Om Prakash Agrawal

Presence of two alternative kdr -like mutations, L1014F and L1014S, and a novel mutation, V1010L, in the voltage gated Na + channel of Anopheles culicifacies from Orissa, India

BackgroundKnockdown resistance in insects resulting from mutation(s) in the voltage gated Na+ channel (VGSC) is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common kdr mutation in insects, was reported in Anopheles culicifacies-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an An. culicifacies population from Malkangiri district of Orissa, India.MethodsAnopheles culicifacies sensu lato (s.l.) samples, collected from a population of Malkangiri district of Orissa (India), were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR) was developed for the detection of the new mutation L1014S. The An. culicifacies population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS) and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing.ResultsDNA sequencing of An. culicifacies individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA)-to-Phe (TTT) or -Ser (TCA) changes, respectively. A third and novel substitution, Val (GTG)-to-Leu (TTG or CTG), was identified at residue V1010 resulting from either of the two transversions–3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the identification of the new mutation L1014S was found specific as evident from DNA sequencing results of respective samples. Since L1014S was found tightly linked to V1010L, no separate assay was developed for the latter mutation. Screening of population using PIRA-PCR assays for 1014S and ARMS for 1014F alleles revealed the presence of all the three amino acid substitutions in low frequency.ConclusionsThis is the first report of the presence of L1014S (homologous to the kdr-e in An. gambiae) and a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC of An. culicifacies in addition to the previously described mutation L1014F. The V1010L substitution was tightly linked to L1014S substitution. A new PIRA-PCR strategy was developed for the detection of L1014S mutation and the linked V1010L mutation.

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection

BackgroundKnockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection.MethodsPart of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s).ResultsAnalysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TC A) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples.ConclusionsTwo alternative kdr- like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

Pharmacological intervention of tiferron and propolis to alleviate beryllium-induced hepatorenal toxicity

Intervention of chelating agent tiferron (sodium‐4,5‐dihydroxy‐1,3‐benzene disulfonate; 300 mg/kg, intraperitoneal) with propolis (honey beehive product; 200 mg/kg, p.o.) was evaluated to encounter the characteristic biochemical and ultra‐morphological alterations following subchronic exposure to beryllium. Female albino rats were challenged with beryllium nitrate (1 mg/kg, i.p.) daily for 28 days followed by treatment of the above‐mentioned therapeutic agents either individually or in combination for five consecutive days. Exposure to beryllium increased its concentration in the serum, liver and kidney, and caused significant alterations in cytochrome P450 activity, microsomal lipid peroxidation and proteins. Activities of alkaline phosphatase, lactate dehydrogenase, γ‐glutamyl transpeptidase, bilirubin, creatinine and urea in the serum and activity of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, glucose‐6‐phophatase and succinic dehydrogenase in the liver and kidney were significantly altered after beryllium administration. Beryllium exposure also induced severe hepatorenal alterations at histopathological and ultra‐morphological level. Tiferron along with propolis dramatically reversed the alterations in all the variables more towards control rather than their individual treatment. The study concludes that pharmacological intervention of tiferron and propolis is beneficial in attenuating beryllium‐induced systemic toxicity.

Variations in life tables of geographically isolated strains of the mosquito Culex quinquefasciatus

Variations in the life tables and other biological attributes of four strains of Culex quinquefasciatus Say (Diptera: Culicidae) from geographically isolated regions of India that had been reared to the fifth generation in the laboratory were assessed under a standardized rearing regime under constant laboratory conditions. Two strains from arid habitats [Jodhpur (JD) and Bikaner (BKN)], one from a semi‐arid inland habitat [Bathinda (BTH)], one from a semi‐arid coastal habitat [Jamnagar (JMN)] and a standard laboratory strain (LAB) were compared. Horizontal life‐table parameters were measured for each strain. Egg mortality ranged from 4.4% (JD and BTH) to 19.5% (BKN). The lowest rate of adult emergence and highest female : male ratio were found in BKN, and the highest rate of adult emergence and lowest female : male ratio were recorded in BTH. The egg‐hatching period was longest in BTH and shortest in LAB. The duration from oviposition to adult emergence was longest in JD and shortest in LAB. Females lived longer than males in all strains. The net reproductive rates (R0) of all field‐derived strains (122.9–162.2) differed significantly between strains and were significantly greater than that of LAB (107.6). Similarly, both the intrinsic rate of increase (rm) and finite rate of increase (λ) were found to be lower in LAB than in the field strains, but the mean generation time (T) and doubling time (DT) were longest in LAB. For several life‐table attributes, JD and BTH clustered together and were more similar to JMN than to BKN and LAB. The results indicate that BTH, BKN and JD can be characterized as r‐strategists, more so than JMN. Overall fecundity increased with age. Differences in annual temperature ranges and mean annual rainfall between locations were positively correlated (r = 0.46–0.97) with egg production, female life expectancy, R0, rm, λ and T. The results suggest that strains of Cx. quinquefasciatus from different geographical areas with contrasting habitats vary in their survival and reproductive strategies accordingly.

Comparative Toxic Effect of Nitrogen Mustards (HN-1, HN-2, and HN-3) and Sulfur Mustard on Hematological and Biochemical Variables and Their Protection by DRDE-07 and Its Analogues

The chemical warfare agents sulfur mustard (SM) and nitrogen mustards (HN-1, HN-2, and HN-3) are highly reactive vesicants. The study was planned to investigate the protective efficacy of amifostine, DRDE-07 and their analogues, and few conventional antidotes (30 minutes pretreatment) against dermally applied SM and nitrogen mustards in preventing hematological and biochemical changes in mice. Mustard agents (1.0 median lethal dose [LD50]) induced a significant decrease in the body weight and spleen weight. A significant decrease in the white blood cell (WBC) count and an increase in serum transaminases and alkaline phosphatases (ALPs) were observed. A significant decrease in reduced (GSH) and oxidized glutathione (GSSG) and an increase in thiobarbituric acid reactive substances were also observed. All the mustard agents increased DNA fragmentation. The effects of SM were significantly ameliorated by DRDE-07 analogues, and with nitrogen mustards the protection was partial. Overall, DRDE-30 (propyl analogue) followed by DRDE-35 (butyl analogue) are favored as safer and better compounds.

Hepatic endogenous defense potential of propolis after mercury intoxication

Exposure to mercuric chloride (HgCl(2) ; 5 mg kg(-1) body weight; i.p.) induced oxidative stress in mice and substantially increased lipid peroxidation (LPO) and oxidized glutathione (GSSG) levels, decreased the level of reduced glutathione (GSH) and various antioxidant enzymes in liver and also increased the activities of liver marker enzymes in serum. Therapy with propolis extract, a resinous wax-like beehive product (200 mg kg(-1) orally, after mercury administration), for 3 days inhibited LPO and the formation of GSSG and increased the level of GSH in the liver. Release of serum transaminases, alkaline phosphatase, lactate dehydrogenase and γ-glutamyl transpeptidase were significantly restored after propolis treatment. The activities of antioxidant enzymes, that is, superoxide dismutase, catalase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase, were also concomitantly restored towards normal levels after propolis administration. These observations clearly demonstrate that propolis treatment augments antioxidant defense against mercury-induced toxicity and provide evidence that propolis has therapeutic potential as a hepatoprotective agent.

Knockdown resistance (kdr) mutations in Indian Anopheles culicifacies populations

BackgroundAnopheles culicifacies s.l. is one of the primary vectors of malaria in India responsible for the highest number of malaria cases. This vector is resistant to DDT in most parts of the country with indication of emerging resistance to pyrethroids. Since knockdown resistance (kdr) is known to confer cross-resistance between DDT and pyrethroids owing to a common target site of action, knowledge of prevalence of knockdown resistance (kdr) alleles is important from insecticide resistance management point of view.MethodsNine populations of An. culicifacies belonging to five states of India, representing northern, western and central-east India, were screened for the presence of two alternative kdr mutations L1014F and L1014S using PCR-based assays. Dead and alive mosquitoes, following WHO standard insecticide susceptibility test against deltamethrin and DDT, were tested for allelic association.ResultsL1014F mutation was recorded in all populations studied except from Haryana and Rajasthan states in northern India, with low frequencies ranging between 0.012 and 0.076; whereas presence of L1014S mutation was recorded in five populations only belonging to central-east India, with allelic frequencies ranging between 0.010 and 0.046. Both the kdr mutant alleles were found mostly in heterozygous condition without deviating from Hardy-Weinberg equilibrium. Both mutations showed protection against deltamethrin whereas only L1014S mutation showed protection against DDT when tested using additive model.ConclusionsThe two L1014-kdr mutations, L1014F and L1014S, co-occurred in five populations belonging to Chhattisgarh and Odisha states of India whereas L1014F was present in all populations studied except populations from northern states. Both kdr mutations were found with very low allelic frequencies mostly in heterozygous condition and exhibited protection against deltamethrin.

Degradation of cuticle during larval-pupal and pupal-adult development of the housefly, Musca domestica

Abstract. The patterns of changes in cuticle weight, its chitin content and chitinase activity have been studied during postembryonic development of the housefly, Musca domestica L. During pupariation the larval cuticle loses weight. During the early part of this weight‐loss the decline in chitin content parallels the overall change in cuticle weight. A simultaneous elevation in chitinase activity suggests that at this time the larval cuticle is being enzymatically degraded. Later weight loss may be due to sclerotization. No significant changes in cuticle weight or its chitin content occur in pharate cuticle until one day before eclosion. However, a peak of chitinase activity found at mid‐late pupal stage suggests the timing of pupal cuticle breakdown.

Population genetic structure of Culex quinquefasciatus in India by ISSR marker

OBJECTIVE To characterize the genetic structure of various populations of Culex quinquefasciatus (Cx. quinquefasciatus) from India representing different geoclimatic locations. METHODS Inter simple sequence repeat (ISSR) markers were used. A set of 20 primers were screened with the laboratory populations of mosquito species. Finally the IS 40 primer was chosen based on the scorable banding pattern showing 100 percent polymorphism among the various populations. The statistical analysis was done using POPGENE 1.31 software. The consensus tree was generated based on UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5. RESULTS The cluster analysis shows the main cluster which is divided into two sub cluster representing all the populations separated as per their phylogeographic and geoclimatic condition. CONCLUSIONS The findings will be helpful in understanding the population variation under different ecological conditions and development of effective vector management strategies.

Malaria vector Anopheles culicifacies sibling species differentiation using egg morphometry and morphology

BackgroundThe malaria vector Anopheles culicifacies (sensu lato) is an important malaria vector in Southeast Asia which comprises of five sibling species namely A, B, C, D and E. However, only a few forms have been identified as malaria vectors in various endemic countries. Currently, for the first time egg morphometry and morphology has been used to differentiate the three known vector sibling species of Anopheles culicifacies collected from malaria endemic Madhya Pradesh state of central India.MethodsThe adult An. culicifacies (s.l.) was collected from five districts using standard mosquito collection methods. Adult female mosquitoes were allowed to lay eggs individually. The emerged mosquitoes were identified using allele specific polymerase chain reaction (AS-PCR) to sibling species. Eggs of sibling species A, D and E were studied using scanning electron microscopy (SEM) for morphometric and morphological characteristics.ResultsCurrently AS-PCR identified four known sibling species (B, C, D and E) of An. culicifacies in the study area. The surface morphology and morphometric attributes of the sibling species A, D and E eggs considerably differed from each other. An. culicifacies E had a narrow deck as compared to A and D, while An. culicifacies A had a bigger micropyle with 6–7 sectors as compared to D and E that had 6 sectors. An. culicifacies D had the smallest float (the structure present on sides of the egg surface in which air is filled that help in floating) and the number of ribs was also fewer than for An. culicifacies A and E.ConclusionsThe present study provides the first evidence that in addition to PCR assay, sibling species of An. culicifacies can also be differentiated using morphological and morphometric characteristics of the egg stage. The results also advocate that the sibling species of An. culicifacies are morphologically dissimilar and can be resolved using advanced microscopy.