Mosquito/microbiota interactions: from complex relationships to biotechnological perspectives.

To date around 3500 different species of mosquito have been described, several tens of which are vectors of pathogens of remarkable interest in public health. Mosquitoes are present all around the world showing a great ability to adapt to very different types of habitats where they play relevant ecological roles. It is very likely that components of the mosquito microbiota have given the mosquito a great capacity to adapt to different environments. Current advances in understanding the mosquito-microbiota relationships may have a great impact in a better understanding of some traits of mosquito biology and in the development of innovative mosquito-borne disease-control strategies aimed to reduce mosquito vectorial capacity and/or inhibiting pathogen transmission

Chapter Facing Malaria Parasite with Mosquito Symbionts

Optical physic

Facing Malaria Parasite with Mosquito Symbionts

Optical physic

Inhibition of Asaia in adult mosquitoes causes male-specific mortality and diverse transcriptome changes

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

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

Interactions between Asaia, Plasmodium and Anopheles: new insights in mosquito symbiosis and implications in Malaria Symbiotic Control

The recent identification of acetic acid bacteria belonging to the genus Asaia, that are stably associated with larvae and adults of different species of malaria transmitting mosquitoes, in particular Anopheles stephensi, indicates this bacterium as a suitable candidate for the paratransgenic or symbiotic control of the malaria infection. The presence of Asaia in the mosquito reproductive system represents an additional intriguing feature, particularly important towards the development of Paratransgenetically Modified (PM) mosquitoes that can mix with natural populations. By the means of Real Time quantitative PCR I have investigated the relative amount of Asaia and Plasmodium in co-infected mosquitoes thus providing evidence that the malaria infection does not interfere with the blooming of Asaia that follows a blood meal. Furthermore, by immunological studies we provided evidences that Asaia induces ‘in vitro’ the expression of antimicrobial peptides (AMPs) in the mosquito and that, the adaptation of Asaia to the life into the insect, doesn’t appear to be related with a reduced immunogenicity, but with a resistance to the immune reaction. In addition, by fluorescent recombinant strains of both Asaia and Plasmodium, we were able to precisely define the co-localisation of these organisms at the level of mosquito midgut and salivary glands. It was also considered necessary to verify the Asaia role about the vitality of adults mosquitoes. For this aim, has been adopted a novel strategy using monoclonal antibodies against Asaia, to interfere with the function of these bacteria by antibody opsonization. \ud These studies while confirming the potential suitable use of Asaia as microbial agent for the paratransgenic control of malaria infection. \ud Beyond bacteria, yeast-like organisms were also present in the mosquito midgut and in reproductive systems. We have identified Wickerhamomyces anomalus (Saccharomycetales) as an important component of the yeast microbiome in this mosquito species, suggesting multiple transmission patterns. The possibility to easily manipulate yeast, and the chance to express effector molecules in a eukaryotic organism renders W. anomalus another good potential candidate for paratransgenesis.\u

Denaturing Gradient Gel Electrophoresis Analysis of Bacteria in Italian Ticks and First Detection of Streptococcus equi in Rhipicephalus bursa from the Lazio Region

Tick-borne diseases are an increasing problem for the community. Ticks harbor a complex microbial population acquired while feeding on a variety of animals. Profiling the bacterial population by 16S rDNA amplification and denaturing gradient gel electrophoresis enables detection of the broad spectrum of bacteria that settles in the ticks. This study identified known and unknown tick-infecting bacteria in samples from Italy. Seven adult ticks from different hosts and origins were analyzed: two Rhipicephalus sanguineus ticks from dogs (Lombardia), two Rhipicephalus bursa ticks from bovines (Lazio), and three Ixodes ricinus ticks from humans (Marche). The major result was the first report of the zoonotic agent Streptococcus equi in ticks. S. equi is a species complex of highly contagious pathogens. Subsequent to S. equi detection in a R. bursa tick removed from a bovine of Lazio in 2012, we studied 95 R. bursa samples collected from 3 bovines, 3 ponies, and 1 sheep grazing in the same area in 2012 and from 6 ponies grazing there in 2017. The results of a specific PCR assay indicated a not sporadic occurrence of S. equi in ticks. This finding provides a basis for assessing the potential of ticks to harbor and disperse S. equi

Mosquito/microbiota interactions: from basic research to biotechnological perspectives in mosquito borne diseases control.

Mosquito/microbiota interactions: from basic research to biotechnological perspectives in mosquito borne diseases contro