1 INTRODUCTION

Understanding interspecific interactions is important to assess the specific roles of species in complex ecological networks (Tylianakis & Morris, 2017) and to develop effective strategies for biodiversity conservation (Genes et al., 2017). Recent advances in molecular techniques have significantly improved our knowledge of ecological networks (Evans et al., 2016). Molecular-based techniques, such as metabarcoding of insects or their intestinal contents (invertebrate-derived DNA [iDNA]), have emerged as a subset of environmental DNA (eDNA) applications (Massey et al., 2022), allowing us to identify and quantify the strength of ecological interactions within trophic networks (Ji et al., 2022).

Many dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae) are coprophages with obligatory trophic associations with mammalian dung (Bogoni et al., 2019; Halffter & Halffter, 2009). Adult dung beetles utilize mammalian dung as a food source and build and relocate brood balls to lay eggs and shelter their larvae (Scholtz et al., 2009). Dung beetles can be classified into three functional groups, namely telecoprids (rollers), paracoprids (tunnelers), and endocoprids (dwellers), based on their dung-relocation behavior (Tonelli, 2021). Telecoprids relocate dung and bury it away from the original dung pad. Paracoprids excavate tunnels beneath dung pads. Endocoprids reside within the dung pad itself. The relocation and consumption of dung by functionally variable beetles assist ecological processes and services such as nutrient cycling, secondary seed dispersal, soil excavation, and parasite and pest control (Nichols et al., 2008).

Gillett et al. (2016) used the iDNA approach to extract mammalian DNA from dung beetle guts and demonstrated the potential to employ molecular techniques to explore dung beetle–mammal interactions. The methodological details of the iDNA approach on dung beetles were also explored by Gómez and Kolokotronis (2017), who used Sanger sequencing to show that the detection probability of mammalian DNA depends on beetle body size. Subsequently, Drinkwater et al. (2021) demonstrated that the use of iDNA on dung beetles can serve as a temporal window to recover iDNA from their mammalian diet, providing a tool to monitor mammalian communities. These studies, however, used live beetles. Live beetles captured by manual hand collection may be taxonomically biased, as this method generally captures endocoprids from dung pads (Mora-Aguilar et al., 2023). On the other hand, pitfall trapping of live beetles can capture different beetle species and functional groups; however, captured live specimens have a higher chance of losing iDNA due to excretion.

The application of the iDNA approach on dung beetles has great potential if mammalian DNA can be detected from specimens collected by conventional pitfall traps. Conventional pitfall traps consist of a bait—derived from human or other mammalian dung, or alternative potential food sources for dung beetles (e.g., rotting fruits and mushrooms), and a collection pot with a killing and preservative solution (e.g., ethanol, propylene glycol, and water). Traps are left in the field for 24–48 h, depending on the spacing between traps and other environmental conditions (Larsen & Forsyth, 2005; Silva & Hernández, 2015). Ethanol effectively preserves collected specimens; however, pitfall traps with only ethanol solution cannot be left in the field for an extended duration due to its quick evaporation (Aristophanous, 2010). Consequently, a 75% propylene glycol and 25% ethanol solution is commonly used, as this can be kept in the field longer (Scholtz et al., 2009). The effectiveness of the iDNA approach hinges on the possibility of extracting mammalian DNA from dung beetles captured and preserved by such a method, but this has yet to be investigated.

This study aims to demonstrate the practicality of using baited pitfall traps with the conventionally used preservative solution to extract mammalian DNA from dung beetle guts. Such an approach not only offers insights into dung beetle–mammal interactions but also introduces an additional method for monitoring mammalian communities.

2 MATERIALS AND METHODS

3 RESULTS

3.1 Sequencing summary and mammalian DNA identification in the dung beetle gut

The study included 18 dung beetle specimens captured using pitfall traps. These specimens represent three species (paracoprid Onthopagus diabolicus, endocoprid Onthopagus cf. gracilipes, and telecoprid Paragymnopleurus sp.). However, from five beetles (two Onthopagus cf. gracilipes and three Paragymnopleurus sp.), nonhuman mammalian DNA could not be extracted, resulting in a recovery rate of 72%. After filtering and removing low-quality reads from the remaining successfully processed gut samples, we retrieved 744,688 reads. The number of retained sequences per sample varied between 37,184 and 48,153, averaging 41,372 reads per specimen. The rarefaction curves indicated that the paracoprids exhibited a greater sequence depth than the endocoprids and telecoprids (Figure 1). The rarefaction curve of paracoprids showed a steeper slope than those of endocoprids and telecoprids, but all curves reached asymptotes.

Based on published literature and other sources, we listed a total of 39 mammal species (excluding bats) known to occur in Xishuangbanna (Supporting Information: Table S2). We recovered eight nonhuman mammal species from dung beetle guts (Figure 2a). The identified mammals represented four orders and eight families, including common species (wild boar: Sus scrofa and the common muntjac: Muntiacus muntjac) and rare species (Asian black bear: Ursus thibetanus) known to occur in the habitat. Paracoprids yielded the highest number of mammal species (seven out of eight), with two or more mammal species detected from many individual specimens (Figure 2b). In contrast, endocoprids and telecoprids yielded lower numbers of mammal species. Most individual specimens from these groups detected only one or two mammal species, and notably, five specimens did not yield any nonhuman mammal species at all. Two mammal species were found uniquely in paracoprids, and one species in endocoprids (Figure 2b).

4 DISCUSSION

Until our study, the possibility of extracting dung beetle iDNA from conventionally used baited pitfall traps was uncertain. The amplified sequences in our study identified eight nonhuman mammal species. The composition of the detected species differed among the three functional groups but shared locally common wild boar (Sus scrofa) and domestic cat (Felis catus). Paracoprids preserved the highest number of mammal species (seven out of eight total recorded), including rare species such as the Asian black bear (Ursus thibetanus), southern red muntjac (Muntiacus muntjac), and the northern pig-tailed macaque (Macaca leonine). Telecoprids preserved the least mammal species, perhaps because they tend to rely on a single dung source and make brood balls from large and nutritious dung pads often derived from large omnivores (e.g., wild boars) (Batilani-Filho & Hernandez, 2017; Carpaneto et al., 2005). On the contrary, paracoprids represented diverse mammal diets, including those detected by telecoprids. Paracoprids' diverse diet may be related to their feeding preference for different types of mammal dung with highly variable dung sizes (Holter et al., 2002). The fact that endocoprids have a diet similar to both telecoprids and paracoprids is related to their primary behavior of feeding on smaller dung particles that have already been processed by telecoprids and paracoprids, which helps them minimize direct competition for resources (Holter et al., 2002; Sabu et al., 2006).

This approach provides an additional sampling tool for not only the discovery of mammals but also the studies of dung beetle–mammal interactions. We demonstrate that dung beetle specimens captured by conventionally used baited pitfall traps can preserve the DNA of mammal species in their guts. Conventionally used baited pitfall traps can capture diverse dung beetle species with various mammalian diets over an extended period in the field. As a result, it enables the detection of a broader range of mammalian species through iDNA compared to live beetles collected by hand. Notably, we detected the presence of the Asian black bear, which had not been recorded in previous camera trap studies specific to this location, although it has been listed in the greater Xishuangbanna region (Tongkok et al., 2020). Additionally, this approach detected two arboreal species, the masked palm civet (Paguma larvata) and the northern pig-tailed macaque (Macaca leonine), demonstrating that the beetles preserved DNA from a diverse array of mammals in their gut, encompassing common, rare, and even arboreal species. The conventional dung beetle–mammal interaction experiments, such as cafeteria experiments involving selected dung sources (Raine et al., 2018) and laboratory dietary trials (Jones et al., 2012), may overlook potential interactions with rare or niche-specialized mammal species. Nonetheless, the number of mammal species identified using dung beetle iDNA was lower than the number of species known to occur at the study site. The sequencing depth for each species reached asymptote, indicating that we obtained enough sequences to capture mammalian DNA. Consequently, the detected disparity between observed and listed mammalian species could be attributed to the small sample size, the specific choice of beetle species for investigation, or the possibility that some mammal species no longer inhabit the studied forest area. We expect that the inclusion of more dung beetle species would yield more mammal species, as our recent survey in the same region found over 50 species (Nimalrathna et al., unpublished data). Our methods do not replace other mammal monitoring methods (e.g., camera traps) but complement other tools for monitoring mammal populations.

Our study showed that dung beetles collected from conventionally used baited pitfall traps preserved mammal DNA in their guts, demonstrating the use of dung beetle iDNA as an additional tool for biodiversity assessment. Our study suggests that paracoprids are an ideal focal group to survey mammal species effectively. Paracoprids are commonly collected by baited pitfall traps (Mora-Aguilar et al., 2023), thus reinforcing our approach as an effective method to monitor mammals. Compared to live dung beetles, the use of preserved dung beetles captured by baited pitfall traps makes sampling easier and more cost-effective while minimizing sampling bias. This method may also enable us to detect mammal species that are already rare and threatened by anthropogenic disturbances (e.g., the Asian black bear). Similar to other iDNA applications designed to understand insect-plant (Kajtoch et al., 2015) and predator-prey (Cuff et al., 2023) interactions, the proposed approach broadens the capacity of the dung beetle community and trophic interaction studies.

AUTHOR CONTRIBUTIONS

Thilina S. Nimalrathna: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; software; visualization; writing—original draft. Huan Fan: Methodology; software; validation; visualization; writing—review and editing. Rui-Chang Quan: Resources. Akihiro Nakamura: Funding acquisition; project administration; resources; supervision; validation; visualization; writing—review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.