TAXONOMIC, GENETIC, AND FUNCTIONAL DIVERSITY IN LUCINID-BACTERIA CHEMOSYMBIOSES

Michelle Baldassare, Shen Jean Lim, Erin Walker, Alvee Ahmed, Barbara J. Campbell

Department of Biological Sciences, Clemson University, Clemson, SC

Objective

-The overall goal is to see how the symbionts are related and what genes they possess.

-Use metagenomic analysis to examine symbiont diversity within and between hosts, identify gene clusters, and generate functional annotations.

-Generate phylogenetic trees and pangenomes for the clam symbionts.

-Examine gene clusters from each group for core and unique functions to understand what is required for symbiosis (core) and what genes may confer other benefits (unique) to the symbiotic relationship.

Background

-Lucinid clams have an intimate symbiotic relationship with chemoautotrophic endosymbionts, mainly Gammaproteobacteria.

-The clams analyzed and studied in this experiment were Codakia orbicularis, Lucina pensylvanica, Ctena orbiculata, and Stewartia floridana from the Bahamas and Florida seen in Figure 1 and Figure 4.

-The symbionts contained genes that perform key processes for the host, are horizontally acquired, and live in extremely close contact with their host.

-These symbionts are found within the cells, known as bacteriocytes, always found in the gills of the clam.

Figure 1: Examples of Codakia orbicularisLucina pensylvanicaCtena orbiculata, and Stewartia floridana clams 

Materials and Methods

  1. The clams foot and gill tissues were dissected
  2. DNA and RNA were extracted from the gills and feet of the four species seen in Figure 1
  3. The DNA libraries were sequenced using Illumina HiSeq platform
  4. The sequences were assembled into core genes and pangenome analysis

Results

-All symbionts contained genes which were involved in the sulfur, nitrogen, and carbon cycles.

-The groups did not cluster according to host species or location which indicates their horizontal acquisition by the host. This was seen in the phylogenetic tree, in Figure 2, as each clade contained symbionts from diverse host species and different locations.

Figure 2: Phylogenomic tree (10 ribosomal genes) made using FigTree showing four distinct clades of symbionts

*ASV- amplified sequence variant

-Four pangenomes were identified in Anvio, indicated by the blue, pink, red and orange colored MAGs in Figure 3.

-These main clusters were not dependent on location or species.

-The pangenomes were analyzed for shared and unique gene clusters.

-All four clusters had an abundance of genes involved in the sulfur cycle.

Figure 3: The four pangenomes identified by Anvio

Conclusions

-The endosymbionts shared core genomic traits, involving the sulfur, nitrogen, and carbon cycles, but also had their own unique genes which indicated that they were highly variable.

-There was an abundance of Ca. Thiodiazotropha species among the samples forming four major clades independent of their host and geographical location.

-These results are due to the probable effect of environmentally acquired genes and horizontal gene transfer.

-More genomic sequencing must be done on Lucinid clams to further study these host-symbiont relationships by seeing what genes are involved.

-The results direct us to study the differential expression of the genes in the symbionts based on their species and locations.

Figure 4: Pictures from the Bahamas sampling sites (Moon Rock Pond, Crescent Pond, Pain Pond, and Pigeon Creek)