Eva Nováková

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

BackgroundThe genus Arsenophonus 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.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.ResultsThe analysis of 110 Arsenophonus sequences (incl. Riesia and Phlomobacter), provides a robust monophyletic clade, characterized by unique molecular synapomorphies. On the other hand, unstable inner topology indicates that complete understanding of Arsenophonus evolution cannot be achieved with 16S rDNA. Moreover, taxonomically restricted Sampling matrices prove sensitivity of the phylogenetic signal to sampling; in some cases, Arsenophonus 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.ConclusionThe sample of currently available molecular data presents the genus Arsenophonus 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 Arsenophonus lineages as independent genera (e.g. Riesia and Phlomobacter). The resolution of the evolutionary questions encountered within the Arsenophonus clade will thus require identification of new molecular markers suitable for the low-level phylogenetics.

Reconstructing the phylogeny of aphids (Hemiptera: Aphididae) using DNA of the obligate symbiont Buchnera aphidicola

Reliable phylogenetic reconstruction, as a framework for evolutionary inference, may be difficult to achieve in some groups of organisms. Particularly for lineages that experienced rapid diversification, lack of sufficient information may lead to inconsistent and unstable results and a low degree of resolution. Coincidentally, such rapidly diversifying taxa are often among the biologically most interesting groups. Aphids provide such an example. Due to rapid adaptive diversification, they feature variability in many interesting biological traits, but consequently they are also a challenging group in which to resolve phylogeny. Particularly within the family Aphididae, many interesting evolutionary questions remain unanswered due to phylogenetic uncertainties. In this study, we show that molecular data derived from the symbiotic bacteria of the genus Buchnera can provide a more powerful tool than the aphid-derived sequences. We analyze 255 Buchnera gene sequences from 70 host aphid species and compare the resulting trees to the phylogenies previously retrieved from aphid sequences, only. We find that the host and symbiont data do not conflict for any major phylogenetic conclusions. Also, we demonstrate that the symbiont-derived phylogenies support some previously questionable relationships and provide new insights into aphid phylogeny and evolution.

Diversification of Genes for Carotenoid Biosynthesis in Aphids following an Ancient Transfer from a Fungus

The pea aphid genome was recently found to harbor genes for carotenoid biosynthesis, reflecting an ancestral transfer from a fungus. To explore the evolution of the carotene desaturase gene family within aphids, sequences were retrieved from a set of 34 aphid species representing numerous deeply diverging lineages of aphids and analyzed together with fungal sequences retrieved from databases. All aphids have at least one copy of this gene and some aphid species have up to seven, whereas fungal genomes consistently have a single copy. The closest relatives of aphids, adelgids, also have carotene desaturase; these sequences are most closely related to those from aphids, supporting a shared origin from a fungal to insect transfer predating the divergence of adelgids and aphids. Likewise, all aphids, and adelgids, have carotenoid profiles that are consistent with their biosynthesis using the acquired genes of fungal origin rather than derivation from food plants. The carotene desaturase was acquired from a fungal species outside of Ascomycota or Basidiomycota and closest to Mucoromycotina among sequences available in databases. In aphids, an ongoing pattern of gene duplication is indicated by the presence of both anciently and recently diverged paralogs within genomes and by the presence of a high frequency of pseudogenes that appear to be recently inactivated. Recombination among paralogs is evident, making analyses of patterns of selection difficult, but tests of selection for a nonrecombining region indicates that duplications tend to be followed by bouts of positive selection. Species of Macrosiphini, which often show color polymorphisms, typically have a larger number of desaturase copies relative to other species sampled in the study. These results indicate that aphid evolution has been accompanied by ongoing evolution of carotenogenic genes, which have undergone duplication, recombination, and occasional positive selection to yield a wide variety of carotenoid profiles in different aphid species.

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

Bacteria of the genus Sodalis live in symbiosis with various groups of insects. The best known member of this group, a secondary symbiont of tsetse flies Sodalis glossinidius, 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 Sodalis species which could provide a useful comparative model to the tsetse symbiont. It lives in association with Melophagus ovinus, an insect related to tsetse flies, and resembles S. glossinidius in several important traits. Similar to S. glossinidius, it cohabits the host with another symbiotic bacterium, the bacteriome-harbored primary symbiont of the genus Arsenophonus. As a typical secondary symbiont, Candidatus 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, groEL and spaPQR region) we show that the bacterium branched within the genus Sodalis, but originated independently of the two previously described symbionts of hippoboscoids. We propose the name Candidatus Sodalis melophagi for this new bacterium.

Arsenophonus and Sodalis Symbionts in Louse Flies: an Analogy to the Wigglesworthia and Sodalis System in Tsetse Flies.

ABSTRACT Symbiosis between insects and bacteria result in a variety of arrangements, genomic modifications, and metabolic interconnections. Here, we present genomic, phylogenetic, and morphological characteristics of a symbiotic system associated with Melophagus ovinus, a member of the blood-feeding family Hippoboscidae. The system comprises four unrelated bacteria representing different stages in symbiosis evolution, from typical obligate mutualists inhabiting bacteriomes to freely associated commensals and parasites. Interestingly, the whole system provides a remarkable analogy to the association between Glossina and its symbiotic bacteria. In both, the symbiotic systems are composed of an obligate symbiont and two facultative intracellular associates, Sodalis and Wolbachia. In addition, extracellular Bartonella resides in the gut of Melophagus. However, the phylogenetic origins of the two obligate mutualist symbionts differ. In Glossina, the mutualistic Wigglesworthia appears to be a relatively isolated symbiotic lineage, whereas in Melophagus, the obligate symbiont originated within the widely distributed Arsenophonus cluster. Although phylogenetically distant, the two obligate symbionts display several remarkably similar traits (e.g., transmission via the hosts “milk glands” or similar pattern of genome reduction). To obtain better insight into the biology and possible role of the M. ovinus obligate symbiont, “Candidatus Arsenophonus melophagi,” we performed several comparisons of its gene content based on assignments of the Cluster of Orthologous Genes (COG). Using this criterion, we show that within a set of 44 primary and secondary symbionts, “Ca. Arsenophonus melophagi” is most similar to Wigglesworthia. On the other hand, these two bacteria also display interesting differences, such as absence of flagellar genes in Arsenophonus and their presence in Wigglesworthia. This finding implies that a flagellum is not essential for bacterial transmission via milk glands.

Mosquito Microbiome Dynamics, a Background for Prevalence and Seasonality of West Nile Virus

Symbiotic microbial communities augment host phenotype, including defense against pathogen carriage and infection. We sampled the microbial communities in 11 adult mosquito host species from six regions in southern Ontario, Canada over 3 years. Of the factors examined, we found that mosquito species was the largest driver of the microbiota, with remarkable phylosymbiosis between host and microbiota. Seasonal shifts of the microbiome were consistently repeated over the 3-year period, while region had little impact. Both host species and seasonal shifts in microbiota were associated with patterns of West Nile virus (WNV) in these mosquitoes. The highest prevalence of WNV, with a seasonal spike each year in August, was in the Culex pipiens/restuans complex, and high WNV prevalence followed a decrease in relative abundance of Wolbachia in this species. Indeed, mean temperature, but not precipitation, was significantly correlated with Wolbachia abundance. This suggests that at higher temperatures Wolbachia abundance is reduced leading to greater susceptibility to WNV in the subsequent generation of C. pipiens/restuans hosts. Different mosquito genera harbored significantly different bacterial communities, and presence or abundance of Wolbachia was primarily associated with these differences. We identified several operational taxonomic units (OTUs) of Wolbachia that drive overall microbial community differentiation among mosquito taxa, locations and timepoints. Distinct Wolbachia OTUs were consistently found to dominate microbiomes of Cx. pipiens/restuans, and of Coquilletidia perturbans. Seasonal fluctuations of several other microbial taxa included Bacillus cereus, Enterococcus, Methylobacterium, Asaia, Pantoea, Acinetobacter johnsonii, Pseudomonas, and Mycoplasma. This suggests that microbiota may explain some of the variation in vector competence previously attributed to local environmental processes, especially because Wolbachia is known to affect carriage of viral pathogens.

Genome sequence of Candidatus Arsenophonus lipopteni, the exclusive symbiont of a blood sucking fly Lipoptena cervi (Diptera: Hippoboscidae)

Candidatus Arsenophonus lipopteni (Enterobacteriaceae, Gammaproteobacteria) is an obligate intracellular symbiont of the blood feeding deer ked, Lipoptena cervi (Diptera: Hippoboscidae). The bacteria reside in specialized cells derived from host gut epithelia (bacteriocytes) forming a compact symbiotic organ (bacteriome). Compared to the closely related complex symbiotic system in the sheep ked, involving four bacterial species, Lipoptena cervi appears to maintain its symbiosis exclusively with Ca. Arsenophonus lipopteni. The genome of 836,724xa0bp and 24.8xa0% GC content codes for 667 predicted functional genes and bears the common characteristics of sequence economization coupled with obligate host-dependent lifestyle, e.g. reduced number of RNA genes along with the rRNA operon split, and strongly reduced metabolic capacity. Particularly, biosynthetic capacity for B vitamins possibly supplementing the host diet is highly compromised in Ca. Arsenophonus lipopteni. The gene sets are complete only for riboflavin (B2), pyridoxine (B6) and biotin (B7) implying the content of some B vitamins, e.g. thiamin, in the deer blood might be sufficient for the insect metabolic needs. The phylogenetic position within the spectrum of known Arsenophonus genomes and fundamental genomic features of Ca. Arsenophonus lipopteni indicate the obligate character of this symbiosis and its independent origin within Hippoboscidae.

Legionella Becoming a Mutualist: Adaptive Processes Shaping the Genome of Symbiont in the Louse Polyplax serrata

Abstract Legionellaceae are intracellular bacteria known as important human pathogens. In the environment, they are mainly found in biofilms associated with amoebas. In contrast to the gammaproteobacterial family Enterobacteriaceae, which established a broad spectrum of symbioses with many insect taxa, the only instance of legionella-like symbiont has been reported from lice of the genus Polyplax. Here, we sequenced the complete genome of this symbiont and compared its main characteristics to other Legionella species and insect symbionts. Based on rigorous multigene phylogenetic analyses, we confirm this bacterium as a member of the genus Legionella and propose the name Candidatus Legionella polyplacis, sp.n. We show that the genome of Ca. Legionella polyplacis underwent massive degeneration, including considerable size reduction (529.746u2009bp, 484 protein coding genes) and a severe decrease in GC content (23%). We identify several possible constraints underlying the evolution of this bacterium. On one hand, Ca. Legionella polyplacis and the louse symbionts Riesia and Puchtella experienced convergent evolution, perhaps due to adaptation to similar hosts. On the other hand, some metabolic differences are likely to reflect different phylogenetic positions of the symbionts and hence availability of particular metabolic function in the ancestor. This is exemplified by different arrangements of thiamine metabolism in Ca. Legionella polyplacis and Riesia. Finally, horizontal gene transfer is shown to play a significant role in the adaptive and diversification process. Particularly, we show that Ca. L. polyplacis horizontally acquired a complete biotin operon (bioADCHFB) that likely assisted this bacterium when becoming an obligate mutualist.

Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species

RNA interference (RNAi) has emerged as a promising method for validating gene function; however, its utility in nonmodel insects has proven problematic, with delivery methods being one of the main obstacles. This study investigates a novel method of RNAi delivery in aphids, the aerosolization of short interfering RNA (siRNA)–nanoparticle complexes. By using nanoparticles as a siRNA carrier, the likelihood of cellular uptake is increased, when compared to methods previously used in insects. To determine the efficacy of this RNAi delivery system, siRNAs were aerosolized with and without nanoparticles in three aphid species: Acyrthosiphon pisum, Aphis glycines and Schizaphis graminum. The genes targeted for knockdown were carotene dehydrogenase (tor), which is important for pigmentation in Ac. pisum, and branched chain‐amino acid transaminase (bcat), which is essential in the metabolism of branched‐chain amino acids in all three aphid species. Overall, we observed modest gene knockdown of tor in Ac. pisum and moderate gene knockdown of bcat in Ap. glycines along with its associated phenotype. We also determined that the nanoparticle emulsion significantly increased the efficacy of gene knockdown. Overall, these results suggest that the aerosolized siRNA–nanoparticle delivery method is a promising new high‐throughput and non‐invasive RNAi delivery method in some aphid species.

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.