The diet of major predators of forest soils: A first analysis on syntopic species of Chilopoda through DNA metabarcoding

https://doi.org/10.1016/j.soilbio.2021.108264Get rights and content

Highlights

  • Chilopoda are major predators in forest soils but their diet is almost unknown.

  • Prey spectrum of centipede species has been assessed by DNA-metabarcoding of guts.

  • Dominant syntopic centipede species have a broad prey spectrum.

  • Some of the dominant syntopic centipede species have a slightly different diet.

Abstract

Centipedes (Chilopoda) represent a dominant group of predators in temperate forest soils, where multiple species often coexist in syntopy. However, their trophic ecology is largely unknown because traditional methodologies are hindered by the secretive habits of these animals and their partial fluid-feeding. We applied a DNA-metabarcoding approach with COI to assess and compare the prey spectrum of three centipede species (Clinopodes flavidus, Eupolybothrus tridentinus, Lithobius validus) living in a forest plot in the Southern Alps. We obtained DNA sequences from the guts of 79 specimens through Next-Generation Sequencing technology and applied a suite of stringent criteria of data filtering and taxonomic assignment. We found evidence for a similar rate of prey detection between predator species and for a broad prey spectrum, including most taxa of invertebrates living in the local soil biocoenosis and having subduable body size. We did not find evidence of diet variation in relation to sex and annual season, and instead slight but significant differentiation of diet between the geophilomorph C. flavidus and the other two lithobiomorph species.

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

References (88)

  • M.J. Anderson

    A new method for non-parametric multivariate analysis of variance

    Austral Ecology

    (2001)
  • B. Balech et al.

    Tackling critical parameters in metazoan meta-barcoding experiments: a preliminary study based on coxI DNA barcode

    PeerJ

    (2018)
  • R.D. Bardgett

    The Biology of Soil: a Community and Ecosystem Approach

    (2005)
  • N.A. Bokulich et al.

    Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2's q2-feature-classifier plugin

    Microbiome

    (2018)
  • E. Bolyen et al.

    Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2

    Nature Biotechnology

    (2019)
  • L. Bonato et al.

    Phylogeny of Geophilomorpha (Chilopoda) inferred from new morphological and molecular evidence

    Cladistics

    (2014)
  • L. Bonato et al.

    ChiloKey, an interactive identification tool for the geophilomorph centipedes of Europe (Chilopoda, Geophilomorpha)

    ZooKeys

    (2014)
  • S. Boyer et al.

    Using next-generation sequencing to analyse the diet of a highly endangered land snail (Powelliphanta augusta) feeding on endemic earthworms

    PLoS One

    (2013)
  • S.G. Brade-Birks

    The economic status of Diplopoda and Chilopoda and their allies, especially of forms occurring in the British Isles, particularly those of importance in agriculture and horticulture. A critical examination of some records with an account of original observations and experiments

    Journal of the South-Eastern Agricultural College, Wye, Kent

    (1929)
  • G.J. Brandon-Mong et al.

    DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

    Bulletin of Entomological Research

    (2015)
  • D.S. Brown et al.

    Dietary competition between the alien Asian Musk Shrew (Suncus murinus) and a re‐introduced population of Telfair's Skink (Leiolopisma telfairii)

    Molecular Ecology

    (2014)
  • F.M. Calabrese et al.

    NUMTs colonization in mammalian genomes

    Scientific Reports

    (2017)
  • B.J. Callahan et al.

    DADA2: high-resolution sample inference from Illumina amplicon data

    Nature Methods

    (2016)
  • Y.C. Cheng et al.

    Dietary niche partitioning of Euphaea formosa and Matrona cyanoptera (Odonata: zygoptera) on the basis of DNA barcoding of larval feces

    Journal of Insect Science

    (2016)
  • E.L. Clare

    Molecular detection of trophic interactions: emerging trends, distinct advantages, significant considerations and conservation applications

    Evolutionary Applications

    (2014)
  • L.J. Clarke et al.

    Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias

    Molecular Ecology Resources

    (2014)
  • B.E. Deagle et al.

    Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

    Frontiers in Zoology

    (2006)
  • B.E. Deagle et al.

    DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match

    Biology Letters

    (2014)
  • B.E. Deagle et al.

    Counting with DNA in metabarcoding studies: how should we convert sequence reads to dietary data?

    Molecular Ecology

    (2019)
  • B. Eitzinger et al.

    Assessing changes in arthropod predator–prey interactions through DNA‐based gut content analysis—variable environment, stable diet

    Molecular Ecology

    (2019)
  • B. Eitzinger et al.

    Testing the validity of functional response models using molecular gut content analysis for prey choice in soil predators

    Oikos

    (2018)
  • V. Elbrecht et al.

    Validation of COI metabarcoding primers for terrestrial arthropods

    PeerJ

    (2019)
  • V. Elbrecht et al.

    Sorting things out: assessing effects of unequal specimen biomass on DNA metabarcoding

    Ecology and Evolution

    (2017)
  • V. Elbrecht et al.

    Assessing strengths and weaknesses of DNA metabarcoding‐based macroinvertebrate identification for routine stream monitoring

    Methods in Ecology and Evolution

    (2017)
  • N.A. Fahner et al.

    Large-scale monitoring of plants through environmental DNA metabarcoding of soil: recovery, resolution, and annotation of four DNA markers

    PloS One

    (2016)
  • O. Folmer et al.

    Conserved primers for PCR amplification of mitochondrial DNA from different invertebrate phyla

    Molecular Marine Biology and Biotechnology

    (1994)
  • T.G. Frøslev et al.

    Algorithm for post-clustering curation of DNA amplicon data yields reliable biodiversity estimates

    Nature Communications

    (2017)
  • M. Galan et al.

    Metabarcoding for the parallel identification of several hundred predators and their prey: application to bat species diet analysis

    Molecular Ecology Resources

    (2018)
  • N.J. Gotelli et al.

    EcoSimR: null model analysis for ecological data

    R package version 0.1.0

    (2015)
  • E.T. Grau et al.

    Survey of mitochondrial sequences integrated into the bovine nuclear genome

    Scientific Reports

    (2020)
  • B. Günther et al.

    Variations in prey consumption of centipede predators in forest soils as indicated by molecular gut content analysis

    Oikos

    (2014)
  • M. Hajibabaei et al.

    Environmental barcoding: a next-generation sequencing approach for biomonitoring applications using river benthos

    PloS One

    (2011)
  • E. Hazkani-Covo et al.

    Evolutionary dynamics of large Numts in the human genome: rarity of independent insertions and abundance of post-insertion duplications

    Journal of Molecular Evolution

    (2003)
  • E. Hazkani-Covo et al.

    Molecular Poltergeists: mitochondrial DNA copies (NUMTs) in sequenced nuclear genomes

    PLoS Genetics

    (2010)
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