A Time Point Proteomic Analysis Reveals Protein Dynamics of Plasmodium Oocysts.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.

Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission.

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

A yeast strain associated to Anopheles mosquitoes produces a toxin able to kill malaria parasites.

BACKGROUND: Malaria control strategies are focusing on new approaches, such as the symbiotic control, which consists in the use of microbial symbionts to prevent parasite development in the mosquito gut and to block the transmission of the infection to humans. Several microbes, bacteria and fungi, have been proposed for malaria or other mosquito-borne diseases control strategies. Among these, the yeast Wickerhamomyces anomalus has been recently isolated from the gut of Anopheles mosquitoes, where it releases a natural antimicrobial toxin. Interestingly, many environmental strains of W. anomalus exert a wide anti-bacterial/fungal activity and some of these 'killer' yeasts are already used in industrial applications as food and feed bio-preservation agents. Since a few studies showed that W. anomalus killer strains have antimicrobial effects also against protozoan parasites, the possible anti-plasmodial activity of the yeast was investigated. METHODS: A yeast killer toxin (KT), purified through combined chromatographic techniques from a W. anomalus strain isolated from the malaria vector Anopheles stephensi, was tested as an effector molecule to target the sporogonic stages of the rodent malaria parasite Plasmodium berghei, in vitro. Giemsa staining was used to detect morphological damages in zygotes/ookinetes after treatment with the KT. Furthermore, the possible mechanism of action of the KT was investigated pre-incubating the protein with castanospermine, an inhibitor of ß-glucanase activity. RESULTS: A strong anti-plasmodial effect was observed when the P. berghei sporogonic stages were treated with KT, obtaining an inhibition percentage up to around 90%. Microscopy analysis revealed several ookinete alterations at morphological and structural level, suggesting the direct implication of the KT-enzymatic activity. Moreover, evidences of the reduction of KT activity upon treatment with castanospermine propose a ß-glucanase-mediated activity. CONCLUSION: The results showed the in vitro killing efficacy of a protein produced by a mosquito strain of W. anomalus against malaria parasites. Further studies are required to test the KT activity against the sporogonic stages in vivo, nevertheless this work opens new perspectives for the possible use of killer strains in innovative strategies to impede the development of the malaria parasite in mosquito vectors by the means of microbial symbionts.

A rapid qPCR method to investigate the circulation of the yeast Wickerhamomyces anomalus in humans.

The yeast Wickerhamomyces anomalus has been proposed for many biotechnological applications in the food industry. However, a number of opportunistic pathogenic strains have been reported as causative agents of nosocomial fungemia. Recognition of potentially pathogenic isolates is an important challenge for the future commercialization of this yeast. The isolation of W. anomalus from different matrices and, recently, from mosquitoes, requires further investigations into its circulation in humans. Here we present a qPCR protocol for the detection of W. anomalus in human blood samples and the results of a screening of 525 donors, including different classes of patients and healthy people.

Dietary fiber supplementation increases Drosophila melanogaster lifespan and gut microbiota diversity.

Dietary fiber has been shown to have multiple health benefits, including a positive effect on longevity and the gut microbiota. In the present study, Drosophila melanogaster has been chosen as an in vivo model organism to study the health effects of dietary fiber supplementation (DFS). DFS extended the mean half-life of male and female flies, but the absolute lifespan only increased in females. To reveal the underlying mechanisms, we examined the effect of DFS on gut microbiota diversity and abundance, local gut immunity, and the brain proteome. A significant difference in the gut microbial community was observed between groups with and without fiber supplementation, which reduced the gut pathogenic bacterial load. We also observed an upregulated expression of dual oxidase and a modulated expression of Attacin and Diptericin genes in the gut of older flies, possibly delaying the gut dysbiosis connected to the age-related gut immune dysfunction. Brain proteome analysis showed that DFS led to the modulation of metabolic processes connected to mitochondrial biogenesis, the RhoV-GTPase cycle, organelle biogenesis and maintenance, membrane trafficking and vesicle-mediated transport, possibly orchestrated through a gut-brain axis interaction. Taken together, our study shows that DFS can prolong the half-life and lifespan of flies, possibly by promoting a healthier gut environment and delaying the physiological dysbiosis that characterizes the ageing process. However, the RhoV-GTPase cycle at the brain level may deserve more attention in future studies.

Wolbachia and Asaia Distribution among Different Mosquito Vectors Is Affected by Tissue Localization and Host Species.

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.

Tripartite interactions comprising yeast-endobacteria systems in the gut of vector mosquitoes.

It is shown that bacteria use yeast as a niche for survival in stressful conditions, therefore yeasts may act as temporary or permanent bacterial reservoirs. Endobacteria colonise the fungal vacuole of various osmotolerant yeasts which survive and multiply in sugar-rich sources such as plant nectars. Nectar-associated yeasts are present even in the digestive system of insects and often establish mutualistic symbioses with both hosts. Research on insect microbial symbioses is increasing but bacterial-fungal interactions are yet unexplored. Here, we have focused on the endobacteria of Wickerhamomyces anomalus (formerly Pichia anomala and Candida pelliculosa), an osmotolerant yeast associated with sugar sources and the insect gut. Symbiotic strains of W. anomalus influence larval development and contribute digestive processes in adults, in addition to exerting wide antimicrobial properties for host defence in diverse insects including mosquitoes. Antiplasmodial effects of W. anomalus have been shown in the gut of the female malaria vector mosquito Anopheles stephensi. This discovery highlights the potential of utilizing yeast as a promising tool for symbiotic control of mosquito-borne diseases. In the present study, we have carried out a large Next Generation Sequencing (NGS) metagenomics analysis including W. anomalus strains associated with vector mosquitoes Anopheles, Aedes and Culex, which has highlighted wide and heterogeneous EB communities in yeast. Furthermore, we have disclosed a Matryoshka-like association in the gut of A stephensi that comprises different EB in the strain of W. anomalus WaF17.12. Our investigations started with the localization of fast-moving bacteria-like bodies within the yeast vacuole of WaF17.12. Additional microscopy analyses have validated the presence of alive intravacuolar bacteria and 16S rDNA libraries from WaF17.12 have identified a few bacterial targets. Some of these EB have been isolated and tested for lytic properties and capability to re-infect the yeast cell. Moreover, a selective competence to enter yeast cell has been shown comparing different bacteria. We suggested possible tripartite interactions among EB, W. anomalus and the host, opening new knowledge on the vector biology.

Anopheline mosquito saliva contains bacteria that are transferred to a mammalian host through blood feeding.

Introduction: Malaria transmission occurs when Plasmodium sporozoites are transferred from the salivary glands of anopheline mosquitoes to a human host through the injection of saliva. The need for better understanding, as well as novel modes of inhibiting, this key event in transmission has driven intense study of the protein and miRNA content of saliva. Until now the possibility that mosquito saliva may also contain bacteria has remained an open question despite the well documented presence of a rich microbiome in salivary glands. Methods: Using both 16S rRNA sequencing and MALDI-TOF approaches, we characterized the composition of the saliva microbiome of An. gambiae and An. stephensi mosquitoes which respectively represent two of the most important vectors for the major malaria-causing parasites P. falciparum and P. vivax. Results: To eliminate the possible detection of non-mosquito-derived bacteria, we used a transgenic, fluorescent strain of one of the identified bacteria, Serratiamarcescens, to infect mosquitoes and detect its presence in mosquito salivary glands as well as its transfer to, and colonization of, mammalian host tissues following a mosquito bite. We also showed that Plasmodium infection modified the mosquito microbiota, increasing the presence of Serratia while diminishing the presence of Elizabethkingia and that both P. berghei and Serratia were transferred to, and colonized mammalian tissues. Discussion: These data thus document the presence of bacteria in mosquito saliva, their transfer to, and growth in a mammalian host as well as possible interactions with Plasmodium transmission. Together they raise the possible role of mosquitoes as vectors of bacterial infection and the utility of commensal mosquito bacteria for the development of transmission-blocking strategies within a mammalian host.

De novo genome assembly of the invasive mosquito species Aedes japonicus and Aedes koreicus.

BACKGROUND: Recently, two invasive Aedes mosquito species, Ae. japonicus and Ae. koreicus, are circulating in several European countries posing potential health risks to humans and animals. Vector control is the main option to prevent mosquito-borne diseases, and an accurate genome sequence of these mosquitoes is essential to better understand their biology and to develop effective control strategies. METHODS: A de novo genome assembly of Ae. japonicus (Ajap1) and Ae. koreicus (Akor1) has been produced based on a hybrid approach that combines Oxford Nanopore long-read and Illumina short-read data. Their quality was ascertained using various metrics. Masking of repetitive elements, gene prediction and functional annotation was performed. RESULTS: Sequence analysis revealed a very high presence of repetitive DNA and, among others, thermal adaptation genes and insecticide-resistance genes. Through the RNA-seq analysis of larvae and adults of Ae. koreicus and Ae. japonicus exposed to different temperatures, we also identified genes showing a differential temperature-dependent activation. CONCLUSIONS: The assembly of Akor1 and Ajap1 genomes constitutes the first updated collective knowledge of the genomes of both mosquito species, providing the possibility of understanding key mechanisms of their biology such as the ability to adapt to harsh climates and to develop insecticide-resistance mechanisms.

Horizontal transmission of the symbiotic bacterium Asaia sp. in the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae).

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.

Delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts.

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

Bacterial Symbionts in Ceratitis capitata.

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.

Wolbachia in Aedes koreicus: Rare Detections and Possible Implications.

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.

The yeast Wickerhamomyces anomalus (Pichia anomala) inhabits the midgut and reproductive system of the Asian malaria vector Anopheles stephensi.

While symbiosis between bacteria and insects has been thoroughly investigated in the last two decades, investments on the study of yeasts associated with insects have been limited. Insect-associated yeasts are placed on different branches of the phylogenetic tree of fungi, indicating that these associations evolved independently on several occasions. Isolation of yeasts is frequently reported from insect habitats, and in some cases yeasts have been detected in the insect gut and in other organs/tissues. Here we show that the yeast Wickerhamomyces anomalus, previously known as Pichia anomala, is stably associated with the mosquito Anopheles stephensi, a main vector of malaria in Asia. Wickerhamomyces anomalus colonized pre-adult stages (larvae L(1)-L(4) and pupae) and adults of different sex and age and could be isolated in pure culture. By a combination of transmission electron microscopy and fluorescent in situ hybridization techniques, W. anomalus was shown to localize in the midgut and in both the male and female reproductive systems, suggesting multiple transmission patterns.

Different mosquito species host Wickerhamomyces anomalus (Pichia anomala): perspectives on vector-borne diseases symbiotic control.

The genetic manipulation of the microbial community associated with hematophagus insects is particularly relevant for public health applications. Within mosquito populations, this relationship has been overlooked until recently. New advances in molecular biotechnology propose the genetic manipulation of mosquito symbionts to prevent the transmission of pathogens to humans by interfering with the obligatory life cycle stages within the insect through the use of effector molecules. This approach, defined as 'paratransgenesis', has opened the way for the investigation and characterization of microbes residing in the mosquito body, particularly those localised within the gut. Some interesting bacteria have been identified as candidates for genetic modification, however, endosymbiotic yeasts remain largely unexplored with little information on the symbiotic relationships to date. Here we review the recent report of symbiotic relationship between Wickerhamomyces anomalus (Pichia anomala) and several mosquito vector species as promising methods to implement control of mosquito-borne diseases.

Formulation and Safety Tests of a Wickerhamomyces anomalus-Based Product: Potential Use of Killer Toxins of a Mosquito Symbiotic Yeast to Limit Malaria Transmission.

Wickerhamomyces anomalus strain WaF17.12 is a yeast with an antiplasmodial property based on the production of a killer toxin. For its symbiotic association with Anopheles mosquitoes, it has been proposed for the control of malaria. In an applied view, we evaluated the yeast formulation by freeze-drying WaF17.12. The study was carried out by comparing yeast preparations stored at room temperature for different periods, demonstrating that lyophilization is a useful method to obtain a stable product in terms of cell growth reactivation and maintenance of the killer toxin antimicrobial activity. Moreover, cytotoxic assays on human cells were performed, showing no effects on the cell viability and the proinflammatory response. The post-formulation effectiveness of the killer toxin and the safety tests indicate that WaF17.12 is a promising bioreagent able to impair the malaria parasite in vector mosquitoes.

Biting midge dynamics and bluetongue transmission: a multiscale model linking catch data with climate and disease outbreaks.

Bluetongue virus (BTV) serotype 8 has been circulating in Europe since a major outbreak occurred in 2006, causing economic losses to livestock farms. The unpredictability of the biting activity of midges that transmit BTV implies difficulty in computing accurate transmission models. This study uniquely integrates field collections of midges at a range of European latitudes (in Sweden, The Netherlands, and Italy), with a multi-scale modelling approach. We inferred the environmental factors that influence the dynamics of midge catching, and then directly linked predicted midge catches to BTV transmission dynamics. Catch predictions were linked to the observed prevalence amongst sentinel cattle during the 2007 BTV outbreak in The Netherlands using a dynamic transmission model. We were able to directly infer a scaling parameter between daily midge catch predictions and the true biting rate per cow per day. Compared to biting rate per cow per day the scaling parameter was around 50% of 24 h midge catches with traps. Extending the estimated biting rate across Europe, for different seasons and years, indicated that whilst intensity of transmission is expected to vary widely from herd to herd, around 95% of naïve herds in western Europe have been at risk of sustained transmission over the last 15 years.

Phylogenomics Reveals that Asaia Symbionts from Insects Underwent Convergent Genome Reduction, Preserving an Insecticide-Degrading Gene.

The mosquito microbiota is composed of several lineages of microorganisms whose ecological roles and evolutionary histories have yet to be investigated in depth. Among these microorganisms, Asaia bacteria play a prominent role, given their abundance in the gut, reproductive organs, and salivary glands of different mosquito species, while their presence has also been reported in several other insects. Notably, Asaia has great potential as a tool for the control of mosquito-borne diseases. Here, we present a wide phylogenomic analysis of Asaia strains isolated from different species of mosquito vectors and from different populations of the Mediterranean fruit fly (medfly), Ceratitis capitata, an insect pest of worldwide economic importance. We show that phylogenetically distant lineages of Asaia experienced independent genome reductions, despite following a common pattern, characterized by the early loss of genes involved in genome stability. This result highlights the role of specific metabolic pathways in the symbiotic relationship between Asaia and the insect host. Finally, we discovered that all but one of the Asaia strains included in the study possess the pyrethroid hydrolase gene. Phylogenetic analysis revealed that this gene is ancestral in Asaia, strongly suggesting that it played a role in the establishment of the symbiotic association between these bacteria and the mosquito hosts. We propose that this gene from the symbiont contributed to initial pyrethroid resistance in insects harboring Asaia, also considering the widespread production of pyrethrins by several plants.IMPORTANCE We have studied genome reduction within several strains of the insect symbiont Asaia isolated from different species/strains of mosquito and medfly. Phylogenetically distant strains of Asaia, despite following a common pattern involving the loss of genes related to genome stability, have undergone independent genome reductions, highlighting the peculiar role of specific metabolic pathways in the symbiotic relationship between Asaia and its host. We also show that the pyrethroid hydrolase gene is present in all the Asaia strains isolated except for the South American malaria vector Anopheles darlingi, for which resistance to pyrethroids has never been reported, suggesting a possible involvement of Asaia in determining resistance to insecticides.

Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species.

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

Acetic acid bacteria, newly emerging symbionts of insects.

Recent research in microbe-insect symbiosis has shown that acetic acid bacteria (AAB) establish symbiotic relationships with several insects of the orders Diptera, Hymenoptera, Hemiptera, and Homoptera, all relying on sugar-based diets, such as nectars, fruit sugars, or phloem sap. To date, the fruit flies Drosophila melanogaster and Bactrocera oleae, mosquitoes of the genera Anopheles and Aedes, the honey bee Apis mellifera, the leafhopper Scaphoideus titanus, and the mealybug Saccharicoccus sacchari have been found to be associated with the bacterial genera Acetobacter, Gluconacetobacter, Gluconobacter, Asaia, and Saccharibacter and the novel genus Commensalibacter. AAB establish symbiotic associations with the insect midgut, a niche characterized by the availability of diet-derived carbohydrates and oxygen and by an acidic pH, selective factors that support AAB growth. AAB have been shown to actively colonize different insect tissues and organs, such as the epithelia of male and female reproductive organs, the Malpighian tubules, and the salivary glands. This complex topology of the symbiosis indicates that AAB possess the keys for passing through body barriers, allowing them to migrate to different organs of the host. Recently, AAB involvement in the regulation of innate immune system homeostasis of Drosophila has been shown, indicating a functional role in host survival. All of these lines of evidence indicate that AAB can play different roles in insect biology, not being restricted to the feeding habit of the host. The close association of AAB and their insect hosts has been confirmed by the demonstration of multiple modes of transmission between individuals and to their progeny that include vertical and horizontal transmission routes, comprising a venereal one. Taken together, the data indicate that AAB represent novel secondary symbionts of insects.