Insights into the earthworm gut multi-kingdom microbial communities
Graphical abstract
Introduction
Earthworms consists of >800 genera and are the dominant soil fauna in natural as well as cultivated soil. They are often known as engineers of soil ecosystem, due to their activities and their interactions with microorganisms that regulate the biogeochemistry of terrestrial soils (Blouin et al., 2013). By moving in and between the soil horizons, earthworms accelerate the organic matter turnover by excreting readily available nutrients in the soil. Their digestive systems host an abundance of microbes that form a complex interdependence with the host, and play important regulatory roles in host nutrient metabolism, immune system and other physiological functions (Liu et al., 2018). In recent years, gut microbiomes has been of growing interest as their composition and activity has been reported to be vital for the host (Drake et al., 2006). The earthworm tube-like gut provides a unique anoxic micro-environment with stable conditions of moisture and different nutrient pools that can act as a biological filter for ingested microbial communities, selecting and/or favouring specific groups of microorganism pools (Drake and Horn, 2007). This process may enrich the soil in certain microbial taxa, for example in bacteria able to decompose the organic matter that earthworms feed on or in denitrifying bacteria able to survive in the reduced oxygen conditions of the earthworm gut (Byzov et al., 2007; Medina-Sauza et al., 2019). Thus, understanding of the gut microbiome will widen our knowledge on the direct and indirect effect of earthworms on the soil ecosystem functioning.
Based on burrowing and feeding habits, earthworms are broadly categorized into three life forms: epigeic, anecic and endogeic. Epigeic species of earthworm live on top soils and feed on organic matter; anecic species move between soils layers making vertical burrows, and often use the burrows as link between top layers used for feeding and lower layer used as shelter; finally, endogeic species not only make temporary, but also, random burrows (horizontally or vertically) which are rarely reused (Bouché, 1977). The different life forms of earthworms are known to have different impacts on the diversity of soil microbes, explained by the changes in the earthworm cast, and thus with origin in the gut process (Aira et al., 2015; Medina-Sauza et al., 2019; Winding et al., 1997). They might also be responsible for favouring or having co-evolved with certain core taxa in the gut by regulating microbial communities via gut fluid, which limits the growth of some microbes while creating the environment for proliferation of others (Byzov et al., 2007). Clearly, the food source was found to affect the earthworm gut microbiome, however, the core microbiomes associated with earthworms remained largely unchanged (Liu et al., 2018). This suggests that specific microbial communities in the earthworm gut have evolved in each earthworm genus and life form.
Bacterial communities in the earthworm gut have been studied using culturing methods (Toyota and Kimura, 2000), but also recently using culture independent methods such as 16S ribosomal RNA gene amplicon sequencing (Liu et al., 2018). However, gut microbiome studies have exclusively focused on bacteria, ignoring the large proportion of eukaryotes, like fungi, protists and even small metazoans being part of the earthworm diet. These microeukaryotes and bacteria occupy very different positions in the trophic network and therefore, can be differently affected by the earthworm gut habitat. Indeed, gut studies corroborate a strong structuring of the gut microbiome in consequence of the cascading effects of eukaryotes modulating the relative abundance of many species, by their grazing activity (Laforest-Lapointe and Arrieta, 2018). Therefore, in microbiome studies, it is of utmost importance to describe the patterns of diversity, how they are influenced by inter-kingdom interactions, and how these community patterns are influenced by the host.
Identification of unicellular as well as multicellular eukaryotes are often challenging and requires taxonomic expertise to resolve the taxa. DNA metabarcoding tools have enabled researchers to profile communities to a high degree of taxonomic resolution from macro-environments and even micro-environments such as the earthworm gut. Using DNA sequence-based assessment, the aim of this study was to characterize the earthworm gut prokaryotic and eukaryotic microbiome; to assess if it is a result of the different earthworm genera or if the life form including different feeding habits is a stronger predictor of the microbiome composition; and finally explore the cross kingdom gut microbiome associations in an attempt to identify keystone species. We hypothesized that earthworm life form is a stronger predictor of microbiome richness and composition than the genera.
To explore the earthworm gut microbiome, we included three distinct earthworm genera found in Danish grassland soil. The earthworms were extracted from the same geographical location and were characterized into two distinct life forms, epigeic and anecic that both mainly feed on the top-soil. Gut microbial communities were characterized by high throughput amplicon sequencing with universal primers and the correlations between prokaryotic and eukaryotic communities were used to describe the relationship in the earthworm gut microbiomes and tentatively identify indicator species.
Section snippets
Earthworms and gut sampling
A total of 23 earthworms of the genera Aporrectodea, Allolobophora and Lumbricus were collected from a grassland soil at Risø, Denmark (N: 55.6845; E: 12.0928). Earthworm species and number of individuals of each can be found in Table 1. Gut content was manually forced out of the earthworms and stored at −20 °C until DNA extraction.
DNA extraction
Total DNA from collected gut content was extracted using PowerLyzer UltraClean Microbial DNA isolation kit (Qiagen), according to the manufacturer's instructions.
Earthworm 16S mitochondrial gene sequencing
While comparing the results for taxonomic identification using morphological and molecular technique, 21 out of 23 earthworms matched two life forms, covering three different earthworm genera (Allolobophora, Aporrectodea, Lumbricus) and three species within Aporrectodea (Table 1, Fig. S1). The gut microbiomes of the single Aporrectodea longa and the two earthworm samples with unmatched morphological and DNA barcode taxonomy were excluded from the downstream analysis allowing us to work with
Discussion
Our study of earthworm gut microbiome using DNA metabarcoding provided detailed descriptions of the diverse group of prokaryotic and eukaryotic communities in different earthworm species living in the same grassland soil. This allowed us to a first attempt of studying the effects of genotype and their ecology and feeding modes (life form) on gut microbiome diversity and show that each factor modulates different aspects of diversity. The impact of earthworms on soil microbiomes has mainly been
Conclusions
By characterizing variation of the gut microbiota of prokaryotes and eukaryotes within and among earthworm species using DNA amplicon sequencing we have determined the richness and diversity of the gut microbiomes of earthworms. This provides the foundation for a mechanistic understanding of how the gut microbiome is affected by its host and life form. This study highlights the structure of prokaryotic and eukaryotic diversity in the earthworm gut. The distinct structure and richness observed
Author statements
Rumakanta Sapkota: Data curation, analysed all data using bioinformatic and statistical analyses, wrote the original draft.
Susana Santos: Heading field sampling and processing of all analyses in the laboratory, including PCR and sequencing. Did the initial bioinformatic and statistical analyses. Reviewed and edited manuscript.
Pedro Farias: Participated in the field sampling, laboratory analyses and analyses of sequencing data.
Paul Henning Krogh: Conceptualized the experiment and secured funding
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
Tina Thane and Tanja Begovic are acknowledged for technical assistance.
Funding
Pedro Farias received an STSM grant from the EU COST Action ES1406 “Soil fauna - Key to Soil Organic Matter Dynamics and Modelling (KEYSOM)”. Rumakanta Sapkota, Susana Santos, Anne Winding and Paul Henning Krogh were supported by the eDNA Center at Aarhus University through funding from the Danish Centre for Environment and Energy (DCE).
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- 1
Present address: Department of Agroecology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark.
- 2
Present address: Department of Life Sciences, FCTUC, University of Coimbra, Portugal.