Immature mosquitoes are aquatic, and their distribution, abundance, and individual fitness in a particular breeding habitat are known to be dependent on mainly three factors: biotic factors, abiotic factors, and their interaction between each other and with other associated taxa. Mosquito breeding habitats harbor a diversified naturally occurring microbiota assemblage, and the biota have different types of interactions with mosquito larvae in those habitats. Those interactions may include parasitism, pathogenism, predation, and competition which cause the mortality of larvae, natural reduction of larval abundance, or alterations in their growth. Many microbiota species serve as food items for mosquito larvae, and there are also some indigestible or toxic phytoplanktons to larvae. However, when there is coexistence or mutualism of different mosquito species along with associated microbiota, they form a community sharing the habitat requirements. With the available literature, it is evident that the abundance of mosquito larvae is related to the densities of associated microbiota and their composition in that particular breeding habitat. Potential antagonist microbiota which are naturally occurring in mosquito breeding habitats could be used in integrated vector control approaches, and this method rises as an ecofriendly approach in controlling larvae in natural habitats themselves. To date, this aspect has received less attention; only a limited number of species of microbiota inhabiting mosquito breeding habitats have been recorded, and detailed studies on microbiota assemblage in relation to diverse vector mosquito breeding habitats and their association with mosquito larvae are few. Therefore, future studies on this important ecological aspect are encouraged. Such studies may help to identify field characteristic agents that can serve as mosquito controlling candidates in their natural habitats themselves.

The pool of microbiota associated with mosquito breeding habitats varies with the habitat type and its characteristic features. The pool of microbiota in a given mosquito breeding habitat can include free living, symbiotic, noncompetitive, parasitic, predatory, and toxin producing species. However, in Sri Lanka the studies on the microbiota associated with mosquito breeding habitats are scarce. The present study was conducted to identify microbiota species/taxa associated with a variety of mosquito breeding habitats in selected areas of the Kurunegala district in Sri Lanka to determine the relationship, if any, the microbiota has with mosquito larvae breeding. A total of 44 microbiota species/taxa belonging to 10 phyla, namely, Bacillariophyta, Charophyta, Chlorophyta, Cyanobacteria/Cyanophyta, Ochrophyta/Heterokontophyta, Amoebozoa, Euglenozoa, Ciliophora, Arthropoda, and Rotifera were identified. Vorticella microstoma (Ciliophora) showed a constant occurrence frequency in rice field habitats occupied mainly by Culex tritaeniorhynchus while the rest of the species had an accidental or rare frequency of occurrence. Nineteen species/taxa were identified as common species. Trophont stages of Vorticella microstoma and Zoothamnium spp. were found attached to the cuticle of mosquito larvae but only V. microstoma caused a lethal effect. The autotrophic protist, Euglena geniculate, Closterium spp., and Pinnularia spp. served as the diet items to mosquito larvae. The majority of the microbiota identified had no observable effect on mosquito larvae breeding.

Background. Larval and adult mosquito stages harbor different extracellular microbes exhibiting various functions in their digestive tract including host-parasite interactions. Midgut symbiotic bacteria can be genetically exploited to express molecules within the vectors, altering vector competency and potential for disease transmission. Therefore, identification of mosquito gut inhabiting microbiota is of ample importance before developing novel vector control strategies that involve modification of vectors. Method. Adult mosquitoes of Culex tritaeniorhynchus, Culex gelidus, and Mansonia annulifera were collected from selected Medical Officer of Health (MOH) areas in the Gampaha district of Sri Lanka. Midgut lysates of the field-caught non-blood-fed female mosquitoes were cultured in Plate Count Agar medium, and Prokaryotic 16S ribosomal RNA partial genes of the isolated bacteria colonies were amplified followed by DNA sequencing. Diversity indices were used to assess the diversity and richness of the bacterial isolates in three mosquito species. The distribution pattern of bacterial isolates between different mosquito species was assessed by Distance-Based Redundancy Analysis (dbRDA). Results. A total of 20 bacterial species (Staphylococcus pasteuri, Bacillus megaterium, Staphylococcus cohnii, Pantoea dispersa, Staphylococcus chromogenes, Bacillus aquimaris, Staphylococcus arlettae, Staphylococcus sciuri, Staphylococcus warneri, Moraxella osloensis, Enterobacter sp., Klebsiella michiganensis, Staphylococcus hominis, Staphylococcus saprophyticus, Streptomyces sp., Bacillus niacin, Cedecea neteri, Micrococcus luteus, Lysinibacillus sphaericus, and Bacillus licheniformis) were identified. All of these species belonged to three phyla, Proteobacteria, Firmicutes, and Actinobacteria, out of which phylum Firmicutes (71.1%) was the most prominent. The least number of species was recorded from Actinobacteria. The relative distribution of midgut microbes in different mosquito species differed significantly among mosquito species (Chi-square, χ2=486.091; df=36; P≤0.001). Midgut microbiota of Cx. tritaeniorhynchus and Cx. gelidus indicated a similarity of 21.51%, while Ma. annulifera shared a similarity of 6.92% with the cluster of above two species. The gut microbiota of Cx. tritaeniorhynchus was also significantly more diverse and more evenly distributed compared to Ma. annulifera. Simpson’s diversity, Margalef’s diversity, and Menhinick’s diversity indices were higher in Cx. gelidus. Of the recorded species, P. dispersa and strains of nonpathogenic species in Bacillaceae family (B. megaterium, B. niacini, B. licheniformis, and L. sphaericus) can be recommended as potential candidates for paratransgenesis. Conclusion. The relative distribution of midgut microbes in different mosquito species differed significantly among the three studied adult mosquito species. The present data strongly encourage further investigations to explore the potential usage of these microbes through paratransgenic approach for novel eco-friendly vector control strategies.

Background The midgut microbiota of mosquitoes maintain basal immune activity and immune priming. In recent years, scientists have focused on the use of microbial communities for vector control interventions. In the present study, the midgut bacteria of larvae and adults of Aedes aegypti and Ae. albopictus were assessed using both field-collected and laboratory-reared mosquitoes from Sri Lanka. Methods Adults and larvae of Ae. aegypti and Ae. albopictus were collected from three selected areas in Gampaha Medical Officer of Health area, Gampaha District, Western Province, Sri Lanka. Bacterial colonies isolated from mosquito midgut dissections were identified by PCR amplification and sequencing of partial 16S rRNA gene fragments. Results Adults and larvae of Ae. aegypti and Ae. albopictus harbored 25 bacterial species. Bacillus endophyticus and Pantoea dispersa were found more frequently in field-collected Ae. aegypti and Ae. albopictus adults, respectively. The midgut bacteria of Ae. aegypti and Ae. albopictus adults (X2 = 556.167, df = 72, P < 0.001) and larvae (X2 = 633.11, df = 66, P < 0.001) were significantly different. There was a significant difference among the bacterial communities between field-collected adults (X2 = 48.974, df = 10, P < 0.001) and larvae (X2 = 84.981, df = 10, P < 0.001). Lysinibacillus sphaericus was a common species in adults and larvae of laboratory-reared Ae. aegypti. Only P. dispersa occurred in the field-collected adults of Ae. aegypti and Ae. albopictus. Species belonging to genera Terribacillus, Lysinibacillus, Agromyces and Kocuria were recorded from Aedes mosquitoes, in accordance with previously reported results. Conclusions This study generated a comprehensive database on the culturable bacterial community found in the midgut of field-collected (Ae. aegypti and Ae. albopictus) and laboratory-reared (Ae. aegypti) mosquito larvae and adults from Sri Lanka. Data confirm that the midgut bacterial diversity in the studied mosquitoes varies according to species, developmental stage and strain (field vs laboratory). Graphical abstract

A mosquito species has its own favourable requirements of abiotic and biotic characteristics including microbiota, in a breeding habitat. Some of the microbiota may cause parasitic or pathogenic effects to mosquito larvae such as species of viruses, parasitic bacteria, fungi, protists, entomopathogenic nematodes, and filamentous fungi. In Sri Lanka, there is a scarcity of information on microbiota associated with mosquito breeding habitats and their effect on mosquito larvae. Hence, the present study was conducted to determine microbiota species/taxa associated with a variety of mosquito breeding habitats in selected areas of the Gampaha District in Sri Lanka and the relationship, if any, the microbiota has with mosquito larva survival and breeding. Forty-five microbiota species belonging to 11 phyla were found from different mosquito breeding habitats with the highest percentage belonging to phylum Euglenozoa (27.89%). Species that belonged to the phylum Amoebozoa (1.22%) and Sarcodina (1.17%) had the lowest abundance, and each of its species richness was recorded as one. Philodina citrina followed by Monostyla bulla comprised 30.8% and 16.59%, respectively, of the total rotifer population. From the total microbiota, 25-50% existed as accidental while less than 25% rare, in the habitat type according to their abundance. Paddy fields had the highest species richness (17), evenness (23.52), Shannon-Weiner (66.64), and beta diversity (0.65) over 50% indicating high heterogeneity in microbiota composition among the habitats. Ciliated protists, namely, Vorticella microstoma, Zoothamnium spp., and Chilodinella sp., were identified as naturally occurring microbiota associated with Culex mosquito larvae that inhabited in paddy fields and associated irrigation canals. Only Vorticella microstoma caused a significant lethal effect on mosquito larvae. This study revealed that species of Cx. gelidus, Cx. pseudovishnui, Cx. tritaeniorhynchus, Cx. quinquefasciatus, and Cx. whitmorei served as hosts for V. microstoma where infectivity rate in Cx. tritaeniorhynchus reached 73.22. Chilodinella sp. selectively served as endoparasitic to Cx. gelidus larvae causing only 4.58% mortality, and invasive cysts of the pathogen were observed in the subcuticular layer of the host body. Even though Zoothamnium spp. were found on Cx. tritaeniorhynchus larvae, there was no lethal effect due to the attachment of the parasitic agent. The potential of these microbiotas in integrated vector controlling approaches in future perspectives is recommended.