Development, standardization and validation of molecular techniques for malaria vector species identification, trophic preferences, and detection of Plasmodium falciparum

Background & objectives: Knowledge on prevalence of malaria vector species of a certain area provides important information for implementation of appropriate control strategies. The present study describes a rapid method for screening of major Anopheline vector species and at the same time detection of Plasmodium falciparum sporozoite infection and blood meal preferences/trophic preferences. Methods: The study was carried from February 2012 to March 2013 in three seasons, i.e. rainy, winter and summer in Jhumpura PHC of Keonjhar district, Odisha, India. Processing of mosquitoes was carried out in two different methods, viz. mosquito pool (P1) and mosquito DNA pool (P2). Pool size for both the methods was standardized for DNA isolation and multiplex PCR assay. This PCR based assay was employed to screen the major vector composition in three different seasons of four different ecotypes of Keonjhar district. Pearson’s correlation coefficient was determined for a comparative analysis of the morphological identification with the pool prevalence assay for each ecotype. Results: A pool size of 10 was standardized for DNA isolation as well as PCR. PCR assay revealed that the average pool prevalence for all ecotypes was highest for An. annularis in winter and summer whereas for An. culicifacies it was rainy season. Foothill and plain ecotypes contributed to highest and lowest vectorial abundance respectively. The results of the prevalence of vector species in pool from PCR based assay were found to be highly correlated with that of the results of morphological identification. Interpretation & conclusion: Screening by pool based PCR assay is relatively rapid as compared to conventional morphological identification and can be employed as an important tool in malaria control programmes

Newly Identified Wild Rice Accessions Conferring High Salt Tolerance Might Use a Tissue Tolerance Mechanism in Leaf

Cultivated rice (Oryza sativa\ua0L.) is very sensitive to salt stress. So far a few rice landraces have been identified as a source of salt tolerance and utilized in rice improvement. These tolerant lines primarily use Na+\ua0exclusion mechanism in root which removes Na+\ua0from the xylem stream by membrane Na+\ua0and K+\ua0transporters, and resulted in low Na+\ua0accumulation in shoot. Identification of a new donor source conferring high salt tolerance is imperative. Wild relatives of rice having wide genetic diversity are regarded as a potential source for crop improvement. However, they have been less exploited against salt stress. Here, we simultaneously evaluated all 22 wild\ua0Oryza\ua0species along with the cultivated tolerant lines including Pokkali, Nona Bokra, and FL478, and sensitive check varieties under high salinity (240 mM NaCl). Based on the visual salt injury score, three species (O.\ua0alta, O.\ua0latifolia, and\ua0O.\ua0coarctata) and four species (O.\ua0rhizomatis, O.\ua0eichingeri, O.\ua0minuta, and\ua0O.\ua0grandiglumis) showed higher and similar level of tolerance compared to the tolerant checks, respectively. All three CCDD genome species exhibited salt tolerance, suggesting that the CCDD genome might possess the common genetic factors for salt tolerance. Physiological and biochemical experiments were conducted using the newly isolated tolerant species together with checks under 180 mM NaCl. Interestingly, all wild species showed high Na+\ua0concentration in shoot and low concentration in root unlike the tolerant checks. In addition, the wild-tolerant accessions showed a tendency of a high tissue tolerance in leaf, low malondialdehyde level in shoot, and high retention of chlorophyll in the young leaves. These results suggest that the wild species employ tissue tolerance mechanism to manage salt stress. Gene expression analyses of the key salt tolerance-related genes suggested that high Na+\ua0in leaf of wild species might be affected by\ua0OsHKT1;4-mediated Na+\ua0exclusion in leaf and the following Na+\ua0sequestration in leaf might be occurring independent of tonoplast-localized OsNHX1. The newly isolated wild rice accessions will be valuable materials for both rice improvement to salinity stress and the study of salt tolerance mechanism in plants