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.

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.

Electroantennogram, flight orientation and oviposition responses of Anopheles stephensi and Aedes aegypti to a fatty acid ester-propyl octadecanoate.

Studies were carried out to evaluate the role of a C(21)-fatty acid ester; propyl octadecanoate (PO) for olfaction-mediated behavioral responses of urban malaria vector, Anopheles stephensi and dengue vector, Aedes aegypti mosquitoes using electroantennogram (EAG), flight orientation and oviposition experiments. Dose dependent electrophysiological responses were recorded for PO from the antenna of both mosquito species in which 10(-5) g elicited significant EAG response. An. stephensi exhibited 2.4, 4.2 and 5.5 fold increased EAG response compared to control, while Ae. aegypti showed 1.9, 4.6 and 5.8 fold EAG responses respectively at 10(-7) g, 10(-6) g and 10(-5) g doses. In the Y-tube olfactometer, 77-80% gravid females of An. stephensi, and 64-77% of Ae. aegypti were caught in the chambers releasing 10(-6) g and 10(-5) g plume of PO. The synthetic fatty acid ester loaded onto an effervescent tablet at 0.1 mg/L, 1 mg/L and 10 mg/L elicited increased ovipositional responses from gravid mosquitoes compared to control. The oviposition activity indices (OAI) of An. stephensi females were +0.40, +0.51 and +0.58, whereas the OAI for Ae. aegypti females were +0.05, +0.36 and +0.57 respectively in 0.1, 1, 10 mg/L of PO; indicated concentration dependent increased egg deposition. Similarly, in the residual activity studies, oviposition substrates treated with PO on effervescent tablet at 1mg/L and 10mg/L received significantly increased egg deposition by gravid females of both mosquito species for up to 1 week compared to control substrates. PO can potentially be used in ovitraps to monitor An. stephensi and Ae. aegypti populations in the vector surveillance programs.

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.

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.

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.

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.

Molecular phylogenetic study of Culex quinquefasciatus mosquito from different geographical regions of India using 16S rRNA gene sequences.

Culex quinquefasciatus is a major vector of filariasis and various encephalitis in India and worldwide. Vector control remains the most successful strategy for the suppression of mosquito borne diseases. The genetic structure of vector populations in terms of insecticide resistance and susceptibility or refractoriness to infection may possibly vary. To exploit the genetic variability in vector population could pave the path for the alternative strategies in vector management. The sequences of ribosomal RNA molecules have been widely used for such studies. Here, we examined the molecular phylogenetic relationship among the Cx. quinquefasciatus collected from different geographical regions of India, using 16S ribosomal RNA (16S rRNA) gene nucleotide sequences. The distances among the species were measured using Pearson correlation; the Neighbor-Joining (NJ) method was used for the clustering with appropriate bootstrap values using Data Analysis in Molecular Biology and Evolution (DAMBE) software. The results revealed that the populations are genetically diverse. Based on the distance values and the tree topology on the basis of 16S rRNA sequences reflected the clear biogeographical and geoclimatic pattern among the different geographical populations from India.

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.

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.

Genetic variability in geographical populations of Culex quinquefasciatus Say (Diptera: Culicidae) from India based on random amplified polymorphic DNA analysis.

Genetic variability and environmental factors may influence the refractiveness, propagation of pathogen and transmission of disease. Random amplified polymorphic DNA (RAPD) is one of the widely used molecular markers for population genetic diversity studies. In present study, RAPD is used to ascertain the genetic variability in Culex quinquefasciatus populations collected from various Indian geographical locations. Out of 50 RAPD primers screened, 14 primers exhibited clear, concrete and distinct banding pattern showing up to 100% polymorphism. Primer OPBD3 was tested with DNA of 14 geographical populations from India (including one laboratory population) showed 21 loci representing 14 populations with 100% polymorphism. The genetic diversity among the populations indicated the Shannon index (I) and gene diversity index (H(ST)), 0.48 and 0.31, respectively among the population, displaying rich genetic variation among the Cx. quinquefasciatus populations. Consensus tree showed two clusters indicating the genetic variation among the various geographical populations. The findings of this study may be useful to understand the population variation under different ecological conditions and development of effective vector management strategies.

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.

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.

Electroantennogram, flight orientation, and oviposition responses of Aedes aegypti to the oviposition pheromone n-heneicosane.

Oviposition pheromones specifically influence the females of many insects to lay eggs in the sites resulting in more egg deposition. A previous report describes the principal role of n-heneicosane (C(21)) identified and characterized from the larval cuticle of Aedes aegypti (L.) in attracting the gravid mosquitoes to oviposit in treated substrates among other chemical components. However, the means by which this compound is perceived by the females for oviposition has not been reported. In this study, we have recorded the peripheral olfactory responses from the antenna of Ae. aegypti from 10(-7) g to 10(-3) g doses of n-heneicosane. The EAG response of female mosquitoes increased in a dose-dependent manner with increasing stimulus strength. In the orientation assay using Y-maze olfactometer, female mosquitoes were attracted to the odor plume of 10(-6) g and 10(-5) g dose, while the higher dose of 10(-3) g plume enforced repellency to gravid mosquitoes. The response to oviposition substrates by gravid Ae. aegypti females differed across the range of concentrations of n-heneicosane under multiple choice conditions, larger number of eggs were deposited in 10 ppm (10 mg/l) solutions compared to lower and higher concentrations indicating 10 ppm was most attractive. Application of n-heneicosane at 10 ppm in breeding habitats will be a useful method to attract the gravid mosquitoes using ovitraps for surveillance and monitoring. The possible use of this compound in monitoring of mosquito population in endemic areas in relevance to integrated vector management strategies is discussed in detail.

Mediation of oviposition responses in the malaria mosquito Anopheles stephensi Liston by certain fatty acid esters.

The chemical factors involved in oviposition site selection by mosquitoes have become the focus of interest in recent years, and considerable attention is paid to the chemical cues influencing mosquito oviposition. Studies on synthetic oviposition attractants/repellents of long-chain fatty acid esters against Anopheles stephensi are limited. Screening and identification of chemicals which potentially attract/repel the gravid females to/or from oviposition site could be exploited for eco-friendly mosquito management strategies. The ester compounds demonstrated their ability to repel and attract the gravid A. stephensi females in the treated substrates. Significant level of concentration-dependent negative oviposition response of mosquitoes to octadecyl propanoate, heptadecyl butanoate, hexadecyl pentanoate, and tetradecyl heptanoate were observed. In contrast, decyl undecanoate, nonyl dodecanoate, pentyl hexadecanoate, and propyl octadecanoate elicited concentration-dependent positive oviposition responses from the gravid mosquitoes. Forcing a female to retain her eggs due to unavailability of a suitable oviposition site and attracting them to lay the eggs in a baited ovitraps shall ensure effective control of mosquito breeding and population buildup because the oviposition bioassay target the most susceptible stage of an insect life cycle. Treating relatively smaller natural breeding sites with an effective repellent and placing ovitraps containing an attractant in combination with insect-growth regulator (IGR)/insecticide would be a promising method of mosquito management.

Variation in morphology and morphometrics of eggs of Culex quinquefasciatus mosquitoes from different ecological regions of India.

Variation in egg surface morphology and morphometrics of Culex quinquefasciatus mosquitoes of the Jodhpur, Bikaner, Jamnagar, and Bathinda strains were correlated with geographical distribution in different ecological regions of India. We report the geographic variation in Cx. quinquefasciatus based on 44 attributes of micropylar and conical-shaped regions of eggs, including micropylar apparatus (corolla, disc, and mound), micropylar tubercles, and the exochorionic tubercle, pores, and network in anterior, middle, and posterior regions. No remarkable differences were observed in the surface morphology of eggs of these strains except the absence of small tubercles in the anterior and middle region of the JMN strain. However, a statistical analysis indicated significant morphometric variations in 66% of the attributes of the eggs. The cluster analysis of all egg attributes showed that the JD, BKN, and BTH strains are closer to each other than the JMN strain. The positive correlation (r = 0.95) also indicated an effect of geographical distribution on morphometry of various egg attributes of these strains. The present study suggests that ecological variation may have affected the morphometric attributes of the egg of four strains of Cx. quinquefasciatus from different geographical areas.