Search

Aims A wide range of vector control programmes rely on the efficient production and release of male mosquito. Asaia bacteria are described as potential symbionts of several mosquito species but their relationship with Aedes aegypti has never been rigorously tested. Here, we aimed to quantify the benefits of three Asaia species on host development in Ae. aegypti , and the ability of these bacteria to form a stable symbiotic association with growing larvae. Methods and results In order to disentangle direct and indirect effects of Asaia inoculation on host development, experiments used insects with an intact microbiome and those reared in near-aseptic conditions, while we characterized bacterial communities and Asaia densities with culture dependent and independent methods (16S rRNA amplicon sequencing). Neonate larvae were inoculated with Asaia spp. for 24 h, or left as uninoculated controls, all were reared on sterile food. Aseptic larvae were produced by surface sterilization of eggs. Although all Asaia were transient members of the gut community, two species accelerated larval development relative to controls. The two mutualistic species had lasting impacts on the larval microbiome, largely by altering the relative abundance of dominant bacteria, namely Klebsiella and Pseudomonas. Axenic larvae were dominated by Asaia when inoculated with this species but showed slower development than conventionally reared insects, indicating that Asaia alone could not restore normal development. Conclusions Our results reveal Asaia as a poor mutualist for Ae. aegypti , but with a species-specific positive effect on improving host performance mediated by interactions with other bacteria. [ABSTRACT FROM AUTHOR]

ABSTRACT The widespread prevalence of Asaia in mosquitoes makes it a potential candidate for paratrangenic control in Anopheles. To better understand whether this bacterium could be used for malaria control, we quantified Asaia in An. gambiae s.l populations in malaria endemic regions examining co-infection with Plasmodium falciparum. Adult Anopheles mosquitoes were collected across two different eco-geographical localities in Cameroon, during both the dry and wet seasons. DNA was extracted from whole individual mosquitoes, and real time-qPCR amplification of the 16S ribosomal RNA was used to quantify Asaia in both An. gambiae and An. coluzzii samples. We also detected and quantified P. falciparum infection in the same mosquitoes. The density of Asaia was successfully quantified in a total of 864 field mosquitoes, comprising of 439 An. gambiae from Bankeng and 424 An. coluzii collected from Gounougou. Interestingly, a higher prevalence of Asaia in An. gambiae (88.3%) compared to An. coluzzii (80.9%) was observed. Moreover, the density of Asaia in both species was significantly affected by seasonal changes in the two localities. Furthermore, a significant difference between the infection densities of Asaia and the Plasmodium infection status in the two species was recorded. However, no correlation was observed between the number of Asaia and P. falciparum infections. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii. Nevertheless, further studies incorporating experimental infections are required to better investigate the correlation between Anopheles mosquitoes, Asaia, and Plasmodium.IMPORTANCEThe symbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but further analysis of its biology and genetics across Africa is necessary. In this study, we investigated and quantified the influence of Asaia in naturally infected An. gambiae s.l. populations with the malaria parasite Plasmodium falciparum. Genomic DNA was extracted from whole individual mosquitoes collected from two localities, and Asaia was quantified using real-time qPCR by amplification of the 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in the same mosquitoes and established the correlation between Asaia and Plasmodium coinfection. This study provides evidence that naturally occurring Asaia infection is not correlated with P. falciparum development within An. gambiae and An. coluzzii mosquitoes.

Microbial communities play an important role in the fitness of mosquito hosts. However, the factors shaping microbial communities in wild populations, with regard to interactions among microbial species, are still largely unknown. Previous research has demonstrated that two of the most studied mosquito symbionts, the bacteria Wolbachia and Asaia, seem to compete or not compete, depending on the genetic background of the reference mosquito host. The large diversity of Wolbachia–Asaia strain combinations that infect natural populations of mosquitoes may offer a relevant opportunity to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction. We surveyed Wolbachia and Asaia in 44 mosquito populations belonging to 11 different species of the genera Anopheles, Aedes, and Culex using qualitative PCR. Through quantitative PCR, the amounts of both bacteria were assessed in different mosquito organs, and through metagenomics, we determined the microbiota compositions in some selected mosquito populations. We show that variation in microbial community structure is likely associated with the species/strain of mosquito, its geographical position, and tissue localization. Together, our results shed light on the interactions among different bacterial species in the microbial communities of mosquito vectors, and this can aid the development and/or improvement of methods for symbiotic control of insect vectors. [ABSTRACT FROM AUTHOR]

Microbial communities play an important role in the fitness of mosquito hosts. However, the factors shaping microbial communities in wild populations, with regard to interactions among microbial species, are still largely unknown. Previous research has demonstrated that two of the most studied mosquito symbionts, the bacteria Wolbachia and Asaia, seem to compete or not compete, depending on the genetic background of the reference mosquito host. The large diversity of Wolbachia–Asaia strain combinations that infect natural populations of mosquitoes may offer a relevant opportunity to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction. We surveyed Wolbachia and Asaia in 44 mosquito populations belonging to 11 different species of the genera Anopheles, Aedes, and Culex using qualitative PCR. Through quantitative PCR, the amounts of both bacteria were assessed in different mosquito organs, and through metagenomics, we determined the microbiota compositions in some selected mosquito populations. We show that variation in microbial community structure is likely associated with the species/strain of mosquito, its geographical position, and tissue localization. Together, our results shed light on the interactions among different bacterial species in the microbial communities of mosquito vectors, and this can aid the development and/or improvement of methods for symbiotic control of insect vectors.

Mosquitoes (Diptera: Culicidae) are found widely throughout the world. Several species can transmit pathogens to humans and other vertebrates. Mosquitoes harbor great amounts of bacteria, fungi, and viruses. The bacterial composition of the microbiota of these invertebrates is associated with several factors, such as larval habitat, environment, and species. Yet little is known about bacterial interaction networks in mosquitoes. This study investigates the bacterial communities of eight species of Culicidae collected in Vale do Ribeira (Southeastern São Paulo State) and verifies the bacterial interaction network in these species. Sequences of the 16S rRNA region from 111 mosquito samples were analyzed. Bacterial interaction networks were generated from Spearman correlation values. Proteobacteria was the predominant phylum in all species. Wolbachia was the predominant genus in Haemagogus leucocelaenus. Aedes scapularis, Aedes serratus, Psorophora ferox, and Haemagogus capricornii were the species that showed a greater number of bacterial interactions. Bacterial positive interactions were found in all mosquito species, whereas negative correlations were observed in Hg. leucocelaenus, Ae. scapularis, Ae. serratus, Ps. ferox, and Hg. capricornii. All bacterial interactions with Asaia and Wolbachia were negative in Aedes mosquitoes. [ABSTRACT FROM AUTHOR]

Abstract Background Malaria control relies mainlyon insecticide-based tools. However, the effectiveness of these tools is threatened by widespread insecticide resistance in malaria vectors, highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated the prevalence of Asaia and its maternal transmission in the natural population of Anopheles mosquitoes in Cameroon. Methods Indoor-resting adult mosquitoes belonging to four species (An. coluzzii, An. arabiensis, An. funestus and An. gambiae) were collected from eight localities across Cameroon from July 2016 to February 2020. PCR was performed on the Asaia-specific 16S ribosomal RNA gene, and samples positive by PCR for Asaia were confirmed by Sanger sequencing and phylogenetic analysis. The vertical transmission of Asaia was investigated by screening F 1 mosquitoes belonging to F 0 Asaia-positive females. Results A total of 895 mosquitoes were screened. We found 43% (384) Asaia infection prevalence in four mosquito species. Phylogenetic analysis revealed that Asaia from Cameroon clustered together with the strains of Asaia isolated from other parts of the world. In addition, seven nucleotide sequence variants were found with low genetic diversity (π = 0.00241) and nucleotide sequence variant diversity (Hd = 0.481). Asaia was vertically transmitted with high frequency (range from 42.5 to 100%). Conclusions This study provides field-based evidence of the presence of Asaia in Anopheles mosquitoes in Cameroon for exploitation as a symbiont in the control of malaria in sub-Saharan Africa. Graphical abstract

Simple Summary: The Mediterranean fly (Medfly), Ceratitis capitata, is considered one of the world's most destructive fruit pests, as it can attack commercially important fruit, thus causing considerable economic damages, estimated to be more than 2 billion dollars annually. The yield reductions are mainly due to the damage incurred when larvae feed directly on the pulp, inducing the premature fruit drop. Additionally, oviposition holes facilitate secondary fungal and bacterial infections, further reducing the yields. Integrated pest management (IPM) strategies for medfly control are highly dependent on the use of insecticides, which, however, pose environmental concerns. Alternative strategies include the Sterile Insect Technique (SIT), which aims to eliminate or suppress pest insects without using pesticides. Lately, the medfly microbiota has been explored to develop new control strategies for insect pests and insect vectors. Here, we report the characterization of the microbial communities associated with selected organs of three different populations of C. capitata to identify possible candidates for a Symbiotic Control approach. Our findings provide new knowledge about the microbiota associated with C. capitata and stress the characterization of microbial symbionts as possible tools for a Symbiotic Control approach to implementing the pest management programs. Ceratitis capitata (Diptera: Tephritidae) is responsible for extensive damage in agriculture with important economic losses. Several strategies have been proposed to control this insect pest including insecticides and the Sterile Insect Technique. Traditional control methods should be implemented by innovative tools, among which those based on insect symbionts seem very promising. Our study aimed to investigate, through the 16S Miseq analysis, the microbial communities associated with selected organs in three different medfly populations to identify possible candidates to develop symbiont-based control approaches. Our results confirm that Klebsiella and Providencia are the dominant bacteria in guts, while a more diversified microbial community has been detected in reproductive organs. Concertedly, we revealed for the first time the presence of Chroococcidiopsis and Propionibacterium as stable components of the medfly's microbiota. Additionally, in the reproductive organs, we detected Asaia, a bacterium already proposed as a tool in the Symbiotic Control of Vector-Borne Diseases. A strain of Asaia, genetically modified to produce a green fluorescent protein, was used to ascertain the ability of Asaia to colonize specific organs of C. capitata. Our study lays the foundation for the development of control methods for C. capitata based on the use of symbiont bacteria. [ABSTRACT FROM AUTHOR]

Blueberry production is affected by fungal postharvest pathogens, including Botrytis cinerea and Alternaria alternata, the causative agents of gray mold disease and Alternaria rot, respectively. Biocontrol agents adapted to blueberries and local environments are not known to date. Here, we report on the search for and the identification of cultivable blueberry epiphytic bacteria with the potential to combat the aforementioned fungi. Native, blueberry-borne bacterial strains were isolated from a plantation in Tucumán, Argentina and classified based on 16S rRNA gene sequences. Antagonistic activities directed at B. cinerea and A. alternata were studied in vitro and in vivo. The 22 bacterial strains obtained could be attributed to eleven different genera: Rosenbergiella, Fictibacillus, Bacillus, Pseudomonas, Microbacterium, Asaia, Acinetobacter, Curtobacterium, Serratia, Sphingomonas and Xylophilus. Three strains displaying antagonistic impacts on the fungal pathogens were identified as Bacillus velezensis (BA3 and BA4) and Asaia spathodeae (BMEF1). These strains are candidates for biological control agents of local blueberry production and might provide a basis for the development of eco-friendly, sustainable alternatives to synthetic pesticides. [ABSTRACT FROM AUTHOR]

Copyright of Postępy Nauki i Technologii Przemysłu Rolno-Spożywczego is the property of Institute of Agricultural & Food Biotechnology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Asaia spp. has recently been reported to cause opportunistic infections in humans and is becoming an emerging hospital pathogen. To our knowledge, this is the first report on  Asaia  spp. in Malaysia. bacteremia in an infant. A girl with underlying Hirschsprung's disease, who was on parenteral feeding via a central venous catheter, developed persistent multidrug-resistant Gram-negative bacteremia. Routine automated identification methods failed to identify the organism, which was later identified by 16S ribosomal RNA sequencing. Bacterial clearance was achieved after the removal of the catheter and initiation of IV amikacin. This case highlights the role of molecular identification and the clinical importance of Asaia spp. in causing infections in humans, especially in patients with indwelling devices., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Mohammad et al.)

Background: Malaria control relies mainlyon insecticide-based tools. However, the effectiveness of these tools is threatened by widespread insecticide resistance in malaria vectors, highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated the prevalence of Asaia and its maternal transmission in the natural population of Anopheles mosquitoes in Cameroon. Methods: Indoor-resting adult mosquitoes belonging to four species (An. coluzzii, An. arabiensis, An. funestus and An. gambiae) were collected from eight localities across Cameroon from July 2016 to February 2020. PCR was performed on the Asaia-specific 16S ribosomal RNA gene, and samples positive by PCR for Asaia were confirmed by Sanger sequencing and phylogenetic analysis. The vertical transmission of Asaia was investigated by screening F1 mosquitoes belonging to F0Asaia-positive females. Results: A total of 895 mosquitoes were screened. We found 43% (384) Asaia infection prevalence in four mosquito species. Phylogenetic analysis revealed that Asaia from Cameroon clustered together with the strains of Asaia isolated from other parts of the world. In addition, seven nucleotide sequence variants were found with low genetic diversity (π = 0.00241) and nucleotide sequence variant diversity (Hd = 0.481). Asaia was vertically transmitted with high frequency (range from 42.5 to 100%). Conclusions: This study provides field-based evidence of the presence of Asaia in Anopheles mosquitoes in Cameroon for exploitation as a symbiont in the control of malaria in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract 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.

Mosquitoes vector many pathogens that cause human disease, such as malaria that is caused by parasites in the genus Plasmodium. 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, called paratransgenesis, 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 is Asaia bogorensis, a Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. However, common secretion signals from E. coli and closely related species do not function in Asaia. Here, we report evaluation of 20 native Asaia N-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 the Asaia bogorensis SF2.1 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 the Asaia bacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine. Further testing in mosquitoes demonstrated that these three Asaia strains were able to suppress the number of oocysts formed after a blood meal containing P. berghei to a significantly greater degree than wild-type Asaia, 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.

Mosquitoes vector many pathogens that cause human disease, such as malaria that is caused by parasites in the genus Plasmodium. 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, called paratransgenesis , 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 is Asaia bogorensis , a Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. However, common secretion signals from E. coli and closely related species do not function in Asaia. Here, we report evaluation of 20 native Asaia N-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 the Asaia bogorensis SF2.1 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 the Asaia bacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine. Further testing in mosquitoes demonstrated that these three Asaia strains were able to suppress the number of oocysts formed after a blood meal containing P. berghei to a significantly greater degree than wild-type Asaia , 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. [ABSTRACT FROM AUTHOR]

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.

The emerging distribution of new alien mosquito species was recently described in Europe. In addition to the invasion of Aedes albopictus, several studies have focused on monitoring and controlling other invasive Aedes species, as Aedes koreicus and Aedes japonicus. Considering the increasing development of insecticide resistance in Aedes mosquitoes, new control strategies, including the use of bacterial host symbionts, are proposed. However, little is known about the bacterial communities associated with these species, thus the identification of possible candidates for Symbiotic Control is currently limited. The characterization of the natural microbiota of field-collected Ae. koreicus mosquitoes from North-East Italy through PCR screening, identified native infections of Wolbachia in this species that is also largely colonized by Asaia bacteria. Since Asaia and Wolbachia are proposed as novel tools for Symbiotic Control, our study supports their use for innovative control strategies against new invasive species. Although the presence of Asaia was previously characterized in Ae. koreicus, our study characterized this Wolbachia strain, also inferring its phylogenetic position. The co-presence of Wolbachia and Asaia may provide additional information about microbial competition in mosquito, and to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction in Ae. koreicus.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is a destructive pest of rice. Bacterial symbionts play an important role in insect hosts, especially hemipteran hosts. This study was designed to examine the bacterial symbionts of the WBPH using 16S rDNA high-throughput sequencing. A total of 63 and 177 operational taxonomic units (OTUs) were identified in females and males of three WBPH populations, respectively. These OTUs included bacteria of 75 genera from 11 phyla, where Wolbachia, Cardinium, and Asaia were the dominant genera, accounting for over 97.99% of all the symbiotic bacteria. Fluorescence in situ hybridization detected Wolbachia, Cardinium, and Asaia in the salivary glands, guts, testes, and eggs of the WBPH, indicating the potential for both horizontal and vertical transmission. Moreover, the infection pattern of the three dominant bacterial symbionts was detected in six WBPH populations. The frequencies of Wolbachia infection of females and Cardinium infection of both sexes were over 96.7%. Wolbachia infection of males ranged between 46.7 and 63.3%, which was significantly lower than that observed for females. Asaia infection of both sexes varied substantially among the populations. These results indicate that the complex host-symbiotic bacteria interaction is influenced by host sex and geographical origin and potentially by the transmission modes of the symbionts. [ABSTRACT FROM AUTHOR]

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 Horváth (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 dorée 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The white-backed planthopper (WBPH), Sogatella furcifera Horváth (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. [ABSTRACT FROM AUTHOR]

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 dorée 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. [ABSTRACT FROM AUTHOR]

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Abstract Background Phlebotomine sand flies transmit the haemoflagellate Leishmania, the causative agent of human leishmaniasis. The Leishmania promastigotes are confined to the gut lumen and are exposed to the gut microbiota within female sand flies. Here we study the colonisation resistance of yeast and bacteria in preventing the establishment of a Leishmania population in sand flies and the ability of Leishmania to provide colonisation resistance towards the insect bacterial pathogen Serratia marcescens that is also pathogenic towards Leishmania. Methods We isolated microorganisms from wild-caught and laboratory-reared female Lutzomyia longipalpis, identified as Pseudozyma sp. Asaia sp. and Ochrobactrum intermedium. We fed the females with a sugar meal containing the microorganisms and then subsequently fed them with a bloodmeal containing Leishmania mexicana and recorded the development of the Leishmania population. Further experiments examined the effect of first colonising the sand fly gut with L. mexicana followed by feeding with, Serratia marcescens, an insect bacterial pathogen. The mortality of the flies due to S. marcescens was recorded in the presence and absence of Leishmania. Results There was a reduction in the number of flies harbouring a Leishmania population that had been pre-fed with Pseudozyma sp. and Asaia sp. or O. intermedium. Experiments in which L. mexicana colonised the sand fly gut prior to being fed an insect bacterial pathogen, Serratia marcescens, showed that the survival of flies with a Leishmania infection was significantly higher compared to flies without Leishmania infection. Conclusions The yeast and bacterial colonisation experiments show that the presence of sand fly gut microorganisms reduce the potential for Leishmania to establish within the sand fly vector. Sand flies infected with Leishmania were able to survive an attack by the bacterial pathogen that would have killed the insect and we concluded that Leishmania may benefit its insect host whilst increasing the potential to establish itself in the sand fly vector. We suggest that the increased ability of the sand fly to withstand a bacterial entomopathogen, due to the presence of the Leishmania, may provide an evolutionary pressure for the maintenance of the Leishmania-vector association.

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. [ABSTRACT FROM AUTHOR]

The ability of bacteria to attach to a surface and develop a biofilm has been of considerable interest for many groups in the food industry. Biofilms may serve as a chronic source of microbial contamination and the research into biofilms and cells interactions might help to improve general understanding of the biofilm resistance mechanisms. Multitude of factors, including surface conditioning, surface charge and roughness and hydrophobicity, are thought to be involved in the initial attachment. Hydrophobic interactions have been widely suggested as responsible for much of the adherence of cells to surfaces. Cell-surface hydrophobicity is an important factor in the adherence and subsequent proliferation of microorganisms on solid surfaces and at interfaces. In the present study, we have estimated the cell-surface characteristics of Asaia bogorensis - isolated contamination of flavoured bottled water and compared its ability to colonise surfaces which are typical in the beverage production - stainless steel, glass and plastic materials.

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 (Asaiagfp), to diffuse in Anopheles stephensi and Anopheles gambiae target mosquito populations. Asaiagfp 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. [ABSTRACT FROM AUTHOR]

The genus Asaia has gained much interest lately owing to constant new species discoveries and its role as a potential opportunistic pathogen to humans. Here we describe a transient bacteremia due to Asaia lannensis in a patient with a psychiatric disorder (compulsive self-injection of different substances). Common phenotypic methods of identification failed to identify this organism, and only restriction fragment lenght polymorphism of PCR-amplified 16S rRNA gene allowed for proper identification. The isolate was highly resistant to most antibiotics. The paper also discusses the currently available medical literature, acknowledges the potential problems linked to the isolation of these strains and proposes an approach to species identification that can be applied in a clinical microbiology laboratory. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Investigations of microbiota within mosquitoes continue to widen the spectrum of possible symbiont-based applications against vector-borne diseases. In this context, α-proteobacteria of the genus Asaia (Rhodospirillales: Acetobacteraceae) are emerging as possible endosymbiotic candidates, particularly in paratransgenic approaches aimed at interrupting pathogen transmission. Previous studies have shown that Asaia spp. distribution among Anopheles gambiae and Anopheles stephensi (Diptera: Culicidae) mosquitoes displayed positive rates of infection in isolated midguts, salivary glands and reproductive tissues. Similarly, Asaia has been detected in Aedes albopictus ( Stegomyia albopicta) and Aedes aegypti ( Stegomyia aegypti) (Diptera: Culicidae) populations. Within the Culex pipiens complex (Diptera: Culicidae), Asaia infection is still largely unexplored. Here, we summarize a preliminary survey of laboratory-reared Cx. pipiens complex and field-collected Culex quinquefasciatus for the presence of Asaia spp., and present the first identification of Asaia in some of the members of the Cx. pipiens complex and the first description in West African populations of Cx. quinquefasciatus. [ABSTRACT FROM AUTHOR]

Background: Phlebotomine sand flies transmit the haemoflagellate Leishmania, the causative agent of human leishmaniasis. The Leishmania promastigotes are confined to the gut lumen and are exposed to the gut microbiota within female sand flies. Here we study the colonisation resistance of yeast and bacteria in preventing the establishment of a Leishmania population in sand flies and the ability of Leishmania to provide colonisation resistance towards the insect bacterial pathogen Serratia marcescens that is also pathogenic towards Leishmania. Methods: We isolated microorganisms from wild-caught and laboratory-reared female Lutzomyia longipalpis, identified as Pseudozyma sp. Asaia sp. and Ochrobactrum intermedium. We fed the females with a sugar meal containing the microorganisms and then subsequently fed them with a bloodmeal containing Leishmania mexicana and recorded the development of the Leishmania population. Further experiments examined the effect of first colonising the sand fly gut with L. mexicana followed by feeding with, Serratia marcescens, an insect bacterial pathogen. The mortality of the flies due to S. marcescens was recorded in the presence and absence of Leishmania. Results: There was a reduction in the number of flies harbouring a Leishmania population that had been pre-fed with Pseudozyma sp. and Asaia sp. or O. intermedium. Experiments in which L. mexicana colonised the sand fly gut prior to being fed an insect bacterial pathogen, Serratia marcescens, showed that the survival of flies with a Leishmania infection was significantly higher compared to flies without Leishmania infection. Conclusions: The yeast and bacterial colonisation experiments show that the presence of sand fly gut microorganisms reduce the potential for Leishmania to establish within the sand fly vector. Sand flies infected with Leishmania were able to survive an attack by the bacterial pathogen that would have killed the insect and we concluded that Leishmania may benefit its insect host whilst increasing the potential to establish itself in the sand fly vector. We suggest that the increased ability of the sand fly to withstand a bacterial entomopathogen, due to the presence of the Leishmania, may provide an evolutionary pressure for the maintenance of the Leishmania-vector association. [ABSTRACT FROM AUTHOR]

• We model interspecific Asaia-Wolbachia competition among malaria vectors. • Parametrization from published lab studies is incongruous with field observations. • Wolbachia tends towards fixation when it blocks environmental infection with Asaia. • Asaia blocking Wolbachia's maternal transmission threatens this biocontrol strategy. Releasing mosquitoes transinfected with the endosymbiotic bacterium Wolbachia is a novel strategy for interrupting vector-borne pathogen transmission. Following its success in controlling arboviruses spread by Aedes aegypti , this technology is being adapted for anopheline malaria vectors. However, antagonistic interactions between Wolbachia and naturally resident Asaia bacteria in malaria vectors have been demonstrated experimentally, potentially jeopardising Wolbachia biocontrol. We developed the first mathematical model accounting for interspecific competition between endosymbionts to assess the feasibility of this novel strategy for controlling malaria. First, Asaia prevalences among natural mosquito populations were compared with simulations parametrized with rates of Asaia transmission reported from laboratory studies. Discrepancies between projections and natural Asaia prevalences indicated potential overestimation of Asaia transmissibility in artificial laboratory settings. With parametrization that matches natural Asaia prevalence, simulations identified redundancies in Asaia's many infection routes (vertical, sexual and environmental). This resilience was only overcome when Wolbachia conferred very high resistance to environmental infection with Asaia, resulting in Wolbachia fixation and Asaia exclusion. Wolbachia's simulated spread was prevented when its maternal transmission was impeded in coinfected mosquitoes and the pre-control Asaia prevalence was beyond a threshold of 60–75%. This theoretical assessment highlights critical next steps in laboratory experiments to inform this strategy's feasibility. [ABSTRACT FROM AUTHOR]

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 Anopheles. 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. [ABSTRACT FROM AUTHOR]