Biology subjects

Singh, O. P.

Publications and source records attributed to Singh, O. P..

11 recordsLinked to original sources

bioRxiv2025

Is Aedes nr. albopictus synonymous with Aedes pseudalbopictus?

A recent study reported a putative cryptic species of Aedes albopictus, provisionally named Aedes nr. albopictus, from Tripura, India, based on molecular data from mitochondrial cytochrome c oxidase subunit I (COI) and nuclear internal transcribed spacer 2 (ITS2) regions. However, our reanalysis, incorporating COI and ITS2 sequences of the co-occurring species Aedes pseudalbopictus and employing maximum likelihood phylogenetic analysis, albeit with a limited number of available sequences, suggests that this lineage is conspecific with Ae. pseudalbopictus. Given the morphological similarities and overlapping habitat use of Ae. albopictus and Ae. pseudalbopictus in India and Southeast Asia, we hypothesize that the reported cryptic species could potentially be attributed to Ae. pseudalbopictus. Our findings underscore the need for comprehensive taxonomic assessments, integrating morphological and molecular analyses of all sympatric Stegomyia species, to ensure accurate species delimitation and support effective vector surveillance and control.

zoology↗

bioRxiv2024

Erythrocytic plasma membrane calcium ATPase (PMCA4b) variations mediate redox imbalance to determine artemisinin sensitivity in malaria

Artemisinin resistance in Plasmodium falciparum remains a major public health challenge for the ongoing malaria elimination programs. Till now, artemisinin resistance is linked with the variations in the genome of P. falciparum which are mainly clustered in the Pfkelch13 gene. Several studies reported artemisinin resistance in the malaria endemic areas without finding any evidence of genetic variation in parasite for this occurrence. This study interrogated whether host genetic variations have any association with artemisinin sensitivity of malaria parasite. We investigated the relationship of 1) genetic variations in the human ATP2B4 gene and 2) the resultant surface expression of plasma membrane calcium ATPase (PMCA4b) with the intraerythrocytic levels of calcium, reactive oxygen species (ROS) and the resistance towards artemisinin in the intraerythrocytic parasite. This study found negative correlation of PMCA4b expression level with intraerythrocytic calcium and ROS levels. Further in-vitro growth assays revealed that artemisinin sensitivity is reduced in the parasites growing within the RBCs having low PMCA4b and high oxidative stress. Overall, this study highlights the strong association of host PMCA4b in cellular calcium mediated redox imbalance, which significantly contributes to artemisinin resistance in malaria parasite. Hence, this is the first study to document the effect of host variations on artemisinin sensitivity of the parasite. We further emphasize that many redox modulating RBC polymorphisms that are prevalent in malaria endemic areas could influence artemisinin resistance and can serve as potential biomarkers for predicting therapeutic response. Thus, detailed population specific research may provide new insights for personalized malaria treatment and may inform future drug development strategies. SignificanceThe human host and malaria parasite share a closely interconnected relationship hence host variations could influence parasite drug resistance. This study demonstrates that reduced PMCA4b expression, the primary calcium efflux pump in RBCs, significantly increases the erythrocytic calcium and reactive oxygen species (ROS) levels, thereby decreasing the P. falciparum growth as well as artemisinin sensitivity. Many ROS inducing RBC polymorphisms, such as sickle cell, thalassemia, G6PD deficiency, PMCA4b variations etc. have emerged as strong malaria protective traits in several studies in malaria endemic areas. But these traits could also influence artemisinin resistance and can serve as potential biomarkers for predicting therapeutic response and highlights the importance of host erythrocytes oxidative microenvironment surveillance in monitoring antimalarial drug resistance.

microbiology↗

bioRxiv2024

Knockdown-resistance (kdr) mutations in Indian Aedes aegypti populations: lack of recombination among haplotypes bearing V1016G, F1534C, and F1534L kdr alleles

BackgroundKnockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene are a key mechanism of insecticide resistance in mosquitoes. In Asian Aedes aegypti populations two main VGSC haplogroups with kdr mutations have been identified: one carrying the F1534C mutation and another with V1016G and/or S989P mutations. Functional studies have demonstrated that these three mutations on a single haplotype confer up to a 1100-fold increase in pyrethroid resistance, underscoring the need to monitor these triple mutations within distinct populations. This study investigates the prevalence of kdr mutations in Indian populations and explores the linkage association between these mutations and two distinct conserved types of introns located between exons 20 and 21. MethodsAe. aegypti specimens collected from eight different locations were genotyped for kdr alleles and intron (between exons 20 and 21) haplotypes using PCR-based assays. Representative samples underwent DNA sequencing of VGSC regions. ResultsFive kdr mutations namely S989P, V1016G, T1520I, F1534C, and F1534L, were identified, each exhibiting varying distribution and frequencies across different geographical regions. Two distinct and stably-diverged intron haplotypes, designated as intron-A and intron-B, were identified between exons 20 and 21. Seven haplotypes, including two wild-type variants, were observed among Indian populations. The kdr-bearing haplotypes can be classified into three distinct haplogroups: haplogroup G (V1016G with/or without S989P and with intron-A), haplogroup L (F1534L and intron-A), and haplogroup C (F1534C with/or without T1520I and with intron-B). Importantly, no evidence of recombination within Indian populations was detected among these three haplogroups. ConclusionsFive kdr mutations were identified in the VGSC of Indian Ae. aegypti populations, each showing a definitive linkage with one of the two types of intron haplotypes. The lack of recombination among haplogroups bearing 1016G with 989P, 1534C and 1534L mutations suggests that the most potent insecticide resistance haplotype, bearing the triple kdr mutation, is currently absent. This finding has significant operational implications, as it may indicate that current vector control measures remain effective against these populations, potentially delaying the emergence of highly resistant phenotypes

zoology↗

bioRxiv2023

Circadian oscillation of perireceptor events influence olfactory sensitivity in diurnal and nocturnal mosquitoes

Olfaction and circadian rhythm gate different behaviors in mosquitoes that are important for their capacity to transmit disease. However, the mechanisms of odor detection, and the circadian-guided changes in olfactory sensitivity across different mosquito species, remain largely unexplored. To this end, we performed a circadian-dependent RNA-sequencing study of the peripheral olfactory- and brain tissues of female Anopheles culicifacies and Aedes aegypti mosquitoes. Data analysis revealed a significant upregulation of genes encoding: (a) odorant binding proteins (OBPs), required for transportation of odorant molecules towards the olfactory receptors, and (b) xenobiotic-metabolizing enzymes (XMEs) during the day time in Aedes aegypti and during the dusk-transition phase in Anopheles culicifacies. While XMEs primarily function in the elimination of toxic xenobiotics, concurrent elevation of XMEs and OBPs are hypothesized to act cumulatively to regulate perireceptor events and odorant sensitivity. Electroantennographic analysis with both Anopheles gambiae and Aedes aegypti against diverse behaviorally relevant odorants, combined with XMEs inhibitors and RNA interference, establish the proof-of-concept that XMEs function in perireceptor events during odorant detection and influence the odorant sensitivity in mosquitoes. Additionally, the RNA-sequencing and RNAi-mediated knockdown data revealed that daily temporal modulation of neuronal serine proteases may facilitate the consolidation of the brain function, and influence the odor detection process in both diurnal and nocturnal mosquitoes. These findings provide the impetus to further explore the species-specific rhythmic expression pattern of the neuro-olfactory encoded molecular factors, which could pave the way to develop and implement successful mosquito control methods. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/563057v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@181b845org.highwire.dtl.DTLVardef@168f22dorg.highwire.dtl.DTLVardef@f3994aorg.highwire.dtl.DTLVardef@d4fc06_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICircadian oscillation of perireceptor proteins possibly influences time-of-day dependent change olfactory sensitivity in diurnal and nocturnal mosquitoes C_LIO_LIDiurnal and nocturnal mosquitoes depict distinct dynamic change in perireceptor proteins C_LIO_LIInhibition of cytochrome P450 gene minimizes antennal response to different odorants C_LIO_LINeuronal serine protease may consolidate brain function and odor detection C_LI

molecular biology↗

bioRxiv2022

Molecular tools for early detection of invasion of malaria vector Anopheles stephensi

There are increasing reports of the expansion of the Asian malaria vector Anopheles stephensi in new geographical areas posing a threat to the elimination of urban malaria. Efficient surveillance of this vector in affected areas and early detection in new geographical areas is key for the containment and control of this species. To overcome the practical difficulties associated with the morphological identification of immature stages and adults of An. stephensi, a species-specific PCR and a Real-Time PCR assay were developed for their identification targeting a unique segment of ITS2 lacking homology to any other organism. Both PCRs can be used to identify An. stephensi individually or in pooled samples of mixed species, including when present in extremely low proportion. This study also reports a method for selective amplification and sequencing of An. stephensi for their confirmation in pooled samples of mixed species.

molecular biology↗

bioRxiv2022

First report of classical knockdown resistance (kdr) mutation, L1014F, in human head louse Pediculus humanus capitis (Phthiraptera: Anoplura)

There are at least three known knockdown resistance (kdr) mutations reported globally in the human head louse Pediculus humanus capitis (Phthiraptera: Anoplura) that are associated with reduced sensitivity to pyrethroids. However, prevalence of kdr mutation is not known in the Indian subcontinent. To identify kdr mutations in the Indian population, the genomic region of the voltage-gated sodium channel gene encompassing IIS1-2 linker to IIS6 segments was PCR-amplified and sequenced from P. humanus capitis samples collected from different geographic localities of India. DNA sequencing revealed the presence of four kdr mutations, i.e., M827I, T929I, L932F and L1014F. The presence of a classical kdr mutation, L1014F, a most widely reported mutation across insect-taxa associated with kdr-trait, is being reported for the first time in P. humanus capitis.

molecular biology↗

bioRxiv2021

Evaluation of odorant-binding protein-1 as a molecular marker for identifying biological forms and delimitating sibling species of Anopheles stephensi

BackgroundAnopheles stephensi, an invasive malaria vector, has been reported to have three biological forms identifiable mainly based on the number of ridges present on the eggs 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. MethodsField 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 Sangers 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. ResultsAsteObp1 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. ConclusionsAsteObp1 cannot serve as a marker for identifying biological forms of An. stephensi or putative sibling species in Indian populations.

molecular biology↗

bioRxiv2021

Are members of the Anopheles fluviatilis complex conspecific?

Anopheles fluviatilis sensu lato, a primary malaria vector in India, was 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. This conclusion is concerning because of the differential public health implications of members of the Fluviatilis Complex. 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.

molecular biology↗

bioRxiv2021

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.

genomics↗

bioRxiv2020

Molecular forms of Anopheles subpictus s.l. and Anopheles sundaicus s.l. in the Indian subcontinent

BackgroundAnopheles subpictus s.l. and Anopheles sundaicus s.l. are closely related species, each comprising of several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of An. subpictus complex. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance. MethodsDNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II of morphologically identified specimens of two nominal species, An. subpictus s.l. and An. sundaicus s.l. collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses. ResultsMolecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. (identified as An. epiroticus s.s. and An. sundaicus D) and three molecular forms of An. subpictus s.l. (provisionally designated as Form A, B and C) in the Indian subcontinent. Phylogenetic analyses revealed two distinct clades: (i) subpictus clade, with a single molecular form of An. subpictus (Form A) prevalent in mainland India and Sri Lanka, and (ii) sundaicus clade, comprising of members of Sundaicus Complex., two molecular forms of An. subpictus s.l., (Form B and C) prevalent in coastal areas or islands, and molecular forms reported from Thailand and Indonesia. Based on the number of float-ridges on eggs, all An. subpictus molecular Form B were classified as Species B whereas majority (80%) of the molecular Form A were classified as sibling species C. Fixed intragenomic sequence variation in ITS2 with the presence of two haplotypes was found in molecular Form A throughout its distribution. ConclusionA total of three molecular forms of An. subpictus s.l. and two molecular forms of An. sundaicus s.l. were recorded in the Indian subcontinent. Phylogenetically, two forms of An. subpictus s.l., (Form B and C) prevalent in coastal areas or islands in the Indian subcontinent and molecular forms reported from Southeast Asia are members of Sundaicus Complex. Molecular Form A of An. subpictus is distantly related to all other forms and deserve a distinct specific status. Presence of An. epiroticus in Indian territory is recorded for the first time.

zoology↗

bioRxiv2019

A new knockdown resistance (kdr) mutation F1534L in Aedes aegypti associated with insecticide resistance

BackgroundAedes aegypti is a primary vector of dengue, chikungunya and zika infections in India. In the absence of specific drugs or safe and effective vaccines for these infections, their control relies mainly on vector control measures. The emergence of insecticide resistance in vectors, especially against pyrethroids, is a serious threat to the insecticide-based vector control programme. This study reports the presence of multiple knockdown resistance (kdr) mutations present in an Ae. aegypti population from Bengaluru (India), including a new mutation F1534L. MethodsAedes aegypti collected from Bengaluru were subjected to insecticide susceptibility tests with DDT, deltamethrin and permethrin. The DNA sequencing of partial domain II, III and IV of the voltage-gated sodium channel (VGSC) was performed to screen kdr mutations present in the population and PCR-based assays were developed for their detection. Genotyping of kdr mutations was done using PCR-based assays, allelic frequencies were determined, and tests of genetic association of kdr mutations with the insecticide resistance phenotype were performed. ResultsThe Ae. aegypti population were resistant to DDT, deltamethrin and permethrin. The DNA sequencing of the VGSC revealed the presence of four kdr mutations, i.e., S989P and V1016G in domain II and two alternative kdr mutations F1534C and F1534L in domain III. Allele-specific PCR assays (ASPCR) were developed for the detection of kdr mutations S989P and V1016G and an existing PCR-RFLP based strategy was modified for the genotyping of all three known kdr mutations in domain III (F1534L, F1534C and T1520I). Genotyping of Ae. aegypti samples collected between October 2014 and April 2015 revealed a moderate frequency of S989P/V1016G (18.27%) and F1534L (17.48%), a relatively high frequency of F1534C (50.61%) and absence of T1520I in the population. Mutations S989P and V1016G were in complete linkage disequilibrium in this population while they were in linkage equilibrium with kdr mutations F1534C and F1534L. The alleles F1534C and F1534L are genetically associated with permethrin resistance. ConclusionsA new kdr mutation, F1534L, was found in an Ae. aegypti population from Bengaluru (India), co-occurring with the other three mutations S989P, V1016G and F1534C. The findings of a new mutation and development of PCR-based diagnostics have implications for insecticide resistance management. Monitoring of F1534L-kdr in other populations and studies on their functional role in altering neuro-sensitivity is warranted.

molecular biology↗