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Mosquitoes, the major vectors of viruses like dengue, are naturally host to diverse microorganisms, which play an important role in their development, fecundity, immunity, and vector competence. The composition of their microbiota is strongly influenced by the environment, particularly their aquatic larval habitat. In this study, we used 2×300 bp 16s Illumina sequencing to compare the microbial profiles of emerging adult Aedes aegypti mosquitoes and the water collected from common types of aquatic habitat containers in Puerto Rico, which has endemic dengue transmission. We sequenced 141 mosquito and 46 water samples collected from plastic containers, septic tanks, discarded tires, underground trash cans, tree holes, or water meters. We identified 9 bacterial genera that were highly prevalent in the mosquito microbiome, and 77 for the microbiome of the aquatic habitat. The most abundant mosquito-associated bacterial OTUs were from the families Burkholderiaceae, Pseudomonadaceae, Comamonadaceae, and Xanthomonadaceae. Microbial profiles varied greatly between mosquitoes, and there were few major differences explained by container type; however, the microbiome of mosquitoes from plastic containers was more diverse and contained more unique taxa than the other groups. Container water was significantly more diverse than mosquitoes, and our data suggest that mosquitoes filter out many bacteria, with Alphaproteobacteria in particular being far more abundant in water. These findings provide novel insight into the microbiome of mosquitoes in the region and provide a platform to improve our understanding of the fundamental mosquito-microbe interactions.

Mosquito-transmitted diseases, including malaria and dengue, are a major threat to human health around the globe, affecting millions each year. A diverse array of next-generation tools has been designed to eliminate mosquito populations or to replace them with mosquitoes that are less capable of transmitting key pathogens. Many of these new approaches have been built on recent advances in CRISPR/Cas9-based genome editing. These initiatives have driven the development of pathogen-resistant lines, new genetics-based sexing methods, and new methods of driving desirable genetic traits into mosquito populations. Many other emerging tools involve microorganisms, including two strategies involving Wolbachia that are achieving great success in the field. At the same time, other mosquito-associated bacteria, fungi, and even viruses represent untapped sources of new mosquitocidal or antipathogen compounds. Although there are still hurdles to be overcome, the prospect that such approaches will reduce the impact of these diseases is highly encouraging.

Vector control plays a key role in reducing the public health burden of mosquito-borne diseases. Today’s vector control strategies largely rely on synthetic insecticides that can have a negative environmental impact when applied outdoors and often become inefficient because of the mosquitoes’ ability to develop resistance. An alternative and promising approach to circumvent these challenges involves the implementation of insecticides derived from nature (biopesticides) for vector control. Biopesticides can constitute naturally occurring organisms or substances derived from them that have lifespan-shortening effects on disease vectors such as mosquitoes. Here we present the discovery and evaluation of natural product-based biological control agents that can potentially be developed into biopesticides for mosquito control. We screened a natural product collection comprising 390 compounds and initially identified 26 molecules with potential ability to kill the larval stages of the yellow fever mosquito Aedes aegypti, which is responsible for transmitting viruses such as dengue, Zika, chikungunya and yellow fever. Natural products identified as hits in the screen were further evaluated for their suitability for biopesticide development. We show that a selection of the natural product top hits, bactobolin, maytansine and ossamycin, also killed the larval stages of the malaria-transmitting mosquito Anopheles gambiae as well as the adult form of both species. We have further explored the usefulness of crude extracts and preparations from two of the best candidates’ sources (organisms of origin) for mosquitocidal activity, that is extracts from the two bacteria Burkholderia thailandensis and Streptomyces hygroscopicus var. ossamyceticus. Graphical abstract

Insect odorant-binding proteins (OBPs) are small soluble proteins that have been assigned roles in olfaction, but their other potential functions have not been extensively explored. Using CRISPR/Cas9-mediated disruption of Aedes aegypti Obp10 and Obp22, we demonstrate the pleiotropic contribution of these proteins to multiple processes that are essential for vectorial capacity. Mutant mosquitoes have impaired host-seeking and oviposition behavior, reproduction, and arbovirus transmission. Here, we show that Obp22 is linked to the male-determining sex locus (M) on chromosome 1 and is involved in male reproduction, likely by mediating the development of spermatozoa. Although OBP10 and OBP22 are not involved in flavivirus replication, abolition of these proteins significantly reduces transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. These results extend our current understanding of the role of insect OBPs in insect reproduction and transmission of human pathogens, making them essential determinants of vectorial capacity. IMPORTANCE Aedes aegypti is the major vector for many arthropod-borne viral diseases, such as dengue, Zika, and chikungunya viruses. Previous studies suggested that odorant-binding proteins (OBPs) may have diverse physiological functions beyond the olfactory system in mosquitoes; however, these hypothesized functions have not yet been demonstrated. Here, we have used CRISPR/Cas9-based genome editing to functionally delete (knock out) Obp10 and Obp22 in Aedes aegypti. We showed that disruption of Obp10 or Obp22 significantly impairs female and male reproductive capacity by adversely affecting blood feeding, oviposition, fecundity and fertility, and the development of spermatozoa. We also showed that disruption of Obp10 or Obp22 significantly reduces the transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. Thus, our study is not only significant in understanding the functions of OBPs in mosquito biology, but also shows that OBPs may represent potent flavivirus transmission-blocking targets. Our study is in this regard particularly timely and important from a translational and public health perspective. Published version

Mosquitoes naturally harbor a diverse community of microorganisms that play a crucial role in their biology. Mosquito-microbiota interactions are abundant and complex. They can dramatically alter the mosquito immune response, and impede or enhance a mosquito's ability to transmit medically important arboviral pathogens. Yet critically, given the massive public health impact of arboviral disease, few such interactions have been well characterized. In this review, we describe the current state of knowledge of the role of microorganisms in mosquito biology, how microbial-induced changes to mosquito immunity moderate infection with arboviruses, cases of mosquito-microbial-virus interactions with a defined mechanism, and the molecular interactions that underlie the endosymbiotic bacterium Wolbachia's ability to block virus infection in mosquitoes.

Mosquito anti-pathogen immune responses, including those controlling infection with arboviruses are regulated by multiple signal transduction pathways. While the Toll pathway is critical in the defense against arboviruses such as dengue and Zika viruses, the factors and mechanisms involved in virus recognition leading to the activation of the Toll pathway are not fully understood. In this study we evaluated the role of virus-produced double-stranded RNA (dsRNA) intermediates in mosquito immune activation by utilizing the synthetic dsRNA analog polyinosinic-polycytidylic acid (poly I:C). Poly I:C treatment of Aedes aegypti mosquitoes and Aag2 cells reduced DENV infection. Transcriptomic analyses of Aag2 cell responses to poly I:C indicated putative activation of the Toll pathway. We found that poly I:C is translocated to the endosomal compartment of Aag2 cells, and that the A. aegypti Toll 6 receptor is a putative dsRNA recognition receptor. This study elucidates the role of dsRNAs in the immune activation of non-RNAi pathways in mosquitoes.

Paratransgenesis targeting the gut protozoa is being developed as an alternative method for the control of the Formosan subterranean termite (FST). This method involves killing the cellulose-digesting gut protozoa using a previously developed antiprotozoal peptide consisting of a target specific ligand coupled to an antimicrobial peptide (Hecate). In the future, we intend to genetically engineer termite gut bacteria as "Trojan Horses" to express and spread ligand-Hecate in the termite colony. The aim of this study was to assess the usefulness of bacteria strains isolated from the gut of FST as "Trojan Horses." We isolated 135 bacteria from the guts of workers from 3 termite colonies. Sequencing of the 16S rRNA gene identified 20 species. We tested 5 bacteria species that were previously described as part of the termite gut community for their tolerance against Hecate and ligand-Hecate. Results showed that the minimum concentration required to inhibit bacteria growth was always higher than the concentration required to kill the gut protozoa. Out of the 5 bacteria tested, we engineered Trabulsiella odontotermitis, a termite specific bacterium, to express green fluorescent protein as a proof of concept that the bacteria can be engineered to express foreign proteins. Engineered T. odontotermitis was fed to FST to study if the bacteria are ingested. This feeding experiment confirmed that engineered T. odontotermitis is ingested by termites and can survive in the gut for at least 48 h. Here we report that T. odontotermitis is a suitable delivery and expression system for paratransgenesis in a termite species.

We sequenced the genome of the novel Enterobacter bacteriophage Tyrion isolated from termite gut. The bacteriophage is temperate in nature and belongs to the Podoviridae family. Many predicted bacteriophage genes showed similarity to prophage regions of the genomes of Enterobacteriaceae bacteria. This is the second bacteriophage isolated from termite gut.

We isolated and sequenced the novel Enterobacter bacteriophage Arya from termite gut. The genome showed synteny to lytic bacteriophage genomes; however, the genome encodes a truncated, putatively nonfunctional integrase pseudogene. Lysogeny-related genes were previously observed in certain lytic phages, but their role and evolution remain unclear.

The Formosan subterranean termite; Coptotermes formosanus is nutritionally dependent on the complex and diverse community of bacteria and protozoa in their gut. Although, there have been many studies to decipher the taxonomic and functional diversity of bacterial communities in the guts of termites, their bacteriophages remain unstudied. We sequenced the metavirome of the guts of Formosan subterranean termite workers to study the diversity of bacteriophages and other associated viruses. Results showed that the termites harbor a virome in their gut comprised of varied and previously unknown bacteriophages. Between 87-90% of the predicted dsDNA virus genes by Metavir showed similarity to the tailed bacteriophages (Caudovirales). Many predicted genes from the virome matched to bacterial prophage regions. These data are suggestive of a virome dominated by temperate bacteriophages. We predicted the genomes of seven novel Caudovirales bacteriophages from the termite gut. Three of these predicted bacteriophage genomes were found in high proportions in all the three termite colonies tested. Two bacteriophages are predicted to infect endosymbiotic bacteria of the gut protozoa. The presence of these putative bacteriophages infecting endosymbionts of the gut protozoa, suggests a quadripartite relationship between the termites their symbiotic protozoa, endosymbiotic bacteria of the protozoa and their bacteriophages. Other than Caudovirales, ss-DNA virus related genes were also present in the termite gut. We predicted the genomes of 12 novel Microviridae phages from the termite gut and seven of those possibly represent a new proposed subfamily. Circovirus like genomes were also assembled from the termite gut at lower relative abundance. We predicted 10 novel circovirus genomes in this study. Whether these circoviruses infect the termites remains elusive at the moment. The functional and taxonomical annotations suggest that the termites may harbor a core virome comprised of the bacteriophages infecting endosymbionts of the gut protozoa.

Background The Formosan subterranean termite, Coptotermes formosanus is an invasive urban pest in the Southeastern USA. Paratransgenesis using a microbe expressed lytic peptide that targets the termite gut protozoa is currently being developed for the control of Formosan subterranean termites. In this study, we evaluated Trabulsiella odontotermitis, a termite-specific bacterium, for its potential to serve as a ‘Trojan Horse’ for expression of gene products in termite colonies. Results We engineered two strains of T. odontotermitis, one transformed with a constitutively expressed GFP plasmid and the other engineered at the chromosome with a Kanamycin resistant gene using a non- disruptive Tn7 transposon. Both strains were fed to termites from three different colonies. Fluorescent microscopy confirmed that T. odontotermitis expressed GFP in the gut and formed a biofilm in the termite hindgut. However, GFP producing bacteria could not be isolated from the termite gut after 2 weeks. The feeding experiment with the chromosomally engineered strain demonstrated that T. odontotermitis was maintained in the termite gut for at least 21 days, irrespective of the termite colony. The bacteria persisted in two termite colonies for at least 36 days post feeding. The experiment also confirmed the horizontal transfer of T. odontotermitis amongst nest mates. Conclusion Overall, we conclude that T. odontotermitis can serve as a ‘Trojan Horse’ for spreading gene products in termite colonies. This study provided proof of concept and laid the foundation for the future development of genetically engineered termite gut bacteria for paratransgenesis based termite control. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0822-4) contains supplementary material, which is available to authorized users.

Paratransgenesis targeting the gut protozoa is being developed as an alternative method for the control of the Formosan subterranean termite (FST). This method involves killing the cellulose-digesting gut protozoa using a previously developed antiprotozoal peptide consisting of a target specific ligand coupled to an antimicrobial peptide (Hecate). In the future, we intend to genetically engineer termite gut bacteria as "Trojan Horses" to express and spread ligand-Hecate in the termite colony. The aim of this study was to assess the usefulness of bacteria strains isolated from the gut of FST as "Trojan Horses." We isolated 135 bacteria from the guts of workers from 3 termite colonies. Sequencing of the 16S rRNA gene identified 20 species. We tested 5 bacteria species that were previously described as part of the termite gut community for their tolerance against Hecate and ligand-Hecate. Results showed that the minimum concentration required to inhibit bacteria growth was always higher than the concentration required to kill the gut protozoa. Out of the 5 bacteria tested, we engineered Trabulsiella odontotermitis, a termite specific bacterium, to express green fluorescent protein as a proof of concept that the bacteria can be engineered to express foreign proteins. Engineered T. odontotermitis was fed to FST to study if the bacteria are ingested. This feeding experiment confirmed that engineered T. odontotermitis is ingested by termites and can survive in the gut for at least 48 h. Here we report that T. odontotermitis is a suitable delivery and expression system for paratransgenesis in a termite species.

The complete genome of bacteriophage CVT22 infecting Citrobacter sp. strain TM1552 is reported here. Both the bacteriophage and Citrobacter sp. TM1552 were isolated from the gut of the Formosan subterranean termite, Coptotermes formosanus . This is the first report of a genome sequence of a bacteriophage isolated from the termite gut.

Dengue viruses are transmitted to humans through the bites of infected female aedine mosquitoes. Differences in the composition and structure of bacterial communities in the midguts of mosquitoes may affect the vector's ability to transmit the disease. To investigate and analyse the role of midgut bacterial communities in viral transmission, midgut bacteria from three species, namely Stegomyia aegypti (= Aedes aegypti), Fredwardsius vittatus (= Aedes vittatus) and Stegomyia albopicta (= Aedes albopictus) (all: Diptera: Culicidae), from dengue-endemic and non-endemic areas of Rajasthan, India were compared. Construction and analyses of six 16S rRNA gene libraries indicated that Serratia spp.-related phylotypes dominated all clone libraries of the three mosquito species from areas in which dengue is not endemic. In dengue-endemic areas, phylotypes related to Aeromonas, Enhydrobacter spp. and uncultivated bacterium dominated the clone libraries of S. aegypti, F. vittatus and S. albopicta, respectively. Diversity indices analysis and real-time TaqMan polymerase chain reaction assays showed bacterial diversity and abundance in the midguts of S. aegypti to be higher than in the other two species. Significant differences observed among midgut bacterial communities of the three mosquito species from areas in which dengue is and is not endemic, respectively, may be related to the vectorial capacity of mosquitoes to carry dengue viruses and, hence, to the prevalence of disease in some areas.

The redbanded stink bug, Piezodorus guildinii (Westwood) (Hemiptera: Heteroptera: Pentatomidae), is a rapidly growing pest damaging southern US agriculture. Pentatomid stink bugs are known to vector bacterial, fungal, and viral plant diseases. However, bacteria associated with redbanded stink bugs and their vector potential have not yet been assessed. In this study, we 1) cultured and identified bacteria transmitted by feeding of redbanded stink bug and 2) described bacteria from guts of redbanded stink bug individuals using next-generation sequencing of 16S rRNA genes. Nineteen bacteria transmitted by feeding of redbanded stink bug on soybean agar were isolated and identified via Sanger sequencing of near full length 16S RNA genes. The transmitted bacteria belonged to at least a dozen species in eight genera and included potential plant pathogens (Phaseolibacter flectens), plant beneficials (Bacillus atropheus), and possible insect beneficials (Acinetobacter sp. and Citrobacter farmeri). A total of 284,448 reads were captured from Illumina MiSeq sequencing of the uncultured gut bacteria community. Fifty-one putative bacteria species (74% of the estimated total species richness) were identified via matches to NCBI databases. The bacteria metagenome contained potential plant and insect pathogens (Erwinia persicina, E. rhaponici, Brenneria nigrifluens, Ralstonia picketti, and Serratia marcescens) and beneficials (Pantoea dispersa, Klebsiella oxytoca, Clostridium butyricum, and Citrobacter farmeri).