Sarala K. Subbarao

EGG-FLOAT RIDGE NUMBER IN ANOPHELES STEPHENSI: ECOLOGICAL VARIATION AND GENETIC ANALYSIS

Abstract. Eight Indian laboratory stocks of Anopheles stephensi Liston could be grouped into three categories with, respectively, 14–22, 12–17 and 9–15 ridges on the egg‐floats. The mode number of ridges among the eggs laid by individual females in these stocks was 16–19,13‐16 and 10–14, respectively. The category with the highest egg‐float ridge number corresponded with the type‐form and the lowest with var. mysorensis Sweet and Rao; the new egg‐float category with ridge number modes of thirteen to sixteen was designated as ‘intermediate’. All three forms, i.e. type‐form, intermediate and mysorensis were observed in semi‐urban areas while only intermediate and mysorensis were seen in rural areas. Breeding experiments indicated no post‐copulatory barriers between the populations. Likelihood analysis of the results of crosses and back crosses indicated that variation in ridge number is controlled by more than one genetic factor. The stocks with different ridge numbers are best considered as ‘ecological variants’.

Susceptibility of Anopheles culicifacies species A and B to Plasmodium vivax and Plasmodium falciparum as determined by immunoradiometric assay

We have used a two-site immunoradiometric assay and species-specific antisporozoite monoclonal antibodies to determine the relative roles that sibling species A and B of the Anopheles culicifacies complex play in malaria transmission in western Uttar Pradesh, India. The results unequivocally establish species A as the primary vector of both Plasmodium vivax and P. falciparum in this area. Our results indicate active transmission of P. vivax from May to October and of P. falciparum from August to December. The identification of species A as the primary malaria vector in northern India will now allow suitable malaria control strategies to be designed.

GIS based malaria information management system for urban malaria scheme in India

A GIS based information management system has been developed to help Urban Malaria Control in India. The basic objective is to develop a model to assist planning and implementation of a suitable control measure. The system can help in: (i) identifying high receptive areas in time and space domain; (ii) identifying risk factors for high receptivity; (iii) monitoring and evaluating control measures. To demonstrate this system, information on 33 parameters and malaria cases has been attached to a digitised map of Dindigul, an urban town in Tamil Nadu. Functionalities of the system and its utility are described in this paper. A GIS based information management system ensures that if a localised spurt of the disease occurs, it can be associated rapidly with a likely cause, a specific vector, and a probable human source, so that appropriate preventive action can be taken to arrest any rising trend.

Plasmodium vivax: enzyme polymorphism in isolates of Indian origin

185 isolates of Plasmodium vivax were collected from patients visiting the malaria clinic run by the National Malaria Eradication Programme, Delhi, India. Percoll gradient centrifugation was used to concentrate P. vivax parasites from 0.4 to 0.5 ml of blood collected by finger prick. The parasite concentrate from each isolate was electrophoretically analysed for lactate dehydrogenase (LDH), NADP-dependent glutamate dehydrogenase (GDH), glucose phosphate isomerase (GPI) and adenosine deaminase (ADA). Variations were observed in GPI, GDH and ADA systems. Four electrophoretic forms of GPI and 5 each of GDH and ADA were observed. Electrophoretic mobilities of the different isoenzymic forms in P. vivax were identical to those reported for P. falciparum, indicating that the 2 species cannot be differentiated on the basis of electrophoretic patterns of the 4 enzyme systems studied.

An allele-specific polymerase chain reaction assay for the differentiation of members of the Anopheles culicifacies complex

Anopheles culicifacies, the principal vector of malaria in India, is a complex of five cryptic species which are morphologically indistinguishable at any stage of life. In view of the practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, an allele-specific polymerase chain reaction (ASPCR) assay targeted to the D3 domain of 28S ribosomal DNA was developed. The assay discriminatesAn. culicifacies species A and D from species B, C and E. The assay was validated using chromosomally-identified specimens ofAn. culicifacies from different geographical regions of India representing different sympatric associations. The assay correctly differentiates species A and D from species B, C and E. The possible use of this diagnostic assay in disease vector control programmes is discussed.

PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

BackgroundAnopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F) in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping.MethodsThe IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped.ResultsThe genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium.ConclusionThe Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at kdr locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.

Cytogenetic Characterization of Anopheles sundaicus (Diptera: Culicidae) Population from Car Nicobar Island, India

Abstract In India, Anopheles sundaicus is now found abundantly and widely only in the Andaman and Nicobar islands, where this species is the sole malaria vector. Recent studies in Thailand and Indonesia have established An. sundaicus as a complex of three isomorphic species (species A, B, and C) identifiable on the basis of cytological variations together with enzyme polymorphism analysis. A study was conducted to examine cytologically An. sundaicus from Car Nicobar island, district Nicobar, India. More than 300 samples screened for ovarian polytene chromosomes had X chromosome of Xa type as reported in case of species A and chromosome arm (2b) similar to that in species C. This combination suggests the existence of a new cytogenetic variant, i.e., cytotype D, in the Indian subcontinent that has not been reported so far. Examination of male and female mitotic karyotypes further substantiated these results. Only cytotype D was prevalent in the fresh water and brackish water areas of the island, indicating wider adaptability of this form to different habitats.

On the conspecificity ofAnopheles fluviatilis species S withAnopheles minimus species C

Anopheles fluviatilis andAn. minimus complexes, each comprising of at least three sibling species, are closely related and important malaria vectors in Oriental Region. RecentlyAn. fluviatilis species S, which is a highly efficient malaria vector in India, has been made conspecific withAn. minimus species C (senior synonym) on the basis of homology in 335 base pair nucleotide sequence of D3 domain of 28S ribosomal DNA(rDNA). We examined the conspecificity of these two nominal species by obtaining and analysing the DNA sequences of nuclear ribosomal loci internal transcribed spacer 2 (ITS2) and D2-D3 domain of 28S rDNA (28S-D2/D3) from those ofAn. fluviatilis S andAn. minimus C. We found that the sequences ofAn. fluviatilis S are appreciably different from those ofAn. minimus C with pair-wise distance (Kimura-2-parametre model) of 3.6 and 0.7% for loci ITS2 and 28S-D2/D3, respectively. Pair-wise distance and phylogenetic analyses using ITS2 sequences of members of Minimus and Fluviatilis Complexes revealedthat An. fluviatilis S is distantly related toAn. minimus C as compared to any other members of the Fluviatilis Complex. These findings suggest that the two nominal species,An. fluviatilis S andAn. minimus C, do not merit synonymy. The study also confirms that the reported speciesAn. fluviatilis X is synonym with species S.

Genetic structure of Plasmodium vivax isolates in India

Variations in the allelic composition of glucose phosphate isomerase (GPI), NADP-dependent glutamate dehydrogenase (GDH) and adenosine deaminase (ADA) enzyme systems of Plasmodium vivax were observed in isolates of Indian origin in 1985-1993. No significant difference was observed in allelic frequencies in different years. The data indicated random distribution of GPI, GDH and ADA alleles among the isolates, suggesting that loci for these enzymes were not linked. A high proportion of the isolates comprised at least 2 genetically distinct clones, the mean number of clones per isolate being 1.4. There was no significant difference in the number of oocysts in Anopheles stephensi fed on uniclonal and multiclonal isolates. No difference was observed in the proportions of uniclonal and multiclonal isolates during low and high transmission periods.

Lactate dehydrogenase allozyme differentiation of species in the Anopheles culicifacies complex

Abstract Genetically controlled enzyme variation exists within and between four sibling species of the Anopheles culicifacies complex of malaria vectors in India. A study on electrophoretic variation of nine enzymes in An.culicifacies sibling species revealed that the lactate dehydrogenase (Ldh) locus has Fast (F) and Slow (S) allozymes distinguishing species A+D from species B+C with a probability of c. 95%.