Moving the corpse to hide the evidence: horizontal as well as vertical movement is important when burying beetles cache a carcass

Being the first to discover a resource can provide a competitive advantage (priority effect), even for an animal that is inferior in aggressive contests. Nicrophorus spp. (burying beetles) are known for caching a small vertebrate carcass as provision for their young, reducing volatile cues available to rivals by burying the carcass (vertical movement) and by altering the microbial community. A decomposing carcass, however, can leave cues (residues of decay) on soil and leaf litter that a burying beetle has less opportunity to neutralize. I investigated whether horizontal movement of the carcass by burying beetles, separating the carcass from soil at the site of death, might reduce competition from congeners. When fresh carcasses were placed in the field on top of soil with residues of decay, akin to no horizontal displacement, carcasses were discovered within 24 h by free-flying competitors more frequently (58.2%) than when the carcass was placed 1 m from treated soil (8.3%). In a second experiment, carcasses were more likely to be discovered by burying beetles when a chemical attractant (methyl thiocyanate) was placed near a carcass (0.03 and 0.25 m) than when it was placed more distant (1 and 5 m) or for controls (no attractant). The results suggest that horizontal displacement of a carcass after discovery serves not only to locate a suitable spot for burial but also to reduce information available to rivals searching for the resource.


Introduction
Species have diverse strategies, even when feeding on the same resource, for obtaining an adequate quantity of food. A fundamental distinction is between exploitative competitors that efficiently consume resources and convert them into offspring, and interference competitors that disrupt rivals' access to food (Birch 1957;Case and Gilpin 1974). When a resource has a high value and must be used over a period of time, we expect well developed contest ability (a form of interference) to evolve, as can be observed in insects that fight to provision a nest with dung or carrion for their young (Trumbo 1990;Simmons and Ridsdill-Smith 2011). Such resources can put individuals with less contest ability at a disadvantage, even if they can rapidly discover a resource, a faculty that is common in subordinate animals (Barta and Giraldeau 1998). Being the first to discover a resource, however, may allow individuals to employ interference mechanisms that do not involve direct aggression.
The priority effects of early colonizers include reducing resource-revealing cues that rivals will use. Caching behavior (hiding and storing a resource) is common among vertebrates (Smith and Reichman 1984;Teurlings et al. 2020;Allen et al. 2021), but it is not always clear if the benefit is preservation against decomposition or prevention of usurpation (van der Veen et al. 2020). Carrion that is buried is slower to be discovered by scavengers (Lundt 1964;Payne 1965;Shubeck and Blank 1982;Shubeck 1985;Teurlings et al. 2020), especially when there are no holes in the carrion made by trauma or by feeding scavengers (Connell and Slatyer 1977;Mann et al. 1990;Trumbo 2017;Trumbo and Sikes 2021). Invertebrates that use manipulable resources such as a small carcass provide an amenable experimental system for investigating the adaptive nature of moving, storing and caching resources.
Burying beetles have a complex search strategy for resources that depends on their age and nutritional condition. When they first emerge as adults they search for well-rotted carcasses (in bloat or active decay) of any size on which to feed, and avoid small, fresh carcasses (von Hoermann et al. 2013). After reaching reproductive maturity, their preference switches, as they require a small carcass that is not too decomposed for breeding (Trumbo and Steiger 2020). Their chemical preferences also switch, as breeding beetles are attracted to methyl thiocyanate (MeSCN) but are repelled by chemicals associated with active decay (e.g., dimethyl trisulfide, Trumbo and Steiger 2020) that can be attractive for feeding beetles von Hoermann et al. 2013). Very little is known about MeSCN, as studies investigating animals' responses to it have been initiated only recently (Trumbo and Steiger 2020;. It is produced early in decomposition as a byproduct of bacterial metabolism and is likely used in competition among microbes (Ohga et al. 1993;Armstrong et al. 2016). The primary adaptive explanation for burial of a breeding resource, which includes digging beneath the resource while stripping it of hair or feathers, is thought to be to reduce competition (Pukowski 1933;Trumbo and Sikes 2021). Resident beetles also apply anal and oral secretions to the exposed carcass skin, altering the microbial community, slowing decomposition, and reducing volatile cues used by rivals (Trumbo 2017;Duarte et al. 2018;Shukla et al. 2018;. While burial (vertical displacement) and carcass preservation have been well studied in burying beetles, there has been little work on the horizontal movement of the resource following discovery. Milne (1944, 1976) noted that horizontal movement of a carcass of more than a meter can occur when the carcass is located at a spot unsuitable for burial, such as on a walking path. If the soil is loose, it may be buried on the spot or transported across the ground prior to interment (Muths 1991;Trumbo 1992). Movement of a carcass horizontally is also used to locate a rodent hole for burial (Smith et al. 2000;Ito 2021).
I investigated whether horizontal movement of a carcass might reduce the chance of discovery by rival burying beetles, on the assumption that residues of decay in soil and leaf litter might cue rivals to a nearby carcass. Carcasses that burying beetles use for breeding are not always discovered within 24 h, and some may be used after three or more days of exposure (Trumbo 2016). A carcass that lies in situ for a period of several days will impart residues of decay to the underlying substrate. The chemistry, microbial community and volatile profile of the underlying soil are altered for considerable periods after decomposition of a carcass (Perrault et al. 2015;Weiss et al. 2016;Quaggiotto et al. 2019), which can be useful in forensic applications when a corpse is re-located (Cobaugh et al. 2015). While burying beetles alter cues released directly from the carcass, they have little control over cues imparted to the underlying substrate. Choice of a burial location that is horizontally displaced from the site of death, however, might reduce discovery by rivals.
I tested the hypothesis that residual cues of decay in soil might direct free-flying beetles to a carcass that was emitting few cues. I also used a known volatile attractant for breeding burying beetles (methyl thiocyanate) (Trumbo and Steiger 2020) to examine how the separation of a carcass from the cue, as well as carcass age, affect discovery by rivals.

Discovery of carcasses aided by soil of decay (Exp. 1)
The ability of free-flying beetles to discover a carcass when aided by soil with residues of decay was assessed in Exp. 1. The field trials were carried out at the Whittemore Preserve in Woodbury, Connecticut, USA (277 hectares, a mixed deciduous woodland, N41 0 31′48″ W N73 0 10′12″) from 12 August to 7 September 2022. Loose topsoil was obtained from the field site. Prior to each field trial, the soil was treated in the laboratory by placing a thawed 20-22 g mouse carcass (Rodent Pro®, Inglefield, Indiana, USA) on 2 cm of soil in each of two containers (5 cm height, 8.5 cm diameter). After 72 h, the carcasses were discarded and the soil was retained in its container for field trials.
The two containers with treated soil were taken to the field along with two 15-18 g mouse carcasses that had been thawed at ambient temperature for 10 h by the start of the beetles' active period. Relatively fresh carcasses, which emit fewer cues, were used for field trials because beetles typically reduce cues during carcass preparation by burial and altering the microbial community (Trumbo 2017;Duarte et al. 2018;Shukla et al. 2018;), leaving soil as a possible important source of cues. Two holes dug into the ground, approximately 25 m apart, were used to insert plastic cups (12 cm height, 10 cm diameter) so that the rim of the cups was flush with the ground. For the first treatment (in-cup), a cup was initially filled with untreated loose field soil to a depth of 8 cm. The experimental soil of decay from one container, chosen at random from the two containers of soil brought from the laboratory, was placed on top of the field soil and patted down, adding another 1 cm of depth. A mouse carcass was placed on top of the soil and the entire trap was protected with a rain cover, propped up on one side to allow easy access for beetles. The soil level was kept 3 cm below the rim of the cup to prevent movement of the carcass out of the cup by burying beetles.
For the second (displacement) treatment, a cup was filled with untreated loose field soil to a depth of 9 cm and a mouse carcass was placed on top, protected with a rain cover, as above. The second container with experimental soil of decay (same soil volume as the in-cup trial) was placed on the ground, open at the top but protected by a rain cover, 1 m distant from the buried cup with the carcass. The direction of the displaced container of soil was rotated among the four cardinal directions for successive replicates. After 24 h, the cups were emptied, any beetles in the cups were counted and sexed, and the carcasses and trap materials were removed from the field. Trap materials were cleaned before re-use (see Trumbo and Dicapua 2020). The beetles were released at a minimum of 500 m from the experimental area. There were three replicates per day on 8 days for a total of 24 replicates (48 carcasses in total). Replicates (each consisting of two cups each with a mouse carcass, 25 m apart) were placed at least 100 m from other replicates. The location of the in-cup and displacement trials was alternated for successive replicates, and each set of three replicates were at least three days apart, to avoid location and priming biases.

Discovery of carcasses aided by a chemical supplement (Exp. 2)
To investigate how carcass age and the distance of a cue from the carcass affects discovery by beetles, methyl thiocyanate (MeSCN), a strong attractant for breeding burying beetles (Trumbo and Steiger 2020), was used as a chemical supplement. There were five treatments per replicate, and each replicate used either all fresh carcasses or all older carcasses (N = 25 replicates each for fresh and older carcasses, 250 data points in all). There was one replicate per night either using fresh or old carcasses (10 August to 16 August 2021 and 10 June to 2 September 2022). Cups were buried in the field and 9 cm of loose field soil was added to the cup and an 8-12 g mouse carcass was placed on top (fresh carcass, thawed for 10 h by the start of the beetles' active period, or old carcass, placed on soil for 48 h in the laboratory prior to a field trial). Twenty µl of MeSCN was placed in a microcentrifuge tube at various distances from the carcass (0.03 m [in cup], 0.25 m, 1 m and 5 m). There was a fifth treatment (control) without chemical supplement. A hypodermic syringe was used to pierce the microcentrifuge tube to allow volatiles of MeSCN to escape (23 g needle, Exelint ®). Pilot trials demonstrated that this quantity of MeSCN and hole size were sufficient to ensure that the chemical would be present during the entire 24 h period on the warmest expected days. The cardinal direction for placement of the microcentrifuge tubes was the same for each treatment on a given night, but was rotated between nights (replicates). Treatments within a replicate were separated by at least 100 m. Trials lasted for 24 h and all trap materials were removed after each trial. There were two sets of 5 trap locations so that no trap location had to be used on consecutive nights, to avoid priming biases. Trapped beetles were released at least 200 m from the experimental site. Exp. 2 was conducted in Bethany, Connecticut, USA (41°27′36 N, 72°57′37"W), 19 km from the location of Exp. 1, in a similar mixed deciduous woodland. The field site for Exp. 2 was different than Exp. 1 so that trials carried out on the same night would not interfere with each other. Other procedures were as in Exp. 1.

Discovery of carcasses aided by soil of decay (Exp. 1)
The effect of the location of soil of decay (Exp. 1) was assessed using two statistics. The binary outcome of discovered by a beetle or not was compared using Fisher's Exact test, two-tailed, for the in-cup treatment and the displacement treatment. Carcasses scavenged by vertebrates were excluded. For 18 of the 24 replicates, neither carcass was scavenged. For these replicates, the numbers of beetles per trap were assessed for each replicate using a paired test. Numbers were not normally distributed, contained many zero values and were highly skewed; standard transformations did not result in near-normal distributions. A nonparametric paired statistic, W (Wilcoxon's Matched Pairs Signed Ranks test, WMPSRT, twotailed) was therefore used to compare the number of beetles coming to the traps in the two treatments.

Discovery of carcasses aided by a chemical supplement (Exp. 2)
Explanatory variables for Exp. 2 were carcass age (fresh versus 2-day), distance between the carcass and the MeSCN supplement, and their interaction. The binary outcome of discovery of the carcass or not was initially evaluated using logistic regression. The control treatment distance was set at 100 m, as this was the approximate distance to other trials using the chemical supplement. The results of the logistic regression were qualitatively the same if the control treatment was dropped from the analysis; only the analysis including the control treatment is reported. Because there was no effect of carcass age or the interaction between carcass age and distance to the supplement, the two age classes of carcasses (fresh and old) were combined for further analysis. Pairwise comparisons of the effect of distance to the MeSCN supplement were examined for discovery (binary outcome, Fisher's Exact test, two-tailed) and the number of beetles per trap night (WMPSRT, two-tailed), as for Exp. 1. The second experiment trapped enough burying beetles for a meaningful test of a 1:1 sex ratio (Binomial test, one-tailed). All analyses were carried out using JMP 7 (SAS Institute Inc 2007).

Discovery of carcasses aided by soil of decay (Exp. 1)
When soil with residues of decay was placed directly underneath a fresh carcass, the carcass was discovered within 24 h by free-flying burying beetles significantly more often than when the soil was placed at a distance of 1 m (Fig. 1). The number of beetles per trap night was also significantly greater for trials with the treated soil placed underneath the carcass than for treated soil placed 1 m distant (1.26 ± 0.27 vs. 0.21 ± 0.14, W = 32, p = 0.007, n = 18 pairs, WMP-SRT). Nicrophorus orbicollis Say was more common than N. tomentosus Weber in traps (57.1% to 42.9%).

Discovery of carcasses aided by a chemical supplement (Exp. 2)
The distance of the supplemental MeSCN cue from the carcass affected whether a carcass was discovered by burying beetles, but carcass age and the interaction between carcass age and distance did not affect discovery (distance: χ 2 = 8.65, p < 0.003; carcass age: χ 2 = 1.48, p = 0.22; interaction: χ 2 = 0.26, p = 0.61, N = 195, logistic regression). Pairwise comparisons among distance treatments (results from the two age classes of carcasses combined) revealed that a carcass was more likely to be discovered when the supplement was next to the carcass (0.03 m) or nearby (0.25 m) than for controls (no supplement) (Fig. 2). Carcasses with supplements placed at distances of 1 m and 5 m were no more or less likely to be Vol.: (0123456789) discovered than control carcasses (Fig. 2). The number of beetles per trap-night showed a similar pattern, with more burying beetles arriving at carcasses with supplements placed close to the carcass (0.03 and 0.25 m) than to carcasses with more distant supplements (1 and 5 m) or to controls (Fig. 3). Nicrophorus orbicollis was the most common species (93.1%), with the remainder N. tomentosus. The sex ratio was not significantly different from 1:1 (96 males, 106 females, p = 0.26, Binomial test).

Discussion
Although caching behavior is well known in both vertebrates and invertebrates, its benefits have been difficult to quantify (but see Teurlings et al. 2020), especially the breakdown for burying (or covering) versus horizontal displacement. The first burying beetle to arrive at a small carcass can manipulate the resource to reduce competition from congeners (priority effect) by burial and microbial control, a form of interference competition that does not involve aggression and can be employed by all competitors (Shubeck and Blank 1982;Hall et al. 2011;Trumbo 2017;Vogel et al. 2017;Duarte et al. 2018;Shukla et al. 2018;. The present study demonstrates that horizontal movement of a carcass can contribute to the ability to hide a resource from rivals, by separating cues left on the substrate at the site of death from the carcass. Horizontal movement of a carcass may be the only effective way to cope with cues that cannot be controlled as easily as those originating from the microbial community on the carcass. Cues of decay that were separated from a carcass by as little as 1 m (treated soil in Exp. 1 or MeSCN in Exp. 2) did not aid beetles searching for a resource, while a chemical supplemental cue placed close to the carcass (≤ 0.25 m) increased discovery by free-flying burying beetles.
The lack of a positive effect on discovery from the attractant MeSCN placed 1 or 5 m from a carcass was surprising. This finding has implications for understanding burying beetle search behavior and for trap designs employing chemical volatiles as attractants (see Podskalska et al. 2009). Burying beetles are known for their sensitivity to odors of decay (Kalinova et al. 2009; von Hoermann et al.  Fig. 2 The percent of carcasses discovered within 24 h by free-flying burying beetles for control carcasses (no MeSCN supplemental cue), and for carcasses with the supplement placed 0.03, 0.25, 1 and 5 m from the carcass (results were combined from the fresh and 2-day carcass treatments; carcasses scavenged by vertebrates excluded). Different letters above the bars represent significant differences in pairwise comparisons (p < 0.025, Fisher's Exact test) 1 3 Vol:. (1234567890) 2016), with antennal clubs that are packed with olfactory receptors (Waldow 1973). The inclusion of a no-chemical control in Exp. 2 allows us to address the question of whether a separated cue is an aid to discovery, irrelevant, or a distraction. Because carcasses separated from the chemical cue by 1 or 5 m were no more difficult to discover than control carcasses, it does not appear that MeSCN at these distances was a distraction. Rather, it was simply irrelevant. Perhaps beetles searching near a carcass require multiple cues to be emitted from a common point of origin to elicit landing and further exploration. Many behavioral responses to odors depend on a suite of molecules that must be sensed in combination (Ohsugi et al. 1985;Hammack 2001;Landolt et al. 2007), and this includes burying beetles searching for a well-rotted carcass that is used for feeding Trumbo and Dicapua 2020). Future work employing complex blends of volatiles, as occur in treated soil, can further test whether displaced cues could distract beetles searching for a breeding resource (fresh carcass). It was also unexpected that carcass age would have no effect on discovery. Previous work demonstrates that carcass age can be important; Trumbo (2016) found that carcasses were approximately twice as likely to be discovered on the second or third day of exposure compared to the first day, in the same woodland where Exp. 2 was conducted. In Exp. 2 there was a trend for older carcasses to attract more beetles than fresh carcasses when the MeSCN cue was ineffective (control, 1 and 5 m distance treatments) (Fig. S1). Carcass age, however, clearly was not important when MeSCN was an effective cue (0.03 and 0.25 m distance treatments, Fig. S1). When MeSCN is placed close to a fresh carcass, the carcass is discovered overnight at a very high frequency (85-95%) (Trumbo and Steiger 2020; this study, compared to 19-69% for control fresh carcasses in six experiments reported in Trumbo and Steiger 2020). When MeSCN is placed next to a carcass, cues emanating from an older carcass may add little to the volatile profile to make it more attractive to breeding burying beetles.
One aspect of the experimental design that does not simulate the natural world is that the displaced cue (treated soil or MeSCN) was placed at a distance (e.g., 1 m) from the carcass without any trace of cues left by the dragging behavior of beetles moving a carcass. The preponderance of displaced cues would be at the site of death when a carcass has lain in situ for several days and then moved 1 m within a few hours. There would seem to be few intervening cues available, but it cannot be ruled out that a competitor might use a pathfollowing search technique.
Horizontal movement of a carcass prior to burial might be a more important component of caching in some competitive contexts than others. Burying beetles are known to move a carcass horizontally when the underlying soil is difficult to work Milne 1944, 1976). This might be important even if the final destination offers no better conditions for burial, as carcasses are buried more shallowly in difficult-to-work soil and burial by itself would offer less protection against escaping volatiles. It would also be of interest to know whether burying beetles are more likely to move a carcass horizontally when the burying beetle is smaller in body size or of a behaviorally subordinate species.  Fig. 3 The number of burying beetles per trap arriving at control carcasses (no MeSCN supplemental cue), and at carcasses with the supplement placed 0.03, 0.25, 1 and 5 m from the carcass (results were combined from the fresh and 2-day carcass treatments; carcasses scavenged by vertebrates excluded). Shown are medians (thick horizontal lines), the middle quartiles (boxes), and outliers (open circles). The upper stem and cap bars represent the upper quartile + 1.5*interquartile distance. Different letters above the bars represent significant differences in pairwise comparisons (p < 0.025, WMPSRT) Older carcasses (but still usable for breeding) might also be transported further horizontally, as there would likely be more residual cues left in the soil and leaf litter than from a fresh carcass. An alternative hypothesis for further movement of older carcasses is that such carcasses might result in a microbial community in the underlying soil that is less amenable for egg development (Jacobs et al. 2014). A single female or male-female pair might also be more likely to move a carcass horizontally (or vertically) than a single male that needs a searching female to locate the carcass. Lastly, the perception of high population density, an indicator of competition that affects critical reproductive decisions (Creighton 2005) might influence caching behavior. These factors would be expected to increase the value of horizontal displacement of a carcass, separating substrate cues from the hidden resource, which the present study demonstrates can reduce discovery by rivals. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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