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.

Behavioral and electrophysiological responses of Aedes albopictus to certain acids and alcohols present in human skin emanations.

Human skin emanations attract hungry female mosquitoes toward their host for blood feeding. In this study, we report the flight orientation and electroantennogram response of Aedes albopictus females to certain unsaturated acids and alcohols found in human skin. In the Y-tube olfactometer, odors of lactic acid and 2-methyl-3-pentanol attracted 54-65% of Ae. albopictus females at all doses in a dose-dependent manner. However, at the highest dose (10(-2) g), the acids repelled 40-45% females. Attractancy (ca. 62-68%) at lower doses and repellency (ca. 30-45%) at higher doses were recorded for 3-methyl-3-pentanol and 1-octen-3-ol, while 5-hexen-1-ol, cis-2-hexen-1-ol, and trans 2-hexen-1-ol odor repelled ca. 55-65% of Ae. albopictus females at all doses. Antenna of female Ae. albopictus exhibited a dose-dependent EAG response up to 10(-3) g of L-lactic acid, trans-2-methyl-2-pentenoic acid, 2-octenoic acid, trans-2-hexen-1-ol and 1-octen-3-ol stimulations; however, the highest dose (10(-2) g) caused a little decline in the EAG response. EAG response of 9-10-fold was elicited by lactic acid, 2-octenoic acid, trans-2-hexenoic acid, and 3-methyl-3-pentanol, while cis-2-hexen-1-ol and trans-2-methyl pentenoic acid elicited 1-5-fold responses compared to solvent control. A blend of attractive compounds could be utilized in odor-baited trap for surveillance and repellent molecules with suitable formulation could be used to reduce the biting menace of mosquitoes.

Olfaction in Asian tiger mosquito Aedes albopictus: flight orientation response to certain saturated carboxylic acids in human skin emanations.

The flight orientation response of nonblood-fed and hungry Aedes albopictus females was studied in a Y-tube olfactometer at 10(-6) to 10(-2) g odor plumes of saturated carboxylic acids (C1-C20), in which C2-C18 were the main constituents of human skin emanations. Thirteen acids viz C1, C2, C3, C5, C6, C8 C9, C10, C12, C14, C16, C18, and C20 showed attractance at odor plumes ranging from 10(-5) to 10(-3) g doses, while five acids viz C4, C7, C11, C15, and C19 showed repellence at 10(-4) to 10(-2) g to test mosquitoes. Tridecanoic acid (C13) showed attractance only at 10(-4) g dose while higher doses caused repellence. Dose-dependent reversal of orientation behavior from attractance to repellence was observed at 10(-2) g plumes of C5, C9, C10, C13, C17, C19, and C20 acids. The outcome of the study will help in the identification of odoriferous acids as potential attractants, repellents, or attraction inhibitors, which may find their application in the repellent formulations and odor-baited traps for surveillance and control of mosquitoes.

Oviposition deterrent activity of three mosquito repellents diethyl phenyl acetamide (DEPA), diethyl m toluamide (DEET), and diethyl benzamide (DEB) on Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus.

Oviposition deterrent activity of three mosquito repellents namely diethyl phenyl acetamide (DEPA), diethyl benzamide (DEB) along with diethyl toluamide (DEET) was studied in the laboratory against Aedes aegypti, A. albopictus, and Culex quinquefasciatus mosquitoes. All the repellents evaluated at three log concentrations 0.1, 0.01, and 0.001% in comparison with control by dual choice method. The oviposition response and oviposition activity index (OAI) was estimated by counting number of eggs laid in both the treatment and control. Mean OAI determined for A. aegypti was in the range of -0.23 to +0.22, -0.3 to +0.27, and +0.04 to +0.33 for DEPA, DEET, and DEB, respectively, whereas it was -0.77 to -0.035, -0.92 to +0.001, and -0.77 to -0.07 for A. albopictus and -0.927 to -0.251, -1 to -0.41, and -0.94 to -0.17 for C. quinquefasciatus. The oviposition deterrent activity was observed in the order of DEET > DEPA > DEB. Similarly, the response of species toward the repellents as oviposition deterrent was in the trend of C. quinquefasciatus > A. albopictus > A. aegypti. The finding suggests potential role of mosquito repellents as oviposition deterrent.

Oviposition and flight orientation response of Aedes aegypti to certain aromatic aryl hydrazono esters.

Aedes aegypti is a day-biting, highly anthropophilic mosquito and a potential vector of dengue and chikungunya in India. A. aegypti is a container breeder, generally oviposit in the stored and fresh water bodies, and discarded containers near residential areas that provide suitable habitats for oviposition by gravid females. The diurnal activity and endophilic nature of these mosquitoes have increased the frequency of contact with human being. Assured blood meal from human host in an infested area leads to increased disease occurrence. Gravid mosquitoes can potentially be lured to attractant-treated traps and could subsequently be killed with insecticides or growth regulators. In this direction, oviposition by A. aegypti females to aryl hydrazono esters (AHE)-treated bowls at 10 ppm concentration was tested in dual choice experiment, and their orientation response to these ester compounds was studied in Y-tube olfactometer. Among the esters tested, AHE-2, AHE-11 and AHE-12 elicited increased egg deposition with oviposition activity indices (OAI) of +0.39, +0.24 and +0.48, respectively, compared to control; in contrast, AHE-8, AHE-9 and AHE-10 showed negative oviposition response with OAI of -0.46, -0.35 and -0.29, respectively, at 10 mg/L. In the Y-tube olfactometer bioassay, AHE-2 attracted 60 % females compared to control, while to the odour of AHE-11 and AHE-12, about 70 % of the females were trapped in treated chambers. In contrast, only 27-30 % of gravid females entered the chamber releasing AHE-8, AHE-9 and AHE-10 odour plumes, while 70 % entered control chamber, evincing a possible non-preference of treatment odours as well as interference with olfactory receptors. These compounds have the potential for application as oviposition stimulants or deterrents for surveillance and control of mosquito population using ovitraps.

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.

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 (R(0) ) 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 (r(m) ) 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, R(0) , r(m) , 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.

Synthesis and bio-evaluation of aryl hydrazono esters for oviposition responses in Aedes albopictus.

A novel series of aryl hydrazono esters (AHE) (1-13) were synthesized (yield 76-98%) to study the oviposition responses in Aedes albopictus (Skuse) mosquitoes for the first time. At a concentration of 10µgml(-1) in dual choice experiment, among the screened compounds, AHE-12 showed remarkable oviposition attractant activity with an oviposition activity index (OAI) of +0.299 (greater than 95% confidence limit) comparable to p-cresol (OAI +0.320) which is well-reported oviposition attractant for Aedes aegypti. Conversely, AHE-10 exhibited highest oviposition deterrent activity with OAI -0.247. The possible utilization of these compounds will be in integrated vector management strategies.

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.

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.

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.

Scanning electron microscopic studies on egg surface morphology and morphometrics of Culex tritaeniorhynchus and Culex quinquefasciatus (Diptera: Culicidae).

Culex tritaeniorhynchus Giles, 1901 and Culex quinquefasciatus Say, 1823 is an important vector of Japanese encephalitis and Bancroftian filariasis, respectively in India and South East Asian countries. In this paper, we are describing the surface morphology and morphometrics of the egg of C. tritaeniorhynchus in comparison with C. quinquefasciatus for the first time. The results indicated that eggs of both the species appears to be similar to great extent in surface morphology, however, morphometrics provide 56.81% demarking attributes out of 44 attributes at various significant levels (p < 0.05-0.001), i.e., egg length, width and ratio of length/width, attributes of micropylar apparatus including corolla, disc, mound, tubercles size, and length of tubercular rows in micropylar region, size and density of tubercles, exochorionic pore in conical-shaped regions of eggs, and size of tubercular wheel units. Structurally, the additional presence of large tubercles strengthens the micropylar region to bear various collapsing forces in these species.

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.

Susceptibility of immature stages of Aedes (Stegomyia) aegypti; vector of dengue and chikungunya to insecticides from India.

Susceptibility of Aedes aegypti to some insecticides in different geographic areas was conducted during dengue and chikungunya outbreak. At present, the only method of preventing dengue and chikungunya is to control the vector, which is the weakest link in vector-borne diseases. In our study, the susceptibility of A. aegypti collected from urban areas of Delhi, Mumbai, Jodhpur, Chennai and Coimbatore was evaluated against temephos, fenthion, malathion and DDT. The A. aegypti from different locations exhibited 0.33-7.11, 0.36-3.00, 0.65-2.84 and 2.16-20.8 fold more lethal concentration value of 50% (LC50) to temephos, fenthion, malathion and DDT, respectively, compared to susceptible reference strain. The result reveals that A. aegypti from various locations studied are still susceptible to temephos, fenthion and malathion, whereas low level of DDT resistance was noticed in field-collected A. aegypti. Amongst the insecticides tested, temephos was found to be relatively more effective in controlling A. aegypti, followed by fenthion, malathion and DDT.

Molluscicidal effect of nicotinanilide and its intermediate compounds against a freshwater snail Lymnaea luteola, the vector of animal schistosomiasis.

The molluscicidal effect of nicotinanilide was evaluated and compared with niclosamide (2',5-dichloro-4'-nitrosalicylanilide, ethanolamide salt) against different stages of the freshwater snail Lymnaea luteola i.e., eggs, immature, young mature, and adults. Calculated values of lethal concentrations (LC50 and LC90) showed that both nicotinanilide and niclosamide as toxic against eggs, immature, and adults. The young mature stage of the snails was comparatively more tolerant to both molluscicides than the other stages. The toxicity of the intermediate compounds of nicotinanilide against the young mature stage of the snails showed them as ineffective. The mortality pattern of the snails exposed to LC90 concentration of these molluscicides showed niclosamide to kill faster (within 8 to 9 h) than nicotinanilide (26 to 28 h). In view of the above studies it may be concluded that both molluscicides are toxic against all the stages of the L. luteola snails.

Molluscicidal effect of nicotinanilide and its intermediate compounds against a freshwater snail Lymnaea luteola, the vector of animal schistosomiasis

The molluscicidal effect of nicotinanilide was evaluated and compared with niclosamide (2',5-dichloro-4'-nitrosalicylanilide, ethanolamide salt) against different stages of the freshwater snail Lymnaea luteola i.e., eggs, immature, young mature, and adults. Calculated values of lethal concentrations (LC50 and LC90 ) showed that both nicotinanilide and niclosamide as toxic against eggs, immature, and adults. The young mature stage of the snails was comparatively more tolerant to both molluscicides than the other stages. The toxicity of the intermediate compounds of nicotinanilide against the young mature stage of the snails showed them as ineffective. The mortality pattern of the snails exposed to LC90 concentration of these molluscicides showed niclosamide to kill faster (within 8 to 9 h) than nicotinanilide (26 to 28 h). In view of the above studies it may be concluded that both molluscicides are toxic against all the stages of the L. luteola snails.