Matteo Montagna

Phylogenomic evidence for the presence of a flagellum and cbb3 oxidase in the free-living mitochondrial ancestor

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives-generally considered to be the closest living relatives of mitochondria-indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb(3)-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral α-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.

Molecular Evidence for Multiple Infections as Revealed by Typing of Asaia Bacterial Symbionts of Four Mosquito Species

ABSTRACT The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

Candidatus Midichloriaceae fam. nov. (Rickettsiales), an ecologically widespread clade of intracellular alphaproteobacteria.

ABSTRACT “Candidatus Midichloria mitochondrii” is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus. Bacteria phylogenetically related to “Ca. Midichloria mitochondrii” (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales. This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family “Candidatus Midichloriaceae.” In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

Phylogenomics and Analysis of Shared Genes Suggest a Single Transition to Mutualism in Wolbachia of Nematodes

Wolbachia, endosymbiotic bacteria of the order Rickettsiales, are widespread in arthropods but also present in nematodes. In arthropods, A and B supergroup Wolbachia are generally associated with distortion of host reproduction. In filarial nematodes, including some human parasites, multiple lines of experimental evidence indicate that C and D supergroup Wolbachia are essential for the survival of the host, and here the symbiotic relationship is considered mutualistic. The origin of this mutualistic endosymbiosis is of interest for both basic and applied reasons: How does a parasite become a mutualist? Could intervention in the mutualism aid in treatment of human disease? Correct rooting and high-quality resolution of Wolbachia relationships are required to resolve this question. However, because of the large genetic distance between Wolbachia and the nearest outgroups, and the limited number of genomes so far available for large-scale analyses, current phylogenies do not provide robust answers. We therefore sequenced the genome of the D supergroup Wolbachia endosymbiont of Litomosoides sigmodontis, revisited the selection of loci for phylogenomic analyses, and performed a phylogenomic analysis including available complete genomes (from isolates in supergroups A, B, C, and D). Using 90 orthologous genes with reliable phylogenetic signals, we obtained a robust phylogenetic reconstruction, including a highly supported root to the Wolbachia phylogeny between a (A + B) clade and a (C + D) clade. Although we currently lack data from several Wolbachia supergroups, notably F, our analysis supports a model wherein the putatively mutualist endosymbiotic relationship between Wolbachia and nematodes originated from a single transition event.

Effects of the Diet on the Microbiota of the Red Palm Weevil (Coleoptera: Dryophthoridae)

Rhynchophorus ferrugineus, also known as the red palm weevil, is regarded as the major pest of palm trees. Although studies of the microbiota associated with this species have been performed in recent years, little attention has been dedicated to the influence of the diet in shaping the host bacterial community. Here, we investigated the influence of food sources (i.e. palm tissues vs apple based substrate) on the microbial diversity associated with RPW, which was compared with the microbiota associated with wild individuals of the sister species Rhynchophorus vulneratus. The bacterial characterization was performed using a culture independent approach, i.e. the 16S rRNA pyrotag, and a culture dependent approach for a subset of the samples, in order to obtain bacterial isolates from RPW tissues. The bacterial community appeared significantly influenced by diet. Proteobacteria resulted to be the most abundant clade and was present in all the specimens of the three examined weevil groups. Within Proteobacteria, Enterobacteriaceae were identified in all the organs analysed, including hemolymph and reproductive organs. The apple-fed RPWs and the wild R. vulneratus showed a second dominant taxon within Firmicutes that was scarcely present in the microbiota associated with palm-fed RPWs. A comparative analysis on the bacteria associated with the palm tissues highlighted that 12 bacterial genera out of the 13 identified in the plant tissues were also present in weevils, thus indicating that palm tissues may present a source for bacterial acquisition.

The integration of multiple independent data reveals an unusual response to Pleistocene climatic changes in the hard tick Ixodes ricinus

In the last few years, improved analytical tools and the integration of genetic data with multiple sources of information have shown that temperate species exhibited more complex responses to ice ages than previously thought. In this study, we investigated how Pleistocene climatic changes affected the current distribution and genetic diversity of European populations of the tick Ixodes ricinus, an ectoparasite with high ecological plasticity. We first used mitochondrial and nuclear genetic markers to investigate the phylogeographic structure of the species and its Pleistocene history using coalescent‐based methods; then we used species distribution modelling to infer the climatic niche of the species at last glacial maximum; finally, we reviewed the literature on the I. ricinus hosts to identify the locations of their glacial refugia. Our results support the scenario that during the last glacial phase, I. ricinus never experienced a prolonged allopatric divergence in separate glacial refugia, but persisted with interconnected populations across Southern and Central Europe. The generalist behaviour in host choice of I. ricinus would have played a major role in maintaining connections between its populations. Although most of the hosts persisted in separate refugia, from the point of view of I. ricinus, they represented a continuity of ‘bridges’ among populations. Our study highlights the importance of species‐specific ecology in affecting responses to Pleistocene glacial–interglacial cycles. Together with other cases in Europe and elsewhere, it contributes to setting new hypotheses on how species with wide ecological plasticity coped with Pleistocene climatic changes.

Metamicrobiomics in herbivore beetles of the genus Cryptocephalus (Chrysomelidae): toward the understanding of ecological determinants in insect symbiosis.

The Cryptocephalus marginellus (Coleoptera: Chrysomelidae) complex is composed by six species that are supposed to have originated by events of allo‐ or parapatric speciation. In the present study we investigated the alternative hypotheses that the bacterial communities associated with six populations of this species complex are shaped by environmental factors, or reflect the proposed pattern of speciation. The microbiota associated with the six populations, from five species of the complex, have been characterized through 16S rRNA pyrotag sequencing. Based on a 97% sequence similarity threshold, data were clustered into 381 OTUs, which were analyzed using a variety of diversity indices. The microbiota of C. acquitanus and C. marginellus (Calanques) were the most diverse (over 100 OTUs), while that from C. zoiai yielded less bacterial diversity (45 OTUs). Taxonomic assignment revealed Proteobacteria, Tenericutes and Firmicutes as the dominant components of these beetles’ microbiota. The most abundant genera were Ralstonia, Sphingomonas, Rickettsia, and Pseudomonas. Different strains of Rickettsia were detected in C. eridani and C. renatae. The analysis of β‐diversity revealed high OTU turnover among the populations of C. marginellus complex, with only few shared species. Hierarchical clustering taking into account relative abundances of OTUs does not match the phylogeny of the beetles, therefore we hypothesize that factors other than phylogenetic constraints play a role in shaping the insects’ microbiota. Environmental factors that could potentially affect the composition of bacterial communities were tested by fitting them on the results of a multi‐dimensional scaling analysis. No significant correlations were observed towards the geographic distances or the host plants, while the composition of the microbiota appeared associated with altitude. The metabolic profiles of the microbiotas associated with each population were inferred from bacterial taxonomy, and interestingly, the obtained clustering pattern was consistent with the host phylogeny.

Localization of the bacterial symbiont Candidatus Midichloria mitochondrii within the hard tick Ixodes ricinus by whole-mount FISH staining.

Here, we present an investigation on the spatial distribution of the bacterial symbiont Candidatus Midichloria mitochondrii within Ixodes ricinus, by whole mount fluorescence in situ hybridization (FISH). M. mitochondrii is a peculiar, recently discovered bacterium that resides in the mitochondria of female ticks. We applied a rapid and specific FISH protocol with oligonucleotide probes targeted on the 16S rRNA of M. mitochondrii, 12S rRNA of tick mitochondria, and a probe revealing active mitochondria. In this report that represents the first application of whole mount FISH on ticks, we observed strong, specific fluorescence signals in all the examined life stages, as the optimized protocol allowed us to overcome the autofluorescence interference of the cuticle. Cellular localization and quantification of the symbionts were also assessed with electron microscopy and specific real-time PCR, respectively.

Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi

In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps.

Tick-box for 3'-end formation of mitochondrial transcripts in Ixodida, basal chelicerates and Drosophila.

According to the tRNA punctuation model, the mitochondrial genome (mtDNA) of mammals and arthropods is transcribed as large polycistronic precursors that are maturated by endonucleolytic cleavage at tRNA borders and RNA polyadenylation. Starting from the newly sequenced mtDNA of Ixodes ricinus and using a combination of mitogenomics and transcriptional analyses, we found that in all currently-sequenced tick lineages (Prostriata, Metastriata and Argasidae) the 3′-end of the polyadenylated nad1 and rrnL transcripts does not follow the tRNA punctuation model and is located upstream of a degenerate 17-bp DNA motif. A slightly different motif is also present downstream the 3′-end of nad1 transcripts in the primitive chelicerate Limulus polyphemus and in Drosophila species, indicating the ancient origin and the evolutionary conservation of this motif in arthropods. The transcriptional analyses suggest that this motif directs the 3′-end formation of the nad1/rrnL mature RNAs, likely working as a transcription termination signal or a processing signal of precursor transcripts. Moreover, as most regulatory elements, this motif is characterized by a taxon-specific evolution. Although this signal is not exclusive of ticks, making a play on words it has been named “Tick-Box”, since it is a check mark that has to be verified for the 3′-end formation of some mt transcripts, and its consensus sequence has been here carefully characterized in ticks. Indeed, in the whole mtDNA of all ticks, the Tick-Box is always present downstream of nad1 and rrnL, mainly in non-coding regions (NCRs) and occasionally within trnL(CUN). However, some metastriates present a third Tick-Box at an intriguing site - inside the small NCR located at one end of a 3.4 kb translocated region, the other end of which exhibits the nad1 Tick-Box - hinting that this motif could have been involved in metastriate gene order rearrangements.