Cost analyses for malaria molecular diagnosis for research planners in India and beyond.

BACKGROUND: Malaria elimination mandates early and accurate diagnosis of infection. Although malaria diagnosis is programmatically dependent on microscopy/RDTs, molecular diagnosis has much better diagnostic accuracy. Higher cost of molecular diagnoses is a recognized challenge for use at the point of care. Because funding is always a recognized constraint, we performed financial cost-analyses of available molecular platforms for better utilization of available budget. METHODS: Two strategies were applied to deduce the cost per sample. Strategy 1 included recurring components (RC) in minimum pack size, and biologist's time whereas strategy 2 included only RC and non-recurring components and costs are calculated for sample sizes (1-1,000,000) to infer the sample size effect. RESULTS: Spin column-based manual DNA extraction (US$ 3.93 per sample) is the lowest-cost method, followed by magnetic bead-based automated, semi-automated, and PCI-based manual method. Further, DNA extraction cost per sample via spin column-based manual method and semi-automated method decreases with an increase in sample size up to 10,000. Real-time PCRs are ~ 2-fold more economical than conventional PCR, regardless of sample size. CONCLUSIONS: This study is the first for malaria to estimate systematic molecular diagnosis financial costs. Kit-based and automated methods may replace conventional DNA extraction and amplification methods for a frugal high-throughput diagnosis.

Enhanced anti-malarial efficacy of mefloquine delivered via cationic liposome in a murine model of experimental cerebral malaria.

Malaria is a longstanding global health challenge that continues to afflict over 90 countries located in tropical and subtropical regions of the globe. The rise of drug-resistant malarial parasites has curtailed the therapeutic efficacy of a number of once-effective anti-malarials, including mefloquine. In the present study, we have taken advantage of drug encapsulation approach to elevate the anti-malarial potential of mefloquine. Encouragingly, our findings unveil that liposomal formulations of mefloquine outperform equivalent doses of free mefloquine, both in laboratory cultures and in a murine model of malaria. Intriguingly, a cationic liposomal mefloquine formulation, administered at four successive doses of 3 mg/kg body weight, achieves complete resolution of cerebral malaria in the murine model while avoiding noticeable toxic repercussions. Altogether, our study furnishes pre-clinical validation for a therapeutic strategy that can remarkably enhance the drug efficacy, offering a revitalizing solution for failing anti-malarials.

Meta-analysis on Plasmodium falciparum sulfadoxine-pyrimethamine resistance-conferring mutations in India identifies hot spots for genetic surveillance.

BACKGROUND: India is on track to eliminate malaria by 2030 but emerging resistance to first-line antimalarials is a recognised threat. Two instances of rapid development, spread, and natural selection of drug-resistant mutant parasites in India (chloroquine across the country and artesunate + sulfadoxine-pyrimethamine [AS+SP] in the northeastern states) translated into drug policy changes for Plasmodium falciparum malaria in 2010 and 2013, respectively. Considering these rapid changes in the SP drug resistance-conferring mutation profile of P. falciparum, there is a need to systematically monitor the validated mutations in Pfdhfr and Pfdhps genes across India alongside AS+SP therapeutic efficacy studies. There has been no robust, systematic countrywide surveillance reported for these parameters in India, hence the current study was undertaken. METHODS: Studies that reported data on WHO-validated SP resistance markers in P. falciparum across India from 2008 to January 2023 were included. Five major databases, PubMedⓇ, Web of ScienceTM, ScopusⓇ, EmbaseⓇ, and Google Scholar, were exhaustively searched. Individual and pooled prevalence estimates of mutations were obtained through random- and fixed-effect models. Data were depicted using forest plots created with a 95% confidence interval. The study is registered with PROSPERO (CRD42021236012). RESULTS: A total of 37 publications, and 533 Pfdhfr and 134 Pfdhps National Centre of Biotechnology Information (NCBI) DNA sequences were included from >4000 samples. The study included information from 80 districts, 21 states and 3 union territories (UTs) from India. The two PfDHFR mutations, C59R (62%) and S108N (74%), were the most prevalent mutations (pooled estimates 61% and 71%, respectively) and appeared to be stabilised/fixed. Although rarest overall, the prevalence of I164L was observed to be as high as 32%. The PfDHFR double mutants were the most prevalent overall (51%; pooled 42%). The prevalence of triple and quadruple mutations was 6% and 5%, respectively, and is an immediate concern for some states. The most prevalent PfDHPS mutation was A437G (39%), followed by K540E (25%) and A581G (12%). There was a low overall prevalence of PfDHFR/PfDHPS quintuple and sextuple mutations but surveillance for these mutations is critical for some areas. CONCLUSION: The analyses span the two critical policy changes, highlight the areas of concern, and guide policymakers in strategising and refining the anti-malaria drug policy for malaria elimination. The results of the analyses also highlight the SP-resistance hot spots, critical gaps and challenges, and indicate that focal and local malaria genetic surveillance (including drug-resistance markers) is needed until malaria is successfully eliminated.

Evaluation of the Effects of Topical Fluoride Varnish and Fluoride Releasing Adhesive on Shear Bond Strength of Orthodontic Brackets: An In Vitro Study.

Aim: The aim of this study was to evaluate the in vitro effects of topical fluoride varnish and fluoride-releasing adhesive on the shear bond strength (SBS) of orthodontic brackets. Materials and methods: A total of 60 extracted premolars were bonded to 0.022, stainless steel brackets and equally divided into three groups (n = 20) based on the adhesive used-Group I- Transbond XT Plus color change (3M Unitek), Group II- Transbond XT followed by application of fluoride varnish, and Group III- Transbond XT (3M Unitek) adhesive and their bond strengths were compared. Brackets were debonded with a universal testing machine. The modified adhesive remnant index (ARI) was also recorded. Data were analyzed by using an analysis of variance, and a post hoc test was performed for multiple comparisons among the groups. Results: There were no significant differences between the SBSs (p = 0.91) between the groups. Also, no significant difference was found in the modified ARI (p = 0.093). Conclusion: The orthodontic adhesives used in our study, with or without the application of topical fluoride varnish, did not have a significant effect on the bond strengths of brackets. Clinical significance: Adhesives evaluated in this study can be successfully used for bonding brackets. How to cite this article: Chauhan C, Mangla R, Gandhi G, et al. Evaluation of the Effects of Topical Fluoride Varnish and Fluoride Releasing Adhesive on Shear Bond Strength of Orthodontic Brackets: An In Vitro Study. Int J Clin Pediatr Dent 2023;16(1):37-41.

Salivary AsHPX12 influences pre-blood meal associated behavioral properties in Anopheles stephensi.

BACKGROUND & OBJECTIVES: A successful blood meal acquisition process by an adult female mosquito is accomplished through salivary glands, which releases a cocktail of proteins to counteract the vertebrate host's immune homeostasis. Here, we characterize a salivary-specific Heme peroxidase family member HPX12, originally identified from Plasmodium vivax infected salivary RNAseq data of the mosquito Anopheles stephensi. METHODS: To demonstrate we utilized a comprehensive in silico and functional genomics approach. RESULTS: Our dsRNA-mediated silencing experiments demonstrate that salivary AsHPX12 may regulate pre-blood meal-associated behavioral properties such as probing time, probing propensity, and host attraction. Altered expression of the salivary secretory and antennal proteins expression may have accounted for salivary homeostasis disruption resulting in the unusual fast release of salivary cocktail proteins and delayed acquisition of blood meal in the AsHPX12 knockdown mosquitoes. We also observed a significant parallel transcriptional modulation in response to blood feeding and P. vivax infection. INTERPRETATION & CONCLUSION: With this work, we establish a possible functional correlation of AsHPX12 role in the maintenance of salivary physiological-homeostasis, and Plasmodium sporozoites survival/transmission, though the mechanism is yet to unravel.

An Augmented Method for Collecting PLGA Nanoparticles and the Fabrication of 1, 3, 4, 6-Tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (Ac42AzGlc)-Loaded PLGA Nanoparticles for Efficient and Prospective in Vivo Metabolic Processing.

We investigated two ways for fabricating 1, 3, 4, 6-tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (Ac42AzGlc)-loaded poly (lactic-co-glycolic acid) PLGA nanoparticles in this article : 1) single emulsion solvent evaporation and 2) the nanoprecipitation method. Among the available methods of collecting nanoparticles using an ultra-high-speed centrifuge, we improvised a less-known method for collecting synthesized nanoparticles without a high-speed centrifuge, based on molecular weight (MW)-dependent centrifugal filters. These nanoparticles were collected in a tabletop centrifuge at a meager centrifugal force in the range of 200-300 xg whereas the conventional high-speed centrifuge method for nanoparticle recovery results in a hard nanoparticle pellet with poor resuspendability which hampers the yield and outcomes of the product. The Ac42AzGlc-loaded PLGA nanoparticles were spherical in shape with consistent and reliable nanometric particle size. The polydispersity indices were well within the acceptable limits. The preliminary studies in RAW 264.7 cell and C57BL/6 mice advocated efficient engineering in the former; however, the latter needs further confirmatory investigations. Preliminary in vivo studies with un-encapsulated Ac42AzGlc showed poor engineering of cardiac glycoproteins, opening up avenues for Ac42AzGlc-loaded nanoparticles for improved bioavailability and efficient metabolic engineering.

Bidirectional Microbiome-Gut-Brain-Axis Communication Influences Metabolic Switch-Associated Responses in the Mosquito Anopheles culicifacies.

The periodic ingestion of a protein-rich blood meal by adult female mosquitoes causes a drastic metabolic change in their innate physiological status, which is referred to as a 'metabolic switch'. While understanding the neural circuits for host-seeking is modestly attended, how the gut 'metabolic switch' modulates brain functions, and resilience to physiological homeostasis, remains unexplored. Here, through a comparative brain RNA-Seq study, we demonstrate that the protein-rich diet induces the expression of brain transcripts related to mitochondrial function and energy metabolism, possibly causing a shift in the brain's engagement to manage organismal homeostasis. A dynamic mRNA expression pattern of neuro-signaling and neuro-modulatory genes in both the gut and brain likely establishes an active gut-brain communication. The disruption of this communication through decapitation does not affect the modulation of the neuro-modulator receptor genes in the gut. In parallel, an unusual and paramount shift in the level of neurotransmitters (NTs), from the brain to the gut after blood feeding, further supports the idea of the gut's ability to serve as a 'second brain'. After blood-feeding, a moderate enrichment of the gut microbial population, and altered immunity in the gut of histamine receptor-silenced mosquitoes, provide initial evidence that the gut-microbiome plays a crucial role in gut-brain-axis communication. Finally, a comparative metagenomics evaluation of the gut microbiome highlighted that blood-feeding enriches the family members of the Morganellaceae and Pseudomonadaceae bacterial communities. The notable observation of a rapid proliferation of Pseudomonas bacterial sp. and tryptophan enrichment in the gut correlates with the suppression of appetite after blood-feeding. Additionally, altered NTs dynamics of naïve and aseptic mosquitoes provide further evidence that gut-endosymbionts are key modulators for the synthesis of major neuroactive molecules. Our data establish a new conceptual understanding of microbiome-gut-brain-axis communication in mosquitoes.

Functional disruption of transferrin expression alters reproductive physiology in Anopheles culicifacies.

BACKGROUND: Iron metabolism is crucial to maintain optimal physiological homeostasis of every organism and any alteration of the iron concentration (i.e. deficit or excess) can have adverse consequences. Transferrins are glycoproteins that play important role in iron transportation and have been widely characterized in vertebrates and insects, but poorly studied in blood-feeding mosquitoes. RESULTS: We characterized a 2102 bp long transcript AcTrf1a with complete CDS of 1872bp, and 226bp UTR region, encoding putative transferrin homolog protein from mosquito An. culicifacies. A detailed in silico analysis predicts AcTrf1a encodes 624 amino acid (aa) long polypeptide that carries transferrin domain. AcTrf1a also showed a putative N-linked glycosylation site, a characteristic feature of most of the mammalian transferrins and certain non-blood feeding insects. Structure modelling prediction confirms the presence of an iron-binding site at the N-terminal lobe of the transferrin. Our spatial and temporal expression analysis under altered pathophysiological conditions showed that AcTrf1a is abundantly expressed in the fat-body, ovary, and its response is significantly altered (enhanced) after blood meal uptake, and exogenous bacterial challenge. Additionally, non-heme iron supplementation of FeCl3 at 1 mM concentration not only augmented the AcTrf1a transcript expression in fat-body but also enhanced the reproductive fecundity of gravid adult female mosquitoes. RNAi-mediated knockdown of AcTrf1a causes a significant reduction in fecundity, confirming the important role of transferrin in oocyte maturation. CONCLUSION: All together our results advocate that detailed characterization of newly identified AcTrf1a transcript may help to select it as a unique target to impair the mosquito reproductive outcome.

A testis-expressing heme peroxidase HPX12 regulates male fertility in the mosquito Anopheles stephensi.

In vertebrates dysregulation of the antioxidant defense system has a detrimental impact on male fertility and reproductive physiology. However, in insects, especially mosquitoes the importance of sperm quality has been poorly studied. Since long-term storage of healthy and viable sperm earmarks male reproductive competency, we tested whether the heme peroxidase, a member of antioxidant enzyme family proteins, and abundantly expressed in the testis, also influence male fertility in the mosquito An. stephensi. Here, we show that a heme peroxidase 12 (HPX12), is an important cellular factor to protect the sperms from oxidative stress, and maintains semen quality in the male mosquito reproductive organ. We demonstrate that knockdown of the HPX12 not only impairs the sperm parameters such as motility, viability but also causes a significant down-regulation of MAG expressing transcripts such as ASTEI02706, ASTEI00744, ASTEI10266, likely encoding putative Accessory gland proteins. Mating with HPX12 knockdown male mosquitoes, resulted in ~ 50% reduction in egg-laying, coupled with diminished larval hatchability of a gravid female mosquito. Our data further outlines that increased ROS in the HPX12 mRNA depleted mosquitoes is the ultimate cause of sperm disabilities both qualitatively as well as quantitatively. Our data provide evidence that testis expressing AsHPX12 is crucial for maintaining optimal homeostasis for storing and protecting healthy sperms in the male mosquito's reproductive organs. Since, high reproductive capacity directly influences the mosquito population, manipulating male mosquito reproductive physiology could be an attractive tool to combat vector-borne diseases.

Hemocyte RNA-Seq analysis of Indian malarial vectors Anopheles stephensi and Anopheles culicifacies: From similarities to differences.

Anopheles stephensi and Anopheles culicifacies are dominant malarial vectors in urban and rural India, respectively. Both species carry significant biological differences in their behavioral adaptation and immunity, but the genetic basis of these variations are still poorly understood. Here, we uncovered the genetic differences of immune blood cells, that influence several immune-physiological responses. We generated, analyzed and compared the hemocyte RNA-Seq database of both mosquitoes. A total of 5,837,223,769 assembled bases collapsed into 7,595 and 3,791 transcripts, originating from hemocytes of laboratory-reared 3-4 days old naïve (sugar-fed) mosquitoes, Anopheles stephensi and Anopheles culicifacies respectively. Comparative GO annotation analysis revealed that both mosquito hemocytes encode similar proteins. Furthermore, while An. stephensi hemocytes showed a higher percentage of immune transcripts encoding APHAG (Autophagy), IMD (Immune deficiency pathway), PRDX (Peroxiredoxin), SCR (Scavenger receptor), IAP (Inhibitor of apoptosis), GALE (galactoside binding lectins), BGBPs (1,3 beta D glucan binding proteins), CASPs (caspases) and SRRP (Small RNA regulatory pathway), An. culicifacies hemocytes yielded a relatively higher percentage of transcripts encoding CLIP (Clip domain serine protease), FREP (Fibrinogen related proteins), PPO (Prophenol oxidase), SRPN (Serpines), ML (Myeloid differentiation 2-related lipid recognition protein), Toll path and TEP (Thioester protein), family proteins. However, a detailed comparative Interproscan analysis showed An. stephensi mosquito hemocytes encode proteins with increased repeat numbers as compared to An. culicifacies. Notably, we observed an abundance of transcripts showing significant variability of encoded proteins with repeats such as LRR (Leucine rich repeat), WD40 (W-D dipeptide), Ankyrin, Annexin, Tetratricopeptide and Mitochondrial substrate carrier repeat-containing family proteins, which may have a direct influence on species-specific immune-physiological responses. Summarily, our deep sequencing analysis unraveled that An. stephensi evolved with an expansion of repeat sequences in hemocyte proteins as compared to An. culicifacies, possibly providing an advantage for better adaptation to diverse environments.

Genetic changes of Plasmodium vivax tempers host tissue-specific responses in Anopheles stephensi.

Recently, we showed how an early restriction of gut flora proliferation by Plasmodium vivax favors immune-suppression and Plasmodium survival in the gut lumen (Sharma et al., 2020). Here, we asked post gut invasion how P. vivax interacts with individual tissues such as the midgut, hemocyte, and salivary glands, and manages its survival in the mosquito host. Our data from tissue-specific comparative RNA-Seq analysis and extensive temporal/spatial expression profiling of selected mosquito transcripts in the uninfected and P. vivax infected mosquito's tissues indicated that (i) a transient suppression of gut metabolic machinery by early oocysts; (ii) enriched expression of nutritional responsive proteins and immune proteins against late oocysts, together may ensure optimal parasite development and gut homeostasis restoration; (iii) pre-immune activation of hemocyte by early gut-oocysts infection via REL induction (p â€‹< â€‹0.003); and altered expression of hemocyte-encoded immune proteins may cause rapid removal of free circulating sporozoites from hemolymph; (iv) while a strong suppression of salivary metabolic activities, and elevated expression of salivary specific secretory, as well as immune proteins together, may favor the long-term storage and survival of invaded sporozoites. Finally, our RNA-Seq-based discovery of 4449 transcripts of Plasmodium vivax origin, and their developmental stage-specific expression modulation in the corresponding infected mosquito tissues, predicts a possible mechanism of mosquito responses evasion by P. vivax. Conclusively, our system-wide RNA-Seq analysis provides the first genetic evidence of direct mosquito-Plasmodium interaction and establishes a functional correlation.

Molecular and Functional Characterization of Trehalase in the Mosquito Anopheles stephensi.

Like other insects, in blood-feeding mosquitoes, trehalase (TRE; EC 3.2.1.28), an enzyme that metabolizes trehalose, may influence a wide array of functions including flight, survival, reproduction, and vectorial capacity, but its role has not been investigated in detail. Here, we characterized a 1,839-bp-long transcript, encoding a 555-aa-long trehalase-2 homolog protein from the mosquito Anopheles stephensi. With a conserved insect homology, and in silico predicted membrane-bound protein, we tested whether trehalase (As-TreH) also plays a role in mosquito physiologies. Constitutive expression during aquatic development or adult mosquito tissues, and a consistent upregulation until 42 h of starvation, which was restored to basal levels after sugar supply, together indicated that As-TreH may have a key role in stress tolerance. A multifold enrichment in the midgut (p < 0.001819) and salivary glands (p < 4.37E-05) of the Plasmodium vivax-infected mosquitoes indicated that As-TreH may favor parasite development and survival in the mosquito host. However, surprisingly, after the blood meal, a consistent upregulation until 24 h in the fat body, and 48 h in the ovary, prompted to test its possible functional correlation in the reproductive physiology of the adult female mosquitoes. A functional knockdown by dsRNA-mediated silencing confers As-TreH ability to alter reproductive potential, causing a significant loss in the egg numbers (p < 0.001), possibly by impairing energy metabolism in the developing oocytes. Conclusively, our data provide initial evidence that As-TreH regulates multiple physiologies and may serve as a suitable target for designing novel strategies for vector control.

Hemocyte-specific FREP13 abrogates the exogenous bacterial population in the hemolymph and promotes midgut endosymbionts in Anopheles stephensi.

The immune blood cells "hemocytes" of mosquitoes impart a highly selective immune response against various microorganisms/pathogens. Among several immune effectors, fibrinogen-related proteins (FREPs) have been recognized as key modulators of cellular immune responses; however, their physiological relevance has not been investigated in detail. Our ongoing comparative RNA-sequencing analysis identified a total of 13 FREPs originating from naïve sugar-fed, blood-fed, bacterial challenged and Plasmodium vivax-infected hemocytes in Anopheles stephensi. Transcriptional profiling of the selected seven FREP transcripts showed distinct responses against different pathophysiological conditions, where an exclusive induction of FREP12 after 10 days of P. vivax infection was observed. This represents a possible role of FREP12 in immunity against free circulating sporozoites and needs to be explored in the future. When challenged with live bacterial injection in the thorax, we observed a higher affinity of FREP13 and FREP65 toward Gram-negative and Gram-positive bacteria in the mosquito hemocytes, respectively. Furthermore, we observed increased bacterial survival and proliferation, which is likely compromised by the downregulation of TEP1, in FREP13 messenger RNA-depleted mosquito hemolymph. In contrast, after blood-feeding, we also noticed a significant delay of 24 h in the enrichment of gut endosymbionts in the FREP13-silenced mosquitoes. Taken together, we conclude that hemocyte-specific FREP13 carries the unique ability of tissue-specific regulation, having an antagonistic antibacterial role in the hemolymph, and an agonistic role against gut endosymbionts.

Altered Gut Microbiota and Immunity Defines Plasmodium vivax Survival in Anopheles stephensi.

Blood-feeding enriched gut-microbiota boosts mosquitoes' anti-Plasmodium immunity. Here, we ask how Plasmodium vivax alters gut-microbiota, anti-Plasmodial immunity, and impacts tripartite Plasmodium-mosquito-microbiota interactions in the gut lumen. We used a metagenomics and RNAseq strategy to address these questions. In naïve mosquitoes, Elizabethkingia meningitis and Pseudomonas spp. are the dominant bacteria and blood-feeding leads to a heightened detection of Elizabethkingia, Pseudomonas and Serratia 16S rRNA. A parallel RNAseq analysis of blood-fed midguts also shows the presence of Elizabethkingia-related transcripts. After, P. vivax infected blood-meal, however, we do not detect bacterial 16S rRNA until circa 36 h. Intriguingly, the transcriptional expression of a selected array of antimicrobial arsenal cecropins 1-2, defensin-1, and gambicin remained low during the first 36 h-a time frame when ookinetes/early oocysts invaded the gut. We conclude during the preinvasive phase, P. vivax outcompetes midgut-microbiota. This microbial suppression likely negates the impact of mosquito immunity which in turn may enhance the survival of P. vivax. Detection of sequences matching to mosquito-associated Wolbachia opens a new inquiry for its exploration as an agent for "paratransgenesis-based" mosquito control.

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 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 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.