Kuldeep Gupta

Molecular characterization of SOCS gene and its expression analysis on Plasmodium berghei infection in Anopheles culicifacies.

Anopheles culicifacies mosquitoes are able to transmit both falciparum and vivax malaria in India. More than 65% of malaria cases reported annually spread through this vector. Despite the fact that it poses major vectorial burden in India, the molecular basis of its immune role against Plasmodium development has not been explored intensively. Here, we characterized An. culicifacies SOCS (suppressor of cytokine signaling) gene, a regulator of STAT pathway and its expression analysis upon Plasmodium infection. Our analysis has demonstrated that An. culicifacies SOCS gene shares strikingly high level of sequence similarity in SH2 domain and SOCS box region with other mosquito species. However, its N-terminal identity is limited to Anophelines mosquito only, suggesting its genus specific role. SOCS mRNA is expressed in all developmental stages of mosquito and its expression is higher in male than female adults. SOCS mRNA is significantly induced after Plasmodium infection in midgut tissue indicating its involvement in the immune defense responses. This is the first evidence of involvement of SOCS as an immune gene in Indian malaria vector An. culicifacies.

Identification of an Anopheles Lineage-Specific Unique Heme Peroxidase HPX15: A Plausible Candidate for Arresting Malaria Parasite Development

Background: Human malaria parasite Plasmodium falciparum is transmitted by several species of Anopheles mosquito. The advancement of drug-resistant parasites and insecticide resistance in mosquito vectors are major hurdles in the malaria control. Alternatively, the manipulation of mosquito immunity is also an ideal way to block Plasmodium development inside the insect host. This approach demands the identification of key mosquito molecules that regulate anti-plasmodial immunity. Our previous findings revealed that the silencing of Anopheles gambiae heme peroxidase 15 (AgHPX15, AGAP013327) induced mosquito innate immunity and drastically suppressed the development of human and rodent malaria parasites. Further, we aim to characterize HPX15 orthologs in Indian malaria vectors and other worldwide-distributed anophelines to understand the novelty of this molecule as a plausible target to block Plasmodium development. Method: AgHPX15 orthologs were cloned from major Indian malaria vectors A. stephensi and A. culicifacies and their conserve domains were determined by CDD search tool. The sequence homology and phylogenetic relationship of these clones with other heme peroxidases was analysed using Mega5.2 software. Results and conclusion: We found that A. stephensi AsHPX15 and A. culicifacies AcHPX15 clones are close orthologs of A. gambiae AgHPX15. The phylogenetic relationship of these anopheline HPX15 with other animal and plant heme peroxidases revealed that they form a separate lineage-specific cluster and their orthologs are not found in human, nematodes or other related arthropods such as, Drosophila, Aedes and Culex mosquitoes. However, their putative orthologs are present in 16 other globally distributed anophelines and exhibit a highly conserved amino acids identity in the range of 70-99%. Based on these findings we propose that the anopheline-specific and evolutionary conserved heme peroxidase HPX15 may serve as a unique target for designing transmission-blocking strategies to block Plasmodium-mosquito cycle. These findings will generate new frontiers in the field of malaria research and disease control.

A Fine-Tuned Management between Physiology and Immunity Maintains the Gut Microbiota in Insects

The association of microbial community with the digestive system is a distinct phenomenon and the insect gut is an excellent model to understand these interactions. Insects are omnivorous, feed on all kinds of food and encounter a variety of microbes. The diversity of these large and varied microbial communities inhabiting the gut depends on the feeding behavior of insects. Insect gut is also the foremost immune organ that encounters foreign food particles and exogenous pathogenic/non pathogenic microbes. Thus, it should be equipped by some mechanism that can distinguish between the food and pathogens. In most of the insects, the synthesis of an acellular chitinous peritrophic matrix (PM) around the ingested food compartmentalizes the gut to keep exogenous/endogenous microbes containing food bolus detached from the immunoreactive gut epithelium. This barrier-like functioning of the PM blocks the induction of insect immunity against the microbes present in the gut bolus. In addition to the PM, an extensively cross-linked mucin barrier also suppresses gut immunity against soluble microbial elicitors in the mosquito. Eventually, these acellular barriers maintain ‘low immunity zone’ in the gut to support the survival and proliferation of endosymbiotic microbes. In this review, we discuss that the ‘fine-tuned’ regulation of physiological state of digestion and immunity maintains the fitness-relevant traits such as growth and fecundity in insects.

Identification of the Temperature Induced Larvicidal Efficacy of Agave angustifolia against Aedes, Culex, and Anopheles Larvae.

Synthetic insecticides are generally employed to control the mosquito population. However, their injudicious over usage and non-biodegradability are associated with many adverse effects on the environment and mosquitoes. The application of environment-friendly mosquitocidals might be an alternate to overcome these issues. In this study, we found that organic or aqueous extracts of Agave angustifolia leaves exhibited a strong larvicidal activity (LD50 28.27 μg/ml) against Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi larvae within a short exposure of 12 h. The larvicidal activity of A. angustifolia is inherited and independent of the plants vegetative growth. Interestingly, the plant larvicidal activity was observed exclusively during the summer season (April–August, when outside temperature is between 30 and 50°C) and it was significantly reduced during winter season (December–February, when the outside temperature falls to ~4°C or lower). Thus, we hypothesized that the larvicidal components of A. angustifolia might be induced by the manipulation of environmental temperature and should be resistant to the hot conditions. We found that the larvicidal activity of A. angustifolia was induced when plants were maintained at 37°C in a semi-natural environment against the controls that were growing outside in cold weather. Pre-incubation of A. angustifolia extract at 100°C for 1 h killed 60% larvae in 12 h, which gradually increased to 100% mortality after 24 h. In addition, the dry powder formulation of A. angustifolia, also displayed a strong larvicidal activity after a long shelf life. Together, these findings revealed that A. angustifolia is an excellent source of temperature induced bioactive metabolites that may assist the preparedness for vector control programs competently.

Characterization and expression analysis of gene encoding heme peroxidase HPX15 in major Indian malaria vector Anopheles stephensi (Diptera: Culicidae)

The interaction of mosquito immune system with Plasmodium is critical in determining the vector competence. Thus, blocking the crucial mosquito molecules that regulate parasite development might be effective in controlling the disease transmission. In this study, we characterized a full-length AsHPX15 gene from the major Indian malaria vector Anopheles stephensi. This gene is true ortholog of Anopheles gambiae heme peroxidase AgHPX15 (AGAP013327), which modulates midgut immunity and regulates Plasmodium falciparum development. We found that AsHPX15 is highly induced in mosquito developmental stages and blood fed midguts. In addition, this is a lineage-specific gene that has identical features and 65-99% amino acids identity with other HPX15 genes present in eighteen worldwide-distributed anophelines. We discuss that the conserved HPX15 gene might serve as a common target to manipulate mosquito immunity and arresting Plasmodium development inside the vector host.

Apolipophorin-III Acts as a Positive Regulator of Plasmodium Development in Anopheles stephensi

Apolipophorin III (ApoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune responses of insects. Here we report the molecular and functional characterization of Anopheles stephensi Apolipophorin-III (AsApoLp-III) gene. This gene consists of 679 nucleotides arranged into two exons of 45 and 540 bp that give an ORF encoding 194 amino acid residues. Excluding a putative signal peptide of the first 19 amino acid residues, the 175-residues in mature AsApoLp-III protein has a calculated molecular mass of 22 kDa. Phylogenetic analysis revealed the divergence of mosquitoes (Order Diptera) ApoLp-III from their counterparts in moths (Order: Lepidoptera). Also, it revealed a close relatedness of AsApoLp-III to ApoLp-III of An. gambiae. AsApoLp-III mRNA expression is strongly induced in Plasmodium berghei infected mosquito midguts suggesting its crucial role in parasite development. AsApoLp-III silencing decreased P. berghei oocysts numbers by 7.7 fold against controls. These effects might be due to the interruption of AsApoLp-III mediated lipid delivery to the developing oocysts. In addition, nitric oxide synthase (NOS), an antiplasmodial gene, is also highly induced in AsApoLp-III silenced midguts suggesting that this gene acts like an agonist and protects Plasmodium against the mosquito immunity.

Silencing of Anopheles stephensi Heme Peroxidase HPX15 Activates Diverse Immune Pathways to Regulate the Growth of Midgut Bacteria

Anopheles mosquito midgut harbors a diverse group of endogenous bacteria that grow extensively after the blood feeding and help in food digestion and nutrition in many ways. Although, the growth of endogenous bacteria is regulated by various factors, however, the robust antibacterial immune reactions are generally suppressed in this body compartment by a heme peroxidase HPX15 crosslinked mucins barrier. This barrier is formed on the luminal side of the midgut and blocks the direct interactions and recognition of bacteria or their elicitors by the immune reactive midgut epithelium. We hypothesized that in the absence of HPX15, an increased load of exogenous bacteria will enormously induce the mosquito midgut immunity and this situation in turn, can easily regulate mosquito-pathogen interactions. In this study, we found that the blood feeding induced AsHPX15 gene in Anopheles stephensi midgut and promoted the growth of endogenous as well as exogenous fed bacteria. In addition, the mosquito midgut also efficiently regulated the number of these bacteria through the induction of classical Toll and Imd immune pathways. In case of AsHPX15 silenced midguts, the growth of midgut bacteria was largely reduced through the induction of nitric oxide synthase (NOS) gene, a downstream effector molecule of the JAK/STAT pathway. Interestingly, no significant induction of the classical immune pathways was observed in these midguts. Importantly, the NOS is a well known negative regulator of Plasmodium development, thus, we proposed that the induction of diverged immune pathways in the absence of HPX15 mediated midgut barrier might be one of the strategies to manipulate the vectorial capacity of Anopheles mosquito.

Characterization of Anopheline unique peroxidase and its role in the regulation of Plasmodium development

Background Malaria is major health problem in tropical and subtropical countries of the world. The WHO reported, 207 million malaria cases and 627,000 deaths in 2013 [1]. Malaria is caused by Plasmodium, which completes its asexual cycle in human host and sexual cycle in the female Anopheline mosquito. In order to combat malaria several strategies are in progress, blocking Plasmodium development inside mosquitos is one of them. For this transmission blocking approach we need to understand mosquito immune system and its interaction with Plasmodium at molecular levels. In the African malaria vector, Anopheles gambiae, a peroxidase HPX15 (IMPer) is reported to modulate mosquito immunity against Plasmodium [2]. Furthermore we are interested in understanding the regulation of Plasmodium development by its orthologous peroxidase-mediated immune responses in major Indian malaria vectors An. stephensi and An. culicifacies.

Anopheles stephensi Heme Peroxidase HPX15 Suppresses Midgut Immunity to Support Plasmodium Development

The heme peroxidase HPX15 is an evolutionary conserved anopheline lineage-specific gene. Previously, we found that this gene is present in the genome of 19 worldwide distributed different species of Anopheles mosquito and its orthologs are absent in other mosquitoes, insects, or human. In addition, 65–99% amino acid identity among these 19 orthologs permitted us to hypothesize that the functional aspects of this gene might be also conserved in different anophelines. In this study, we found that Anopheles stephensi AsHPX15 gene is mainly expressed in the midgut and highly induced after uninfected or Plasmodium berghei-infected blood feeding. RNA interference-mediated silencing of midgut AsHPX15 gene drastically reduced the number of developing P. berghei oocysts. An antiplasmodial gene nitric oxide synthase was induced 13-fold in silenced midguts when compared to the unsilenced controls. Interestingly, the induction of antiplasmodial immunity in AsHPX15-silenced midguts is in absolute agreement with Anopheles gambiae. In A. gambiae, AgHPX15 catalyzes the formation of a dityrosine network at luminal side of the midgut that suppresses the activation of mosquito immunity against the bolus bacteria. Thus, a low-immunity zone created by this mechanism indirectly supports Plasmodium development inside the midgut lumen. These indistinguishable functional behaviors and conserved homology indicates that HPX15 might be a potent target to manipulate the antiplasmodial immunity of the anopheline midgut, and it will open new frontiers in the field of malaria control.

The evolutionary divergence of STAT transcription factor in different Anopheles species

Anopheles mosquito transmits Plasmodium, the malaria causing parasite. Different species of Anopheles mosquito dominate in a particular geographical location and are capable of transmitting specific strains of Plasmodium. It is important to understand the biology of different anophelines to control the parasite transmission. STAT is an evolutionary conserved transcription factor that regulates the parasite development in African malaria vector Anopheles gambiae. Unlike Drosophila and Aedes aegypti, where a single STAT gene plays an important role in immunity, An. gambiae contains one evolutionary conserved STAT-A and another retro-duplicated, introns-less STAT-B gene. To find out whether other species of Anopheles also have two STATs, the available genomic data of different anophelines were used to annotate their STATs through in silico analyses. Our results revealed that Indian malaria vector An. stephensi genome contains two STATs, AsSTAT-A and AsSTAT-B genes. These genes were cloned and confirmed by sequencing. Both AsSTATs were found to be expressed in different development stages of mosquito. However, the relative mRNA levels of evolutionary conserved AsSTAT-A gene were always higher than the retroduplicated AsSTAT-B gene. STAT pathway was activated upon Plasmodium berghei infection, indicated its role in immunity. Furthermore, comparative in silico analysis of eighteen Anopheles species revealed that five species: An. sinensis, An. albimanus, An. darlingi, An. dirus andAn. farauti do not contain STAT-B gene in their genome. Interestingly, thirteen species of the subgenus Anopheles and Cellia that contain both STATs were also mutually diverged. This consequence leads to sequence variability in some significant protein motifs within the STAT-B genes. Phylogenetic analyses indicated that an independent, lineage-specific duplication occurred in the subgenus Cellia after the diversification of series Neomyzomyia from its last common ancestor. In An. atroparvus (subgenus Anopheles), STAT gene underwent recent lineage-specific duplication and give rise to a highly similar STAT-B gene. This suggested that the genetic divergence in various Anopheles species might appeared due to their adaptations to the altered environmental conditions or pathogen encounters.