Insights into the earthworm gut multi-kingdom microbial communities

Highlights

• We studied the gut microbiome of four earthworm species from the same grassland soil.

• A distinct microbiome in different earthworm species and life forms was identified.

• Gut microbiome structure and richness is earthworm species-specific.

• Cross-kingdom interactions revealed bacteriovore protists as keystone species.

Abstract

Earthworms are widely known to impact soil health, having a key role in nutrient cycling and are often referred to as soil engineers. They are vital for soil microbial assemblages particularly through their feeding and burrowing activity in soil. Earthworms feed on soil organic matter and litter, and the resulting casts alter the soil microbial community. However, the gut microbiome of earthworms remains less known. In this study, we used amplicon sequencing of the 16S rRNA gene for bacteria and 18S rRNA gene for eukaryotes to assess the gut community assemblages of earthworm species within three genera Aporrectodea, Allolobophora and Lumbricus that represent different life forms sharing the same habitat. The objective was to compare the gut microbiome profiles of eukaryotic and prokaryotic organisms to assess significance of earthworm life forms, and to explore the cross kingdom networks in an attempt to identify keystone species. We found a high eukaryotic diversity with a dominance of the SAR supergroup along with fungi and metazoan in the earthworm gut. The bacterial community were dominated by members of Proteobacteria, Acidobacteria, Actinobacteria, Firmicutes, and Verrucomicrobia. The eukaryotic and prokaryotic communities showed similar differences in alpha diversity, being lowest in Lumbricus herculeus. The beta diversity showed earthworm species as a key factor in shaping gut microbiomes with L. herculeus harboring distinct microbiomes compared to species of Aporrectodea caliginosa, A. longa, A. tuberculata and Allolobophora chlorotica. Cross kingdom networks showed high interactions between several protist and bacterial OTUs. In conclusion, this study suggested that the community assemblages of gut microbiomes were shaped by earthworm species and life form, and such assemblage consists of cross kingdom interactions among eukaryotes and prokaryotes.

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.