Metagenomic Exploration of Bacterial Community Structure of Earthworms’ Gut

Living organisms are naturally bestowed with unique and imitable qualities for maintaining ecological balance and earthworms are no exceptions. These so-called keystone species of terrestrial ecosystems are equipped with wonderful machinery, allowing them to nurture soil beautifully. Earthworm gut represents a potential microbial reservoir, having a complex interdependence with the host. The study aimed to profile bacterial community structure of three earthworm species belonging to two different life forms; Perionyx excavatus and Eudrilus eugeniae (epigeic), Polypheretima elongata (endogeic) respectively. Diversity analysis using 16S amplicon sequencing revealed that the dominant phyla were Proteobacteria (34.17-77.88) followed by Actinobacteria (13.43-35.54%), Firmicutes (1.69-15.45%) and Bacteroidetes (0.51-8.12%). The alpha diversity indices explicit similar gut microbiota of Perionyx excavatus and Eudrilus eugeniae and while higher alpha diversity was recorded in comparison to Polypheretima elongata gut. The taxonomic to the phenotypic annotation of 16S rRNA metagenomes revealed that dominance of Gram-negative bacterial community in all earthworm species while, Polypheretima elongata comprises higher percentage (78%) of Gram-negative bacterial community to Perionyx excavatus (32.3%) and Eudrilus eugeniae (38.3%). The oxygen requirement phenotypic analysis showed that all earthworm species were abundant with aerobic followed by anaerobic bacterial groups. Furthermore, functional metabolism phenotypic analysis revealed that a high abundance of ammonia oxidizers (29.3-80.2%), the gut microbiomes showed the relative abundance of sulphate reducer (22.6-78.7%), nitrite reducer (19.8-73.2%), dehalogenators (12.6-25.1%), illustrating in the role of these microbial communities in various degradation and bioremediation processes. The present study signifies the intrinsic gut microbiota of earthworm species for intensified biodegradation.


Molecular identification of earthworms' species
One adult earthworm was randomly chosen from each sterile polythene bag and characterized using mitochondrial molecular marker COI following standard protocol 16 . Table 1 depicts the accession number of studied samples. All the COI sequences of the present study are accessible on BOLD web portal under the research project 'Diversity studies in earthworms of India' (IEW). In addition, 22 COI sequences including outgroup were retrieved from NCBI and BOLD database for the molecular analysis.

Sequence alignment and data analysis for earthworm species identification
25 COI sequences were analysed on MEGA software using the Kimura twoparameter 17 . COI dataset was searched on NCBI, the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov) to blast three query COI sequences and were aligned using Multiple sequence alignment program MUSCLE v3.8.31 (Multiple Sequence Comparison by Log-Expectation) 18 . Their phylogenetic estimation were inferred using neighbour-joining tree method following 1000 bootstraps using Molecular Evolutionary Genetics Analysis (MEGA X) 19 .

DNA extraction of gut microbiome
The same earthworm from each group was selected for gut metagenomic study, that tissue used for molecular identification of earthworms species. The worms were washed three time with distilled water and placed on separate sterile petri dishes (one per dish), after the couple of minutes worms were dissected to take out their gut. The metagenomic DNA was extracted using Qiagen Blood and Tissue Kit (Qiagen, USA) following provided protocol. Their quality was assessed on the 0.85% agarose gel and quantified on NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).
In order to differentiate all samples, the reverse primer was labelled with a specific barcode for each sample. For PCR reaction, total of 20 µl reaction mixture was prepared comprising 17 μl reaction buffer, AccuPrime Supermix, 1 μl of DNA template and 1 μl of each forward and reverse primer. The PCR Thermocycler was set at 95 °C for 5 min as initial denaturation step, followed by 35 cycles of 94 °C for 30s, 52 °C for 40s and 72 °C for 60 s, with a final extension step at 72 °C for 5 min. The same parameters were used for negative control of PCR product. Their integrity were analysed after mixing equal volume of 1X loading buffer ran on agarose gel electrophoresis.
Subsequently, the intact and sharp bands between 400-500 bp were used for construction of library.

Construction of library and sequencing
The Qiagen Gel Extraction Kit was used to purify the PCR products and the library prepared using TruSeq DNA PCR-Free Sample Preparation Kit following protocol provided by manufacturer. The quality of library products were evaluated on Qubit 2.0 Fluorometer and Agilent Bioanalyzer 2100 system. The sequencing of constructed library was performed on an Illumina HiSeq 2500 platform using standard protocol 21 at Nucleome Informatics Pvt. Ltd.

Data processing and In silico analysis
Paired-end raw sequences were filtered using FastQC 22 on CosmosID's bioinformatics pipeline (https://app.cosmosid.com; Rockville, MD, USA). Where, raw sequence files were uploaded to the CosmosID cloud application without set parameters or modified parameters. As reported earlier the application uses highperformance k-mer based algorithms and curated taxonomy databases (GenBook®) enable via the cloud interface [23][24][25][26][27][28] . Using CosmosID bioinformatics pipeline software the taxonomic community profiling, alpha diversity analysis (Chao1, Simpson and Shannon), Hierarchical clustering heatmap analysis at phylum and genus level was evaluated and plotted, to reveal microbial community composition in earthworms' gut.

Taxonomic to phenotypic analysis
T h e t a x o n o m i c a b u n d a n c e t a b l e g e n e r a t e d b y C o s m o s I D (https://app.cosmosid.com) was uploaded on METAGENassist (http://www.metagenassist. ca/) 29 for taxonomic to phenotypic profiling. The generated data were normalised following Paul et al 30 . Further analysis of phenotypic subsets, Gram stating oxygen requirement and metabolism having various phenotypic characteristics were corelated with given taxa, pie charts and bar graphs were plotted to depict the fraction of percent of taxa characteristic. The supervised pie chart and bar graph were employed for each metabolic phenotype analysis 29 .

Molecular identification of the species using the COI gene
Based on the molecular identification methods, these specimens were identified with the help of cytochrome oxidase subunit 1 (COI) gene partial sequence. The phylogenetic position of three query COI sequences was based on the BLASTN homology against the nucleotide sequence collection of the NCBI GenBank and BOLD sequence database and were identified as Perionyx excavatus (S1), Eudrilus eugeniae (S2), and Polypheretima elongata (S3). Obtained sequences showed 99% similarity with the available sequence in NCBI GenBank and BOLD databases were distantly related to the outgroup Calomera littoralis (Fig. 1).

Alpha diversity analysis of earthworms' gut microbiota
A total 1298 OTUs were obtained during the evaluation of bacterial diversity o n C o s m o s I D b i o i n fo r m a t i c s p i p e l i n e (https://app.cosmosid.com). A good coverage sequencing depth (99.8%) was found which represents capturing of majority of the bacterial diversity in all samples. The species richness (Chao1) was highest in Perionyx excavatus (947) followed by Eudrilus eugeniae (751) and lowest in Polypheretima elongata (678). Similar trends were found in Shannon index and Simpson index, the Shannon index for Perionyx excavatus (7.6139) was highest followed by Eudrilus eugeniae (7.2667) and lowest in Polypheretima elongata (5.1776 Fig. 4A-C). The alpha diversity indices analysis reflected high diversity in Perionyx excavatus followed by Eudrilus eugeniae and lowest in Polypheretima elongata (Fig. 4A-C).

heatmap clustering analysis
The abundance of clusters and similarities were observed by plotting the heatmap, a graphical display of values in colour gradients of data matrix. Where, vertical clustering represents similarity in abundance of different species in the samples. The lesser distances between two samples represents shorter branch lengths that expressed relatively similar abundance between samples. The colour gradient from blue to red symbolizes low to high relative abundance 31 .

At phylum level
The relative abundance of microbiota in Perionyx excavatus was recorded the highest, followed by Eudrilus eugeniae and the lowest in Polypheretima elongate (Fig. 5A). The upper clustering tree indicates that the bacterial community in Perionyx excavatus and Eudrilus eugeniae, were more similar compare to the Polypheretima elongata.

DISCuSSIoN
Earthworms play a vital role in the overall health and maintenance of soil ecosystems by altering soil texture, regulate water content, maintain the availability of nutrients for the plant; this diverse functionality of earthworms is mainly attributed to their gut microbiome 32 [39][40][41][42] and Perionyx excavatus with metagenomic pyrosequencing 43,44 . We recorded a slight divergence in the diversity across the species, e.g the collective reads derived from the Perionyx sp. has the greatest value of Chao1 (947), followed by Eudrilus sp. (751), and Polypheretima sp. (678). Since there was no significant difference found in Good's coverage value among the samples (> 99%), reflects that sufficient amount of the bacterial diversity were captured in all the samples [45][46][47] . The Shannon index ranged from 5.177 to 7.613 and Simpson index ranged from 0.8985-0.9875 across the samples, indicates Polypheretima elongata had the lowest bacterial diversity, while Perionyx excavatus has the highest diversity, with high species richness. The alpha diversity analysis (which is comprehensive indicator of species richness in community ecology) showed Perionyx excavatus gut had the highest diversity followed by Eudrilus eugeniae while Polypheretima elongata gut was the lowest according to Chao1, Shannon and Simpson values ( Table 2 & Fig. 4A-C). The next generation sequences of soil microbiome with respect to soil depth suggested that the upper layer of topsoil have higher microbial diversity than the lower layer of topsoil, with increase in soil depth, microbiome abundance decreases 48,49 . The topsoil is made up of decomposed material of plants and leaves, which provides a favourable conditions for growth of soil microbes 50 and play a crucial role in formation of humus, nutrients and organic matter 51 . Since epigeic earthworms live and feed in upper layer of topsoil, get high exposure of soil microbiome, humus, nutrient and organic matter. Effect of surrounding environment (available substrate and feeding habit) may not be ignored on counting earthworms' gut microbiome 12,52 . Our observation on alpha diversity in gut microbiome of Perionyx excavates, Eudrilus eugeniae and Polypheretima elongata corresponds to above facts. The worms live in the lower layer of topsoil carry relatively low microbial abundance and less availability of nutrients. In addition, alteration in the relative abundance of bacterial communities in earthworms' gut may be interlinked with variation in their feeding behaviour pattern because of dependence on microbial colonization of host's feeding behaviour pattern 52 . The epigeic species Perionyx excavatus and, Eudrilus eugeniae feeding behaviour ranges at upper layer of topsoil, making them access to feed on soil minerals, humus as well as remains of plant materials, while endogeic species Polypheretima elongata feeds on decomposing litter 53 . The study reported 5 major phyla of bacteria (Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Chloroflexi) in all three earthworm species, irrespective of their different feedings behaviours, which corresponded to the "core microbiota" of earthworms gut, although relative difference were observed at various taxonomical levels when compared for gut microbiota respectively. This could be explained due to co-evolution of certain core taxa via secretions of gut fluids that regulate microbial communities in earthworm gut 54 , that remains largely unchanged with earthworms 12,55 . The progression of a particular microbial community depends on the food source and life forms. Moreover, various microbial communities are selected or favoured over other microbes in the tube-like gut, which is stable in the moisture and nutrient conditions, although it acts as unique anoxic micro-environment filtering agent for ingested microbial communities of microorganism pools 56 .
The gut of Polypheretima elongata, Perionyx excavatus and Eudrilus eugeniae may be viewed as a bioreactor, in which diverse functions (biodegradation, bioremediation and biogeochemical cycling) goes simultaneously. On analysing homology datasets from the various online databases, Proteobacteria was found the predominant phylum, followed by Actinobacteria, Firmicutes and Bacteroidetes. The dominance of Proteobacteria may be due their fast-growing nature and its ability to employ available organic carbon sources and amino acids in the earthworm gut 32 . The predicted phenotypic analysis showed that gram negative bacteria's abundance depicts strong relationship between proteobacteria and ammonia oxidizers, sulfate reducers, nitrite reducers and chitin degraders. The nutrient poor environment carries high abundance of protobacteria 12,57 which play a vital role in the nitrogen cycle 58,59 and cellulose degradation 60 . It is noteworthy that similar trends have been recorded with predicted phenotype metabolism analysis in earthworms 42 and mammals gut microbiome 61,62 . In addition, few low abundance of certain phyla were also present such as Chloroflexi, Acidobacteria, Saccharibacteria and Verrucomicrobia. The earthworm's digestive system is involved in various processes such as oxidation and reduction, emission of N 2 O and N 2 , remediation, nitrogen fixation, denitrification and degradation processes. Deciphering the earthworm gut microbiome may enable researchers in understanding a much better perspective of their metabolic capabilities. Taxonomic to the phenotypic mapping on the basis of metabolism of the three species suggested that the earthworm gut microbiome played vital role in remediation, denitrification, nitrogen fixation, degradation of cellulose, reduction and sulfur oxidation processes. Isolation of such functionally active bacterial communities from earthworm gut may prove as a pearl of essential enzymes to degrade xenobiotic, lignocellulose for production of biofuels, environmental remediation and biogeochemical cycling.

CoNCLuSIoN
The present study revealed Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes were the dominant phyla in earthworm sp. Functional characterization revealed that the majority of the bacterial groups were ammonia oxidizers followed by sulfate reducer and nitrite reducer. The study highlights that next-generation high throughput sequencing provides a much detailed and accurate insight into the gut microbiome than other conventional techniques. It can be hypothesized that the majority of the functional attributions of earthworms in the soil ecosystem may be related to their diverse gut microbiome instead the activity of soil microbiomes.