RT-PCR assay for the detection of infective (L3) larvae of lymphatic filarial parasite, Wuchereria bancrofti, in vector mosquito Culex quinquefasciatus.

BACKGROUND & OBJECTIVES: Periodic monitoring of vector population for infection and infectivity rates is central to the evaluation of the filariasis elimination strategies in endemic areas to monitor the success of MDA and also to establish endpoints for intervention. The main objective of this study was to develop a RT-PCR assay, based on L3 stage-specific primers to detect the presence of infective stage larvae of filarial parasite, Wuchereria bancrofti in the vector Culex quinquefasciatus. MATERIAL & METHODS: Subtracted probe development technique was employed for the identification of infective stage (L3) specific genes. The subtracted cDNA was labeled by non-radioisotopic method and used for screening cDNA library of L3 stage larvae of W. bancrofti constructed in UniZap XR. Recombinants were probed and identified from the library. The inserts of the recombinant clones were purified and sequenced. Primers were designed based on the sequence information of three recombinant clones for detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay. Preliminary laboratory evaluation was carried out to assess the sensitivity and specificity of WbL31 RT-PCR assay. RESULTS: cDNA library of L3 stage of W. bancrofti constructed in UniZap XR vector, constituted 5 x 10(5) phages with 80-90% recombinant phages and the size of inserts varied from 0.1 to 1.0 kb. When subtracted cDNA was random prime labeled and used for screening cDNA library of L3 stage of W. bancrofti constructed in UniZap XR, 18 clones were identified from the library. Three genes were found up-regulated in the L3 stage, out of which WbL31 (cuticular collagen) was found to be useful in detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay with high specificity and sensitivity (98-100%). CONCLUSION: Present paper marks first report on the development of an infective stage-specific RT-PCR assay (WbL31 RT-PCR assay) to detect L3 stage W. bancrofti in the vector. This assay will have potential application in assessing the transmission of infection and hence in decision-making related to elimination programme.

Laboratory evaluation of Ssp I PCR assay for the detection of Wuchereria bancrofti infection in Culex quinquefasciatus.

BACKGROUND & OBJECTIVES: There is a need to delimit the areas of filariasis transmission in view of the Filariasis Elimination Programme launched in India. Infection rate in vectors is an important parameter in determining transmission and it is conventionally assessed by dissection and microscopy. A PCR assay based on Ssp I repeats of Wuchereria bancrofti has shown potential in the detection of infection in vectors. The aim of the present study was to evaluate the specificity and sensitivity of this assay on W. bancrofti and its vector, Culex quinquefasciatus, prevalent in India. METHODS: The DNA from pools of C. quinquefasciatus to which W. bancrofti microfilariae (mf) were added, was extracted by lysing with 0.1 M NaOH and 0.2 per cent sodium dodecyl sulphate (SDS), followed by silica absorption in the presence of guanidinium thiocyanate. The PCR assay of the DNA samples was carried out using NV-1 and NV-2 primers and the species specific SspI band was visualized on agarose gels stained with ethidium bromide. RESULTS: The Ssp I PCR assay was found to be highly species specific, as it did not detect the DNA of a closely related filarial parasite, Brugia malayi. The assay detected as little as 0.04 pg of W. bancrarofti DNA. Minimum number of parasite detectable in pools of mosquitoes was 1 mf. A pool size of 50 mosquitoes was found to be optimum for the PCR assay. INTERPRETATION & CONCLUSION: The Ssp I PCR assay was found to be highly specific and sensitive in detecting filarial parasite in pools of mosquitoes and therefore has potential application in rapid assessment of transmission of filariasis.

Influence of IGR treatment on oviposition of three species of vector mosquitos at sublethal concentrations.

Sublethal effect of hexaflumuron, an insect growth regulator (IGR), on the oviposition of three species of vector mosquitos. Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi was studied. Significant reduction in oviposition was observed in the females of the above three species derived from fourth instar larvae and pupae exposed to sublethal (EI5 and EI50) doses. The reduction in egg laying is proportional to the dose of exposure and was found to be about twice higher in females of three species exposed to EI50 dose than those exposed to EI5 dose. Among the three species exposed at larval and pupal stages, Ae. aegypti showed maximum reduction in egg laying (29.3-46.6%). Blood feeding was also reduced in females exposed to EI50 dose at larval stage and a positive correlation was demonstrated between the quantity of blood meal taken and the proportion of eggs laid. Significant reduction in the quantum of blood ingested by the treated females may be responsible for the reduced egg laying.

Insecticidal activity of some new synthetic compounds against different mosquito species.

Insecticidal activity of an organosilane (HOE 84498, OMS 3055), Organophosphorous (MAT 9460, OMS 3052) and a synthetic pyrethroid compound (Trebon, OMS 3002) was evaluated against adult and larval stages of eight species of mosquitoes. Insecticidal activity of these compounds was limited to larval stages only. Highest activity of HOE 84498 was found against both Cx. tritaeniorhynchus and Anopheles culicifacies (LC50-0.0065 mg/I), MAT 9460 against Armigeres subalbatus (LC50-0.00043 mg/I) and Trebon against Cx. quinuefasciatus (LC50-0.00579 mg/I). Adulticidal effect was observed at higher dosages of 25-100 ug/cm2 of MAT 9460 and HOE 84498 against all the species with LT50 ranging from 22.15-33.76 min. No appreciable residual effect was evident for the three compounds on the surfaces treated at the rate of 1-100 mg/m2 against any mosquito species.

Use of hexaflumuron, an insect growth regulator in the control of Aedes albopictus (Skuse).

An attempt was made to assess the control potency of an insect growth regulator (IGR), hexaflumuron, against Aedes albopictus, a potent dengue vector, both in the laboratory and under field conditions. Emergence inhibition activity of this IGR against laboratory reared and field collected larvae of Ae. albopictus showed EI50 values of 1.9 x 10(-4) and 1.80 x 10(-4) mg(ai)/l respectively. Under field conditions, no appreciable reduction in immature density occurred at the lowest application rate of 0.001 mg(ai)/l whereas at the rate of 0.01 mg(ai)/l 100% reduction could be seen in earthern pots placed in a garden for 9 days. A reduction of 100% in pupal density was observed for 21 and 18 days at an application rate of 0.1 mg(ai)/l in pots and tyres respectively. Considering that this IGR was effective for about three weeks, it can be used successfully at the rate of 0.1 mg(ai)/l for controlling Ae. albopictus breeding in container habitats.

Evaluation of organophosphorus and synthetic pyrethroid insecticides against six vector mosquitoes species

Três compostos organo-fosforados - malation, folition e temefos - e dois piretroides sintéticos - alfametrina e deltametrina - foram usados para controlar o estado da susceptibilidade das larvas e adultos de seis mosquitos vetores na India. Foram utilizadas cepas de laboratório e área de Culex quinquefasciatus (filariasis) e Aedes albopictus (Dengue) e cepas de laboratório de Aedes aegypti (Dengue), Anopheles stephensi e Anopheles culicifacies (Malária) e Culex tritaenorhynchus (encefalite japonesa). Os valores de C1**50 obtidos para esses inseticidas mostram que todas as espécies incluindo as cepas de área foram muito susceptíveis. Nos mosquitos adultos das referidas espécies salvo na cepa da área de Culex quinquefasciatus com o malathion, observou-se 100 por cento da mortalidade às doses discriminatórias recomendadas pela Organizaçäo Mundial de Saúde. O efeito residual da alfametrina, deltametrina, malation e folition a 25 mg(ai)/m2 em diversas superfícies contra seis espécies de mosquitos vetores evidenciou que a alfametrina foi a mais efetiva em todas as superfícies tratadas (argila, plywood, cimento e palha)

Influence of short time exposure to an insect growth regulator, hexaflumuron, on mortality and adult emergence of vector mosquitoes

Hexaflumuron, an insect growth regulator (IGR), was found to greatly affect the development of immatures and emergence of adults of three species of vector mosquitoes, Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi, when larvae were subjected to short time exposure of < or = 1h. This IGR could completely prevent adult emergence even at a minimum exposure time of 10 min at 0.001, 0.01 and 0.1 mg/l. On treatment, larval and pupal mortality as well as varying degrees of morphogenetic abnormalities were induced in immatures and adults of the three species. Four weeks of control achieved in a slow moving sullage canal breeding Culex quinquefasciatus indicates that this IGR can be of use in such breeding habitats

Beta-cyfluthrin, a synthetic pyrethroid for mosquito control.

Beta-cyfluthrin (OMS 3051), a new synthetic pyrethroid and one of the stereoisomers of cyfluthrin, was studied for insecticidal activity against eight mosquito species. Its larvicidal activity with LC50 values of 5.62 x 10(-5) and 1.19 x 10(-4) mg/l respectively for Culex quinquefasciatus and Aedes aegypti was comparable with that of deltamethrin. This pyrethroid was more effective against the larvae of Armigeres subalbatus (LC50 - 7.76 x 10(-7)) and the adults of Anopheles culicifacies LT50 - 27.76 min at 2.0 mu/cm2) than the other species tested. Residual efficacy at 50 mg(ai)/m2 was more persistent (for 14-25 weeks) on thatch and asbestos among the four treated surfaces. This compound also elicited oviposition deterrent activity at 0.001 mg/l against Cx.quinquefasciatus. beta-cyfluthrin is a good insecticide for mosquito control. However, care should be exercised while using it as a larvicide in breeding habitats considering its toxicity to fish.

Adult longevity of certain mosquito species after larval and pupal exposure to sublethal concentration of an insect growth regulator, hexaflumuron.

Longevity of the adults of three vector species, Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti was drastically reduced when they were exposed at larval and pupal stages to sublethal concentrations of an insect growth regulator hexaflumuron. When the three species were exposed to 0.05 mg/l at the pupal stage, males and females of Cx. quinquefasciatus suffered a more shortened life span than other species. Among the females whose feeding activity was adversely affected by IGR treatment at the pupal stage, Ae. aegypti showed the minimum survival duration with LT50 of 2.74 days.

Quantitative structure activity relationship in diphenyl ureas for insect growth regulating activity.

Insect growth regulating (IGR) activity of 29 diphenyl urea compounds was determined against the filariasis vector, Culex quinquefasciatus. The variations in the activity of these compounds were analysed with the physicochemical substituent parameters and regression analysis. Quantitative structure activity relationship (QSAR) studies revealed that the IGR activity was influenced by lipophilicity, electron withdrawing effect of the substituents on the aniline moiety, the steric effect of the substituents in the acidic moiety and the position of substitution. This diphenyl ureas for IGR activity seem to offer a successful model for the application of QSAR method in the development of new vector control agents.

Structure-activity relationships in diphenyl ethers for insect growth regulating activity against mosquitoes.

Out of the 30 substituted diphenyl ethers synthesized and tested for insect growth regulating (IGR) activity against mosquitoes, three compounds viz., DPE-16, 19 and 28 showed promising IGR activity. While DPE-16 and 19 were found to be effective against all three vector species tested viz., Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi, with the respective EI50 values of 0.1485, 0.3650, 0.2225 mg/l and 0.1474, 0.1392, 0.1145 mg/l, DPE-28 was found to be highly effective against C. quinquefasciatus with an EI50 value of 0.0022 mg/l. The structure-activity relationship in the diphenyl ethers with respect to their octanol-water partition coefficients showed an increase of lipophilicity when both the ortho positions of the phenolic moiety are substituted by tertiary butyl group. The simulated field trial carried out with DPE-16, 19 and 28 showed that these compounds were effective for 7, 7 and 9 days respectively at 0.1 mg/l against C. quinquefasciatus.

Establishment of a standard test method for determining susceptibility of Mesocyclops to different insecticides.

A method to determine the susceptibility of Mesocylops sp. to insecticides was devised. Matured adults were used for the bioassays. Mesocyclops sp. (25 in number) were placed in 24.9 ml of sterilized paddy field water in 5 cm diameter petri dish and 0.1 ml of appropriate stock solution of the insecticide was added. Mortality was scored 24 h after treatment. Among the insecticides tested, Permethrin, Dichlorvos, Temephos, DDT, Carbendazim, Fenitrothion, Zolone and Aldrin were effective against Mesocyclops sp.