High genetic diversity and variability of bacterial communities associated with the sandhopper Talitrus saltator (Montagu) (Crustacea, Amphipoda)

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Highlights

  • We analyzed the microbiome profiles of the sandohopper Talitrus saltator.

  • Highly variable microbiomes were found among individuals.

  • T. saltator populations were also differentiated on the basis of their microbiomes.

  • Alpha- and Beta-proteobacteria, Clostridiales and Bacillales were found.

Abstract

The microbiome present in individuals of Talitrus saltator belonging to seven populations distributed along the Tuscan coast (Italy) was assessed by using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Talitrus saltator is one of the key species of the damp band of European sandy beaches and despite of the large interest on animal-associated bacteria, only a few and preliminary data were present. Results showed a high diversity of the microbiome, composed mainly by members of Alphaproteobacteria, Gammaproteobacteria, Bacillales and Clostridiales classes. The microbiome fingerprints were highly variable among individuals, even from the same populations, the inter-individual differences accounting for 88.7% of total fingerprint variance. However, statistically significant population-specific microbiome signatures were detected, and accounted for the remaining 11.3% of total fingerprint variance. These population-specific differences were mainly attributed to sequences from members of known host-associated bacteria such as Gammaproteobacteria and Betaproteobacteria, Cytophagia and Spirochaetia. This study showed the high complexity of the microbiome associated with an amphipod species and on the inter-individual microbiome variation with potential importance for understanding amphipod trophic and ecologic processes.

Introduction

Talitrid amphipods, one of the main components (in terms of biomass) of the damp band of sandy beaches, play an important role in the energy flow within the sandy beach ecosystem because they feed on organic matter of marine and terrestrial origin and provide nourishment for many species of beetles, fishes, birds and mammals (Olabarria et al., 2009). Talitrid amphipods constitute the main animal biomass in sandy beaches and play an important role in the supralittoral environment (Griffiths et al., 1983, Wildish, 1988, McLachlan and Brown, 2006). The sandhopper Talitrus saltator is a well-established model species for biomonitoring human pressure and pollution on sandy beaches (Barca-Bravo et al., 2008, Ugolini et al., 2004, Ugolini et al., 2005, Ugolini et al., 2012; Ungherese et al., 2010a, Ungherese et al., 2010b, 2012) and for ecology and behavior (Pardi and Papi, 1953, Wildish, 1988, Ugolini et al., 1999, Ugolini, 2003, Ugolini, 2006). Despite their key ecological relevance for carbon cycling on the damp band of sandy beaches, to date there are very limited reports on T. saltator-associated gut microbial flora (Nuti et al., 1971, Martineti et al., 1995). In contrast, several reports on Crustacea-associated microorganisms are present, but mainly linked to health and aquaculture issues (Small and Pagenkopp, 2011, Wang, 2011) or to the presence of Wolbachia infections (Cordaux et al., 2012). There are no investigations on the total microbial communities carried by Crustaceans, despite the fact that in recent years, invertebrate microbiology is attracting more attention for its implication in biocontrol (Natrah et al., 2011) and animal–microbe interaction studies (Muller et al., 2008, Olson and Kellogg, 2010, Goffredi, 2011). Moreover, despite several studies concerning the microflora of particular organs (e.g. gut or reproductive organs) (Crotti et al., 2010, Hamdi et al., 2011), as well as the presence and effect of Wolbachia infections (Ben Nasr et al., 2010, Cordaux et al., 2012), no data were reported about the variation of microbial communities among single individuals, nor about possible population-specific microbial communities.

This work aimed to answer two key basic questions on the ecological interactions of dump band amphipods, using Talitrus saltator as model species: 1) What is the composition, in taxonomic terms, of the bacterial community associated with single individuals and populations of T. saltator?; 2) Is there individual or population-specific differentiation of the bacterial community? To address these questions the richness and the variability (both animal-by-animal and population-specific) of the bacterial communities associated with the sandhopper T. saltator were characterized by using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis (Liu et al., 1997, Mengoni et al., 2007) of amplified 16S rRNA genes, to provide a first insight into the microbiology and microbial ecology of this key species of sandy beaches.

Section snippets

Sampling and DNA extraction

Adult individuals of Talitrus saltator were collected from seven populations (FA, AF, AM, RS, FM, C, P, Fig. 1), on the Tuscan coast (Italy), previously characterized in a population genetic analysis (Ungherese et al., 2010b). Live animals were immediately transported to the laboratory, surface cleaned and conserved at −80 °C. From 3 to 7 animals were analyzed per population for a total of 30 animals. Single animals were surface-cleaned by washing with sterile distilled water to remove dust and

T-RFLP bacterial community diversity of Talitrus saltator

The T-RFLP profiling of 30 Talitrus saltator-associated bacterial communities with two restriction enzymes (AluI and TaqI) yielded a total of 56 polymorphic T-RFs, which allowed fingerprinting each single animal-associated bacterial community. Fig. 2a reports the number of Terminal-Restriction Fragments (T-RFs) obtained from each single animal. Values ranged from 7 to 31 T-RFs per animal. The sharing of T-RFs among individuals was relatively low (Fig. 2b), with most of the T-RFs being present

Discussion

In recent years several efforts have explored the biodiversity and understanding the functional role of microbial communities associated with organs and tissues of higher organisms (both plants and animals). The microbiomes carried by higher organisms have an important role in determining the health status of the organism (Hamdi et al., 2011, Kinross et al., 2011), its trophism, and ecological interactions. Recent examples include the human gut microbiome (Arumugam et al., 2011, Schloissnig

Acknowledgments

This work was supported by intramural grants of the University of Florence to AM and AU. We are grateful to Dr. Anna Benedetti (CRA-RPS, Rome, Italy) for the financial support to GB by granting him a PhD fellowship.

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