<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

Composition of SSR-2 and SSR-3 copies of <i>Candidatus</i> Sodalis melophagi TTSS.

<p>Ψ – putative pseudogene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040354#s4" target="_blank">Material and Methods</a>).</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

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

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

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

Data_Sheet_1_Microbiomes of North American Triatominae: The Grounds for Chagas Disease Epidemiology.xlsx

<p>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.</p