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.

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.

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.

Molecular identification of Aedes aegypti mosquitoes from Pilani region of Rajasthan, India.

BACKGROUND & OBJECTIVES: Aedes aegypti is the most important vector of dengue virus infection in humans worldwide. Accurate identification and colonization are the essential requirements to understand vector biology as well as its diseases transmission potential. In this study, we have used molecular approaches for the identification of Ae. aegypti mosquitoes that were collected from the Pilani region of Rajasthan, India Methods: Field collected mosquito larvae were colonized under laboratory conditions. Conserved genetic markers, ITS-2 and mtCOI were used for amplification through species-specific primers to identify the mosquito species/ strain. Sequencing result of this strain was phylogenetically compared with other global strains through MEGA software. RESULTS: A comprehensive multiple sequence alignment and phylogenetic analysis revealed that COI gene of Ae. aegypti has extremely low genetic variability with one of the Indian isolate from Thirumala, Andhra Pradesh region (GenBank: HM807262.1). However, in context of different geographical locations, it indicated close similarity with Thailand's strain and high variability when compared with Madagascar strain. On the other hand, ITS-2 illustrated highest identity with Ae. aegypti of Saudi Arabia (GenBank: JX423807.1) whereas, high divergence was observed from Mayotte, France strain (GenBank: KF135506). INTERPRETATION & CONCLUSION: The findings suggest that this isolate from Rajasthan is similar to other Asian continent strains possibly due to the same origin. Understanding the vectorial capacity of these geographically distributed mosquito strains will enhance our knowledge to improve existing vector surveillance and control programme.

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.