BackgroundMosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.ResultsLab-reared and field-collected A. stephensi male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught A. stephensi mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval A. stephensi, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared A. stephensi midgut, respectively. These OTUs had a Shannon diversity index value of 1.74–2.14 for lab-reared and in the range of 2.75–3.49 for field-caught A. stephensi mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.ConclusionMore than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

BackgroundControl of Aedes aegypti, the mosquito vector of dengue, chikungunya and yellow fever, is a challenging task. Pyrethroid insecticides have emerged as a preferred choice for vector control but are threatened by the emergence of resistance. The present study reports a focus of pyrethroid resistance and presence of two kdr mutations—F1534C and a novel mutation T1520I, in Ae. aegypti from Delhi, India.Methodology/Principal FindingsInsecticide susceptibility status of adult-female Ae. aegypti against DDT (4%), deltamethrin (0.05%) and permethrin (0.75%) was determined using WHO's standard insecticide susceptibility kit, which revealed resistance to DDT, deltamethrin and permethrin with corrected mortalities of 35%, 72% and 76% respectively. Mosquitoes were screened for the presence of kdr mutations including those reported earlier (I1011V/M, V1016G/I, F1534C, D1794Y and S989P), which revealed the presence of F1534C and a novel mutation T1520I. Highly specific PCR-RFLP assays were developed for genotyping of these two mutations. Genotyping using allele specific PCR and new PCR-RFLP assays revealed a high frequency of F1534C (0.41–0.79) and low frequency of novel mutation T1520I (0.13). The latter was observed to be tightly linked with F1534C and possibly serve as a compensatory mutation. A positive association of F1534C mutation with DDT and deltamethrin resistance in Ae. aegypti was established. However, F1534C-kdr did not show significant protection against permethrin.Conclusions/SignificanceThe Aedes aegypti population of Delhi is resistant to DDT, deltamethrin and permethrin. Two kdr mutations, F1534C and a novel mutation T1520I, were identified in this population. This is the first report of kdr mutations being present in the Indian Ae. aegypti population. Highly specific PCR-RFLP assays were developed for discrimination of alleles at both kdr loci. A positive association of F1534C mutation with DDT and deltamethrin resistance was confirmed.

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’.

A five-year epidemiologic study of patients attending a malaria clinic in Delhi was conducted to find the relapse rate of infections with Plasmodium vivax, its seasonal correlation between the primary infection and subsequent relapses, the duration of the incubation period, and the patterns of relapse. By our definition, the relapse rate ranged from 23% to 44% depending on the duration of follow-up. The relapse pattern observed in the study clearly suggests the existence of both tropical and temperate zone types of P. vivax in the population characterized by distinct incubation periods and the possible existence of P. vivax subpopulations characterized by primary long incubation periods. The implication of different incubating forms of P. vivax on the epidemiology and control of malaria is also discussed. Plasmodium vivax malaria constitutes about 60-65% of total malaria cases in India with, pronounced morbidity particularly in the economically weaker sections of the society. 1 The clinicoepidemiologic picture of P. vivax is not well understood due to the phenomenon of latency/relapse. Due to the persistence of the hepatic or hypnozoite form of the parasite, relapses occur in P. vivax infections and it is difficult to predict their timing. 2 Plasmodium vivax exhibits two primary types of incubation/relapse patterns that apparently depend on their tropical or temperate zone origin. 3-5 The classic example of the tropical type is the Chesson strain (New Guinea-South Pacific), characterized by an early primary attack, followed by a short latent period before appearance of frequent relapses during the next year or more, whereas the St. Elizabeth (United States) strain of the temperate type exhibits an early primary attack, followed by a long latent period of 6-14 months, and thereafter succeeded by a series of relapses at short intervals. The present study is an attempt to understand the composition of P. vivax populations exhibiting different types of incubations in relation to the phenomenon of latency and relapses to elucidate their transmission dynamics for planning vector control strategies and chemotherapeutic measures in P. vivax foci. MATERIALS AND METHODS Study site. The malaria clinic of the Malaria Research Centre, at 2-Nanak Enclave, Delhi is located in northeastern Delhi. The clinic attracts patients mostly from 8-9 periurban villages that are 4-5 km from the clinic and have an area of approximately 25 km 2. The Yamuna River is located approximately 3-4 km from these villages. The inhabitants belong mainly to low socioeconomic strata and are employed in small-scale industries as laborers. The climate of Delhi is divided into three distinct seasons: summer (April-June), monsoon (July-October), and winter (November-March). The average temperature, rainfall, and relative humidity during the three seasons are as follows:

The escalating burden, pathogenesis, and clinical sequel of malaria during pregnancy have combinatorial adverse impact on both mother and foetus that further perplexed the situation of diagnosis, treatment, and prevention. This prompted us to evaluate the status of population at risk of MIP in Hazaribag, Jharkhand, India. Cross-sectional study was conducted over a year at Sadar Hospital, Hazaribag. Malaria was screened using blood smear and/or RDT. Anaemia was defined as haemoglobin concentration. Pretested questionnaires were used to gather sociodemographic, clinical, and obstetrical data. The prevalence of MIP was 5.4% and 4.3% at ANC and DU, and 13.2% malaria was in women without pregnancy. Interestingly, majority were asymptomatically infected with P. vivax (over 85%) at ANC and DU. Peripheral parasitemia was significantly associated with fever within past week, rural origin of subjects, and first/second pregnancies in multivariate analysis, with the highest risk factor associated with fever followed by rural residence. Strikingly in cohort, anaemia was prevalent in 86% at ANC as compared to 72% at DU, whereas severe anaemia was 13.6% and 7.8% at ANC and DU. Even more anaemia prevalence was observed in MIP group (88% and 89% at ANC and DU), whereas severe anaemia was 23% and 21%, respectively. In view of observed impact of anaemia, parasitemia and asymptomatic infection of P. vivax during pregnancy and delivery suggest prompt diagnosis regardless of symptoms and comprehensive drug regime should be offered to pregnant women in association with existing measures in clinical spectrum of MIP, delivery, and its outcome.