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The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or the field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, many more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities is associated with variability in host–microbiome interactions and further demonstrate the utility of the microbiome transplantation technique for investigating host–microbe interactions in mosquitoes. [ABSTRACT FROM AUTHOR]

Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naïve regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint shows a number of proteases regulated at 2 and 7 dpi, potentially representative of Toll or melanization pathway activation, and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes. Author summary: Mayaro virus (MAYV) is a mosquito-borne Alphavirus responsible for outbreaks in South America and the Caribbean. In this study we infected Anopheles stephensi with MAYV and sequenced mRNA and small RNA to understand how MAYV infection impacts gene transcription and the expression of small RNAs in the mosquito vector. Genes involved with innate immunity and signaling pathways related to cell death are regulated in response to MAYV infection of An. stephensi, we also discovered novel miRNAs and describe the expression patterns of miRNAs, siRNAs, and piRNAs following bloodmeal ingestion. These results suggest that MAYV does induce a molecular response to infection in its mosquito vector species. [ABSTRACT FROM AUTHOR]

Background: Symbiotic bacteria are pervasive in mosquitoes and their presence can influence many host phenotypes that affect vectoral capacity. While it is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. The CRISPR/Cas9 gene editing system is a powerful tool to alter bacterial genomes facilitating investigations into host-microbe interactions but has yet to be applied to insect symbionts. Methodology/Principal findings: To investigate the role of bacterial genetic factors in mosquito biology and in colonization of mosquitoes we used CRISPR/Cas9 gene editing system to mutate the outer membrane protein A (ompA) gene of a Cedecea neteri symbiont isolated from Aedes mosquitoes. The ompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adult mosquitoes, the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting genetic factors are particularly important for adult gut colonization. We also used the CRISPR/Cas9 system to integrate genes (antibiotic resistance and fluorescent markers) into the symbionts genome and demonstrated that these genes were functional in vitro and in vivo. Conclusions/Significance: Our results shed insights into the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes. The ability to use this technology for site-specific integration of genes into the symbiont will facilitate the development of paratransgenic control strategies to interfere with arboviral pathogens such Chikungunya, dengue, Zika and Yellow fever viruses transmitted by Aedes mosquitoes. Author summary: Microbiota profoundly affect their host but few studies have investigated the role of bacterial genetics in host-microbe interactions in mosquitoes. Here we applied the CRISPR/Cas9 gene editing system to knockout a membrane protein in Cedecea neteri, which is a dominant member of the mosquito microbiome. The mutant strain had an impaired capacity to form biofilms, infected larvae and adults at lower titers, and had a reduced prevalence in adults. The lower prevalence in adults, but not larvae, likely reflects the difference in the modes of bacterial acquisition from the larval water of these two life stages. Importantly from an applied perspective, we also demonstrated that this editing technology can be harnessed for site-specific integration of genes into the bacterial chromosome. In proof-of-principle studies we integrated either a fluorescent protein or gene conferring antibiotic resistance into the bacterial genome and showed these transgenes were functional in mosquitoes. The specificity, flexibility, and simplicity of this editing approach in non-model bacteria will be useful for developing novel symbiotic control strategies to mitigate the burden of arthropod-borne disease. [ABSTRACT FROM AUTHOR]

Microbial interactions are an underappreciated force in shaping insect microbiome communities. Although pairwise patterns of symbiont interactions have been identified, we have a poor understanding regarding the scale and the nature of co-occurrence and co-exclusion interactions within the microbiome. To characterize these patterns in mosquitoes, we sequenced the bacterial microbiome of Aedes aegypti , Ae. albopictus , and Culex quinquefasciatus caught in the field or reared in the laboratory and used these data to generate interaction networks. For collections, we used traps that attracted host-seeking or ovipositing female mosquitoes to determine how physiological state affects the microbiome under field conditions. Interestingly, we saw few differences in species richness or microbiome community structure in mosquitoes caught in either trap. Co-occurrence and co-exclusion analysis identified 116 pairwise interactions substantially increasing the list of bacterial interactions observed in mosquitoes. Networks generated from the microbiome of Ae. aegypti often included highly interconnected hub bacteria. There were several instances where co-occurring bacteria co-excluded a third taxa, suggesting the existence of tripartite relationships. Several associations were observed in multiple species or in field and laboratory-reared mosquitoes indicating these associations are robust and not influenced by environmental or host factors. To demonstrate that microbial interactions can influence colonization of the host, we administered symbionts to Ae. aegypti larvae that either possessed or lacked their resident microbiota. We found that the presence of resident microbiota can inhibit colonization of particular bacterial taxa. Our results highlight that microbial interactions in mosquitoes are complex and influence microbiome composition. [ABSTRACT FROM AUTHOR]

Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37°C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions. [ABSTRACT FROM AUTHOR]

Mycoplasmas are amongst the most successful pathogens of both humans and animals yet the molecular basis of mycoplasma pathogenesis is poorly understood. This is partly due to the lack of classical virulence factors and little similarity to common bacterial pathogenic determinants. Using Mycoplasma agalactiae as a model we initiated research in this direction by screening a transposon mutant library in the natural sheep host using a negative selection method. Having successfully identified putative factors involved in the colonization of local infection and lymphogenic sites, the current study assessed mutants unable to spread systemically in sheep after experimental intramammary infection. Analysis of distant body sites for complete absence of mutants via SSM PCR revealed that additional set of genes, such as pdhB, oppC, oppB, gtsB, MAG1890, MAG5520 and MAG3650 are required for systemic spreading apart from those that were necessary for initial colonization. Additional in vitro studies with the mutants absent at these systemic sites confirmed the potential role of some of the respective gene products concerning their interaction with host cells. Mutants of pdhB, oppC and MAG4460 exhibited significantly slower growth in the presence of HeLa cells in MEM medium. This first attempt to identify genes exclusively required for systemic spreading provides a basis for further in-depth research to understand the exact mechanism of chronicity and persistence of M. agalactiae. [ABSTRACT FROM AUTHOR]

Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae’s induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection. [ABSTRACT FROM AUTHOR]

Mosquito-transmitted arthropod-borne viruses (arboviruses) such as dengue virus, chikungunya virus, and West Nile virus constitute a major public health burden and are increasing in severity and frequency worldwide. The microbiota associated with mosquitoes (comprised of viruses, bacteria, fungi and protozoa) can profoundly influence many host phenotypes including vector competence, which can either be enhanced or suppressed. Thus, the tripartite interactions between the mosquito vector, its microbiota and the pathogens they transmit offer novel possibilities to control arthropod-borne diseases. [ABSTRACT FROM AUTHOR]

The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae. [ABSTRACT FROM AUTHOR]

Cell fusing agent virus (CFAV) is an insect-specific flavivirus (ISF) found in Aedes aegypti mosquitoes. ISFs have demonstrated the ability to modulate the infection or transmission of arboviruses such as dengue, West Nile, and Zika viruses. It is thought that vertical transmission is the main route for ISF maintenance in nature. This has been observed with CFAV, but there is evidence of horizontal and venereal transmission in other ISFs. Understanding the route of transmission can inform strategies to spread ISFs to vector populations as a method of controlling pathogenic arboviruses. We crossed individually reared male and female mosquitoes from both a naturally occurring CFAV-positive Ae. aegypti colony and its negative counterpart to provide information on maternal, paternal, and horizontal transmission. RT-PCR was used to detect CFAV in individual female pupal exuviae and was 89% sensitive, but only 42% in male pupal exuviae. This is a possible way to screen individuals for infection without destroying the adults. Female-to-male horizontal transmission was not observed during this study. However, there was a 31% transmission rate from mating pairs of CFAV-positive males to negative female mosquitoes. Maternal vertical transmission was observed with a filial infection rate of 93%. The rate of paternal transmission was 85% when the female remained negative, 61% when the female acquired CFAV horizontally, and 76% overall. Maternal and paternal transmission of CFAV could allow the introduction of this virus into wild Ae. aegypti populations through male or female mosquito releases, and thus provides a potential strategy for ISFderived arbovirus control. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium smegmatis topoisomerase I (MstopoI) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage in the absence of Mg2+, but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim (topoisomerase–primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+ in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike MstopoI and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage–religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2 +-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors. [Copyright &y& Elsevier]

Wolbachia , a widespread bacterium that can reduce pathogen transmission in mosquitoes, has recently been reported to be present in Anopheles (An.) species. In wild populations of the An. gambiae complex, the primary vectors of Plasmodium malaria in Sub-Saharan Africa, Wolbachia DNA sequences at low density and infection frequencies have been detected. As the majority of studies have used highly sensitive nested PCR as the only method of detection, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Here, we describe high-density Wolbachia infections in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti , and maternal transmission was observed. Genome sequencing of both Wolbachia strains obtained genome depths and coverages comparable to those of other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present, indicating that these strains possess the capacity to induce the cytoplasmic incompatibility phenotype, which allows Wolbachia to spread through host populations. These strains should be further investigated as candidates for use in Wolbachia biocontrol strategies in Anopheles aiming to reduce the transmission of malaria. • High-density Wolbachia strains found in An. moucheti and An. demeilloni mosquitoes • Infections are visualized in the ovaries, and maternal transmission was observed • Sequencing at depths and coverages comparable to other known Wolbachia strains • Homologs of cytoplasmic incompatibility factor genes are present in both genomes Wolbachia is an endosymbiotic bacterium found in a variety of insect species and can prevent pathogen transmission in mosquitoes. Walker et al. provide concrete evidence for high-density Wolbachia infections in the malaria vectors Anopheles moucheti and An. demeilloni and present near-complete genomes of these Wolbachia strains. [ABSTRACT FROM AUTHOR]

The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host-microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems in Serratia sp. Ag1, which was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR/Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a type I-E CRISPR/Cas system and has a single array, CRISPR1. CRISPR2 is a type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequences of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp., including S. marcescens and S. fonticola , whereas CRISPR1 is not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9 % of CRISPR1 (type I-E) targets are in phage sequences and 91 % are in plasmid sequences. Conversely, ~66 % of CRISPR2 (type I-F) targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time., Competing Interests: The authors declare that there are no conflicts of interest., (© 2023 The Authors.)

Bangladesh reported the highest number of annual deaths (n = 281) related to dengue virus infection in 2022 since the virus reappeared in the country in 2000. Earlier studies showed that >92% of the annual cases occurred between the months of August and September. The 2022 outbreak is characterized by late onset of dengue cases with unusually higher deaths in colder months, that is, October-December. Here we present possible hypotheses and explanations for this late resurgence of dengue cases. First, in 2022, the rainfall started late in the season. Compared to the monthly average rainfall for September and October between 2003 and 2021, there was 137 mm of additional monthly rainfall recorded in September and October 2022. Furthermore, the year 2022 was relatively warmer with a 0.71°C increased temperature than the mean annual temperature of the past 20 yr. Second, a new dengue virus serotype, DENV-4, had recently reintroduced/reappeared in 2022 and become the dominant serotype in the country for a large naïve population. Third, the post-pandemic return of normalcy after 2 yr of nonpharmaceutical social measures facilitates extra mosquito breeding habitats, especially in construction sites. Community engagement and regular monitoring and destruction of Aedes mosquitoes' habitats should be prioritized to control dengue virus outbreaks in Bangladesh., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America.)

Zika virus (ZIKV) of the Flaviviridae family is a recently emerged mosquito-borne virus that has been implicated in the surge of the number of microcephaly instances in South America. The recent rapid spread of the virus led to its declaration as a global health emergency by the World Health Organization. The virus is transmitted mainly by the mosquito Aedes aegypti , which is also the vector of dengue virus; however, little is known about the interactions of the virus with the mosquito vector. In this study, we investigated the transcriptome profiles of whole A. aegypti mosquitoes in response to ZIKV infection at 2, 7, and 14 days postinfection using transcriptome sequencing. Results showed changes in the abundance of a large number of transcripts at each time point following infection, with 18 transcripts commonly changed among the three time points. Gene ontology analysis revealed that most of the altered genes are involved in metabolic processes, cellular processes, and proteolysis. In addition, 486 long intergenic noncoding RNAs that were altered upon ZIKV infection were identified. Further, we found changes of a number of potential mRNA target genes correlating with those of altered host microRNAs. The outcomes provide a basic understanding of A. aegypti responses to ZIKV and help to determine host factors involved in replication or mosquito host antiviral response against the virus. IMPORTANCE Vector-borne viruses pose great risks to human health. Zika virus has recently emerged as a global threat, rapidly expanding its distribution. Understanding the interactions of the virus with mosquito vectors at the molecular level is vital for devising new approaches in inhibiting virus transmission. In this study, we embarked on analyzing the transcriptional response of Aedes aegypti mosquitoes to Zika virus infection. Results showed large changes in both coding and long noncoding RNAs. Analysis of these genes showed similarities with other flaviviruses, including dengue virus, which is transmitted by the same mosquito vector. The outcomes provide a global picture of changes in the mosquito vector in response to Zika virus infection.

Arthropods harbor a diverse array of microbes that profoundly influence many aspects of host biology, including vector competence. Additionally, symbionts can be engineered to produce molecules that inhibit pathogens. Due to their intimate association with the host, microbes have developed strategies that facilitate their transmission, either horizontally or vertically, to conspecifics. These attributes make microbes attractive agents for applied strategies to control arthropod-borne disease. Here we discuss the recent advances in microbial control approaches to reduce the burden of pathogens such as Zika, Dengue and Chikungunya viruses, and Trypanosome and Plasmodium parasites. We also highlight where further investigation is warranted.

Mycoplasmas possess complex pathogenicity determinants that are largely unknown at the molecular level. Mycoplasma agalactiae serves as a useful model to study the molecular basis of mycoplasma pathogenicity. The generation and in vivo screening of a transposon mutant library of M. agalactiae were employed to unravel its host colonization factors. Tn4001mod mutants were sequenced using a novel sequencing method, and functionally heterogeneous pools containing 15 to 19 selected mutants were screened simultaneously through two successive cycles of sheep intramammary infections. A PCR-based negative selection method was employed to identify mutants that failed to colonize the udders and draining lymph nodes in the animals. A total of 14 different mutants found to be absent from ≥ 95% of samples were identified and subsequently verified via a second round of stringent confirmatory screening where 100% absence was considered attenuation. Using this criterion, seven mutants with insertions in genes MAG1050, MAG2540, MAG3390, uhpT, eutD, adhT, and MAG4460 were not recovered from any of the infected animals. Among the attenuated mutants, many contain disruptions in hypothetical genes, implying their previously unknown role in M. agalactiae pathogenicity. These data indicate the putative role of functionally different genes, including hypothetical ones, in the pathogenesis of M. agalactiae. Defining the precise functions of the identified genes is anticipated to increase our understanding of M. agalactiae infections and to develop successful intervention strategies against it., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)