Control of Aedes aegypti and Ae. albopictus, the vectors of dengue and chikungunya, by using pheromone C21 with an insect growth regulator: Results of multicentric trials from 2007-12 in India.

BACKGROUND & OBJECTIVES: Aedes mosquito control has gained much importance nowadays in view of rise in number of reported cases of dengue and chikungunya in India and other countries. In the present study, C21 attracticide (containing a pheromone and an insect growth regulator­IGR, developed by Defence Research and Development Establishment (DRDE), Gwalior, India was tested for its feasibility for surveillance and control of Aedes mosquito in a multicentric mode from October 2007 to June 2012 in urban (Delhi, and Bengaluru district, Karnataka) and suburban (Alappuzha district, Kerala) settings of the country in three phases. METHODS: Across the randomly selected households in each study area, two to four containers treated with attracticide (experimental) and untreated (control) were placed and monitored by trained surveillance workers on weekly/ fortnightly basis for determining the presence of eggs, larvae and pupae. Container positivity, percent larvae, egg and pupae collected were determined during different phases and analyzed statistically using SPSS 18.0. RESULTS: Container positivity was found statistically significant at Bengaluru and Alappuzha, Kerala while in Delhi, it was found non-significant. Eggs collected from experimental containers were significantly higher in comparison to control at all the locations except Delhi. Also larvae collected from control containers were significantly higher at all the locations except Bengaluru. Pupae collected from control containers remained significantly higher at all the locations as no pupal formation was recorded from experimental containers. INTERPRETATION & CONCLUSION: The use of C21 attracticide hampered pupal formation, thus inhibiting adult population in the study areas. The study established that C21 attracticide was efficacious in the field conditions and has potential for use in surveillance and management of dengue and chikungunya mosquitoes.

Aggregation responses of Cimex hemipterus F. to semiochemicals identified from their excreta.

BACKGROUND & OBJECTIVES: Bedbug Cimex hemipterus lives in cracks and crevices, and shows aggregation activity in the harbourage containing its fecal matter. Limited information is available on the chemicals influencing this aggregation behaviour in C. hemipterus. Possible components responsible for aggregation, if identified and evaluated can be helpful in designing bedbug management. METHODS: Study on the isolation and identification of chemicals from the excreta extract responsible for aggregation has been carried by GC-MS. The compounds identified were evaluated at different concentrations to C. hemipterus and their attraction index was determined. RESULTS: The GC-MS analysis of the excreta extract resulted in identification of 33 different types of semiochemicals such as esters, carboxylic acids, alcohols, aldehydes, ketones and other hydrocarbons. Among these 33 compounds of various groups, the carboxylic acid (E)-2-hexenoic acid and the aldehyde (E)-2-hexenal found to elicit highest positive aggregation behavioural response in all the stages followed by hexenal. INTERPRETATION & CONCLUSION: The fecal matter influence the aggregation behaviour in C. hemipterus. (E)-2-hexenoic acid, hexanoic acid, (E)-2-hexenal and hexanal found to exhibit aggregation in various stages of bedbug life cycle. These chemical components can be further exploited for designing and development tool for management of bedbugs.

Acute toxicity studies of safer and more effective analogues of N,N-diethyl-2-phenylacetamide.

The present work was designed to evaluate the toxicity of various synthesized aromatic amides that are analogs of N,N-diethyl-2-phenylacetamide, a well known insect repellent. The toxicity profile of these compounds was compared with N,N-diethyl-2-phenylacetamide as well as other registered insect repellents namely N,N-diethyl-3-methyl benzamide and N,N-diethylbenzamide. The primary skin irritation index values of the compounds, dermal toxicity of the chemicals and acute oral toxicity data to assess the safety of the synthesized aromatic amides are reported in this paper. Results of hematological and biochemical studies of these analogues are reported and discussed.

Behavioral responses and bioefficacy of some aromatic amides against Aedes aegypti.

A series of substituted aromatic amides by varying the chain length, substitution of methyl, methoxy, chloro, and fluoro groups at ortho-, meta-, and para-positions of the phenyl ring of N,N-diethyl-2-phenylacetamide were synthesized. Laboratory studies were carried out to observe the behavioral responses and repellent activity of these newly synthesized aromatic amides against Aedes aegypti (L.) mosquitoes. The deterrent activity of these synthetic amides against any vectors has not been reported previously. These aromatic amides were tested for their behavioral responses and compared with the well known insect repellents, namely, N,N-diethyl toluamide; N,N-diethyl phenylacetamide; and N,N-diethylbenzamide. Out of the 14 compounds synthesized, seven compounds were selected on the basis of those showing >75% of repellent response for the bioefficacy test on human volunteers. The potential use of lead compounds in personal protection management is discussed.

Resistance status of the malaria vector mosquitoes, Anopheles stephensi and Anopheles subpictus towards adulticides and larvicides in arid and semi-arid areas of India.

Susceptibility studies of malaria vectors Anopheles stephensi Liston (Diptera: Culicidae) and An. subpictus Grassi collected during 2004-2007 from various locations of Arid and Semi-Arid Zone of India were conducted by adulticide bioassay of DDT, malathion, deltamethrin and larvicide bioassay of fenthion, temephos, chlorpyriphos and malathion using diagnostic doses. Both species from all locations exhibited variable resistance to DDT and malathion from majority of location. Adults of both the species were susceptible to Deltamethrin. Larvae of both the Anopheline species showed some evidence of resistance to chlorpyriphos followed by fenthion whereas susceptible to temephos and malathion.

Amperometric immunosensor based on gold nanoparticles/alumina sol-gel modified screen-printed electrodes for antibodies to Plasmodium falciparum histidine rich protein-2.

We report herein the amperometric immunosensor for antibodies to Plasmodium falciparum histidine rich protein-2 (PfHRP-2). Screen-printed electrodes (SPEs) were modified with alumina sol-gel (Al(2)O(3) sol-gel) derived film and gold nanoparticles i.e. AuNPs/Al(2)O(3)sol-gel/SPE. A thin film was formed by dripping Al(2)O(3) sol on SPE followed by electrochemical deposition of gold nanoparticles (AuNPs). The modified SPEs were characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM-EDAX), Raman spectra and voltammetric experiments. Antibodies in rabbit serum sample were allowed to react with the PfHRP-2 protein that was immobilized on the modified SPE to form antigen-antibody immune complex (PfHRP-2/anti-PfHRP-2). The bound antibodies were quantified by alkaline phosphatase (AP) enzyme labeled secondary antibodies (anti-rabbit immunoglobulins-AP conjugate). Enzymatic substrate, 1-naphthyl phosphate was converted to 1-naphthol by AP and an electroactive product was quantified using amperometry. The performances of the developed immunosensor and Dot-ELISA were tested against different dilutions of hyper immune serum (rabbit anti-PfHRP-2). Dot ELISA and the developed immunosensor (AuNPs/Al(2)O(3)sol-gel/SPE) results for the hyper immune serum containing anti-PfHRP-2 were distinctly positive when diluted upto 8 times (1 : 12800 dilution) and 11 times (1 : 102400 dilution), respectively. The developed immunosensor was applied for antibodies to PfHRP-2 in human clinical samples.

Effect of sublethal dose of diflubenzuron and azadirachtin on various life table attributes of Culex quinquefasciatus (Diptera: Culicidae).

The exposure of sublethal doses (25% emergence inhibition) of diflubenzuron (DFB) (0.0002 mg/liter) and azadirachtin (AZD; 0.0079 mg/liter) insect growth regulators was provided to late third- early fourth instars of Culex quinquefasciatus till emergence of adults to find out the effects on various biological and life table attributes of adults, and comparison was made with control (CTL) as well. Life expectancy of both males and females of DFB survivors was significantly less in comparison with AZD and CTL ones; however, it was nonsignificantly different between CTL and AZD. In all treatments, males were shorter lived than females of their respective treatments. DFB survivors showed significant reduction in number of blood-fed females, resulting in minimum of oviposition days, total number of egg rafts, total number of eggs, eggs per egg raft, eggs per female life span, and eggs per female per day than survivors of both AZD and CTL significantly, but these parameters did not differ significantly between CTL and AZD, except eggs per egg raft. The net reproductive rate, intrinsic rate of increase, and finite rate of increase were significantly less in DFB than rest of the survivors, whereas doubling time for generation was significantly high; however, difference in mean generation was not significant among them. This study reveals that DFB has the potency to affect severely the various life table attributes at sublethal dose, but AZD does not have such effectiveness at similar sublethal dose against Cx. quinquefasciatus.

Electrophysiological, flight orientation and oviposition responses of three species of mosquito vectors to hexadecyl pentanoate: residual oviposition repellent activity.

Understanding on the chemical ecology of mosquito behavior is of paramount importance in developing control programs employing attractants and repellents. Several workers focused on topical repellents and oviposition attractants of mosquitoes, however, only limited work has been accomplished on mosquito oviposition repellents. The present systematic investigation provides evidence on the effectiveness of a C21 fatty acid ester- hexadecyl pentanoate, to stimulate antennal olfactory receptors of Aedes aegypti (L.), Ae. albopictus (Skuse), and Anopheles stephensi (Liston) that mediate their long-range olfaction guided flight orientation behavior by repelling the gravid females of these mosquito vectors in the olfactometer. The compound loaded onto an effervescent tablet retained its repellent property in the treated substrates for up to 1 wk at 10 mg/L. In places, where the mosquito breeding habitats are near to human habitations, could be treated with hexadecyl pentanoate to repel the ovipositing gravid females as a component of the integrated approach for mosquito management by disrupting the mosquito life cycle and population growth.

Temephos-induced resistance in Aedes aegypti and its cross-resistance studies to certain insecticides from India.

A temephos-induced resistance in Aedes aegypti that was developed for 24 generations exhibited 20.3-fold resistance as compared to susceptible strain. The diagnostic dose of temephos 0.02 mg/l exhibited gradual decrease in larval mortality with the progression of generations. An operational dose (1 mg/l) exhibited the LT(50) value of 41.42 min in the susceptible population, whereas the value of the resistant population increased to 72.62, 108.86, 122.34, 182.03, 244.82 and 304.86 min in the fourth, eighth, 12th, 16th, 20th and 24th generation, respectively. The study carried out showed 120 min as the cut-off limit for differentiation between susceptible and resistant A. aegypti. Cross-resistance studies showed a varied degree of cross-resistance to fenthion, chlorpyrifos, malathion and DDT, whereas comparatively higher cross-resistance was observed to chlorpyrifos. Study on diagnostic doses of insecticides to A. aegypti females indicated a gradual decrease in adult mortality at every eighth generation as compared to susceptible population when exposed to malathion 0.8%, fenthion 0.25%, DDT 4%, permethrin 0.25% and temephos 6.5%, whereas little or no change in mortality when exposed to lambda-cyhalothrin 0.03% and propoxur 0.1%, was observed. Thus, the expression of larval resistance was observed in adult stages also.

Surface morphology and morphometric analysis of sensilla of Asian tiger mosquito, Aedes albopictus (Skuse): an SEM investigation.

BACKGROUND & OBJECTIVES: The sensilla and sensory mechanism play a significant role in hostseeking and oviposition behaviour of mosquitoes, which enable them to transmit various diseases to humans. Aedes albopictus (Skuse) has emerged as a major vector of Chikungunya virus in the recent epidemics in most parts of southern India. Studies on the sensory structures of dengue vector, Aedes aegypti (Linn) are comprehensive; whereas information on the sensillary systems of Asian tiger mosquito, Ae. albopictus is inadequate. Therefore, the present study has been carried out to observe various types of sensilla located on the antenna, maxillary palp, labial palp, tarsi and ovipositor of Ae. albopictus using scanning electron microscopy. METHODS: The antennae, maxillary palpi, labellum, tarsi and ovipositor of 10 different female mosquito of Ae. albopictus were fixed individually in 2.5% glutaraldehyde solution, washed twice and dehydrated with ascending grades of ethanol. Samples were cleared with xylene, air-dried, mounted on stubs, gold coated in an ion-sputtering unit and the sensilla were viewed between 5 and 10 KV using FEI-Quanta 400-EDAX scanning electron microscope. ANOVA revealed significant differences in the morphometric features of various sensilla. RESULTS: In the antenna Sensilla trichoidea are numerously distributed in all flagellar segments revealed four distinct subtypes. Two types of grooved peg sensilla were observed. Sensilla coeloconica was observed in the terminal flagellum of antenna and tarsomeres with large variation in diameter. Sensilla chaetica are distributed throughout the body and revealed greater variation in morphology and morphometric parameters. INTERPRETATION & CONCLUSION: The significant difference among various types of sensilla would possibly reveal their functions. The porous sensilla are olfactory and contact chemoreceptors while the aporous sensilla would play the role of mechanoreception. Sensilla coeloconica on the antenna, tarsus showed major differences with Ae. aegypti. The ovipositor sensilla revealed three types of chaetica arranged in rows but has not been reported earlier with other mosquito species.

Highly sensitive amperometric immunosensor for detection of Plasmodium falciparum histidine-rich protein 2 in serum of humans with malaria: comparison with a commercial kit.

A disposable amperometric immunosensor was developed for the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) in the sera of humans with P. falciparum malaria. For this purpose, disposable screen-printed electrodes (SPEs) were modified with multiwall carbon nanotubes (MWCNTs) and Au nanoparticles. The electrodes were characterized by cyclic voltammetry, scanning electron microscopy, and Raman spectroscopy. In order to study the immunosensing performances of modified electrodes, a rabbit anti-PfHRP-2 antibody (as the capturing antibody) was first immobilized on an electrode. Further, the electrode was exposed to a mouse anti-PfHRP-2 antibody from a serum sample (as the revealing antibody), followed by a rabbit anti-mouse immunoglobulin G-alkaline phosphatase conjugate. The immunosensing experiments were performed on bare SPEs, MWCNT-modified SPEs, and Au nanoparticle- and MWCNT-modified SPEs (Nano-Au/MWCNT/SPEs) for the amperometric detection of PfHRP-2 in a solution of 0.1 M diethanolamine buffer, pH 9.8, by applying a potential of 450 mV at the working electrode. Nano-Au/MWCNT/SPEs yielded the highest-level immunosensing performance among the electrodes, with a detection limit of 8 ng/ml. The analytical results of immunosensing experiments with human serum samples were compared with the results of a commercial Paracheck Pf test, as well as the results of microscopy. The specificities, sensitivities, and positive and negative predictive values of the Paracheck Pf and amperometric immunosensors were calculated by taking the microscopy results as the "gold standard." The Paracheck Pf kit exhibited a sensitivity of 79% (detecting 34 of 43 positive samples; 95% confidence interval [CI], 75 to 86%) and a specificity of 81% (correctly identifying 57 of 70 negative samples; 95% CI, 76 to 92%), whereas the developed amperometric immunosensor showed a sensitivity of 96% (detecting 41 of 43 positive samples; 95% CI, 93 to 98%) and a specificity of 94% (correctly identifying 66 of 70 negative samples; 95% CI, 92 to 99%). The developed method is more sensitive and specific than the Paracheck Pf kit.

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase.

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 degrees C with 0.5mM isopropyl beta-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 degrees C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 micromol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k(cat) [(3.2+/-0.02)x10(4)] with 3-acetylpyridine adenine dinucleotide (APAD+). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.

Morphological sexual dimorphism in three species of anopheline mosquito larvae.

Sexual separation at the larval stage in anopheline mosquitoes with the naked eye is difficult. We have identified distinguishing spots visible to the naked eye on the 6th abdominal segment of 3rd and 4th instars of Anopheles stephensi, An. culicifacies, and An. subpictus. Based on this feature, male and female larvae can be differentiated morphologically at 3rd and 4th instars of these species. This is the first report on these characteristic spots that may have a wide application for larval sexing in mosquito taxonomy, physiology, toxicology, genetics, and control.

Electroantennogram and behavioral responses of Culex quinquefasciatus (Diptera: Culicidae) females to chemicals found in human skin emanations.

The southern house mosquito, Culex quinquefasciatus Say (Diptera: Culicidae), is closely associated with humans and is a vector of filariasis. Use of semiochemicals for control of mosquitoes is a novel and potentially ecofriendly control approach. Human skin emanations are known to attract mosquitoes. In the current study, olfactory and behavioral responses of host-seeking female Cx. quinquefasciatus to various chemical components of human skin emanations (carboxylic acids, alcohols, and aldehydes) were evaluated separately at three doses (0.01, 0.1, and 1 microg) for electroantennogram (EAG) and three doses (0.1, 1, and 10 microg) for behavioral assay. Results of EAG studies indicated that all carboxylic acids elicited significant olfactory responses except tetradecanoic acid (C14) and octadecanoic acid (C18). In particular, hexanoic acid (C6) elicited a maximum, eight-fold olfactory response compared with the solvent control. Ethylene glycol and benzyl alcohol exhibited significant EAG and behavioral responses, whereas among aldehydes, nonanal elicited high EAG and behavioral responses, but only at all three doses tested compared with control. Some carboxylic acids elicited significant behavioral responses, attracting approximately 75% of females tested toward chemical stimuli in a Y-tube olfactometer.

Amplification of LDH gene from indian strains of Plasmodium vivax.

BACKGROUND & OBJECTIVES: Plasmodium vivax is geographically widespread and responsible for > 50% of malaria cases in India. Increased drug resistance of the parasite highlights the immediate requirement of early and accurate diagnosis as well as new therapeutics. In view of this, the present study was undertaken to amplify P. vivax (Indian strains) lactate dehydrogenase gene (PvLDH) which has been identified as a good target for antimalarials as well as diagnostics. METHODS: P. vivax infected clinical blood samples were collected from southern part of India and were tested with established diagnostic parameters (ICT, Giemsa staining). Total DNA was extracted from blood samples and subjected to PCR using two sets of primers, one for the amplification of full PvLDH gene (951 bp) and the other for a partial PvLDH gene fragment (422bp), covering a variable antigenic region (140aa) as compared to other plasmodial species. RESULTS & CONCLUSION: PCRs for both the full and partial gene targets were optimised and found to be consistent when tested on several P. vivax positive clinical samples. In addition, full gene PCR was found to specifically detect only P. vivax DNA and could be used as a specific molecular diagnostic tool. These amplified products can be cloned and expressed as a recombinant protein that might be useful for the development and screening of antimalarials as well as for diagnostic purposes.

Bioassay of three sulphur containing compounds as rat attractant admixed in cereal-based bait against Rattus rattus Linn.

Three sulphur containing compounds, carbon disulphide, dimethyl disulphide and dimethyl sulphide were bioassayed for preference after admixing them in cereal base as ready bait block for use against commensal rat, R. rattus (wild type) in four way choice chamber system. Rat preference for different baits was also studied with automatic recording animal activity meter. Rats exhibited attractancy to the baits at 0.005% concentration of all the three compounds while at 0.01% concentration they have showed repellency. Dimethyl sulphide at 0.005% concentration showed better attractancy towards both sexes of rat.

Role of esterases and monooxygenase in the deltamethrin resistance in Anopheles stephensi Giles (1908), at Mysore.

Field collected An. stephensi larvae were colonized in the laboratory for 15 generations and acclimatized. An isofemale line was raised from this colony and the larvae were subjected to continuous deltamethrin selection pressure. LC50 and LC90 values were calculated at every generation. The values indicated that at the end of seventh generation the larvae have developed 87 fold tolerance in terms of LC50 value compared with the first generation. The reason for this kind of resistance was analyzed on the basis of differential activity of A-esterase, B-esterase, glutathione s-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PD). A significant correlation (P < 0.05) was observed with B-esterase and G6PD activity with the rise in the LC50 and LC90 values. However no significant rise were observed in the other enzymes tested such as A-esterase and GST. The isozyme analysis of the A-esterase and B-esterase using polyacrylamide gel electrophoresis (PAGE) have shown differential profiles.

Midgut microbial community of Culex quinquefasciatus mosquito populations from India.

The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a large geographical area.