Biology subjects

Hegde, S.

Publications and source records attributed to Hegde, S..

47 records · Page 3Linked to original sources

bioRxiv2020

Mayaro Virus Infection Elicits an Innate Immune Response and Represses Autophagy in Anopheles stephensi

Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naive 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 suggested activation of the Toll pathway at 7 dpi 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 SUMMARYMayaro 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 discover 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.

microbiology↗

bioRxiv2020

Genomic and microscopic evidence of stable high density and maternally inherited Wolbachia infections in Anopheles mosquitoes

Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has been detected within populations of Anopheles (An.) malaria vectors. In the An. gambiae complex, the primary vectors in Sub-Saharan Africa, Wolbachia strains are at low density and infection frequencies in wild populations. PCR-independent evidence is required to determine whether Wolbachia strains are true endosymbionts in Anopheles given most studies to date have used nested-PCR to identify strains. Here we report high-density strains found 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 strains obtained genome depths and coverages comparable to other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present indicating these strains possess the capacity to induce the phenotype cytoplasmic incompatibility which allows Wolbachia to spread through populations. The characteristics of these two strains suggest they are ideal candidates for Wolbachia biocontrol strategies in Anopheles.

microbiology↗

bioRxiv2020

Regulated distribution of mitochondria in touch receptor neurons of C. elegans influences touch response

Density of mitochondria and their localization at specific sub-cellular regions of the neurons is regulated by molecular motors, their adaptors and the cytoskeleton. However, the regulation of the mitochondrial density, the positioning of mitochondria along the neuronal process and the role of axonal mitochondria in neuronal function remain poorly understood. This study shows that the density of mitochondria in C. elegans touch receptor neuron processes remains constant through development. Simulations show that mitochondrial positioning along parts of the neuronal process that are devoid of synapses is regulated. Additionally, we also demonstrate that axonal mitochondria are necessary for maintaining touch responsiveness.

neuroscience↗

bioRxiv2020

Microbial interactions in the mosquito gut determine Serratia colonization and blood feeding propensity.

How microbe-microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behaviour; Serratia exposure to Ae. aegypti disrupted their feeding behaviour, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host-microbe interactions and provides evidence that microbial interactions influence mosquito behaviour.

microbiology↗

bioRxiv2019

Gut-associated bacteria invade the midgut epithelium of Aedes aegypti and stimulate innate immunity and suppress Zika virus infection in cells.

Microbiota within mosquitoes influence nutrition, immunity, fecundity, and the capacity to transmit pathogens. Despite their importance, we have a limited understanding of host-microbiota interactions, especially at the cellular level. It is evident bacterial symbionts that are localized within the midgut also infect other organs within the mosquito; however, the route these symbionts take to colonize other tissues is unknown. Here, utilizing the gentamicin protection assay, we showed that the bacterial symbionts Cedecea and Serratia have the capacity to invade and reside intracellularly within mosquito cells. Symbiotic bacteria were found within a vacuole and bacterial replication was observed in mosquito cell by transmission electron microscopy, indicating bacteria were adapted to the intracellular milieu. Using gene silencing, we determined that bacteria exploited host factors, including actin and integrin receptors, to actively invade mosquito cells. As microbiota can affect pathogens within mosquitoes, we examined the influence of intracellular symbionts on Zika virus (ZIKV) infection. Mosquito cells harbouring intracellular bacteria had significantly less ZIKV compared to uninfected cells or cells exposed to non-invasive bacteria. Intracellular bacteria were observed to substantially upregulate the Toll and IMD innate immune pathways, providing a possible mechanism mediating these anti-viral effects. Examining mono-axenically infected mosquitoes using transmission electron and fluorescent microscopy revealed that bacteria occupied an intracellular niche in vivo. Our results provided evidence that bacteria that associate with the midgut of mosquitoes have intracellular lifestyles which likely have implications for mosquito biology and pathogen infection. This study expands our understanding of host-microbiota interactions in mosquitoes, which is important as symbiont microbes are being exploited for vector control strategies.

microbiology↗

bioRxiv2019

Dynamic clustering regulates activity of mechanosensitive membrane channels

Mechanosensitive channels regulate the flux of solutes between the cell and its environment in response to mechanical stimuli. Recent in vitro experiments have suggested that bacterial mechanosensitive channels separate into 2D liquid-like clusters, exhibiting cooperative activity. To understand the functional role of the process and its physical mechanism, we develop a coarse-grained model of mechanosensitive channels embedded in a membrane, exposed to an osmotic shock. We find that the channel aggregation into liquid-like clusters promotes the closure of individual channels. The extent of clustering is highly dependent on the channel concentration and membrane stress. We show that this behaviour yields a tightly regulated gating system, whereby at high tensions channels gate individually, and at lower tensions the channels spontaneously aggregate and inactivate. We suggest that this positive feedback protects the cell against excessive loss of cytoplasmic content. Our results point to a novel role of functional micro-phase separation in the cell membrane.

biophysics↗

bioRxiv2018

CRISPR/Cas9-mediated gene deletion of the ompA gene in an Enterobacter gut symbiont impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

BackgroundSymbiotic 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 FindingsTo investigate the role of bacterial 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 an Enterobacter 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 adults 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 bacterial 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 these symbionts genome and demonstrated that these genes were functional in vitro and in vivo. Conclusions/SignificanceOur results shed insights onto 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 summaryMicrobiota 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 knock out a membrane protein in Enterobacter, which is a dominant member of the mosquito microbiome. The mutant strain lacked the 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 control arthropod-borne disease.

microbiology↗

bioRxiv2018

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

Anopheles (An.) mosquitoes contain bacteria that can influence Plasmodium parasites. Wolbachia, a common insect endosymbiont, has historically been considered absent from Anopheles but has recently been found in An. gambiae populations. Here, we assessed a range of Anopheles species from five malaria-endemic countries for Wolbachia and Plasmodium infection. Strikingly, we found Wolbachia infections in An. coluzzii, An. gambiae s.s, An. arabiensis, An. moucheti and An. species A increasing the number of Anopheles species known to be naturally infected by this endosymbiont. Molecular analysis suggests the presence of phylogenetically diverse novel strains, while qPCR and 16S rRNA sequencing indicates that Wolbachia is the dominant member of the microbiota in An. moucheti and An. species A. We found no evidence of Wolbachia/Asaia co-infections, and presence of these endosymbionts did not have significant effects on malaria prevalence. We discuss the importance of novel Wolbachia strains in Anopheles and potential implications for disease control.

microbiology↗

bioRxiv2018

Microbiome interaction networks and community structure from lab-reared and field-collected Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus mosquito vectors.

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.

microbiology↗

bioRxiv2018

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

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 {degrees}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.\n\nAuthor summaryMosquitoes spread many human pathogens and novel approaches are required to reduce the burden of mosquito-borne disease. One promising approach is transferring Wolbachia into Aedes aegypti mosquitoes where it blocks transmission of arboviruses like dengue, Zika and Yellow fever viruses and spreads through mosquito populations. For effective evaluation of this approach, regular surveillance of Wolbachia infections in Ae. aegypti is required, but current diagnostic tools are not well suited to support these critical surveillance needs. To fill this need we developed a simple, robust and inexpensive assay to identify Ae. aegypti mosquitoes and Wolbachia using our unique one-pot assay platform, LAMP-OSD, which uses loop-mediated isothermal amplification to amplify nucleic acid targets at a single temperature. Unlike other LAMP-based tests, our assays assure accuracy by coupling amplification with novel nucleic acid strand displacement (OSD) probes that hybridize to specific sequences in LAMP amplification products and thereby generate simple yes/no readout of fluorescence readable by human eye and by off-the-shelf cellphones. To facilitate field use, we developed our assays so they are compatible with crushed mosquito homogenate as the template, meaning no nucleic acid extraction is required. In blinded tests using field collected mosquitoes, LAMP-OSD-cellphone tests performed robustly to identify 29 of 30 Ae. aegypti even after 3 weeks of storage at 37 {degrees}C while producing only one false positive out of 60 non-specific mosquitoes. Similarly, our assay could identify Wolbachia in field-caught Aedes albopictus without producing any false positives. Our easy to use and easy to interpret assays should facilitate widespread field mosquito surveillance with minimal instrumentation and high accuracy.

molecular biology↗

bioRxiv2017

Global transcriptome analysis of Aedes aegypti mosquitoes in response to Zika virus infection

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 that also vectors 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 Ae. aegypti mosquitoes in response to ZIKV infection at 2, 7, and 14 days post-infection using RNA-Seq. 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 process, cellular process and proteolysis. In addition, 486 long intergenic non-coding RNAs were identified that were altered upon ZIKV infection. Further, we found correlational changes of a number of potential mRNA target genes with that of altered host microRNAs. The outcomes provide a basic understanding of Ae. aegypti responses to ZIKV and helps to determine host factors involved in replication or mosquito host anti-viral response against the virus.\n\nImportanceVector-borne viruses pose great risks on 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 both in coding and long non-coding 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.

microbiology↗