Molecular Tools for Early Detection of Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Reports of the expansion of the Asia malaria vector Anopheles stephensi mosquito into new geographic areas are increasing, which poses a threat to the elimination of urban malaria. Efficient surveillance of this vector in affected areas and early detection in new geographic areas is key to containing and controlling this species. To overcome the practical difficulties associated with the morphological identification of immature stages and adults of An. stephensi mosquitoes, we developed a species-specific PCR and a real-time PCR targeting a unique segment of the second internal transcribed spacer lacking homology to any other organism. Both PCRs can be used to identify An. stephensi mosquitoes individually or in pooled samples of mixed species, including when present in extremely low proportions (1:500). This study also reports a method for selective amplification and sequencing of partial ribosomal DNA from An. stephensi mosquitoes for their confirmation in pooled samples of mixed species.

Modular electromagnetic railgun accelerator for high velocity impact studies.

A modular electromagnetic railgun accelerator facility named "RAFTAR" (i.e., Railgun Accelerator Facility for Technology and Research) has been commissioned and its performance has been characterized for high velocity impact testing on materials in a single-shot mode. In the first tests, RAFTAR demonstrated an acceleration of more than 1000 m/s for an 8 g solid aluminum-7075 armature projectile. The current fed was 220 kA, having a muzzle time of about 1.75 ms. It is a single pulse breech-fed rectangular bore (14 × 13 mm2) railgun, and its 1.15 m long barrel assembly consists of two parallel copper bars with an inter-gap of 13 mm that are encased within 50 mm thick high strength reinforced fiberglass sheets (Garolite G10-FR4) and bolted from both the sides. RAFTAR is powered by two capacitor bank modules that have a maximum stored energy of 160 kJ each (containing eight 178 µF/15 kV capacitors), two high power ignitron switches, and a pulse shaping inductor. To obtain consistent acceleration of the armature inside the barrel, reversal of driving current is prevented, and its pulse duration is stretched by tactical integration of the crowbar switch and bitter coil inductor in the circuit. Armature projectile velocity measurement in-bore and outside in free space was performed by the time-of-flight technique using indigenously made miniature B-dot sensors and a novel shorting-foil arrangement, respectively. The time resolved measurement of the in-bore armature evidenced a velocity-skin-effect in the high acceleration phase. There is good agreement between the experimentally measured and theoretically predicted efficiency, confirming the optimal choice of operating parameters. The conclusion summarizes important experimental findings and analyzes the underlying causes that limit the performance of railguns.

Evaluation of intron-1 of odorant-binding protein-1 of Anopheles stephensi as a marker for the identification of biological forms or putative sibling species.

BACKGROUND: Anopheles stephensi, an invasive malaria vector, has been reported to have three biological forms identifiable mainly based on the number of ridges present on the egg's floats. Recently, the first intron of the odorant-binding protein-1 (AsteObp1) has been introduced as a molecular marker for the identification of these forms, and based on this marker, the presence of three putative sibling species (designated as species A, B and C) has been proposed. However, there is no data on the association of proposed markers with biological form or putative species on field populations. METHODS: Field collected and laboratory-reared An. stephensi were characterized for biological forms based on the number of ridges on the egg's float. DNA sequencing of the partial AsteObp1 gene of An. stephensi individuals were performed by Sanger's method, either directly or after cloning with a plasmid vector. Additionally, AsteObp1 sequences of various laboratory lines of An. stephensi were retrieved from a public sequence database. RESULTS: AsteObp1 intron-1 in Indian An. stephensi populations are highly polymorphic with the presence of more than 13 haplotypes exhibiting nucleotides as well as length-polymorphism (90-to-121 bp). No specific haplotype or a group of closely related haplotypes of intron-1 was found associated with any biological form identified morphologically. High heterozygosity for this marker with a low inbreeding coefficient in field and laboratory populations indicates that this marker is not suitable for the delimitation of putative sibling species, at least in Indian populations. CONCLUSIONS: AsteObp1 cannot serve as a marker for identifying biological forms of An. stephensi or putative sibling species in Indian populations.

Are members of the Anopheles fluviatilis complex conspecific?

Anopheles fluviatilis sensu lato, a primary malaria vector in India, has been identified to be comprised of four cryptic species, provisionally designated as species S, T, U and V. However, Kumar et al. (Mol Ecol Resour, 2013;13:354-61) considered all of the then known three members of this species complex (S, T and U) conspecific. The specific status of species S and T was refuted based on the lack of sufficient barcode gap in mitochondrial-CO1 and the perceived presence of heterozygotes in populations as detected through one of the two species-specific PCR assays employed for the cryptic species identification. The existence of species U was refuted claiming that earlier investigations have already refuted their existence. Here we discuss problems associated with the CO1-based barcode approach for delimitation of cryptic species, the perceived heterozygosity between species S and T based on a species-specific PCR assay, and interpretation of published reports. We demonstrated that fixed differences do exist in the ITS2-rDNA sequence of species S and T with no evidence of heterozygotes in sympatric populations and, that the observed heterozygosity by Kumar et al. in the ITS2-based species diagnostic PCR is due to the high mispriming tendency of the T-specific primer with species S. We infer that mitochondrial DNA-based 'barcoding gap', an arbitrary threshold recommended for species delimitation, alone, is inadequate to delimit the members of An. fluviatilis complex.

Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi.

Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic sequence variation in the ITS2-rDNA of An. stephensi and hypothesizes that observed intra-specific differences in this species may have resulted due to ambiguous DNA sequence-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S-rDNA (d1-d3 domains). Intragenomic variations were found in ITS2 region of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S-rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central, and southern India confirmed the presence of intragenomic variation in ITS2 due to transitions at three loci and two bp indel in a di-nucleotide microsatellite locus. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses.

The burden of submicroscopic and asymptomatic malaria in India revealed from epidemiology studies at three varied transmission sites in India.

Malaria in India, while decreasing, remains a serious public health problem, and the contribution of submicroscopic and asymptomatic infections to its persistence is poorly understood. We conducted community surveys and clinic studies at three sites in India differing in their eco-epidemiologies: Chennai (Tamil Nadu), Nadiad (Gujarat), and Rourkela (Odisha), during 2012-2015. A total of 6,645 subject blood samples were collected for Plasmodium diagnosis by microscopy and PCR, and an extensive clinical questionnaire completed. Malaria prevalence ranged from 3-8% by PCR in community surveys (24 infections in Chennai, 56 in Nadiad, 101 in Rourkela), with Plasmodium vivax dominating in Chennai (70.8%) and Nadiad (67.9%), and Plasmodium falciparum in Rourkela (77.3%). A proportional high burden of asymptomatic and submicroscopic infections was detected in community surveys in Chennai (71% and 71%, respectively, 17 infections for both) and Rourkela (64% and 31%, 65 and 31 infections, respectively). In clinic studies, a proportional high burden of infections was identified as submicroscopic in Rourkela (45%, 42 infections) and Chennai (19%, 42 infections). In the community surveys, anemia and fever were significantly more common among microscopic than submicroscopic infections. Exploratory spatial analysis identified a number of potential malaria hotspots at all three sites. There is a considerable burden of submicroscopic and asymptomatic malaria in malarious regions in India, which may act as a reservoir with implications for malaria elimination strategies.

Knockdown Resistance (kdr) Mutations in Indian Anopheles stephensi (Diptera: Culicidae) Populations.

Knockdown resistance (kdr) in insects resulting from mutation(s) in the voltage-gated sodium channel (VGSC) gene is one of the mechanisms of resistance against DDT and the pyrethroid group of insecticides. Earlier, we reported the presence of two classic kdr mutations, i.e., L1014F and L1014S in Anopheles stephensi Liston, a major Indian malaria vector affecting mainly urban areas. This report presents the distribution of these alleles in different An. stephensi populations. Seven populations of An. stephensi from six states of India were screened for the presence of two alternative kdr mutations L1014F and L1014S using allele-specific polymerase chain reaction assays. We recorded the presence of both kdr mutations in northern Indian populations (Alwar and Gurgaon), with the preponderance of L1014S, whereas only L1014F was present in Raipur (central India) and Chennai (southern India). None of the kdr mutations were found in Ranchi in eastern India and in Mangaluru and Mysuru in southern India. This study provides evidence for a focal pattern of distribution of kdr alleles in India.