Communal breeding, wherein multiple conspecific individuals live and reproduce together during a single breeding event, may generate immediate benefits in terms of defence and reproduction. However, the carry-over effects of events in communal breeding on individual behaviour and fitness remain less studied. We experimentally tested the immediate and carry-over effects of communal breeding on parenting performance and fitness in the burying beetle (Nicrophorus vespilloides). These beetles bury carcasses as food resource for their offspring and themselves, and provide extended care to the developing larvae on the buried carcass. We subjected individuals of varying sizes to communal (i.e. group-breeding) or non-communal breeding (i.e. pair-breeding) experience during their first breeding event, and subsequently to non-communal breeding during their second breeding event, and measured parental effort and reproductive success during both breeding events. In communal groups, large individuals became dominant and monopolized the carcass. At the first breeding attempt, large males in communal groups spent more time providing care than large males in non-communal groups, while such a difference was not observed for large females and small females or males in communal and non-communal groups. Reproductive success was similar for individuals that bred in communal and non-communal groups during their first reproductive event, indicating no significant immediate benefits of communal breeding in terms of reproduction. Compared to males that originated from non-communal groups, males from communal groups produced a similar number, but heavier larvae during their second breeding attempt, whereas such an effect was not observed for females. Our results provide evidence for sex-specific effects of communal breeding experience on parenting performance and fitness. Such observed sex differences in carry-over effects of communal breeding on fitness may generate sexual conflict over parental effort in social animals.

(a) Set-up of laboratory population of burying beetles

Wild beetles were caught during the reproductive season (from late April to September) in 2016 and in 2017 in pitfall traps buried in the forest soil. Beetles were caught at two locations: the estate ‘Vosbergen’, Eelde (53°08´N, 06°35´E), and the University of Groningen Zernike campus, Groningen (53°14´N, 06°32´E), both in The Netherlands. These wild beetles were transplanted to the Animal Facility at University of Groningen under laboratory conditions, and their descended, outbreed, second-generation offspring were used in experiments 1 and 2. During the entire rearing period, four to six adult beetles of the same sex were kept in plastic boxes (23×19 cm and 12.5 cm high), and fed mealworms twice a week. All beetles were reared in small and similarly-sized groups in the absence of a breeding resource (e.g. mouse carcass), and thus the social environment in early life was similar for all beetles. During the entire rearing and experimental period, all beetles were maintained at 20°C with a 16:8 h light to dark photoperiod.

(b) Experimental protocols

Experiment 1: Immediate implications of communal versus non-communal breeding on parental care and reproduction:

Communal versus non-communal breeding

To investigate the immediate implications of communal breeding on parental care and reproductive success, we set-up double-pair (consisting of two pairs, i.e., one large pair and one small pair) and single-pair (one male and one female) treatments to create communal and non-communal breeding (i.e. pair breeding) events, respectively. We selected sexually mature adult beetles aged between 10 – 14 days at post-eclosion for our experiments. Each individual was sexed according to morphological traits, and body size was measured (protonum width; accuracy: 0.01 mm). Before the experiment, similarly-sized, unrelated male and female individuals (mean_{|female-male|} ± SE = 0.11mm ± 0.02) were paired and kept for 8 – 12 h in the same box (10×5 cm and 8.5 cm high) filled with 1 cm of clean peat, to ensure female insemination and partner recognition [49,64]. Then, each pair of beetles was classified as large- and small-pair groups according to their size, and pairs within groups were similar in body size (mean size ± SE for large-pair: 4.92 mm ± 0.04; for small-pair: 4.37 mm±0.05). In burying beetles, adult body size determines individual competitive ability, such that larger individuals are more likely to monopolize a carcass after fights and to have a higher reproductive success compared to smaller individuals. To create the communally breeding treatment, one large pair (dominants) and one small pair (subordinates) of beetles (n = 19) were selected, and we ensured that the two pairs of beetles were unrelated and differed by approximately 10 % in body size, because a stable dominance hierarchy in carcass use is more likely when the size difference between opponents is larger. Previous studies indicated that the double-pair breeding treatment could create a communal breeding event. For these beetles, an individual’s body size has also been found to influence its parental investment towards the current brood, i.e. larger individuals likely invest more in the current brood than smaller individuals. Thus, we randomly selected some pairs of beetles from small-pair (n = 10) or large-pair (n = 10) groups to create the non-communal breeding treatment, which could control for the effect of body size on individual behaviour (i.e. parental investment). We weighed individuals (accuracy: 0.0001 g) immediately prior to the onset of the experiment. We chose a large mouse carcass (25.0 ± 2.0 g) as the breeding resource, because communal breeding is more likely to take place on carcasses larger than 25 g in N. vespilloides. In another study, we tested the parentage of offspring produced in double-pair breeding groups using 5 microsatellite loci, and found that small pairs of individuals produced a relatively small proportion of offspring in a shared brood (9 out of 10 small females reproduced; 5 out of 10 small males reproduced; N_brood = 10, N_offspring = 204), indicating that communal breeding events (i.e. one shared brood) were indeed induced in such double-pair treatment. All breeding events took place in a transparent box (23×19 cm and 12.5 cm high) filled with 3 cm of moist peat. At the onset of each treatment, beetles were placed in breeding boxes, where a thawed mouse was introduced as the breeding resource. To recognize individuals during observation, each individual was marked by making small holes in the elytra with a size 00 insect pin. 

Measurement of parental care and reproductive success

During the entire reproductive period (from the onset of experimentation until larval dispersal), beetle activity on and around the carcass was checked twice daily by visual inspection at 9:00 and 17:00. For each check, beetles were observed for 30 seconds per group continuously to ascertain whether or not they were providing parental care on the carcass. Such continuous observations could exclude any cases where beetles were present on the carcass for other reasons (e.g. wandering on the carcass) than to provide parental care. We recorded parental care behaviour when an individual provided indirect parental care (i.e. carcass guarding and maintenance on the surface of the carcass), and/or provided direct parental care (i.e. larvae provisioning inside the carcass). For each individual, we defined individual parental investment time by calculating the proportion of times that each individual was observed providing parental care on the carcass (i.e. carcass preparation and offspring provisioning) during the entire observation period. We also defined the total amount of time spent on parental care by groups (including communal and non-communal groups) by calculating the proportion of total times that all individuals in groups were found to provide care on the carcass. As a proxy for the cooperation of breeding groups or breeding pairs in parental care, the degree of carcass burial was estimated according to the fraction of the mouse above the ground and carcass roundness. In burying beetles, the degree of carcass burial over time may be associated with resource protection, i.e. a faster rate in carcass burial is likely to reduce the probability of being found or usurped by other intruders (e.g. flies).

In communal breeding events, dominant individuals largely monopolize the carcass, while both dominant and subordinate females are able to reproduce offspring by laying eggs surrounding the carcass. This results in females laying their eggs earlier (which may hatch earlier) and synchronously laying eggs with other female cobreeders. Therefore, we examined the timing of egg-laying and larvae-hatching in communal and non-communal groups. Egg-laying and larvae-hatching time was recorded as the time from the start of the experiment until the onset of egg laying and larvae hatching, respectively. We defined ‘the onset of egg laying’ as the onset observational time when some eggs were found at the bottom of the box, and we defined ‘the onset of larvae-hatching’ as the onset observational time when some newly-hatched larvae were found. We also calculated the egg-laying period (the period of time from the onset of egg laying until the onset of larvae hatching). As measures for offspring development and reproductive output of groups, we used the larvae-dispersing time (the time from the start of the experiment until the onset of larvae dispersing) and the brood size (number of larvae) and the average larval weight (total weight of larvae/brood size) of groups at larval dispersal (i.e. larvae dispersed from the carcass), respectively. We recorded weight change during breeding ([final weight– initial weight]/ initial weight) and survival of adult individuals at the end of the experiment, as parameters for parental investment during the entire breeding period and reproductive costs for individuals, respectively. For burying beetles, an individual’s weight change during the entire breeding period is a mixture of the costs of providing parental care and the benefits of consuming parts of the carcass. After the first breeding event, surviving beetles were kept individually for five days in rearing boxes (10×5 cm and 8.5 cm high) and were not fed with any food (i.e. mealworms) prior to subsequent experimentation. We did not feed beetles during this period to avoid the potential effect of food consumption and weight change on an individual’s behaviour in the subsequent period.

 Experiment 2: Carry-over effects of communal versus non-communal breeding on parental care and reproduction: 

Communal versus non-communal breeding experience

To examine the effects of previous breeding experience (communal versus non-communal breeding) on parental care and reproductive success in the subsequent breeding event, orthogonal experiments were performed using beetles from experiment 1. In contrast to the first breeding events, the second breeding events were carried out solely as pair breeding (one pair of individuals bred on the carcass). For this experiment, beetles that had not formed pairs with each other in the previous experiment, and originated from communal (i.e. double-pair treatment) or non-communal groups (i.e. single-pair treatment), were paired randomly; e.g. large females from communal groups were paired with either large or small males from communal or non-communal groups, and small females from communal groups were paired with either large or small males from communal or non-communal groups (figure 1). For newly-formed breeding pairs in the second breeding event (without other conspecifics), all males may experience some paternity uncertainty because females mated in the first experiment. However, in the second experiment, the uncertainty of paternity was unlikely to have an effect on an individual’s parental care. Then, each pair of individuals were left to induce a pair breeding event on a small mouse carcass (15.0 ± 1.0 g), because single pairs are more likely to utilize small-sized carcasses. Just prior to the experiment, all individuals were weighed.

Measurement of parental care and reproductive success

During the entire reproductive period of this second experiment, burial degree, time spent providing care on the carcass for each individual, and reproductive and developmental timing were recorded following the same protocol as in the first experiment. For the second breeding event, we also defined the total parental investment time of pairs by calculating the proportion of total times that both female and male individuals were found providing care on the carcass. At larval dispersal, brood size (number of larvae) and average larval weight (total weight of larvae/brood size) were measured as indicators of reproductive success of pairs. We recorded weight change and survival of individuals (described below), as parameters for parental investment and reproductive costs for individuals, respectively. 

(c) Statistical analyses

All analyses were performed using R version 3.6.3 (R Core Team 2018). The best-fitting models with the lowest AIC values were selected using the stepAIC function, and only the statistics for the terms that were included in these models are reported. 

Please refer to ReadMe file.