Tanwee Das De

Salivary glands harbor more diverse microbial communities than gut in Anopheles culicifacies

BackgroundIn recent years, it has been well documented that gut flora not only influence mosquito physiology, but also significantly alter vector competency. Although, salivary gland and gut constitute key partners of the digestive system, it is still believed that salivary glands may harbor less flora than gut (Parasit Vectors 6: 146, 2013).MethodsUsing a metagenomic approach, we have identified for the first time the diverse microbial community associated with these two physiologically different tissues of the digestive system in the mosquito Anopheles culicifacies.ResultsA total of 17 different phyla could be assigned to the whole metagenomic dataset, predominated by the phylum Proteobacteria, Firmicutes, Bacteriodetes, Tenericutes and Actinomycetes. Common bacteria included the members of Enhydrobacter, Agromonas, Serratia, Ralsonia, Lactobacillus, Pseudomonas, Streptococcus, Rubrobacter, Anaerococcus, Methylobacterium, Turicibacter, Elizabethkingia etc. in both the tissues representing ‘core microbiota’ of the mosquito digestive system. Salivary associated unique bacterial community included the members of Chloriflexi, Chlorobi, Cyanobacteria, Nitrospira, TM7, Armatimonadetes, Planctomycetes, Fibrobacteres etc.ConclusionWe find that the salivary gland microbial community structure is more diverse than gut of the mosquito, probably due to differential feeding associated engagements such as food acquisition, ingestion and digestion processes.

Hemocytome: deep sequencing analysis of mosquito blood cells in Indian malarial vector Anopheles stephensi.

Hemocytes are tiny circulating blood cells of insects known to play multiple roles in physiological as well as cellular immune responses. However, the molecular nature of hemocytes in blood feeding insects, especially mosquitoes which transmit several deadly diseases such as malaria, dengue etc. is still limited. Therefore, to know the basic molecular composition of naïve mosquito hemocyte encoded proteins, we sequenced RNA-Seq library and analyzed a total of 13,105,858 Illumina sequencing reads in the mosquito Anopheles stephensi, an urban malarial vector in India. Denovo assembly approach yielded a buildup of 3025 contigs, for molecular and functional annotation. A total of 1829 contigs (48%) could be mapped to the mosquito transcript database, while out of remaining 1196 unmatched contigs, at least 1108 contigs i.e. 40% of total contigs, yielded a significant match to the available draft genome. ImmunoDB analysis predicted a total of 88 putative hemocyte transcripts belonging to 11 immune family proteins. A comprehensive molecular analysis of several unique transcripts including novel LRR, Holotricin, OBP, NiFU, that are involved in immunity, chemo sensing, cell-cell communication, nitrogen fixation/metabolism etc. provides initial evidence that mosquito hemocytes carry unique ability to meet and manage cell specific diverse functions of the mosquito blood. An unexpected observation of abundant transcripts encoding hypothetical proteins with unknown functions indicated that a much of the hemocyte biology remains to be understood.

Unraveling dual feeding associated molecular complexity of salivary glands in the mosquito Anopheles culicifacies

ABSTRACT Mosquito salivary glands are well known to facilitate meal acquisition, however the fundamental question on how adult female salivary gland manages molecular responses during sugar versus blood meal uptake remains unanswered. To investigate these responses, we analyzed a total of 58.5 million raw reads generated from two independent RNAseq libraries of the salivary glands collected from 3–4 day-old sugar and blood fed Anopheles culicifacies mosquitoes. Comprehensive functional annotation analysis of 10,931 contigs unraveled that salivary glands may encode diverse nature of proteins in response to distinct physiological feeding status. Digital gene expression analysis and PCR validation indicated that first blood meal significantly alters the molecular architecture of the salivary glands. Comparative microscopic analysis also revealed that first blood meal uptake not only causes an alteration of at least 12–22% of morphological features of the salivary glands but also results in cellular changes e.g. apoptosis, confirming together that adult female salivary glands are specialized organs to manage meal specific responses. Unraveling the underlying mechanism of mosquito salivary gene expression, controlling dual feeding associated responses may provide a new opportunity to control vector borne diseases.

A Synergistic Transcriptional Regulation of Olfactory Genes Drives Blood-Feeding Associated Complex Behavioral Responses in the Mosquito Anopheles culicifacies.

Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in the adult female mosquitoes may provide an opportunity to design new molecular strategy to disrupt human-mosquito interactions. Although there is a great progress in the field of mosquito olfaction and chemo-detection, little is known about the sex-specific evolution of the specialized olfactory system of adult female mosquitoes that enables them to drive and manage the complex blood-feeding associated behavioral responses. A comprehensive RNA-Seq analysis of prior and post blood meal olfactory system of An. culicifacies mosquito revealed a minor but unique change in the nature and regulation of key olfactory genes that may play a pivotal role in managing diverse behavioral responses. Based on age-dependent transcriptional profiling, we further demonstrated that adult female mosquitos chemosensory system gradually learned and matured to drive the host-seeking and blood feeding behavior at the age of 5–6 days. A time scale expression analysis of Odorant Binding Proteins (OBPs) unravels unique association with a late evening to midnight peak biting time. Blood meal-induced switching of unique sets of OBP genes and Odorant Receptors (Ors) expression coincides with the change in the innate physiological status of the mosquitoes. Blood meal follows up experiments further provide enough evidence that how a synergistic and concurrent action of OBPs-Ors may drive “prior and post blood meal” associated complex behavioral events. A dominant expression of two sensory appendages proteins (SAP-1 & SAP2) in the legs of An. culicifacies suggests that this mosquito species may draw an extra advantage of having more sensitive appendages than An. stephensi, an urban malarial vector in the Indian subcontinents. Finally, our molecular modeling analysis predicts crucial amino acid residues for future functional characterization of the sensory appendages proteins which may play a central role in regulating multiple behaviors of An. culicifacies mosquito. SIGNIFICANCE Evolution and adaptation of blood feeding behavior not only favored the reproductive success of adult female mosquitoes but also make them important disease-transmitting vectors. An environmental exposure after emergence may favor the broadly tuned olfactory system of mosquitoes to drive complex behavioral responses. But, how these olfactory derived genetic factors manage female specific “pre and post” blood meal associated complex behavioral responses are not well known. Our findings suggest that a synergistic action of olfactory factors may govern an innate to prime learning strategy to facilitate rapid blood meal acquisition and downstream behavioral activities. A species-specific transcriptional profiling and an in-silico analysis predict that “sensory appendages protein” may be a unique target to design disorientation strategy against the mosquito Anopheles culicifacies.

Interorgan Molecular Communication Strategies of “Local” and “Systemic” Innate Immune Responses in Mosquito Anopheles stephensi

Mosquitoes that transmit many deadly infectious diseases also need to keep fighting against many microbial infections. Constitutive expression of multiple antimicrobial peptides (AMPs) in almost all body tissues is believed to facilitate the effective management of these local infections. When any infection breaches the local barrier, AMPs are induced rapidly in non-target tissues such as hemocytes (HCs) and establish their co-ordination with systemic immune effectors to clear off the body infection. But how interorgan immune communication is managed during local and systemic infections remain largely unknown. To understand this interorgan molecular relationship, we identified, extensively profiled and compared the expression of AMPs in three important mosquito tissues viz. midgut, fat body (FB), and HCs. dsRNA-mediated AMPs silencing suggests that mosquito tissues are able to manage an optimal expression of AMPs at the physiological level. We also examined the possible contribution of two important immune regulator genes relish (REL) and nitric oxide synthase, controlling AMPs expression in these tissues during local or systemic infections. We show that each tissue has a unique ability to respond to local/systemic challenges, but HCs are more specialized to recognize and discriminate-specific antigens than gut and FB. Our investigation also revealed that both REL and NO participate in the overall management of the interorgan immune responses, but at the same time each tissue also has its own ability to maintain the interorgan flow of signals. In our knowledge, this is the first large-scale study examining the interorgan immune relationship in the mosquito.

Sex specific molecular responses of quick-to-court protein in Indian malarial vector Anopheles culicifacies: conflict of mating versus blood feeding behaviour

Understanding the molecular basis of mosquito behavioural complexity plays a central role in designing novel molecular tools to fight against their vector-borne diseases. Although the olfactory system plays an important role in guiding and managing many behavioural responses including feeding and mating, but the sex-specific regulation of olfactory responses remain poorly investigated. From our ongoing transcriptomic data annotation of olfactory tissue of blood fed adult female An. culicifacies mosquitoes; we have identified a 383 bp long unique transcript encoding a Drosophila homolog of the quick-to-court protein. Previously this was shown to regulate courtship behaviour in adult male Drosophila. A comprehensive in silico analysis of the quick-to-court (qtc) gene of An. culicifacies (Ac-qtc) predicts a 1536 bp single copy gene encoding 511 amino acid protein, having a high degree of conservation with other insect homologs. The age-dependent increased expression of putative Ac-qtc correlated with the maturation of the olfactory system, necessary to meet the sex-specific conflicting demand of mating (mate finding) versus host-seeking behavioural responses. Sixteen to eighteen hours of starvation did not alter Ac-qtc expression in both sexes, however, blood feeding significantly modulated its response in the adult female mosquitoes, confirming that it may not be involved in sugar feeding associated behavioural regulation. Finally, a dual behavioural and molecular assay indicated that natural dysregulation of Ac-qtc in the late evening might promote the mating events for successful insemination. We hypothesize that Ac-qtc may play a unique role to regulate the sex-specific conflicting demand of mosquito courtship behaviour versus blood feeding behaviour in the adult female mosquitoes. Further elucidation of this molecular mechanism may provide further information to evaluate Ac-qtc as a key molecular target for mosquito-borne disease management.

Deep sequencing revealed molecular signature of horizontal gene transfer of plant like transcripts in the mosquito Anopheles culicifacies: an evolutionary puzzle.

In prokaryotes, horizontal gene transfer (HGT) has been regarded as an important evolutionary drive to acquire and retain beneficial genes for their survival in diverse ecologies. However, in eukaryotes, the functional role of HGTs remains questionable, although current genomic tools are providing increased evidence of acquisition of novel traits within non-mating metazoan species. Here, we provide another transcriptomic evidence for the acquisition of massive plant genes in the mosquito, Anopheles culicifacies. Our multiple experimental validations including genomic PCR, RT-PCR, real-time PCR, immuno-blotting and immuno-florescence microscopy, confirmed that plant like transcripts (PLTs) are of mosquito origin and may encode functional proteins. A comprehensive molecular analysis of the PLTs and ongoing metagenomic analysis of salivary microbiome provide initial clues that mosquitoes may have survival benefits through the acquisition of nuclear as well as chloroplast encoded plant genes. Our findings of PLTs further support the similar questionable observation of HGTs in other higher organisms, which is still a controversial and debatable issue in the community of evolutionists. We believe future understanding of the underlying mechanism of the feeding associated molecular responses may shed new insights in the functional role of PLTs in the mosquito.

Transcriptional responses of attractin gene in the mosquito Anopheles culicifacies: A synergistic neuro-olfactory regulation

Background & objectives: Attractin, is a large multi-domain protein which has regulatory functions in multiple physiological processes and thus have strong therapeutic potential. In invertebrates, it was first identified as a water-borne protein pheromone that plays important role in chemical communication and coordinates reproductive activities. But its role in mosquitoes/insects remains unknown. Our unexpected discovery of attractin homolog from the olfactory tissue of Anopheles culicifacies mosquito prompted us to investigate the possible role of Ac-attractin (Ac-atrn) in diverse behavioural responses, e.g. feeding, mating and other non-genetic stresses. Methods: A homology search analysis was performed to identify the full length attractin (Ac-atrn) gene of Anopheles culicifacies mosquito. To unravel its molecular function during external and internal stresses, extensive real-time PCR was performed in the neuro-olfactory tissues of the adult mosquitoes as well as in the larval stages. Further, a behavioural assay was conducted to elucidate its role in mosquitoes mating behaviour. Results: The results indicated that Ac-atrn is a 3942 bp long transcript which encodes a 1313 amino acid protein, having multiple domains including CUB, EGF, Keltch, etc, with 80–90% homology to other insect/mosquito homologs. Ac-atrn gene was dominantly expressed in the young larvae and its expression was elevated in response to the fresh food supply in the starved larvae. Cold stress temporarily arrested the expression of Ac-atrn gene. In case of adult mosquitoes, olfactory and brain tissue showed relatively higher expression of Ac-atrn than reproductive organs. Although, starvation did not yield significant changes in olfactory tissues, but aging and nutritional stress modulated Ac-atrn expression in the brain tissue. Furthermore, a circadian rhythm dependent change in the expression of Ac-atrn of virgin and mated mosquitoes (both sexes), indicates that Ac-atrn might also have a pheromone guided role during swarm formation and mating behaviour. Interpretation & conclusion: The relative expression profiling of Ac-atrn gene in the larvae during nutritional and cold stress suggested its possible role in mediating chemical communication towards the food source and in thermal regulation of young larvae. Similarly, it might have crucial regulatory role in the stress management and survival of adult mosquitoes. The results revealed that Ac-atrn gene is a global regulator of many physiological processes in mosquitoes including stress response and mating behaviour and thus might be a potential target to design novel intervention strategy against mosquitoes.

Sex Specific Molecular Responses Of Quick-To-Court In Indian Malarial Vector Anopheles culicifacies: Conflict Of Mating And/Or Blood Feeding?

Understanding the molecular basis of mosquito behavioral complexity is central to the design of novel molecular tool to fight against their vector borne diseases. Although, olfactory system play important role to guide and manage many behavioral co-ordinates including feeding, mating, breeding etc., but the sex specific regulation of olfactory responses remains unanswered. From our ongoing transcriptomic data annotation of blood fed adult female olfactory tissue of A. culicifacies mosquito, we identified a 383 bp long unique transcript encoding Drosophila homolog of Quick-To-Court protein, previously shown to regulate the courtship behavior in adult male Drosophila. A comprehensive in silico analysis predicts Ac-qtc is 1536 bp long single copy gene encoding 511 AA long protein, having high degree of conservation with other insect homolog. Age dependent increased expression of putative Ac-qtc in the naïve mosquitoes correlates the maturation of olfactory system, necessary to meet sex specific conflicting demand of mating (mate finding) vs. host-seeking behavioral responses. Though, 16-18 hour of starvation did not altered Ac-qtc expression in both the sexes, however blood feeding significantly modulated its response in the adult female mosquitoes, confirming that it may not be involved in sugar feeding associated behavioural regulation. Finally, a behavioural-cum-molecular assay indicated that natural dysregulation of Ac-qtc in late evening may promotes key mating event of successful insemination process. We hypothesize that Ac-qtc may play unique role to meet and manage the sex specific conflicting demand of mosquito courtship behaviour and/or blood feeding behaviour in the adult female mosquitoes. A molecular mechanism elucidation may provide new knowledge to consider Ac-qtc as a key molecular target for mosquito borne disease management.

A synergistic transcriptional regulation of olfactory genes derives complex behavioral responses in the mosquito Anopheles culicifacies

Decoding the molecular basis of host seeking and blood feeding behavioral evolution/adaptation in the adult female mosquito may provide an opportunity to design new molecular strategy to disrupt human-mosquito interactions. However, despite the great progress in the field of mosquito olfaction and chemo-detection, little is known that how the sex-specific specialization of the olfactory system enables adult female mosquitoes to derive and manage complex blood feeding associated behavioral responses. A comprehensive RNAseq analysis of prior and post blood meal olfactory system of An. culicifacies mosquito revealed that a minor but unique change in the nature and regulation of key olfactory genes play a pivotal role in managing diverse behavioral responses. Age dependent transcriptional profiling demonstrated that adult female mosquito’s chemosensory system gradually learned and matured to drive the host-seeking and blood feeding behavior at the age of 5-6 days. A zeitgeber time scale expression analysis of Odorant Binding Proteins (OBPs) unravels unique association with a late evening to midnight peak biting time. Blood meal-induced switching of unique sets of OBP genes and Odorant Receptors (ORs) expression coincides with the change in the innate physiological status of the mosquitoes. Blood meal follows up experiments provide enough evidence that how a synergistic and concurrent action of OBPs-ORs may drive ‘prior and post blood meal’ complex behavioral events. Finally, tissue-specific gene expression analysis and molecular modelling predicted two uncharacterized novel sensory appendages proteins (SAP-1 & SAP2) unique to An. culicifacies mosquito and may play a central role in the host-seeking behavior. Significance Evolution and adaptation of blood feeding behavior not only favored the reproductive success of adult female mosquito but also make them an important disease vectors. Immediately after emergence, an environmental exposure may favor the broadly tuned olfactory system of mosquitoes to derive complex behavioral responses. But, how these olfactory derived genetic factors manage female specific ‘pre and post’ blood meal associated complex behavioral responses are not well known. We unraveled synergistic actions of olfactory factors governs an innate to prime learning strategy to facilitate rapid blood meal acquisition and downstream behavioral activities. A species-specific transcriptional profiling and an in-silico analysis predict novel ‘sensory appendages protein’, as a unique target to design disorientation strategy against the mosquito Anopheles culicifacies.