Arsenophonus, an emerging clade of intracellular symbionts with a broad host distribution

<p>Abstract</p> <p>Background</p> <p>The genus <it>Arsenophonus </it>is a group of symbiotic, mainly insect-associated bacteria with rapidly increasing number of records. It is known from a broad spectrum of hosts and symbiotic relationships varying from parasitic son-killers to coevolving mutualists.</p> <p>The present study extends the currently known diversity with 34 samples retrieved mainly from hippoboscid (Diptera: Hippoboscidae) and nycteribiid (Diptera: Nycteribiidae) hosts, and investigates phylogenetic relationships within the genus.</p> <p>Results</p> <p>The analysis of 110 <it>Arsenophonus </it>sequences (incl. <it>Riesia </it>and <it>Phlomobacter</it>), provides a robust monophyletic clade, characterized by unique molecular synapomorphies. On the other hand, unstable inner topology indicates that complete understanding of <it>Arsenophonus </it>evolution cannot be achieved with 16S rDNA. Moreover, taxonomically restricted <it>Sampling </it>matrices prove sensitivity of the phylogenetic signal to sampling; in some cases, <it>Arsenophonus </it>monophyly is disrupted by other symbiotic bacteria. Two contrasting coevolutionary patterns occur throughout the tree: parallel host-symbiont evolution and the haphazard association of the symbionts with distant hosts. A further conspicuous feature of the topology is the occurrence of monophyletic symbiont lineages associated with monophyletic groups of hosts without a co-speciation pattern. We suggest that part of this incongruence could be caused by methodological artifacts, such as intragenomic variability.</p> <p>Conclusion</p> <p>The sample of currently available molecular data presents the genus <it>Arsenophonus </it>as one of the richest and most widespread clusters of insect symbiotic bacteria. The analysis of its phylogenetic lineages indicates a complex evolution and apparent ecological versatility with switches between entirely different life styles. Due to these properties, the genus should play an important role in the studies of evolutionary trends in insect intracellular symbionts. However, under the current practice, relying exclusively on 16S rRNA sequences, the phylogenetic analyses are sensitive to various methodological artifacts that may even lead to description of new <it>Arsenophonus </it>lineages as independent genera (e.g. <it>Riesia </it>and <it>Phlomobacter</it>). The resolution of the evolutionary questions encountered within the <it>Arsenophonus </it>clade will thus require identification of new molecular markers suitable for the low-level phylogenetics.</p

Host-parasite incongruences in rodent Eimeria suggest significant role of adaptation rather than cophylogeny in maintenance of host specificity.

The degree of host specificity, its phylogenetic conservativeness and origin are virtually unknown in Eimeria. This situation is largely due to the inadequate sample of eimerian molecular data available for reliable phylogenetic analyses. In this study, we extend the data set by adding 71 new sequences of coccidia infecting 16 small-mammal genera, mostly rodents. According to the respective feasibility of PCR gene amplification, the new samples are represented by one or more of the following genes: nuclear 18S rRNA, plastid ORF 470, and mitochondrial COI. Phylogenetic analyses of these sequences confirm the previous hypothesis that Eimeria, in its current morphology-based delimitation, is not a monophyletic group. Several samples of coccidia corresponding morphologically to other genera are scattered among the Eimeria lineages. More importantly, the distribution of eimerians from different hosts indicates that the clustering of eimerian species is influenced by their host specificity, but does not arise from a cophylogenetic/cospeciation process; while several clusters are specific to a particular host group, inner topologies within these clusters do not reflect host phylogeny. This observation suggests that the host specificity of Eimeria is caused by adaptive rather than cophylogenetic processes

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

Taxa and sequences included in the phylogenetic analyses.

*<p>: sequences included in the <i>Skeleton</i> matrix.</p><p>•: taxa used as outgroups for the phylogenetic analyses.</p><p>– : the sequence is not available.</p><p>Taxa for which new sequences were obtained in this study and Accession numbers of these sequences are printed in bold.</p

Morphological features and origin of the newly obtained samples within this study.

<p>CZ – Czech Republic, SK – Slovakia, UK – England; OW – oocyst wall, MP – micropyle, OR – oocyst residuum.</p

A <i>Skeleton</i> tree.

<p><i>Skeleton</i> tree (ML and BI) of the taxa for which all 3 genes (<i>18S rDNA</i>, <i>ORF 470</i> and <i>COI</i>) are available.</p

<i>Concatenated</i> ML tree.

<p> Letters A–D indicate clusters delimited according to the <i>Skeleton</i> tree (taxa present in the <i>Skeleton</i> tree are labeled with asterisks). Clades A and B are supported by both BI and ML analyses of the <i>Concatenated</i> and <i>Skeleton</i> matrices. The red node indicates a cluster with weak host specificity. Numbers 1–4 indicate lineages that are also supported by BI analyses of the following matrices: 1, <i>Concatenated</i>; 2, <i>ORF 470</i>; 3, <i>COI</i>; 4, <i>18S rDNA</i>. The newly added samples are printed in bold; coccidia from rodents are printed in blue. To decrease the size of the tree for the printed presentation, we removed several of the most basal outgroups.</p

Microbiomes of North American Triatominae: The Grounds for Chagas Disease Epidemiology

Insect microbiomes influence many fundamental host traits, including functions of practical significance such as their capacity as vectors to transmit parasites and pathogens. The knowledge on the diversity and development of the gut microbiomes in various blood feeding insects is thus crucial not only for theoretical purposes, but also for the development of better disease control strategies. In Triatominae (Heteroptera: Reduviidae), the blood feeding vectors of Chagas disease in South America and parts of North America, the investigation of the microbiomes is in its infancy. The few studies done on microbiomes of South American Triatominae species indicate a relatively low taxonomic diversity and a high host specificity. We designed a comparative survey to serve several purposes: (I) to obtain a better insight into the overall microbiome diversity in different species, (II) to check the long term stability of the interspecific differences, (III) to describe the ontogenetic changes of the microbiome, and (IV) to determine the potential correlation between microbiome composition and presence of Trypanosoma cruzi, the causative agent of Chagas disease. Using 16S amplicons of two abundant species from the southern US, and four laboratory reared colonies, we showed that the microbiome composition is determined by host species, rather than locality or environment. The OTUs (Operational Taxonomic Units) determination confirms a low microbiome diversity, with 12-17 main OTUs detected in wild populations of T. sanguisuga and T. protracta. Among the dominant bacterial taxa are Acinetobacter and Proteiniphilum but also the symbiotic bacterium Arsenophonus triatominarum, previously believed to only live intracellularly. The possibility of ontogenetic microbiome changes was evaluated in all six developmental stages and feces of the laboratory reared model Rhodnius prolixus. We detected considerable changes along the host's ontogeny, including clear trends in the abundance variation of the three dominant bacteria, namely Enterococcus, Acinetobacter, and Arsenophonus. Finally, we screened the samples for the presence of Trypanosoma cruzi. Comparing the parasite presence with the microbiome composition, we assessed the possible significance of the latter in the epidemiology of the disease. Particularly, we found a trend toward more diverse microbiomes in Trypanosoma cruzi positive T. protracta specimens

Arsenophonus and Sodalis replacements shape evolution of symbiosis in louse flies

Symbiotic interactions between insects and bacteria are ubiquitous and form a continuum from loose facultative symbiosis to greatly intimate and stable obligate symbiosis. In blood-sucking insects living exclusively on vertebrate blood, obligate endosymbionts are essential for hosts and hypothesized to supplement B-vitamins and cofactors missing from their blood diet. The role and distribution of facultative endosymbionts and their evolutionary significance as seeds of obligate symbioses are much less understood. Here, using phylogenetic approaches, we focus on the Hippoboscidae phylogeny as well as the stability and dynamics of obligate symbioses within this bloodsucking group. In particular, we demonstrate a new potentially obligate lineage of Sodalis co-evolving with the Olfersini subclade of Hippoboscidae. We also show several likely facultative Sodalis lineages closely related to Sodalis praecaptivus (HS strain) and suggest repeated acquisition of novel symbionts from the environment. Similar to Sodalis, Arsenophonus endosymbionts also form both obligate endosymbiotic lineages co-evolving with their hosts (Ornithomyini and Ornithoica groups) as well as possibly facultative infections incongruent with the Hippoboscidae phylogeny. Finally, we reveal substantial diversity of Wolbachia strains detected in Hippoboscidae samples falling into three supergroups: A, B, and the most common F. Altogether, our results prove the associations between Hippoboscoidea and their symbiotic bacteria to undergo surprisingly dynamic, yet selective, evolutionary processes strongly shaped by repeated endosymbiont replacements. Interestingly, obligate symbionts only originate from two endosymbiont genera, Arsenophonus and Sodalis, suggesting that the host is either highly selective about its future obligate symbionts or that these two lineages are the most competitive when establishing symbioses in louse flies

<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