Abdominal pigmentation in Drosophila melanogaster females from natural Indian populations

Females of Drosophila melanogaster collected from five geographically distant populations in India were analysed for the intensity of pigmentation in the 5th, 6th and 7th segments of the abdomen. In all three segments, this intensity was found to vary among individuals of any given population, and, furthermore, different populations differ with respect to this phenotypic trait. Statistical analysis revealed significant intra- and interpopulational variation. A clinical pattern was detected: females from populations closer to the equator tended to have lighter cuticle, in which case differences between the three segments could not be detected and all three segments responded both independently and jointly to latitudinal variation, as indicated by a statistically significant positive correlation between latitude and pigmentation score. This is the first report on abdominal pigmentation analysis in natural populations of D. melanogaster that provides evidence that phenotypic flexibility reflects temperature differences, as a result of which abdominal pigmentation shows geographic differentiation.     

Proteomics analysis to compare the venom composition between Naja naja and Naja kaouthia from the same geographical location of eastern India: Correlation with pathophysiology of envenomation and immunological cross-reactivity towards commercial polyantivenom

ABSTRACT

Background : Cobra bite is frequently reported across the Indian subcontinent and is associated with a high rate of death and morbidity. In eastern India (EI) Naja naja and Naja kaouthia are reported to be the two most abundant species of cobra.

Research design and methods : The venom proteome composition of N. naja (NnV) and N. kaouthia (NkV) from Burdwan districts of EI were compared by separation of venom proteins by 1D-SDS-PAGE followed by LC-MS/MS analysis of protein bands. The potency of commercial polyantivenom (PAV) was assessed by neutralization, ELISA, immuno-blot and venom-PAV immunoaffinity chromatography studies.

Results : Proteomic analysis identified 52 and 55 proteins for NnV and NkV, respectively, when searched against the Elapidae database. A small quantitative difference in venom composition between these two species of cobra was observed. PAVs exhibited poor cross-reactivity against low molecular mass toxins (<20 kDa) of both cobra venoms, which was substantiated by a meager neutralization of their phospholipase A₂ activity. Phospholipase A₂ and 3FTx, the two major classes of nonenzymatic and enzymatic proteins, respectively, were partially recognized by PAVs.

Conclusions : Efforts must be made to improve immunization protocols and supplement existing antivenoms with antibodies raised against the major toxins of these venoms.     

Inferring the evolutionary history of Indian Plasmodium vivax from population genetic analyses of multilocus nuclear DNA fragments

The human malaria parasite Plasmodium vivax is globally widespread, causing high malaria morbidity. As P. vivax is highly endemic to India, and previous reports indicate genetic homogeneity in population samples, we tested the hypothesis of no genetic structuring in Indian P. vivax. Further, based on the reports of increasing incidence of Plasmodium falciparum infection in comparison with P. vivax in recent years in India, it was important to understand whether reduction in population size has resulted in decrease in P. vivax infection rate in India. For this, we utilized recently developed putatively neutral markers from chromosome 13 of P. vivax to score single nucleotide polymorphisms in 126 P. vivax isolates collected from 10 different places in India. The overall results indicated that Indian P. vivax bears high nucleotide diversity within population samples but moderate amount of genetic differentiation between population samples. STRUCTURE analysis grouped 10 population samples into three clusters based on the proportion of the genetic ancestries in each population. However, the pattern of clustering does not correlate with sampling locations in India. Furthermore, analyses of past demographic events indicated reduction in population size in majority of population samples, but when isolates from all the 10 samples were considered as a single population, the data fit to the demographic equilibrium model. All these observations clearly indicate that Indian P. vivax presents complex evolutionary history but possesses several features of being a part of ancestral distribution range of this species.     

Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes

The fact that malaria is still an uncontrolled disease is reflected by the genetic organization of the parasite genome. Efforts to curb malaria should begin with proper understanding of the mechanism by which the parasites evade human immune system and evolve resistance to different antimalarial drugs. We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7. We found 60 genes of various functions and lengths, majority (61.67%) of them were performing known functions. Almost all the genes have orthologs in other four species of Plasmodium, of which P. chabaudi seems to be the closest to P. falciparum. However, only two genes were found to be paralogous. Interestingly, the drug-resistant gene, pfcrt was found to be surrounded by seven genes coding for several CG proteins out of which six were reported to be responsible for providing drug resistance to P. vivax. The intergenic regions, in this specified region were generally large in size, majority (73%) of them were of more than 500 nucleotide bp length. We also designed primers for amplification of 21 noncoding DNA fragments in the whole region for estimating genetic diversity and inferring the evolutionary history of this region of P. falciparum genome.     

Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India

Inferring the origin and dispersal of the chloroquine-resistant (CQR) malaria parasite, Plasmodium falciparum, is of academic and public health importance. The Pfcrt gene of P. falciparum is widely known as the CQR gene and two major haplotypes of this gene (CVIET and SVMNT) occur widely across CQR-endemic regions of the globe. In India, studies to date of the Pfcrt gene have indicated the widespread prevalence of the SVMNT haplotype (prevalent in the South America and Papua New Guinea), whereas the CVIET haplotype, primarily found in southeast Asia, was not detected at a high frequency in India. This distribution pattern of the two most common CQR-Pfcrt haplotypes in India is quite surprising. Thus, in order to understand probable evolutionary and migration patterns of the CQR-Pfcrt haplotypes into India, we generated new sequence data of exon 2 of the Pfcrt gene and collected published information on the CQR-Pfcrt haplotype data from India, Papua New Guinea, southeast Asia and South America, and performed several population and evolutionary genetic analyses. Among several interesting findings, statistically significant longitudinal clines for the CVIET and SVMNT haplotypes (in opposite directions) in India, and the clustering of India and Papua New Guinea under the SVMNT-specific clade in the phylogenetic tree, are the two most remarkable aspects of the data. It also appears that both the SVMNT and CVIET haplotypes in India have migrated from southeast Asia. In particular, whereas the Indian CVIET haplotype has a southeast Asian origin, the SVMNT haplotype, prevalent in India, seems to have originated in Papua New Guinea and entered India through southeast Asia.     

Does malaria epidemiology project Cameroon as ‘Africa in miniature’?

Cameroon, a west-central African country with a ~20 million population, is commonly regarded as ‘Africa in miniature’ due to the extensive biological and cultural diversities of whole Africa being present in a single-country setting. This country is inhabited by ancestral human lineages in unique eco-climatic conditions and diverse topography. Over 90% Cameroonians are at risk of malaria infection, and ~41% have at least one episode of malaria each year. Historically, the rate of malaria infection in Cameroon has fluctuated over the years; the number of cases was about 2 million in 2010 and 2011. The Cameroonian malaria control programme faces an uphill task due to high prevalence of multidrug-resistant parasites and insecticide-resistant malaria vectors. Above all, continued human migration from the rural to urban areas as well as population exchange with adjoining countries, high rate of ecological instabilities caused by deforestation, poor housing, lack of proper sanitation and drainage system might have resulted in the recent increase in incidences of malaria and other vector-borne diseases in Cameroon. The available data on eco-environmental variability and intricate malaria epidemiology in Cameroon reflect the situation in the whole of Africa, and warrant the need for in-depth study by using modern surveillance tools for meaningful basic understanding of the malaria triangle (host-parasite-vector-environment).     

Molecular Evidence of Plasmodium vivax Mono and Mixed Malaria Parasite Infections in Duffy-Negative Native Cameroonians

The malaria parasite Plasmodium vivax is known to be majorly endemic to Asian and Latin American countries with no or very few reports of Africans infected with this parasite. Since the human Duffy antigens act as receptors for P. vivax to invade human RBCs and Africans are generally Duffy-negative, non-endemicity of P. vivax in Africa has been attributed to this fact. However, recent reports describing P. vivax infections in Duffy-negative Africans from West and Central parts of Africa have been surfaced including a recent report on P. vivax infection in native Cameroonians. In order to know if Cameroonians living in the southern regions are also susceptible to P. vivax infection, we collected finger-prick blood samples from 485 malarial symptomatic patients in five locations and followed PCR diagnostic assays with DNA sequencing of the 18S ribosomal RNA gene. Out of the 201 malaria positive cases detected, 193 were pure P. falciparum, six pure P. vivax and two mixed parasite infections (P. falciparum + P. vivax). The eight P. vivax infected samples (six single + two mixed) were further subjected to DNA sequencing of the P. vivax multidrug resistance 1 (pvmdr1) and the P.vivax circumsporozoite (pvcsp) genes. Alignment of the eight Cameroonian pvmdr1 sequences with the reference sequence showed high sequence similarities, reconfirming P. vivax infection in all the eight patients. DNA sequencing of the pvcsp gene indicated all the eight P. vivax to be of VK247 type. Interestingly, DNA sequencing of a part of the human Duffy gene covering the promoter region in the eight P. vivax-infected Cameroonians to identify the T-33C mutation revealed all these patients as Duffy-negative. The results provide evidence of single P. vivax as well as mixed malaria parasite infection in native Cameroonians and add knowledge to the growing evidences of P. vivax infection in Duffy-negative Africans.     

Quantitative proteomic analysis of venom from Southern India common krait (Bungarus caeruleus) and identification of poorly immunogenic toxins by immune-profiling against commercial antivenom

Objectives: To study the venom proteome composition of Southern India (SI) Common Krait (Bungarus caeruleus) and immunological cross-reactivity between venom against commercial antivenom.

Methods: Proteomic analysis was done by nano LC-MS/MS and toxins were quantitated by label-free analysis. The immunological cross-reactivity of venom towards polyvalent antivenom (PAV) was assessed by ELISA, Immunoblotting, and immuno-chromatographic methods.

Results: A total of 57 enzymatic and non-enzymatic proteins belonging to 12 snake venom protein families were identified. The three finger toxins (3FTx) (48.3%) and phospholipase A₂ (PLA₂) (37.6%) represented the most abundant non-enzymatic and enzymatic proteins, respectively. β-bungarotoxin (12.9%), a presynaptic neurotoxin, was also identified. The venom proteome composition is well correlated with its enzymatic activities, reported pharmacological properties, and clinical manifestations of krait envenomation. Immuno-cross-reactivity studies demonstrated better recognition of high molecular weight proteins (>45 kDa) of this venom by PAVs compared to low molecular weight (<15 kDa) toxins such as PLA₂ and 3FTxs.

Conclusion: The poor recognition of <15 kDa mass SI B. caeruleus venom proteins is of grave concern for the successful treatment of krait envenomation. Therefore, emphasis should be given to improve the immunization protocols and/or supplement of antibodies raised specifically against the <15 kDa toxins of this venom.     

Multilocus nuclear DNA markers reveal population structure and demography of Anopheles minimus

Utilization of multiple putatively neutral DNA markers for inferring evolutionary history of species population is considered to be the most robust approach. Molecular population genetic studies have been conducted in many species of Anopheles genus, but studies based on single nucleotide polymorphism (SNP) data are still very scarce. Anopheles minimus is one of the principal malaria vectors of Southeast (SE) Asia including the Northeastern (NE) India. Although population genetic studies with mitochondrial genetic variation data have been utilized to infer phylogeography of the SE Asian populations of this species, limited information on the population structure and demography of Indian An. minimus is available. We herewith have developed multilocus nuclear genetic approach with SNP markers located in X chromosome of An. minimus in eight Indian and two SE Asian population samples (121 individual mosquitoes in total) to infer population history and test several hypotheses on the phylogeography of this species. While the Thai population sample of An. minimus presented the highest nucleotide diversity, majority of the Indian samples were also fairly diverse. In general, An. minimus populations were moderately substructured in the distribution range covering SE Asia and NE India, largely falling under three distinct genetic clusters. Moreover, demographic expansion events could be detected in the majority of the presently studied populations of An. minimus. Additional DNA sequencing of the mitochondrial COII region in a subset of the samples (40 individual mosquitoes) corroborated the existing hypothesis of Indian An. minimus falling under the earlier reported mitochondrial lineage B.