virgin female burying beetles. Of these 16 females, 13 laid eggs and had larvae hatching. We discarded the second metabolic measurements of five females because the females were active during the entire measurement. The measurement of one additional female was lost because of a computer malfunction.

General Methods All beetles used in experiments were the laboratory-reared descendants of wild-caught beetles. Burying beetles in JH experiments were descended from populations in Pellston, Michigan, U.S.A., beetles in metabolic rate experiments were descended from populations in Douglas County, Nebraska, U.S.A., and beetles in the body mass and longevity experiments were descended from populations in Bethany, Connecticut, U.S.A. All three populations are phenologically and behaviourally similar. Adult beetles were isolated shortly after adult emergence and maintained in small containers (9 cm diameter, 5 cm height) with a moistened paper towel and scraps of chicken liver. Beetles were maintained at 21
C on a 15:9 h light:dark cycle, unless otherwise noted. Adults were bred in 15  29  11 cm containers, two-thirds filled with soil. Breeding chambers were kept in the dark except for inspection. All carcasses were frozen shortly after death and thawed to room temperature overnight before presentation to breeding age adults. Longevity Costs To examine longevity costs of reproductive activity, 84 females were isolated shortly after emergence as adults and divided into four treatments: (1) control females were never mated or presented a carcass; (2) mated females were mated to a different single male on days 19, 21, 41 and 43, on the same days as females in the remaining two treatments, designed to ensure adequate sperm availability; (3) single egg-producing females (previously mated) were presented a 24e27 g mouse carcass on day 26 and again on day 46 but removed 48 h later after eggs had been produced, the carcass had been rounded and the hair of the carcass removed; (4) parental single females were presented a 24e27 g carcass on day 26 and a second carcass on day 46 and kept on the carcass until their young dispersed (11 days after carcass presentation). After each manipulation, females were again isolated and fed until they died. Four females died of dehydration because of caretaker error prior to day 70 and were excluded from the analysis. Body Mass during the Reproductive Cycle We allowed 36 males and 36 females of N. orbicollis to feed ad libitum after adult emergence on scraps of liver provided in excess and weighed on day 26 postemergence to establish a prebreeding body mass. On the following day, 12 maleefemale pairs were established on large carcasses (30e31.5 g) and 12 pairs were established on small carcasses (11.5e13 g). An additional 12 males and females were maintained in isolation as nonbreeding controls. Starting on the second day after carcass presentation, males and females were weighed each day and then returned to their carcass. Because weight can be affected by moisture and release of defensive secretions, beetles were carefully wiped clean before each measurement. Based on prior work (Trumbo, 1991), parents were removed from the breeding chamber when they were expected to desert normally (after weighing on day 11 (females/large carcass), day 10 (females/small carcass), day 7 (males/large carcass), day 5 (males/small carcass)). After removal, adults were isolated, allowed to feed ad libitum and weighed daily until day 11, and again on day

Metabolic Rates of Females during Parental Care

Metabolic Measurements We used a flow-through respiratory system to measure rates of CO2 production. Ambient air was pumped at a rate of 150 ml/min through the respiratory system. The flow rate of the air was controlled with a RiteflowÒ flowmeter (Model 404070075, Bel-Art Products, Pequannock, NJ, U.S.A.). The ambient air was scrubbed of CO2 by soda lime before being pumped through the animal chamber (50 ml). The animal chamber was located in a water bath maintained at 24
C. Water vapour was removed by a Drierite column before the air entered the infrared CO2 analyser (Qubit S151, Kingston, Ontario, Canada) capable of a resolution of 1 ppm. The analyser was calibrated regularly against a precision gas mixture. The CO2 readings were recorded at 10 s intervals by a PC running the software Logger Pro 2.1 (Vernier Software & Technology, Beaverton, OR, U.S.A.). Based on Lighton (2008), the rate of CO2 production (VCO2) was calculated as follows: VCO2 ¼ (FECO2  FICO2)  flow rate, where FECO2 is the fractional concentration of CO2 leaving the animal chamber and FICO2 is the fractional concentration of CO2 entering the animal chamber. FICO2 was zero, as the CO2 of the air entering the animal chamber had been removed. CO2 production, measured as VCO2, was corrected to standard pressure and temperature. We visually observed the activity of the beetles at regular intervals during the metabolic measurements. Activity caused a substantial increase in CO2 production. At the end of each metabolic measurement, we visually inspected the CO2 recordings for peaks of substantial increases in CO2 production, but we excluded these peaks from our calculations of mean CO2 production (VCO2).

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We determined body mass (0.1 mg) before and after each metabolic measurement and used the mean of these two measurements to calculate the mass-specific rate of CO2 production (VCO2).

We established 48 maleefemale pairs of N. orbicollis (26e28 days postemergence) on 18e24 g mouse carcasses. On the fourth day after carcass presentation, we checked each carcass for the presence of first-instar larvae. For experimental manipulation, we used the first 40 set-ups for which the carcass was prepared and five larvae were present. In half the trials, we removed the female parent. We took a haemolymph sample (3e5 ml) on day 5, 6, 7 or 8 from five individual males of each treatment by severing the hindleg, collecting haemolymph with a micropipette, and storing it in acetonitrile at 70
C. Each male was sampled once. Haemolymph samples were assayed for JH titre as in Trumbo et al. (1995) using a chiral-specific radioimmunoassay (RIA) (Hunnicutt, Toong, & Borst, 1989). JH in Parents on Rat and Mouse Carcasses We established maleefemale pairs of N. pustulatus (32e34 days postemergence) on rat (N ¼ 32, 80e100 g) or mouse (N ¼ 30, 18e 20 g) carcasses. On the fourth day after carcass presentation, we checked each carcass for the presence of first-instar larvae. For the experiment, we used the first 25 set-ups from each treatment for which five larvae were present. We took a haemolymph sample (2e 5 ml) immediately (day 4) or on day 5, 6, 7 or 8 from both the male and female of each replicate (five per treatment). Haemolymph samples were handled as above. For trials terminated on days 6, 7 and 8, we counted the larvae, as all young were expected to have hatched by that time.

Control Mated Egg producer Parental

0.8 Proportion surviving

JH in Single and Paired Males

1

0.6

0.4

0.2

0

50

100

150

200

Day Figure 1. KaplaneMeier survival plot (in days) for control, mated, egg-producing and parental female burying beetles.

(parametric survival model: treatment: c23 ¼ 33:96, P < 0.0001; pronotal width: c21 ¼ 0:00, P ¼ 0.98; interaction: c23 ¼ 2:00, P ¼ 0.57). In pairwise tests, only parental females had a significantly different mean life span (42.5%) than controls (log ranks tests: parental versus control: c2 ¼ 27.51, P < 0.0001; mated versus control: 2.9%, c2 ¼ 0.36, P ¼ 0.55; egg-producing versus control: 10.3%, c2 ¼ 3.64, P ¼ 0.056). Parental females also had a significantly shorter life span than egg-producing females (c2 ¼ 19.8, P < 0.0001) and mated females (c2 ¼ 27.66, P < 0.0001). Body Mass during the Reproductive Cycle

Statistical Methods For the longevity experiment, we generated KaplaneMeier survivorship curves for each treatment, and Weibull plots and fits confirmed that a Weibull distribution was appropriate for parametric survival models. We performed pairwise comparisons of treatments using log ranks test with the significance criterion reduced to 0.01 because of multiple comparisons (SAS, 2007). We used two repeated measures ANOVAs to analyse body mass during the breeding cycle. The first one included only nonbreeding beetles; the second one included all beetles. We conducted the analyses using Proc GLM in SAS (2007) and chose the TukeyeKramer test for pairwise comparisons between males and females and between treatment groups at each day of the breeding cycle. We compared JH sample means for each day of the experiment between paired and single males (N. orbicollis) and between parents on rat versus mouse carcasses (separately for male and female N. pustulatus). JH titres were log transformed to reduce skew and evaluated using t tests. Titres were backtransformed for graphical presentation. Details for unplanned statistical contrasts are given in the relevant section. All tests were two tailed unless otherwise indicated. RESULTS Longevity Costs Survivorship plots for control, mated, egg-producing and parental females are shown in Fig. 1. Treatment but not pronotal size had a significant effect on longevity in N. orbicollis females

Body mass of nonbreeding (control) males and females did not differ (F1,22 ¼ 2.60, P ¼ 0.12) and changed very little during the trial, despite increasing significantly over time (time: F11,242 ¼ 9.03, P < 0.0001; time*sex: F1,242 ¼ 2.03, P ¼ 0.07), with the maximum difference from the starting weight being þ2.4% (Fig. 2). Body mass of breeding males and females changed differently over time (time*sex interaction: F11,682 ¼ 5.58, P < 0.0001), but how it changed depended on whether the carcass was large or small (time*sex*treatment: F22,682 ¼ 14.96, P < 0.0001). In the three treatments in which adult N. orbicollis were expected to engage in regurgitation to larvae (females and males on large carcasses, females on small carcasses), the parent gained significant body mass by day 2 after carcass presentation and maintained an elevated mass relative to body mass of nonbreeding beetles through day 4 (4.4e9.0%; Fig. 2). In all three treatments, body mass declined significantly during the period that included elevated JH and active care (day 4 versus day 7) (paired t test: P < 0.0001, one tailed for each treatment). Body mass on the final day of the trial (day 15, postbreeding) was not significantly different from the body mass of nonbreeding beetles for any of the four breeding treatments (Fig. 2). Males on very small carcasses typically provide little active care such as feeding young. The absence of an increase in male body mass early in the breeding cycle during preparation of a small carcass matched the absence of future energy expenditure (Fig. 2), a very different pattern than that shown by parents that were likely to engage in active care. Males on small carcasses did lose significant mass by day 5 even though all males were seen on the carcass during at least two of the four inspections on days 2e5 (Fig. 2).

S. T. Trumbo, C. M. Rauter / Animal Behaviour 92 (2014) 203e211

115

(a)

Nonbreeding males Nonbreeding females

110

F1,9 ¼ 2.42, P ¼ 0.15), leaving the reason for the significant deficit in female body mass late in the cycle on small carcasses uncertain.

105

Metabolic Rates of Females during Parental Care

100

CO2 production, an indirect measure of metabolic rates, was higher in N. orbicollis females in the parental state (with secondinstar larvae) than in the same females when they were breeding competent but not in a parental state (Table 1). CO2 production was over twice as high during care as during the prebreeding period and 81.5% greater on a body mass-adjusted basis.

95 90 Percentage of starting mass

207

0

2

4

6

110 *

105

*

† †

10

12

14

16

Females on large carcass Females on small carcass Nonbreeding females

(b)

115

8

* *

JH in Single and Paired Males

* * *

†

100 95 90

† 0

2

4

6

* * * *

110

† † 10

12

14

16

JH in Parents on Rat and Mouse Carcasses

Males on large carcass Males on small carcass Nonbreeding males

(c)

115

8

105 100 95 90

† 0

2

4

6

8 10 Day of trial

12

14

Nicrophorus orbicollis males that were manipulated into providing care without a female showed significantly higher levels of JH on the second and third day of care (days 5 and 6 after carcass presentation) compared to males with a female partner (Fig. 3). For both treatments, JH levels fell to similarly low levels by day 7, when parental care is known to be reduced.

16

Figure 2. Percentage of starting body mass (mean  SE) of N. orbicollis across days 0e 16 in each treatment: (a) nonbreeding males versus females; (b) breeding females on large versus small carcasses versus nonbreeding females; (c) breeding males on large versus small carcasses versus nonbreeding males. Day 0 is just before discovery of the carcass. Day 4 is the modal time that larvae arrived on the carcass. The modal time for larval dispersal from the nest was day 10 for small carcasses and day 11 for large carcasses. Arrows represent the time at which the parent was removed from the carcass. *Significant difference (P  0.05) between nonbreeding parent and breeding parent on large carcasses. ySignificant difference (P  0.05) between nonbreeding parent and breeding parent on small carcasses.

Eleven of 11 small carcasses were depleted of all usable resource by the end of the trial; this occurred on only 2 of 11 large carcasses (Fisher’s exact test: P < 0.001, two tailed). It is noteworthy that the superabundance of resource of a large carcass affected body mass of parents as early as day 5, when carcasses of both sizes still had substantial resources upon which to feed. On day 5, females on small carcasses had returned to their prebreeding body mass, whereas those on large carcasses had not (Fig. 2). One possible explanation for this result is that females on large carcasses used less energy during care. This does not appear likely because there was a bigger nest to maintain and more larvae to care for on a large carcass (mean  SE: 16.1  1.9 versus 8.2  0.6; ANOVA: F1,20 ¼ 16.06, P < 0.01). An alternative explanation is that females restrained from feeding themselves on small carcasses to preserve more resource for young. This might explain the significant deficit in body mass on days 8e10 relative to prebreeding mass of female parents on small carcasses, mass that was not recovered until the female was removed from the carcass (Fig. 2). The percentage decrease in body mass of females on small carcasses between the prebreeding state and the last day of care, however, was not significantly related to the final brood mass (regression, one tailed:

Nicrophorus pustulatus biparental pairs had significantly more larvae on large (mean  SE ¼ 54.9  6.2) as opposed to small carcasses (7.5  2.4) (ANOVA: F1,28 ¼ 58.26, P < 0.0001) without any overlap of brood sizes between treatments for trials terminated on days 6, 7 and 8. Both male and female N. pustulatus had higher levels of JH when providing care on a rat carcass (80e100 g) than on a mouse carcass (18e20 g) (Fig. 4). The differences in JH profile when utilizing a large versus a small carcass, however, were distinct for the two sexes. Males had significantly higher JH levels on rat carcasses than on mouse carcasses from the time larvae arrived on the carcass until the third day of care (days 4, 5 and 6) (Fig. 4a). Females, on the other hand, had similarly high levels of JH on mouse and rat carcasses during the first 2 days of care (days 4 and 5), but then maintained elevated levels of JH on rat carcasses for days 6 and 7 (Fig. 4b). DISCUSSION This study demonstrates that the early stage of posthatching parental care in N. orbicollis is a time of elevated metabolic rates, high JH titres and a rapid loss in body mass, which correlate temporally with enhanced regurgitation rates determined in earlier studies (Fetherston et al., 1990, 1994; Panaitof et al., 2004). As predicted, the posthatching parental care component of reproduction showed the greatest longevity cost. Although life span in females followed the predicted pattern (control > mating > egg producers > parental), only parental care had a significant negative effect. There have been few manipulation

Table 1 Effect of reproductive state on resting metabolic rate (mean  SE) in Nicrophorus orbicollis (N ¼ 7) at 24
C Sexually mature not reproducing

Providing parental care

Paired t test

Mass (mg)

356.524.7

413.726.7

t6¼7.94 P<0.001

Rate of CO2 production VCO2 (ml CO2/h)

0.2550.023

0.5340.039

0.7150.036

1.2980.071

t6¼6.48 P<0.001 t6¼6.08 P<0.001

VCO2 (ml CO2 per g per h)

208

S. T. Trumbo, C. M. Rauter / Animal Behaviour 92 (2014) 203e211

*

4000

JH titre (ng/ml)

Male-paired Male-single

3000

2000

* 1000 NS 0

NS

5

6 8 7 Days after carcass presentation

Figure 3. Titres of juvenile hormone, JH (mean  SE) in single and paired males of N. orbicollis. Carcass discovery is day 0. The arrow indicates when larvae arrive on the carcass. Comparison of titres in single males versus paired males (N ¼ 5 per treatmentday): day 5: t8 ¼ 3.20, P ¼ 0.04; day 6: t8 ¼ 4.18, P ¼ 0.01; day 7: t8 ¼ 0.22, P ¼ 0.82; day 8: t8 ¼ 0.70, P ¼ 0.50. *P < 0.05.

3500

**

(a)

Male-rat Male-mouse

* 3000

**

2500 NS

2000 NS

1500

JH titre (ng/ml)

1000 500 0 3500 3000

4 (b)

5

6

7

Female-rat Fem-mouse

**

NS NS

8

* NS

2500 2000 1500 1000 500 0

4

6 7 5 8 Days after carcass presentation

Figure 4. Titres of juvenile hormone, JH (mean  SE) during parental care on mouse and rat carcasses in (a) male and (b) female N. pustulatus. Carcass discovery is day 0. The arrow indicates when larvae arrive on the carcass. Comparison of titres on rat versus small carcasses (N ¼ 5 per treatment-day): males: day 4: t8 ¼ 3.56, P ¼ 0.007; day 5: t8 ¼ 3.83, P ¼ 0.008; day 6: t8 ¼ 6.29, P ¼ 0.001; day 7: t8 ¼ 0.25, P ¼ 0.81; day 8: t8 ¼ 0.26, P ¼ 0.82; females: day 4: t8 ¼ 0.66, P ¼ 0.53; day 5: t8 ¼ 1.31, P ¼ 0.25; day 6: t8 ¼ 8.43, P ¼ 0.001; day 7: t8 ¼ 4.13, P ¼ 0.02; day 8: t8 ¼ 1.08, P ¼ 0.29. *P < 0.05; **P < 0.01.

studies to isolate the cost of parental care in insects, and no study of the longevity cost. A burrower bug (Sehirus cinctus) and an earwig (Forficula auricularia) show greater interclutch intervals when providing care versus when only producing eggs (Agrawal, Combs, & Brodie, 2005; Kölliker, 2007) and digger wasp (Ammophila pubescens) females that invest more in provisioning produce fewer burrows (Field, Turner, Fayle, & Foster, 2007). The co-occurrence of high metabolic rates during care and longevity costs in burying beetles might suggest that this would be a good system for examining the effects of oxidative damage on ageing (Monaghan, Metcalfe, & Torres, 2009; Parrella & Longo, 2010). High parental activity can increase oxidative damage in birds (Christe, Glaizot, Strepparava, Devevey, & Fumagalli, 2012) and elevated JH might lead to greater sensitivity to oxidative stress (Salmon et al., 2001). Creighton et al. (2009) demonstrated that the costs of reproduction (life span, number of lifetime breeding attempts) in N. orbicollis were particularly high when females were manipulated into caring for a supernumerary brood where there was greater stress over limited food. The magnitude of reproductive costs may depend critically on the ability of a parent to feed optimally during times of maximum stress (Fletcher et al., 2012). Pathogens and parasites carried by burying beetles might be alternative agents of reproductive costs. Burying beetles host a large number of parasites that coordinate their reproductive cycles with that of their host’s cycle of finding, utilizing and dispersing from the key resource (e.g. Richter, 1993). These potential costs could be independent of the high metabolic rates, or interact with them. Context-dependent costs of defence against pathogens and parasites are common and may be greater for individuals in poor condition (French, DeNardo, & Moore, 2007; Sandland & Minchella, 2003). One advantage of developing burying beetles as a model system for examining the complexity of reproductive costs is that the timing of reproduction can be manipulated (presentation of a carcass) with respect to differences in age, nutrition, parental effort (size of brood or carcass, sex of parent), hormone state (JH will not induce egg production or care without a carcass) and pathogen levels. An intriguing contrast is the presence of significant reproductive costs in some studies (N. orbicollis: Creighton et al., 2009, this study; N. vespilloides: Cotter et al., 2010; Ward et al., 2009) with the lack of such costs in others (Steiger, Meier, et al., 2012). Boncoraglio and Kilner (2012) found a small reproductive cost for N. vespilloides females, but only if females were removed from the carcass during the most stressful period of care and not allowed to recover on the carcass. Mating costs are widespread in both invertebrate and vertebrate taxa and may include costs from exposure to male accessory fluids (Pomiankowski, Denniff, Fowler, & Chapman, 2005), pathogen transfer (Sheldon, 1993), JH-related reductions in immune function (Rolff & Siva-Jothy, 2002) and male harassment (den Hollander & Gwynne, 2009). None of these factors appears to affect longevity in N. orbicollis. The absence of a significant longevity cost for egg production was unexpected, especially as burying beetles can produce a clutch weighing more than 15% of their own body mass (also see Ward et al., 2009). Consistent with a low cost of egg production is the finding that small-bodied females (with presumably smaller energy reserves) lay just as many eggs as larger females (House, Walling, Stamper, & Moore, 2009). It is possible that a study with a larger sample would find a small cost of egg production, but the cost is unlikely to approach that demonstrated for posthatching care. Egg production is a frequent source of reproductive costs (Field et al., 2007; Partridge, Green, & Fowler, 1987), but not always (Davies, Kattel, Bhatia, Petherwick, & Chapman, 2005; Gems & Riddle, 1996; Hodkova, 2008). Costs of producing eggs may be related to searching and competing for food resources or

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oviposition sites (Minkenberg, Tatar, & Rosenheim, 1992; Monaghan, Nager, & Houston, 1998). These costs may be minimal in burying beetles because successful females have procured a large, high-protein, energy-rich resource on which they feed prior to, during and after ovipositing in the nearby soil. The limited cost of egg production/initial carcass preparation in burying beetles may explain otherwise puzzling behaviour. Burying beetles seem profligate in oviposition near small carcasses, typically laying far more eggs than will be reared, as the parent later kills many of its young to reduce brood size (Bartlett, 1987; House et al., 2009). Subordinate females that are driven off a carcass will often lay eggs that have a poor chance of being reared by the dominant female (Müller, Braunisch, Hwang, & Eggert, 2007). Females out of the breeding season or not yet reproductively mature will sometimes manipulate a carcass and lay a few eggs, but then abandon the attempt prior to care. The force of selection against these seemingly wasteful behaviours may be weak because of the low associated costs. Even minimal rewards might allow these behaviours to be selectively maintained. The present study demonstrates that both male and female N. orbicollis gain mass during the preparation of the carcass, then lose mass rapidly and show higher metabolic rates during the period of active care of young. The weight gain after discovery of a carcass is significant by 24 h (Steiger, Meier, et al., 2012). Two results from the present study suggest that this increase in body mass anticipates the energy demands on parents during the early posthatching period, a common phenomenon in birds (Gowaty, 1996; Moreno, 1989). First, the weight loss during provisioning of young occurred despite the considerable amount of time that N. orbicollis ‘ruminates’ on the carcass, a behaviour interpreted by Fetherston et al. (1990) as processing carrion for subsequent regurgitation and not for self-feeding. Our results clearly support this interpretation. Second, in the one experimental treatment in which a parent was not expected to feed young (a paired male on a very small carcass), no gain in body mass was observed during the prehatching period, presumably because extra energy would not be needed to sustain later food provisioning. The size of the small carcass in this experiment (11.5e13 g) is near the lower range of acceptable sizes for this species and males desert before or shortly after larvae arrive. Benowitz, Head, Williams, Moore, and Royle (2013) found that older male parents of N. vespilloides stay longer on a carcass and provide more care than young males. We predict that this would be reflected by different body mass profiles of young and old males during the carcass preparation period. Several explanations, which are not mutually exclusive, are possible for the loss in body mass by males on small carcasses. Males may have been attempting to desert the nest before day 5 (Trumbo, 1991), they may have been chased off by the female partner (Pukowski, 1933), or they may have restrained from feeding to reserve food for their young. The latter explanation could be relevant if (1) the expected value of the male’s present brood is larger than the uncertain value from future reproduction on a scarce resource or (2) the male has little to gain from consuming extra food, as evidenced by the return of body mass to the prebreeding mass within several days of desertion by both males and females, even in the presence of excess food. Because our males were young (27 days), we suggest that the second factor was more important in our study. We do not know the optimal body mass for N. orbicollis during flight, but we suggest that both males and females eventually returned to their prebreeding body mass because the excess weight would not be optimal for searching for a new reproductive opportunity (the flight efficiency hypothesis; see Boyle, Winkler, & Guglielmo, 2012). Burying beetles without a carcass, but with excess food, maintain a near-consistent body mass, or experience a slight increase in mass with time, even though the body mass gained

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during carcass preparation suggests that they could gain substantial weight rapidly if that were adaptive. Levels of JH in N. orbicollis and N. pustulatus map extremely well onto time periods of active caregiving and high resting metabolic rates. JH is higher early in the posthatching period when the larvae are most dependent on parental feedings and JH is upregulated in males that increase provisioning in response to loss of a mate (Panaitof et al., 2004; Scott et al., 2001; Trumbo, 1997; this study). Resting metabolic rate has not been measured in caregiving males, but we predict higher levels in single males than in paired males. We also found that JH is elevated in N. pustulatus males on larger but not smaller carcasses during the time when larvae are being fed. Early in the posthatching period, JH levels are more context dependent in parental males than in parental females, and are facultatively higher on a very large carcass and if the female is removed. This parallels the more variable participation in provisioning behaviour by males found in three species of Nicrophorus (Fetherston et al., 1994; Rauter & Moore, 2004; Smiseth & Moore, 2004; Suzuki & Nagano, 2009). The carcass size effect could be due to a larger resource as well as to a larger brood. Burying beetles (especially males) will stay in the nest longer on larger carcasses, and may provide substantial care even if the brood size is small (Rauter & Moore, 1999). Prior studies of N. orbicollis using smaller differences in carcass size (Scott & Panaitof, 2004) or brood size (Panaitof et al., 2004) found no significant differences in JH titres, although a positive relationship between final brood mass and JH levels in single males was observed. During the first 2 days of care in N. pustulatus females (days 4 and 5 of the breeding cycle), JH levels were similarly high on both small and large carcasses; this is coincident with high resting metabolic rates (Miller, 2011). This parallels study of caregiving intensity in other species of Nicrophorus, in which females initially work close to their maximum capacity with all but the smallest broods, and have little capacity to increase their workload (Rauter & Moore, 2004; Smiseth & Moore, 2004; Smiseth et al., 2005). Interestingly, we found that N. pustulatus females maintained their high JH levels for a greater number of days on larger carcasses. This may have occurred, in part, because larvae arrive later on a large carcass. An additional factor is that the period of regurgitation may have been extended, as late-arriving larvae are not culled on larger carcasses (Müller & Eggert, 1990) and large brood sizes (24e119) may have slowed larval development as young competed for parental feedings. Scott and Panaitof (2004) demonstrated that the period of high JH levels in female N. orbicollis caring for smaller broods could be extended by experimentally substituting begging first-instar larvae for older larvae. The association between JH and the presence of young larvae observed in Nicrophorus is in stark contrast to that observed in the only non-Nicrophorus member of the subtribe Nicrophorini to be studied, Ptomascopus morio Kraatz. In P. morio, which does not regurgitate to young and has minimal parental care (Suzuki & Nagano, 2006), JH levels are lower overall and fall at the time larvae arrive on the carcass (Trumbo, Kon, & Sikes, 2001). This suggests a novel hormonal association related to the evolution of intensive parental care in Nicrophorus. The tight correlations among the regurgitation phase of care, high JH, high metabolic rates and loss of body mass leave unanswered the mechanism of how the costs of parental care are incurred. Whether the costs of care in the present study were caused by parental activity, elevated JH, or both, is uncertain. JH is an important mediator of reproductive costs in many insects (Herman & Tatar, 2001; Tu et al., 2006), although the cost may depend on the underlying condition of the individual (Tibbetts & Banan, 2010). In some cases it may be possible to tease apart the effect of JH itself from the effect of JH-related reproductive

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behaviour on the costs to longevity (Pereira, Sivinski, Teal, & Brockmann, 2010). Further work also needs to be done on the relationship between JH and the metabolic rate. JH might affect metabolic rates directly, affect metabolic rate only in the context of appropriate stimuli from the environment, or covary with metabolic rate in response to a third variable. The co-occurrence of elevated parental activity, high metabolic rates, loss of body mass and significant longevity costs in N. orbicollis seems logically consistent. Why JH should also be high during the early stages of parental care, in contrast to its nearest noncaring relative (Trumbo et al., 2001), and to most other caregiving insects (Rankin, Fox, & Stotsky, 1995; Tallamy et al., 2002; Tobe et al., 1985; but see Kight, 1998), has not been determined. We hypothesize that JH plays a novel role associated with the evolution of energetically demanding parental care in this group. Acknowledgments We thank Valon Mersini, Chanon Boonyavairoje, Katherine Nazario, Justin Sardi and Mohammed Sayeem for assistance in the laboratory. Allen Moore and an anonymous referee provided valuable comments for improving the manuscript. Zachary Huang, Michele Elekonich and David Borst helped with JH radioimmunoassays. Michelle Scott and Per Smiseth graciously provided insight into the role of JH in burying beetles. Gene Robinson has provided guidance for JH burying beetle projects for over 20 years. References Agrawal, A. F., Combs, N., & Brodie, E. D. (2005). Insights into the costs of complex maternal behavior in the burrower bug (Sehirus cinctus). Behavioral Ecology and Sociobiology, 57, 566e574. Bartlett, J. (1987). Filial cannibalism in burying beetles. Behavioral Ecology and Sociobiology, 21, 179e183. Benowitz, K. M., Head, M. L., Williams, C. A., Moore, A. J., & Royle, N. J. (2013). Male age mediates reproductive investment and response to paternity assurance. Proceedings of the Royal Society B: Biological Sciences, 280, 20131124. Blouin-Demers, G., & Weatherhead, P. J. (2000). A novel association between a beetle and a snake: parasitism of Elaphe obsoleta by Nicrophorus pustulatus. Ecoscience, 7, 395e397. Bokony, V., Lendvai, A. Z., Liker, A., Angelier, F., Wingfield, J. C., & Chastel, O. (2009). Stress response and the value of reproduction: are birds prudent parents? American Naturalist, 173, 589e598. Boncoraglio, G., & Kilner, R. M. (2012). Female burying beetles benefit from male desertion: sexual conflict and counter-adaptation over parental investment. PLoS One, 7, e31713. Boyle, W. A., Winkler, D. W., & Guglielmo, C. G. (2012). Rapid loss of fat but not lean mass prior to chick provisioning supports the flight efficiency hypothesis in tree swallows. Functional Ecology, 26, 895e903. Brent, C., Peeters, C., Dietmann, V., Crewe, R., & Vargo, E. (2006). Hormonal correlates of reproductive status in the queenless ponerine ant, Streblognathus peetersi. Journal of Comparative Physiology A, 192, 315e320. Calow, P. (1979). The cost of reproduction: a physiological approach. Biological Reviews, 54, 23e40. Christe, P., Glaizot, O., Strepparava, N., Devevey, G., & Fumagalli, L. (2012). Twofold cost of reproduction: an increase in parental effort leads to higher malarial parasitaemia and to a decrease in resistance to oxidative stress. Proceedings of the Royal Society B: Biological Sciences, 279, 1142e1149. Cotter, S. C., Littlefair, J. E., Grantham, P. J., & Kilner, R. M. (2013). A direct physiological trade-off between personal and social immunity. Journal of Animal Ecology, 82, 846e853. Cotter, S. C., Topham, E., Price, A. J. P., & Kilner, R. M. (2010). Fitness costs associated with mounting a social immune response. Ecology Letters, 13, 1114e1123. Creighton, J. C., Heflin, N. D., & Belk, M. C. (2009). Cost of reproduction, resource quality, and terminal investment in a burying beetle. American Naturalist, 174, 673e684. Davies, S., Kattel, R., Bhatia, B., Petherwick, A., & Chapman, T. (2005). The effect of diet, sex and mating status on longevity in Mediterranean fruit flies (Ceratitis capitata), Diptera: Tephritidae. Experimental Gerontology, 40, 784e792. Denlinger, D., & Tanaka, S. (1989). Cycles of juvenile hormone esterase activity during the juvenile hormone-driven cycles of oxygen consumption in pupal diapause of flesh flies. Experientia, 45, 474e476.  Farkas, R., & Sut’áková, G. (2001). Swelling of mitochondria induced by juvenile hormone in larval salivary glands of Drosophila melanogaster. Biochemistry and Cell Biology, 79, 755e764.

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