Search

Mosquitoes vector many pathogens that cause human disease, such as malaria that is caused by parasites in the genusPlasmodium. Current strategies to control vector-transmitted diseases are hindered by mosquito and pathogen resistance, so research has turned to altering the microbiota of the vectors. In this strategy, calledparatransgenesis, symbiotic bacteria are genetically modified to affect the mosquito’s phenotype by engineering them to deliver antiplasmodial effector molecules into the midgut to kill parasites. One paratransgenesis candidate isAsaia bogorensis, a Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and salivary glands ofAnophelessp. mosquitoes. However, common secretion signals fromE. coliand closely related species do not function inAsaia. Here, we report evaluation of 20 nativeAsaiaN-terminal signal sequences predicted from bioinformatics for their ability to mediate increased levels of antiplasmodial effector molecules directed to the periplasm and ultimately outside the cell. We tested the hypothesis that by increasing the amount of antiplasmodials released from the cell we would also increase parasite killing power. We scanned theAsaia bogorensisSF2.1 genome to identify signal sequences from extra-cytoplasmic proteins and fused these to the reporter protein alkaline phosphatase. Six signals resulted in significant levels of protein released from theAsaiabacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine. Further testing in mosquitoes demonstrated that these threeAsaiastrains were able to suppress the number of oocysts formed after a blood meal containingP. bergheito a significantly greater degree than wild-typeAsaia, although prevalence was not decreased beyond levels obtained with a previously isolated siderophore receptor signal sequence. We interpret these results to indicate that there is a maximum level of suppression that can be achieved when the effectors are constitutively driven due to stress on the symbionts. This suggests that simply increasing the amount of antiplasmodial effector molecules in the midgut is insufficient to create superior paratransgenic bacterial strains and that symbiont fitness must be considered as well.

Asaia is a bacterial symbiont of sugar-feeding insects that has been shown to be vertically transmitted by maternal transmission and paternal transmission mechanism, and to be horizontally transmitted via co-feeding artificial diet and venereal routes. Here, the first case of plant-mediated horizontal transmission of Asaia between white-backed planthoppers (WBPH), Sogatella furcifera, was reported. In Asaia-infected WBPH, Asaia was detected mostly in salivary glands and to a less extent in stylets. The rice leaf sheaths fed by Asaia-infected WBPH for 12 h were all positive with Asaia, where Asaia persisted for at least 30 d but was localized in the feeding sites only. When confined to Asaia-infected leaf sheaths for 7 d at the sites pre-infested by the Asaia-infected WBPH, all Asaia-free WBPH became infected with Asaia and the acquired Asaia could be vertically transmitted to their offspring. Phylogenetic analysis confirmed an identical Asaia strain in the Asaia-infected donor WBPH, the Asaia-infected leaf sheaths, and the newly infected recipient WBPH. Our findings provide direct evidence for the first time that rice plant can mediate horizontal transmission of Asaia between WBPH, which may contribute to the spread of Asaia in the field WBPH populations.

Mosquitoes are one of the deadliest animals on earth because of their ability to transmit a wide range of human pathogens. Traditional mosquito control methods use chemical insecticides, but with dwindling long-term effectiveness and negative effects on the environment, microbial forms of control have become common alternatives. The insecticide Bacillus thuringiensis subspecies israelensis (Bti) is the most popular of these alternatives, although it can also have direct effects on lowering environmental biodiversity and indirect effects on food-web relationships in the ecosystems where it is deployed. In addition, microbial control agents that impede pathogen development or transmission from mosquito to human are under investigation, including Wolbachia and Asaia, but unexpected interactions with mosquito gut bacteria can hinder their effectiveness. Improved characterization of mosquito gut bacterial communities is needed to determine the taxa that interfere with microbial controls and their effectiveness in wild populations. This mini-review briefly discusses relationships between mosquito gut bacteria and microbial forms of control, and the challenges in ensuring their success.

Made available in DSpace on 2019-10-06T16:59:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Paleontological Research Institution Symbiosis is now recognized as a driving force in evolution, a role that finds its ultimate expression in the variety of associations bonding insects with microbial symbionts. These associations have contributed to the evolutionary success of insects, with the hosts acquiring the capacity to exploit novel ecological niches, and the symbionts passing from facultative associations to obligate, mutualistic symbioses. In bacterial symbiont of insects, the transition from the free-living life style to mutualistic symbiosis often resulted in a reduction in the genome size, with the generation of the smallest bacterial genomes thus far described. Here, we show that the process of genome reduction is still occurring in Asaia, a group of bacterial symbionts associated with a variety of insects. Indeed, comparative genomics of Asaia isolated from different mosquito species revealed a substantial genome size and gene content reduction in Asaia from Anopheles darlingi, a South-American malaria vector. We thus propose Asaia as a novel model to study genome reduction dynamics, within a single bacterial taxon, evolving in a common biological niche. Biotechnology Institute (IBTEC) Biosciences Institute Botucatu (IBB) Sao Paulo State University (UNESP) School of Biosciences and Veterinary Medicine University of Camerino Clinical Pediatric Research Center Romeo and Enrica Invernizzi Department of Biosciences University of Milan MRC-University of Glasgow-Centre for Virus Research Biotechnology Institute (IBTEC) Biosciences Institute Botucatu (IBB) Sao Paulo State University (UNESP) Paleontological Research Institution: 2012T85B3R_001 Paleontological Research Institution: 2015JXC3JF_001

Yeast strains and acetic acid bacteria were isolated from spoiled soft drinks with characteristic flocs as a visual defect. Polymerase chain reaction and amplification of a partial region of the LSU rRNA gene identified the bacteria as Asaia spp. Sequence analysis of the D1/D2 region of the 26S rDNA in turn identified the yeast isolates as Wickerhamomyces anomalus, Dekkera bruxellensis and Rhodotorula mucilaginosa. The hydrophobicity and adhesion properties of the yeasts were evaluated in various culture media, taking into account the availability of nutrients and the carbon sources. The highest hydrophobicity and best adhesion properties were exhibited by the R. mucilaginosa cells. Our results suggest that Asaia spp. bacterial cells were responsible for the formation of flocs, while the presence of yeast cells may help to strengthen the structure of co-aggregates.

Background According to scientific recommendations, paratransgenesis is one of the solutions for improving the effectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, affecting the fitness and longevity of vectors or reducing the vectorial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, findings showed that RNase III mutant bacteria are more competent for this aim. Asaia spp. have been introduced as potent paratransgenesis candidates for combating malaria and, based on their specific features for this goal, could be considered as effective dsRNA production and delivery tools to Anopheles spp. Therefore, we decided to characterize the rnc gene and its related protein to provide the basic required information for creating an RNase III mutant Asaia bacterium. Methods Asaia bacteria were isolated from field-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplification of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purified recombinant protein was assayed. Furthermore, Asaia RNaseIII amino acid sequence was analyzed by in silico approaches such as homology modeling and docking to determine its structural properties. Results In this study, the structure of rnc gene and its related operon from Asaia sp. was determined. In addition, by performing superimposition and docking with specific substrate, the structural features of Asaia RNaseIII protein such as critical residues which are involved and essential for proper folding of active site, binding of magnesium ions and double stranded RNA molecule to protein and cleaving of dsRNA molecules, were determined. Conclusions In this study, the basic and essential data for creating an RNase III mutant Asaia sp. strain, which is the first step of developing an efficient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in different medically-important vectors and these data are potentially very helpful for researchers studying paratransgenesis and vector-borne diseases and are interested in applying the RNAi technology in the field.

Background The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera, Tephritidae) is the most significant insect pest of Australian horticulture. Bactrocera tryoni is controlled using a range of tools including the Sterile Insect Technique (SIT). Mass-rearing and irradiation of pupae in SIT can reduce the fitness and quality of the released sterile insects. Studies have also showed reduced microbial gut diversity in domesticated versus wild tephritids. Results Transmission electron microscopy confirmed the presence of the bacterial isolates in the mid-gut of mass-reared larvae, and plate counts from individual larval guts showed increased numbers of bacteria in supplemented larvae. Several developmental and fitness parameters were tested including larval development time (egg-hatch to pupation), pupal weight, emergence, flight ability, sex-ratio, and time to adult eclosion (egg-hatch to adult eclosion). Enterobacter sp. and Asaia sp. shortened larval development time, while this was delayed by Lactobacillus sp., Leuconostoc sp. and a blend of all four bacteria. The mean time from egg hatch to adult eclosion was significantly reduced by Leuconostoc sp. and the blend for males and females, indicating that the individual bacterium and consortium affect flies differently depending on the life stage (larval or pupal). There was no impact of bacterial supplemented larvae on pupal weight, emergence, flight ability, or sex ratio. Conclusions Our findings show that bacteria fed to the larval stage of B. tryoni can impart fitness advantages, but the selection of probiotic strains (individual or a consortium) is key, as each have varying effects on the host. Bacteria added to the larval diet particularly Leuconostoc sp. and the blend have the capacity to reduce costs and increase the number of flies produced in mass-rearing facilities by reducing time to adult eclosion by 1.3 and 0.8 mean days for males, and 1.2 and 0.8 mean days for females.

Since it has been understood that gut microbiota of vector mosquitoes can influence their vector competence, efforts have been undertaken to develop new control strategies based on host microbiota manipulation, and aimed at suppressing the vector population or replacing it with a less competent one. For the proper design of such control strategies it is necessary to know the microbiota composition of the target vector species, how it is acquired, and how it changes throughout the host’s life cycle. In this study, 16S rRNA amplicon high-throughput sequencing was used to characterize the changes in microbiota from the aquatic environment (larval breeding sites) to the different developmental stages of field-collected Aedes koreicus in Italy, an emerging invasive mosquito species in Europe and a potential vector of several pathogens. The bacterial communities of the aquatic breeding sites, larvae, pupae and adults showed distinctive structures to one another. Indeed, 84% of community members were unique to a given sample type. Nevertheless, almost 40% of the sequences generated were assigned to bacteria detected in all sample types, suggesting the importance of bacteria transstadially transmitted from water to the adult stage in constituting mosquito microbiota. Among these, genus C39 largely constituted water microbiota, family Burkholderiaceae was the most abundant in larvae and pupae, and genus Asaia dominated adult communities. In addition, Asaia consitituted a core microbiota across all sample types. Our results suggest that the microbiota of Ae. koreicus mosquitoes is composed by a community which derives from the aquatic bacteria of the larval breeding sites, is then filtered by the larval gut, where only certain members are able to persist, rearranged by metamorphosis and finally modified by the change in diet at the adult stage. Understanding how the microbiota of Ae. koreicus changes through the mosquito life cycle represents a first step in selecting bacterial candidates for use in microbiota-based intervention measures for this species. The properties which Asaia exhibits in this species, such as dominance, high prevalence and transstadial transmission, prevent the use of Wolbachia but make Asaia an ideal candidate for paratransgenesis.

In mosquitoes, the discovery of the numerous interactions between components of the microbiota and the host immune response opens up the attractive possibility of the development of novel control strategies against mosquito borne diseases. We have focused our attention to Asaia, a symbiont of several mosquito vectors who has been proposed as one of the most potential tool for paratransgenic applications; although being extensively characterized, its interactions with the mosquito immune system has never been investigated. Here we report a study aimed at describing the interactions between Asaia and the immune system of two vectors of malaria, Anophelesstephensi and An. gambiae. The introduction of Asaia isolates induced the activation of the basal level of mosquito immunity and lower the development of malaria parasite in An. stephensi. These findings confirm and expand the potential of Asaia in mosquito borne diseases control, not only through paratransgenesis, but also as a natural effector for mosquito immune priming.

The white-backed planthopper (WBPH), Sogatella furcifera Horvath (Hemiptera: Delphacidae), is an economically significant rice insect pest that harbors a primary fungal yeast-like symbiont (YLS), and some secondary bacterial symbionts like Wolbachia and Cardinium. In the present study, an additional bacterial symbiont in WBPH was characterized. Phylogenetic analysis employing the 16S rRNA gene showed a bacterium closely related to Asaia of Nilaparvata lugens and Nysius expressus, and Asaia krungthepensis. TEM observation of the bacterium showed the typical morphology of Asaia sp. with signature filamentous structures in the nucleoid region. These results indicate that the bacterium belongs to Asaia. The Asaia bacterium was detected in all the tested individual adults and tissues of the laboratory WBPH population but showed varying infection rates (ca 45%) in the field collected WBPH populations. Quantitative PCR analysis revealed that Asaia sp. were significantly more abundant in WBPH females than males, and mainly distributed in the guts, fatty bodies, and salivary glands. Asaia-infected WBPH were of shorter nymphal duration and heavier adult weight than Asaia-free WBPH, while Asaia-free WBPH comparatively fed more, indicating that Asaia plays a role in improving WBPH fitness through involvement in host's nutrient supply.

Insect immunity is a crucial process in interactions between host and microorganisms and the presence of pathogenic, commensal, or beneficial bacteria may result in different immune responses. In Hemiptera vectors of phytoplasmas, infected insects are amenable to carrying high loads of phytopathogens, besides hosting other bacterial affiliates, which have evolved different strategies to be retained; adaptation to host response and immunomodulation are key aspects of insect-symbiont interactions. Most of the analyses published to date has investigated insect immune response to pathogens, whereas few studies have focused on the role of host immunity in microbiota homeostasis and vectorial capacity. Here the expression of immune genes in the leafhopper vector of phytoplasmas Euscelidius variegatus was investigated following exposure to Asaia symbiotic bacteria, previously demonstrated to affect phytoplasma acquisition by leafhoppers. The expression of four genes related to major components of immunity was measured, i.e., defensin, phenoloxidase, kazal type 1 serine protease inhibitor and Raf, a component of the Ras/Raf pathway. The response was separately tested in whole insects, midguts and cultured hemocytes. Healthy individuals were assessed along with specimens undergoing early- and late-stage phytoplasma infection. In addition, the adhesion grade of Asaia strains was examined to assess whether symbionts could establish a physical barrier against phytoplasma colonization. Our results revealed a specific activation of Raf in midguts after double infection by Asaia and flavescence doree phytoplasma. Increased expression was observed already in early stages of phytoplasma colonization. Gut-specific localization and timing of Raf activation are consistent with the role played by Asaia in limiting phytoplasma acquisition by E. variegatus, supporting the involvement of this gene in the anti-pathogen activity. However, limited attachment capability was found for Asaia under in vitro experimental conditions, suggesting a minor contribution of physical phytoplasma exclusion from the vector gut wall. By providing evidence of immune modulation played by Asaia, these results contribute to elucidating the molecular mechanisms regulating interference with phytoplasma infection in E. variegatus. The involvement of Raf suggests that in the presence of reduced immunity (reported in Hemipterans), immune genes can be differently regulated and recruited to play additional functions, generally played by genes lost by hemipterans.

Asaia bacteria commonly comprise part of the microbiome of many mosquito species in the genera Anopheles and Aedes, including important vectors of infectious agents. Their close association with multiple organs and tissues of their mosquito hosts enhances the potential for paratransgenesis for the delivery of antimalaria or antivirus effectors. The molecular mechanisms involved in the interactions between Asaia and mosquito hosts, as well as Asaia and other bacterial members of the mosquito microbiome, remain underexplored. Here, we determined the genome sequence of Asaia strain W12 isolated from Anopheles stephensi mosquitoes, compared it to other Asaia species associated with plants or insects, and investigated the properties of the bacteria relevant to their symbiosis with mosquitoes. The assembled genome of strain W12 had a size of 3.94 MB, the largest among Asaia spp. studied so far. At least 3585 coding sequences were predicted. Insect-associated Asaia carried more glycoside hydrolase (GH)-encoding genes than those isolated from plants, showing their high plant biomass-degrading capacity in the insect gut. W12 had the most predicted regulatory protein components comparatively among the selected Asaia, indicating its capacity to adapt to frequent environmental changes in the mosquito gut. Two complete operons encoding cytochrome bo3-type ubiquinol terminal oxidases (cyoABCD-1 and cyoABCD-2) were found in most Asaia genomes, possibly offering alternative terminal oxidases and allowing the flexible transition of respiratory pathways. Genes involved in the production of 2,3-butandiol and inositol have been found in Asaia sp. W12, possibly contributing to biofilm formation and stress tolerance.

Background: Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the Anopheles (An.) genera, but has recently been found in An. gambiae s.l. populations in West Africa. As there are numerous Anopheles species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine Wolbachia prevalence rates, characterise novel Wolbachia strains and determine any correlation between the presence of Plasmodium, Wolbachia and the competing bacterium Asaia. Methods: Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017. Molecular analysis was undertaken using quantitative PCR, Sanger sequencing, Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial 16S rRNA gene. Results: Novel Wolbachia strains were discovered in five species: An. coluzzii, An. gambiae s.s., An. arabiensis, An. moucheti and An. species A, increasing the number of Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with Wolbachia supergroup B strains. We also provide evidence for resident strain variants within An. species A. Wolbachia is the dominant member of the microbiome in An. moucheti and An. species A but present at lower densities in An. coluzzii. Interestingly, no evidence of Wolbachia/Asaia co-infections was seen and Asaia infection densities were shown to be variable and location dependent. Conclusions: The important discovery of novel Wolbachia strains in Anopheles provides greater insight into the prevalence of resident Wolbachia strains in diverse malaria vectors. Novel Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other Anopheles mosquito species, which could be used for population replacement or suppression control strategies.

Background In recent years, the genus Asaia (Rhodospirillales: Acetobacteraceae) has been isolated from different Anopheles species and presented as a promising tool to combat malaria. This bacterium has unique features such as presence in different organs of mosquitoes (midgut, salivary glands and reproductive organs) of female and male mosquitoes and vertical and horizontal transmission. These specifications lead to the possibility of introducing Asaia as a robust candidate for malaria vector control via paratransgenesis technology. Several studies have been performed on the microbiota of Anopheles mosquitoes (Diptera: Culicidae) in Iran and the Middle East to find a suitable candidate for controlling the malaria based on paratransgenesis approaches. The present study is the first report of isolation, biochemical and molecular characterization of the genus Asaia within five different Anopheles species which originated from different zoogeographical zones in the south, east, and north of Iran. Methods Mosquitoes originated from field-collected and laboratory-reared colonies of five Anopheles spp. Adult mosquitoes were anesthetized; their midguts were isolated by dissection, followed by grinding the midgut contents which were then cultured in enrichment broth media and later in CaCO3 agar plates separately. Morphological, biochemical and physiological characterization were carried out after the appearance of colonies. For molecular confirmation, selected colonies were cultured, their DNAs were extracted and PCR was performed on the 16S ribosomal RNA gene using specific newly designed primers. Results Morphological, biochemical, physiological and molecular results indicated that all isolates are members of the genus Asaia. Conclusions Contrary to previous opinions, our findings show that Asaia bacteria are present in both insectary-reared colonies and field-collected mosquitoes and can be isolated by simple and specific methods. Furthermore, with respect to the fact that we isolated Asaia within the different Anopheles specimens from distinct climatic and zoogeographical regions, it is promising and may be concluded that species of this genus can tolerate the complicated environmental conditions of the vector-borne diseases endemic regions. Therefore, it can be considered as a promising target in paratransgenesis and vector control programs. However, we suggest that introducing the new technologies such as next generation sequencing and robust in silico approaches may pave the way to find a unique biomarker for rapid and reliable differentiation of the Asaia species.

We investigated the biological activity of surfactants based on quaternary ammonium compounds: gemini surfactant hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) (C6), synthesized by the reaction of N,N-dimethyl-N-dodecylamine with 1,6-dibromohexane, and its monomeric analogue dodecyltrimethylammonium bromide (DTAB). The experiments were performed with bacteria Asaia lannensis, a common spoilage in the beverage industry. The minimal inhibitory concentration (MIC) values were determined using the tube standard two-fold dilution method. The growth and adhesive properties of bacterial cells were studied in different culture media, and the cell viability was evaluated using plate count method. Both of the surfactants were effective against the bacterial strain, but the MIC of gemini compound was significantly lower. Both C6 and DTAB exhibited anti-adhesive abilities. Treatment with surfactants at or below MIC value decreased the number of bacterial cells that were able to form biofilm, however, the gemini surfactant was more effective. The used surfactants were also found to be able to eradicate mature biofilms. After 4 h of treatment with C6 surfactant at concentration 10 MIC, the number of bacterial cells was reduced by 91.8%. The results of this study suggest that the antibacterial activity of the gemini compound could make it an effective microbiocide against the spoilage bacteria Asaia sp. in both planktonic and biofilm stages.

Background: Malaria still remains a serious health burden in developing countries, causing more than 1 million deaths annually. Given the lack of an effective vaccine against its major etiological agent, Plasmodium falciparum, and the growing resistance of this parasite to the currently available drugs repertoire and of Anopheles mosquitoes to insecticides, the development of innovative control measures is an imperative to reduce malaria transmission. Paratransgenesis, the modification of symbiotic organisms to deliver anti-pathogen effector molecules, represents a novel strategy against Plasmodium development in mosquito vectors, showing the potential to reduce parasite development. However, the field application of laboratory-based evidence of paratransgenesis imposes the use of more realistic confined semi-field environments.Methods: Large cages were used to evaluate the ability of bacteria of the genus Asaia expressing green fluorescent protein (Asaia gfp), to diffuse in Anopheles stephensi and Anopheles gambiae target mosquito populations. Asaia gfp was introduced in large cages through the release of paratransgenic males or by sugar feeding stations. Recombinant bacteria transmission was directly detected by fluorescent microscopy, and further assessed by molecular analysis.Results: Here we show the first known trial in semi-field condition on paratransgenic anophelines. Modified bacteria were able to spread at high rate in different populations of An. stephensi and An. gambiae, dominant malaria vectors, exploring horizontal ways and successfully colonising mosquito midguts. Moreover, in An. gambiae, vertical and trans-stadial diffusion mechanisms were demonstrated.Conclusions: Our results demonstrate the considerable ability of modified Asaia to colonise different populations of malaria vectors, including pecies where its association is not primary, in large environments. The data support the potential to employ transgenic Asaia as a tool for malaria control, disclosing promising perspective for its field application with suitable effector molecules.

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.

Background Wolbachia 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. Methods Different 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. Results Here 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. Conclusions A 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. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0888-0) contains supplementary material, which is available to authorized users.

Background Malaria represents one of the most devastating infectious diseases. The lack of an effective vaccine and the emergence of drug resistance make necessary the development of new effective control methods. The recent identification of bacteria of the genus Asaia, associated with larvae and adults of malaria vectors, designates them as suitable candidates for malaria paratransgenic control. To better characterize the interactions between Asaia, Plasmodium and the mosquito immune system we performed an integrated experimental approach. Methods Quantitative PCR analysis of the amount of native Asaia was performed on individual Anopheles stephensi specimens. Mosquito infection was carried out with the strain PbGFPCON and the number of parasites in the midgut was counted by fluorescent microscopy. The colonisation of infected mosquitoes was achieved using GFP or DsRed tagged-Asaia strains. Reverse transcriptase-PCR analysis, growth and phagocytosis tests were performed using An. stephensi and Drosophila melanogaster haemocyte cultures and DsRed tagged-Asaia and Escherichia coli strains. Results Using quantitative PCR we have quantified the relative amount of Asaia in infected and uninfected mosquitoes, showing that the parasite does not interfere with bacterial blooming. The correlation curves have confirmed the active replication of Asaia, while at the same time, the intense decrease of the parasite. The ‘in vitro’ immunological studies have shown that Asaia induces the expression of antimicrobial peptides, however, the growth curves in conditioned medium as well as a phagocytosis test, indicated that the bacterium is not an immune-target. Using fluorescent strains of Asaia and Plasmodium we defined their co-localisation in the mosquito midgut and salivary glands. Conclusions We have provided important information about the relationship of Asaia with both Plasmodium and Anophele s. First, physiological changes in the midgut following an infected or uninfected blood meal do not negatively affect the residing Asaia population that seems to benefit from this condition. Second, Asaia can act as an immune-modulator activating antimicrobial peptide expression and seems to be adapted to the host immune response. Last, the co-localization of Asaia and Plasmodium highlights the possibility of reducing vectorial competence using bacterial recombinant strains capable of releasing anti-parasite molecules.

Background Bacteria of the genus Asaia have been recently recognized as secondary symbionts of different sugar-feeding insects, including the leafhopper Scaphoideus titanus, vector of Flavescence dorée phytoplasmas. Asaia has been shown to be localized in S. titanus gut, salivary glands and gonoducts and to be maternally transmitted to the progeny by an egg smearing mechanism. It is currently not known whether Asaia in S. titanus is transmitted by additional routes. We performed a study to evaluate if Asaia infection is capable of horizontal transmission via co-feeding and venereal routes. Results A Gfp-tagged strain of Asaia was provided to S. titanus individuals to trace the transmission pathways of the symbiotic bacterium. Co-feeding trials showed a regular transfer of bacterial cells from donors to recipients, with a peak of frequency after 72 hours of exposure, and with concentrations of the administrated strain growing over time. Venereal transmission experiments were first carried out using infected males paired with uninfected females. In this case, female individuals acquired Gfp-labelled Asaia, with highest infection rates 72-96 hours after mating and with increasing abundance of the tagged symbiont over time. When crosses between infected females and uninfected males were conducted, the occurrence of “female to male” transmission was observed, even though the transfer occurred unevenly. Conclusions The data presented demonstrate that the acetic acid bacterial symbiont Asaia is horizontally transmitted among S. titanus individuals both by co-feeding and venereal transmission, providing one of the few direct demonstrations of such a symbiotic transfer in Hemiptera. This study contributes to the understanding of the bacterial ecology in the insect host, and indicates that Asaia evolved multiple pathways for the colonization of S. titanus body.