Sara Epis

Delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts

BackgroundIn recent years, acetic acid bacteria have been shown to be frequently associated with insects, but knowledge on their biological role in the arthropod host is limited. The discovery that acetic acid bacteria of the genus Asaia are a main component of the microbiota of Anopheles stephensi makes this mosquito a useful model for studies on this novel group of symbionts. Here we present experimental results that provide a first evidence for a beneficial role of Asaia in An. stephensi.ResultsLarvae of An. stephensi at different stages were treated with rifampicin, an antibiotic effective on wild-type Asaia spp., and the effects on the larval development were evaluated. Larvae treated with the antibiotic showed a delay in the development and an asynchrony in the appearance of later instars. In larvae treated with rifampicin, but supplemented with a rifampicin-resistant mutant strain of Asaia, larval development was comparable to that of control larvae not exposed to the antibiotic. Analysis of the bacterial diversity of the three mosquito populations confirmed that the level of Asaia was strongly decreased in the antibiotic-treated larvae, since the symbiont was not detectable by PCR-DGGE (denaturing gradient gel electrophoresis), while Asaia was consistently found in insects supplemented with rifampicin plus the antibiotic-resistant mutant in the diet, and in those not exposed to the antibiotic.ConclusionsThe results here reported indicate that Asaia symbionts play a beneficial role in the normal development of An. stephensi larvae.

Mosquito-Bacteria Symbiosis: The Case of Anopheles gambiae and Asaia

The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.

Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

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.

Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiome. They are obligate biotrophs that colonize the roots of most land plants and enhance host nutrient acquisition. Many AMF themselves harbor endobacteria in their hyphae and spores. Two types of endobacteria are known in Glomeromycota: rod-shaped Gram-negative Candidatus Glomeribacter gigasporarum, CaGg, limited in distribution to members of the Gigasporaceae family, and coccoid Mollicutes-related endobacteria, Mre, widely distributed across different lineages of AMF. The goal of the present study is to investigate the patterns of distribution and coexistence of the two endosymbionts, CaGg and Mre, in spore samples of several strains of Gigaspora margarita. Based on previous observations, we hypothesized that some AMF could host populations of both endobacteria. To test this hypothesis, we performed an extensive investigation of both endosymbionts in G. margarita spores sampled from Cameroonian soils as well as in the Japanese G. margarita MAFF520054 isolate using different approaches (molecular phylotyping, electron microscopy, fluorescence in situ hybridization and quantitative real-time PCR). We found that a single AMF host can harbour both types of endobacteria, with Mre population being more abundant, variable and prone to recombination than the CaGg one. Both endosymbionts seem to retain their genetic and lifestyle peculiarities regardless of whether they colonize the host alone or together. These findings show for the first time that fungi support an intracellular bacterial microbiome, in which distinct types of endobacteria coexist in a single cell.

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.

Effects of global changes on the climatic niche of the tick Ixodes ricinus inferred by species distribution modelling.

BackgroundGlobal climate change can seriously impact on the epidemiological dynamics of vector-borne diseases. In this study we investigated how future climatic changes could affect the climatic niche of Ixodes ricinus (Acari, Ixodida), among the most important vectors of pathogens of medical and veterinary concern in Europe.MethodsSpecies Distribution Modelling (SDM) was used to reconstruct the climatic niche of I. ricinus, and to project it into the future conditions for 2050 and 2080, under two scenarios: a continuous human demographic growth and a severe increase of gas emissions (scenario A2), and a scenario that proposes lower human demographic growth than A2, and a more sustainable gas emissions (scenario B2). Models were reconstructed using the algorithm of “maximum entropy”, as implemented in the software Maxent 3.3.3e; 4,544 occurrence points and 15 bioclimatic variables were used.ResultsIn both scenarios an increase of climatic niche of about two times greater than the current area was predicted as well as a higher climatic suitability under the scenario B2 than A2. Such an increase occurred both in a latitudinal and longitudinal way, including northern Eurasian regions (e.g. Sweden and Russia), that were previously unsuitable for the species.ConclusionsOur models are congruent with the predictions of range expansion already observed in I. ricinus at a regional scale and provide a qualitative and quantitative assessment of the future climatically suitable areas for I. ricinus at a continental scale. Although the use of SDM at a higher resolution should be integrated by a more refined analysis of further abiotic and biotic data, the results presented here suggest that under future climatic scenarios most of the current distribution area of I. ricinus could remain suitable and significantly increase at a continental geographic scale. Therefore disease outbreaks of pathogens transmitted by this tick species could emerge in previous non-endemic geographic areas. Further studies will implement and refine present data toward a better understanding of the risk represented by I. ricinus to human health.

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.

Symbiotic control of mosquito borne disease.

Abstract It is well accepted that the symbiotic relationships insects have established with several microorganisms have had a key role in their evolutionary success. Bacterial symbiosis is also prevalent in insects that are efficient disease vectors, and numerous studies have sought to decrypt the basic mechanisms of the host–symbiont relationships and develop ways to control vector borne diseases. ‘Symbiotic control’, a new multifaceted approach that uses symbiotic microorganisms to control insect pests or reduce vector competence, seems particularly promising. Three such approaches currently at the cutting edge are: (1) the disruption of microbial symbionts required by insect pests; (2) the manipulation of symbionts that can express anti-pathogen molecules within the host; and (3) the introduction of endogenous microbes that affect life-span and vector capacity of the new hosts in insect populations. This work reviews current knowledge on microbial symbiosis in mosquitoes that holds promise for development of symbiotic control for mosquito borne diseases.

Mutual exclusion of Asaia and Wolbachia in the reproductive organs of mosquito vectors

BackgroundWolbachia is a group of intracellular maternally inherited bacteria infecting a high number of arthropod species. Their presence in different mosquito species has been largely described, but Aedes aegypti, the main vector of Dengue virus, has never been found naturally infected by Wolbachia. Similarly, malaria vectors and other anophelines are normally negative to Wolbachia, with the exception of an African population where these bacteria have recently been detected. Asaia is an acetic acid bacterium stably associated with several mosquito species, found as a dominant microorganism of the mosquito microbiota. Asaia has been described in gut, salivary glands and in reproductive organs of adult mosquitoes in Ae. aegypti and in anophelines. It has recently been shown that Asaia may impede vertical transmission of Wolbachia in Anopheles mosquitoes. Here we present an experimental study, aimed at determining whether there is a negative interference between Asaia and Wolbachia, for the gonad niche in mosquitoes.MethodsDifferent methods (PCR and qPCR, monoclonal antibody staining and FISH) have been used to address the question of the co-localization and the relative presence/abundance of the two symbionts. PCR and qPCR were performed to qualitatively and quantitatively verify the distribution of Asaia and Wolbachia in different mosquito species/organs. Monoclonal antibody staining and FISH were performed to localize the symbionts in different mosquito species.ResultsHere we provide evidence that, in Anopheles and in other mosquitoes, there is a reciprocal negative interference between Asaia and Wolbachia symbionts, in terms of the colonization of the gonads. In particular, we have shown that in some mosquito species the presence of one of the symbionts prevented the establishment of the second, while in other systems the symbionts were co-localized, although at reduced densities.ConclusionsA mutual exclusion or a competition between Asaia and Wolbachia may contribute to explain the inability of Wolbachia to colonize the female reproductive organs of anophelines, inhibiting its vertical transmission and explaining the absence of Wolbachia infection in Ae. aegypti and in the majority of natural populations of Anopheles mosquitoes.