Origins of bacterial endosymbionts in arthropods

Current bioinformatic methods such as molecular phylogenetics and phylogenomics provide us with good insight to symbiont evolution. Though modern science evolves rapidly, accelerates speed of acquiring novel discoveries and improves their quality, there is still endless row of questions waiting to be answered. This thesis focuses on origins of symbiosis between insects and Enterobacteria, and the mechanisms promoting association of bacteria with arthropods. The main emphasis is put on the secondary symbionts of the genus Sodalis (Enterobacteriaceae) and the pathogenic Anaplasma phagocytophilum (Anaplasmataceae) that seems to be undergoing first steps to become hereditary mutualist

Multiple origins of endosymbiosis within the Enterobacteriaceae (γ-Proteobacteria): convergence of complex phylogenetic approaches

Abstract Background The bacterial family Enterobacteriaceae gave rise to a variety of symbiotic forms, from the loosely associated commensals, often designated as secondary (S) symbionts, to obligate mutualists, called primary (P) symbionts. Determination of the evolutionary processes behind this phenomenon has long been hampered by the unreliability of phylogenetic reconstructions within this group of bacteria. The main reasons have been the absence of sufficient data, the highly derived nature of the symbiont genomes and lack of appropriate phylogenetic methods. Due to the extremely aberrant nature of their DNA, the symbiotic lineages within Enterobacteriaceae form long branches and tend to cluster as a monophyletic group. This state of phylogenetic uncertainty is now improving with an increasing number of complete bacterial genomes and development of new methods. In this study, we address the monophyly versus polyphyly of enterobacterial symbionts by exploring a multigene matrix within a complex phylogenetic framework. Results We assembled the richest taxon sampling of Enterobacteriaceae to date (50 taxa, 69 orthologous genes with no missing data) and analyzed both nucleic and amino acid data sets using several probabilistic methods. We particularly focused on the long-branch attraction-reducing methods, such as a nucleotide and amino acid data recoding and exclusion (including our new approach and slow-fast analysis), taxa exclusion and usage of complex evolutionary models, such as nonhomogeneous model and models accounting for site-specific features of protein evolution (CAT and CAT+GTR). Our data strongly suggest independent origins of four symbiotic clusters; the first is formed by Hamiltonella and Regiella (S-symbionts) placed as a sister clade to Yersinia, the second comprises Arsenophonus and Riesia (S- and P-symbionts) as a sister clade to Proteus, the third Sodalis, Baumannia, Blochmannia and Wigglesworthia (S- and P-symbionts) as a sister or paraphyletic clade to the Pectobacterium and Dickeya clade and, finally, Buchnera species and Ishikawaella (P-symbionts) clustering with the Erwinia and Pantoea clade. Conclusions The results of this study confirm the efficiency of several artifact-reducing methods and strongly point towards the polyphyly of P-symbionts within Enterobacteriaceae. Interestingly, the model species of symbiotic bacteria research, Buchnera and Wigglesworthia, originated from closely related, but different, ancestors. The possible origins of intracellular symbiotic bacteria from gut-associated or pathogenic bacteria are suggested, as well as the role of facultative secondary symbionts as a source of bacteria that can gradually become obligate maternally transferred symbionts.</p

<em>Candidatus</em> Sodalis melophagi sp. nov.: Phylogenetically Independent Comparative Model to the Tsetse Fly Symbiont <em>Sodalis glossinidius</em>

<div><p>Bacteria of the genus <em>Sodalis</em> live in symbiosis with various groups of insects. The best known member of this group, a secondary symbiont of tsetse flies <em>Sodalis glossinidius</em>, has become one of the most important models in investigating establishment and evolution of insect-bacteria symbiosis. It represents a bacterium in the early/intermediate state of the transition towards symbiosis, which allows for exploring such interesting topics as: usage of secretory systems for entering the host cell, tempo of the genome modification, and metabolic interaction with a coexisting primary symbiont. In this study, we describe a new <em>Sodalis</em> species which could provide a useful comparative model to the tsetse symbiont. It lives in association with <em>Melophagus ovinus</em>, an insect related to tsetse flies, and resembles <em>S. glossinidius</em> in several important traits. Similar to <em>S. glossinidius</em>, it cohabits the host with another symbiotic bacterium, the bacteriome-harbored primary symbiont of the genus <em>Arsenophonus</em>. As a typical secondary symbiont, <em>Candidatus</em> Sodalis melophagi infects various host tissues, including bacteriome. We provide basic morphological and molecular characteristics of the symbiont and show that these traits also correspond to the early/intermediate state of the evolution towards symbiosis. Particularly, we demonstrate the ability of the bacterium to live in insect cell culture as well as in cell-free medium. We also provide basic characteristics of type three secretion system and using three reference sequences (16 S rDNA, <em>groEL</em> and <em>spaPQR</em> region) we show that the bacterium branched within the genus <em>Sodalis</em>, but originated independently of the two previously described symbionts of hippoboscoids. We propose the name <em>Candidatus</em> Sodalis melophagi for this new bacterium.</p> </div

Phylogenetic tree derived from <i>spaPQR</i> region by BI in MrBayes.

<p>The numbers at the nodes show the posterior probabilites and bootstrap values from the identical topology obtained by ML in PhyML. New <i>Sodalis</i> lineages added in this study are printed in red.</p

Phylogenetic trees derived from <i>groEL</i> matrices by BI in MrBayes.

<p>New <i>Sodalis</i> lineages added in this study are printed in red. <b>A</b>: The tree inferred from aminoacid matrix. Posterior probabilities are indicated by the numbers at the nodes. <b>B</b>: The tree inferred from nucleotide matrix restricted taxonomically to the <i>Sodalis</i> branch. The numbers at the nodes show the posterior probabilities and bootstrap values from the identical topology obtained by ML in PhyML.</p

16 S rDNA tree derived by BI analysis in MrBayes.

<p>Posterior probabilities are indicated by the numbers at the nodes. New <i>Sodalis</i> lineages added in this study are printed in red.</p

Morphology and ultrastructure of <i>Candidatus</i> Sodalis melophagi.

<p><b>A</b>: <i>In vitro</i> cell culture in Nomarski contrast. <b>B</b>, <b>C</b>: Cells of <i>Candidatus</i> Sodalis melophagi in bacteriome. Black arrows – cells of <i>Candidatus</i> Sodalis melophagi, white arrows – cells of the primary endosymbiont of the genus <i>Arsenophonus</i>.</p