The diet of major predators of forest soils: A first analysis on syntopic species of Chilopoda through DNA metabarcoding
Introduction
DNA-barcoding techniques based on Next-Generation Sequencing technology are being tested and applied to characterise and compare the diet of many animals in different habitats, exploiting either faeces or gut contents (King et al., 2008; Pompanon et al., 2012; Clare, 2014; Alberdi et al., 2019). Focussing on predators, however, applications are strongly biased towards mammals, and especially bats (e.g., Galan et al., 2018), while they are less frequent for other vertebrates and remarkably rare for other animal groups. To the best of our knowledge, only a few dozen published studies have addressed the diet of predator invertebrates in the wild by means of DNA metabarcoding. Moreover, these studies encompass only some of the major groups of predators in aquatic and terrestrial habitats, especially spiders (e.g.: Toju and Baba, 2018; Eitzinger et al., 2019; Kennedy et al., 2019; Lafage et al., 2020) and more rarely dragonflies (e.g.: Cheng and Lin, 2016; Kaunisto et al., 2020), earwigs (e.g.: Mollot et al., 2014; Paula et al., 2016), beetles (e.g.: Paula et al., 2016; Kamenova et al., 2018), ants and wasps (e.g.: Mollot et al., 2014; Hu et al., 2017; Lefort et al., 2020), and carnivorous land snails (Boyer et al., 2013).
Considering soil communities, DNA-metabarcoding techniques are still fully unexploited for exploring the diet of some major groups of predators, like centipedes, mites, and many other arachnids. Breadth and differentiation of the trophic niches of these soil predators are still largely unknown because practical difficulties have hindered other traditional methodologies, like the direct observation of feeding activity in the wild, the morphological analysis of faeces or gut content, or even experimental trials of feeding preferences. Nonetheless, assessing the dietary diversity of the richest predatory components of the soil communities is paramount to adequately understand and model the complex structure and dynamics of soil ecosystems (e.g.: Bardgett, 2005).
Centipedes (Chilopoda) are represented in most temperate forests throughout the world as a dominant group of soil predators, both in terms of species number and biomass, besides ground beetles (Carabidae), spiders (Araneae), predatory mites (Gamasina) and a few other groups. More than 3000 species of centipedes are known globally (Minelli, 2011), all provided with poison claws to catch and envenom other soil-dwelling animals. The ecological role of centipedes as terrestrial predators is invariantly maintained since the Palaeozoic, as highlighted by the fossil record and molecular estimates of divergence times (Murienne et al., 2010). Many species often coexist in single forest plots, only partially separated between microhabitats and soil horizons: up to 26 species have been detected in syntopy (Peretti and Bonato, 2018).
However, despite of the ecological impact of centipedes as predators, little is known about their prey spectrum in the complex biocoenoses of forest soils. Direct observations of predation have been hindered by the fact that centipedes are either endogeic or their epigean activity is strictly nocturnal or limited to morning hours (Ilosvay, 1982; Tuf et al., 2006). On the other hand, satisfactory morphological analyses of gut content have been prevented by the fact that centipedes feed mainly by sucking previously envenomed and pre-digested prey (Poser, 1988; Minelli, 2011). Besides infrequent observations of predation episodes in the field and microscopic inspections of gut content, some hints on the trophic role of selected species have been obtained through indirect methods like fatty acid analysis (Ferlian et al., 2012) and PCR-assays for expected prey taxa in the gut (Eitzinger et al., 2013, 2018; Günther et al., 2014; Bortolin et al., 2018). Most of these investigations have addressed a few species of Lithobius and forest soil communities with a relatively poor species diversity, with the single exception of three species of geophilomorphs living in a sub-Mediterranean wood, but tested only for selected prey taxa (Bortolin et al., 2018).
Most centipede species living in forest soils are considered generalist predators, with some dietary differences between regularly epigeic species (most Lithobiomorpha) and mainly endogeic species (most Geophilomorpha) (Poser, 1988). However, further diet differentiation is expected for the frequent coexistence of many species and for their diversity in morphological features that are functionally related to predation, including the shape of the poison claws (Bonato et al., 2014a; Baiocco et al., 2017), the morphology of sense organs and structures in the pharynx (Koch and Edgecombe, 2012), and the composition of venoms (Undheim et al., 2015). A first assessment through PCR-based assays showed that syntopic and apparently similar species differ significantly in the frequency of consumption of earthworms and dipterans (Bortolin et al., 2018).
We applied a DNA-metabarcoding approach for the first time to assess the prey spectrum of centipede species living in a forest soil community of a temperate forest. Focussing on three of the largest and most abundant centipede species, we explored the efficacy and limitations of a preliminary protocol to investigate the trophic ecology in this neglected group of soil predators.
Section snippets
Specimen sampling and preparation
We sampled specimens of the three centipede species Clinopodes flavidus, Eupolybothrus tridentinus and Lithobius validus (Table 1; Fig. 1) within an area of about 1800 m2 in a silver fir forest of the South-Eastern Prealps (46.2602°N, 12.2062°E, 800 m, Ponte dei Ross, Val del Grisol, Dolomiti Bellunesi National Park, Italy). These species are among the most abundant species of centipedes in the local biocenosis (Peretti and Bonato, 2018) and among those attaining the largest body size in the
Prey taxa detected
The COI amplification and sequencing from the 79 individual gut samples and from the 4 negative controls produced a total of 16,685,300 reads. After alignment, forward sequences were trimmed to 250 bp, while reverse sequences were trimmed to 220 bp because of the lower quality of the latter. In the following, we will report only the results obtained from the forward sequences, as the taxonomic assignment was much less effective for the reverse sequences.
After quality filter, denoise and chimera
Diet analysis through DNA metabarcoding
Despite great methodological progress over a decade, it is universally acknowledged that dietary analysis through DNA metabarcoding has not yet reached a satisfactory level of technical reliability, in terms of reproducibility and replicability of results (e.g.: Elbrecht et al., 2017b; Alberdi et al., 2019; Deagle et al., 2019; Zinger et al., 2019). Many sources of signal noise can flaw the DNA metabarcoding analyses of gut content. Among these are the heterogeneity of the tissue composition of
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Department of Biology of University of Padua (PRID 2016), also through a grant to F. Bortolin. Specific permits have been obtained by the Parco Nazionale Dolomiti Bellunesi, which also facilitated the field work. We are grateful to G. De Zen and C. Cecchin for helping us in the field work. Optimization of the DNA amplification protocol and sequencing were performed by Personal Genomics (Verona). We are also grateful to the anonymous reviewers for their useful
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