Candidate screening of blast resistance donors for rice breeding

Rice blast is one of the most serious diseases in the world. The use of resistant cultivars is the most preferred means to control this disease. Resistance often breaks down due to emergence of new races; hence identification of novel resistance donors is indispensable. In this study, a panel of 80 released varieties from National Rice Research Institute, Cuttack was genotyped with 36 molecular markers that were linked to 36 different blast resistance genes, to investigate the varietal genetic diversity and molecular marker-trait association with blast resistance. The polymorphism information content of 36 loci varied from 0.11 to 0.37 with an average of 0.34. The cluster analysis and population structure categorized the 80 National Rice Research Institute released varieties (NRVs) into three major genetic groups. The principal co-ordinate analysis displays the distribution of resistant and moderately resistant NRVs into different groups. Analysis of molecular variance result demonstrated maximum (97%) diversity within populations and minimum (3%) diversity between populations. Among tested markers, two markers (RM7364 and pi21_79-3) corresponding tothe blast resistance genes (Pi56(t) and pi21) were significantly associated and explained a phenotypic variance of 4.9 to 5.1% with the blast resistance. These associated genes could be introgressed through marker-assisted to develop durable blast resistant rice varieties. The selected resistant NRVs could be good donors for the blast resistance in rice crop improvement research.

Effect of anti-mosquito midgut antibodies on development of malaria parasite, Plasmodium vivax and fecundity in vector mosquito Anopheles culicifacies (Diptera: culicidae).

The effect of anti-mosquito-midgut antibodies on the development of the malaria parasite, P.vivax was studied by feeding the vector mosquito, An. culicifacies with infected blood supplemented with serum from immunized rabbits. In order to get antisera, rabbits were immunized with midgut proteins of three siblings species of Anopheles culicifacies, reported to exhibit differential vectorial capacity.  The mosquitoes that ingested anti-midgut antibodies along with infectious parasites had significantly fewer oocysts compared to the control group of mosquitoes. The immunized rabbits generated high titer of antibodies. Their cross reactivity amongst various tissues of the same species and with other sibling species was also determined. Immunogenic polypeptides expressed in the midgut of glucose or blood fed An. culicifacies sibling species were identified by Western blotting. One immunogenic polypeptide of 62 kDa was exclusively present in the midgut of species A. Similarly, three polypeptides of 97, 94 and 58 kDa and one polypeptide of 23 kDa were present exclusively in species B and C respectively. Immunoelectron microscopy revealed the localization of these antigens on baso-lateral membrane and microvilli. The effects of anti-mosquito midgut antibodies on fecundity, longevity, mortality and engorgement of mosquitoes were studied. Fecundity was also reduced significantly. These observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.

Role of CR1 Knops polymorphism in the pathophysiology of malaria: Indian scenario.

Background & objectives: Plasmodium falciparum is the leading cause of mortality and causes cerebral malaria associated with sequestration caused by cytoadherence of the trophozoite and schizont-infected erythrocytes to the endothelial cells of the deep vascular beds in the brain. Pathophysiology of malaria is complicated by rosetting. Rosetting is a process of binding of uninfected erythrocytes to the erythrocytes infected with mature asexual parasites and is controlled by expression of complement receptor 1 (CR1) on RBC surface. Various polymorphic forms of CR1 are known including molecular weight polymorphism, red blood cell expression levels/density polymorphism and Knops (KN) polymorphism. The Knops blood group includes several allelic pairs; Knops a and b (Kna and Knb), McCoy a and b (McCa, McCb), Swain-Langley (Sla), and Villien (Vil). Knops phenotype Sl (a–) has been found to rosette less effectively than Sl (a+) and hence suggested to be more protective. P. falciparum cases have not reduced much as compared to the reduction in the total number of malaria cases in the past few years. In addition, P. falciparum is the leading cause for all mortality and most of the morbidity in India. We, therefore, investigated the role of CR1 Knops polymorphism in the pathophysiology of malaria in Indian population. Methods: A case control approach was used for this study. CAPS (Cleaved amplified polymorphic sequence) methodology was adopted. A total of 100 normal individuals (free from any ailment) and 100 individuals suffering from P. falciparum infection (uncomplicated malaria) were recruited for this study. Results: We found that in Indian population (normal individuals and P. falciparum-infected individuals), only the wild type allele is present. Interpretation & conclusion: We concluded that the process of rosetting in the Indian context could be occurring independently of the effect of Knops polymorphism and in part could be controlled by other polymorphisms of the CR1 gene (density and structural polymorphism).

On the conspecificity of Anopheles fluviatilis species S with Anopheles minimus species C.

Anopheles fluviatilis and An. minimus complexes,each comprising of at least three sibling species, are closely related and important malaria vectors in Oriental Region. Recently An. fluviatilis species S, which is a highly efficient malaria vector in India, has been made conspecific with An. 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 of An. fluviatilis S and An. minimus C. We found that the sequences of An. fluviatilis S are appreciably different from those of An. 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 revealed that An. fluviatilis S is distantly related to An. minimus C as compared to any other members of the Fluviatilis Complex. These findings suggest that the two nominal species, An. fluviatilis S and An. minimus C, do not merit synonymy. The study also confirms that the reported species An. fluviatilis X is synonym with species S.

Efficacy of IGR compound Starycide 480 SC (Triflumuron) against mosquito larvae in clear and polluted water.

BACKGROUND AND OBJECTIVES: An environmental friendly formulation Starycide 480 SC (Triflumuron-OMS-2015 ), a new insect growth regulator with chitin synthesis inhibitor type mode of action was evaluated against mosquito larvae in laboratory and small-scale field trials carried out in and around Delhi. METHODS: The formulation was tested in laboratory for its bio-efficacy against late III instar mosquito larvae of different species using WHO bioassay procedure. In the field formulation was sprayed at doses of 0.3, 0.5 and 1 ppm (g/m3) in the natural breeding habitats of Anopheles and Culex mosquitoes. The impact was assessed by monitoring densities of larvae by dipper and observing the reduction in larval density and inhibition of adult emergence. RESULTS: In the laboratory, formulation was more effective against larvae of Anopheles stephensi and Aedes aegypti than Culex quinquefasciatus, but it produced 100% inhibition of adult emergence for all mosquito species at a concentration of 0.02 ppm. In the field trials, formulation did not produce 100% reduction in the density of late stage larvae even at 1 ppm (g/m3), the highest dose tested, but it resulted in 100% inhibition of pupal formation of both Anopheles and Culex spp in different types of habitats for 3-7 weeks even at a lower dose of 0.5 ppm. INTERPRETATION AND CONCLUSION: Application of triflumuron in the natural breeding habitats in both clean and polluted water @ 0.5 ppm (g/m3) resulted in complete inhibition of adult emergence of both Anopheles and Culex spp for 3-7 weeks. This formulation may be tested in large-scale field trials for further use in the vector control programme.

Evaluation of malaria endemicity by peptide ELISA.

Interest in mapping the malaria transmission has motivated a need to develop a simple serological assay in a defined population. Evaluation of coded samples by peptide ELISA provided a framework to estimate the malarial impact. Comparison of field data and ELISA OD values in different malariogenic areas shows how the disease has been more correctly interpreted. Here we provide an empirical approach to estimate Annual Parasite Index (API) as well as Equivalent Transmission Index (ETI) using a combination of epidemiological, parasitological and immunological data. We estimate that there were 3 different malariogenic status like low, moderate and high transmission zones based on their' ELISA OD values. Our indigenous developed ETI estimates are 10 fold higher than API reported by the Primary Health Centre. Our record indicates that incidences of malaria will continue to be underestimated unless we adapt an alternative strategy. In order to verify the scope of malaria surveillance, coded samples were tested. Comparison of ELISA OD, API and ETI of the coded samples indicated Rourkela had high, Shahjahanpur had moderate and Bangalore had the lowest malaria transmission. For mass blood slide examination, microscopic method is a tedious process prone to human error while largely automated ELISA could reduce the scope for human error and could be a supplement for microscopic process.

Midgut antibodies reduce the reproductive capacity of Anopheles stephensi (Diptera: Culicidae).

Rabbits immunized with polypeptides of midgut of glucose fed A. stephensi resulted in high titer of antibodies (10(4)-10(6)) as detected by ELISA. Effect of antisera on fecundity, hatchability and engorgement was investigated. Fecundity was reduced drastically (62.4%). Eight polypeptides were recognized by the antisera raised against midgut tissues viz., 92, 85, 55, 52, 45, 38, 29 and 13 kDa. Cross reactivity of these antibodies with different tissues of A. stephensi as well as different species of Anopheles was also analyzed. The results indicated that anti-mosquito midgut antibodies had the potential to disrupt the reproductive physiology of mosquitoes in view of the present study, there is a need for further investigation with target antigens.

Evaluation of bifenthrin treated mosquito nets against anopheline and culicine mosquitoes.

BACKGROUND AND OBJECTIVES: The main rural malaria vector Anopheles culicifacies has developed resistance to dichloro diphenyl trichloroethane (DDT), hexachloro cyclo hexane (HCH) and malathion in the state of Haryana in northern India. An alternative synthetic pyrethroid insecticide bifenthrin was therefore evaluated on mosquito nets against anopheline and culicine mosquitoes, in two villages Jagdishpur and Garh Mirakpur of Community Health Center (CHC) Badhkhalsa in district Sonipat, Haryana state. METHODS: Two formulations of bifenthrin, suspension concentrate (SC) and micro-emulsion (ME) were compared with micro-capsule suspension (CS) of lambdacyhalothrin. The impact of three doses of bifenthrin (10, 25 and 50 mg/m(2)) impregnated on mosquito nets was compared with lambdacyhalothrin (25 mg/m(2)) and untreated control. Quality assessment of treatment on treated nets was carried out by residue analysis and the persistence of the insecticide on nets was determined by contact bioassays. Efficacy of treated nets on mosquito density was assessed by calculating mosquito entry rate, immediate mortality, delayed mortality and excito-repellency to the insecticides. RESULTS: In susceptibility tests An. culicifacies was susceptible to bifenthrin (0.1% test papers) and to lambdacyhalothrin (0.05% test papers). Bioassays on treated nets against A. culicifacies recorded 100 per cent mortality up to tenth fortnight for all the doses of impregnation with bifenthrin (SC and ME) and lambdacyhalothrin (CS). Ring-net bioassays against An. culicifacies showed median knock-down time between 3.1 to 11.4 min. Behavioural indices were also studied for anopheline and culicine mosquitoes. The reduction in entry rates of anopheline and culicine mosquitoes into the rooms with treated nets compared to control indicated good efficacy with all the formulations and doses of the insecticides. INTERPRETAION AND CONCLUSION: Indoor (immediate) mortality of mosquitoes with bifenthrin ME formulation was relatively lower compared to SC fomulation of bifenthrin and based on delayed mortility and continued susceptibility in bioassays, bifenthrin ME at the rate of 10 mg/m(2) dose was found suitable for the impregnation of mosquito nets for phase III trial.

Efficacy of a new formulation of Bacillus thuringiensis var israelensis (Bti) in laboratory and field conditions of Kumaun foothills of Uttaranchal, India.

Efficacy of a new Bti formulation was tested in the laboratory and small scale field trials against mosquito larvae were carried out from March to June, 2001 in different breeding habitats of Kumaun foothill region of Nainital and Udham Singh Nagar districts, Uttaranchal state, India. Laboratory tests revealed increased efficacy against Culex quinquefasciatus (LC50 = 0.035 mg/L) followed by Aedes aegypti (LC50 = 0.0628 mg/L), An. culicifacies sp A (LC50 = 0.184mg/L) and An. stephensi (LC50 - 0.2216mg/L). Results of field trials revealed 100% mortality of mosquito larvae after treatment with Bti @ 0.5 gm/m2 surface area. The impact was similar on different mosquito species breeding in different habitats. However, repeated treatments were required due to reappearance of larvae in the breeding habitats within a week. No side effects of Bti was observed during field trial on non- target organisms (NTOs). Environmental disturbances and man made problems affected the spray impact of Bti.

Alterations in polypeptides pattern in malaria vector Anopheles stephensi, fed upon immunized blood causing fecundity reduction.

Changes in polypeptides pattern of haemolymph, midgut, ovary and salivary glands of female mosquito A. stephensi were studied when fed upon anti-mosquito haemolymph antibodies. The expression of almost all polypeptides was reduced in haemolymph and ovary of the immune fed mosquitoes as compared to control. However, there was no significant difference in case of midgut and salivary glands. Seven polypeptides 100, 90, 84, 80, 62, 19 and 12.5 kDa were absent in haemolymph and five 92, 90, 80, 60 and 55 kDa were absent in ovaries. Changes in the polypeptide pattern have been correlated with the fecundity reduction due to immunized blood feeding.

Comparative toxicity of selected larvicidal formulations against Anopheles stephensi Liston and Aedes aegypti Linn.

Toxicity of selected larvicidal formulations of Bacillus sphaericus, Bacillus thuringiensis H-14 israelensis (Bti) and insect growth regulators was determined against Anopheles stephensi and Aedes aegypti, the two major urban mosquito vectors of malaria and dengue fever in India respectively. The study revealed that the formulations of Bacillus thuringiensis H-14 and IGR compounds were highly toxic against both the mosquito species but Bacillus sphaericus formulation was less toxic against Aedes aegypti and has a limited potential for use against Aedes aegypti. Of the two Bti formulations, 'Vectobac' formulation was more toxic against both the species than 'BMP-1442X' formulation. Bti formulations were more toxic against Aedes aegypti (LC50 = 0.06 and 0.14 mg/Litre) than against An. stephensi (LC50 = 0.14 and 0.81 mg/Litre), while B. sphaericus formulation was more toxic against An. stephensi LC50 = 0.031 mg/Litre than Aedes aegypti LC50 = 0.294 mg/Litre. Among different larvicidal formulations tested in this study, IGR compounds were found to be toxic against both the mosquito species at very low concentrations (EC50 values ranging between 0.0001 and 0.0004 ppm).

Laboratory evaluation of the biocontrol potential of Mesocyclops thermocyclopoides (Copepoda: Cyclopidae) against mosquito larvae.

Biocontrol potential of Mesocyclops thermocyclopoides against first instar larvae of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus was studied under laboratory conditions. It was found that M. thermocyclopoides had the highest predation efficacy against Ae. aegypti followed by An. stephensi and Cx. quinquefasciatus. There was a significant reduction in the predation efficacy of M. thermocyclopoides against Cx. quinquefasciatus in the presence of alternate food (p < 0.01). The cage simulation trial indicated that M. themocyclopoides has the potential to control Ae. aegypti breeding effectively in a container type of habitat.

The biology and predatory potential of notonectid bug, Enithares indica (Fabr) against mosquito larvae.

The biology of a notonectid bug Enithares indica against immatures of Anopheline, Culicine and Aedine mosquitos was studied in the laboratory. The life cycle of the bug consists of the egg and five nymphal stages and takes about 64 +/- 1.54 days for completion. All stages of E. indica have good predatory potential. It can be used as a biological control agent in an integrated disease vector control program.