Larval habitats and biodiversity of anopheline mosquitoes (Diptera: Culicidae) in a malarious area of southern Iran.

Background & objectives: Malaria is the most important mosquito-borne disease in Iran. It is endemic in south to southeastern part of the country. Knowledge about bio-ecology of vectors will support authorities for appropriate management of the disease. Bashagard district is one of the main endemic areas for malaria in south of Iran. This study was conducted to determine anopheline fauna, diversity and affinity in the area, characterization of larval habitats, and mapping their potential distribution across the district. Methods: The potential aquatic habitats for Anopheles larvae were extracted from Indian Remote Sensing Satellite (IRS) image and digital elevation model of the area using GIS. Surface water bodies were sampled monthly during 2009–10 for anopheline larvae, while characteristics of their physical environment were recorded and water samples were analyzed. Results: A total of 4511 Anopheles larvae were found during the year with the lowest and highest frequencies in February and April, respectively. Dominant species was Anopheles culicifacies. The Shanon diversity index ranged from 0.570–0.829 at fixed collection sites, while the affinity index was significant among some vector species. Conclusion: Riversides and riverbeds were the main breeding places which provided sandy, rocky, and clay beds for different species. The potential breeding places as well as distribution of collected species were mapped. Knowledge about ecology of malaria vectors provides information to health sector for effective control programs.

Origin, transfer and distribution of cantharidin-related compounds in the blister beetle Hycleus scabiosae

Cantharidin provides chemical protection for the coleopteran families Meloidae and Oedemeridae. In the present study, it was observed that cantharidin concentration in Hycleus scabiosae was slightly decreased from mated females (mean = 0.011 mg/mg of dry weight) to males (mean = 0.010 mg/mg) and considerably diminished in relation to virgin females (mean = 0.005 mg/mg). Significant concentrations of palasonin (21.69 ng/mg among virgins and 17.49 ng/mg in mated females) and palasoninimide (14.62 ng/mg in virgins and 9.17 ng/mg in mated females) were found in H. scabiosae. Palasonin, palasoninimide and cantharidinimide content of eggs were measured as 5.61, 7.69 and 7.80 ng/mg respectively. Surprisingly, males showed no trace of cantharidin-related compounds (CRCs); therefore CRCs in H. scabiosae could not be transferred from males to females and based on experiments employing its deuterated form, cantharidin is probably independently synthesized in females from the male nuptial transfer. An inseminated female incorporates about 38.5 ng of cantharidin (0.34 percent of the maternal content), 196.35 ng of palasonin (91.82 percent of maternal content) and 269.15 ng of palasoninimide (96.70 percent maternal content) into each egg mass during oviposition. It seems that eggs of this meloid species exploit a different array of protective chemicals by increasing the ratio of CRCs versus cantharidin. CRCs are less toxic than cantharidin; therefore, such compounds might have been deposited in eggs as a safer substitute for cantharidin to provide effective protection, but does not simultaneously harm the susceptible embryo.(AU)

Designing of a laboratory model for evaluation of the residual effects of deltamethrin (K-othrine WP 5%) on different surfaces against malaria vector, Anopheles stephensi (Diptera: Culicidae).

Background & objectives: Deltamethrin plays an important role in controlling malaria vectors, and is used in indoor residual spraying and manufacture of long-lasting insecticidal mosquito nets. The residual activity of deltamethrin (K-othrine WP 5%) @ 25 mg/m2 was studied in laboratory conditions on different surfaces. Methods: The surfaces were made artificially with different building materials such as mud, plaster, cement and wood in the laboratory. The surfaces were mounted inside petri-dishes (diam: 20 cm) and wood surface was prepared separately. The prepared surfaces were attached to the walls and deltamethrin was applied using standard Hudson pump sprayer with a discharge rate of 757 ml/ min or 0.2 gal/min. The spraying was conducted at standard rhythm as recommended by WHO. After application all the sprayed surfaces were transferred into the special wooden boxes designed for this purpose. The surfaces were maintained at laboratory conditions. The WHO’s recommended bioassay kit and method was used during this study. Results: Bioassays on Anopheles stephensi Liston showed that the persistence of deltamethrin on different surfaces (>70% mortality) was around 4 months on plaster (Mortality = 77±6.2%), 2 months on mud (76.9±6.8%), 4.5 months on cement (79±3.2%), 4 months on wood (71.7±6.8%) and 4 months on filter papers (82.3±5.4%). Interpretation & conclusion: The results of this study on residual effects of deltamethrin WP 5% (25 mg/m2) are highly concordant with two field bioassays carried out in a malarious area at south-eastern Iran. This method can be replaced by the field bioassay tests which are time consuming and costlier.

Application of predictive degree day model for field development of sandfly vectors of visceral leishmaniasis in northwest of Iran.

Background & objectives: Temperature plays a significant role in insect’s development where a rise in temperature, accelerates the insect’s metabolic rates, increases egg production and makes blood feeding more frequent. It also shortens the time period required for the development of pathogens within insects. Visceral leishmaniasis (VL) is one of the most important vector-borne diseases transmitted by different sandfly species. In this study, a phenological model was used to estimate the number of generations, peak activity and temporal variability of sandflies in the main VL foci in northwest Iran. Methods: Development requirements of different life stages of a Phlebotomus papatasi laboratory colony were measured and were subjected to the formula for calculation of accumulated degree day (ADD) for field sandflies using the online soft (UC IPM), using horizontal cut-off method and single triangle model. Sandflies population dynamics was monitored in the field during the seasonal activity in the region and its association with the ADD was tested using SAS software. Results: Populations of sandflies accommodated well with the amount of accumulated degree days (ADD) in the region. During the seasonal activity, a total of 639 ADD were produced which was enough to support one complete life cycle and growth of the next generation up to late larval instar. Larvae of the second generation hibernate through winter and the first adult population appears in the mid to late June of the next year when they receive at least 182 ADD from the beginning of the spring. The highest population density of sandflies was observed in early August, followed by a rapid decrease in early September, with the adult population disappearing completely in late September. This is the first degree day model related to sandflies in the most important VL foci of Iran. Interpretation & conclusion: Further studies in various regions with variable climate are recommended in order to better estimate and understand the development time, population dynamics and activities of the vectors which in turn could be used in proper implementation of effective vector control programmes.

Comparative performance of imagicides on Anopheles stephensi, main malaria vector in a malarious area, southern Iran.

BACKGROUND & OBJECTIVES: Jiroft district has subtropical climate and prone to seasonal malaria transmission with annual parasite index (API) 4.2 per 1000 in 2006. Anopheles stephensi Liston is a dominant malaria vector. The monitoring of insecticide susceptibility and irritability was conducted using discriminative dose as described by WHO. METHODS: The IV instar larvae were collected from different larval breeding places and transported to the temporary insectary, fed with Bemax and then 2-3 days-old emerged and sugar-fed adults were used for susceptibility and irritability tests employing WHO methods and kits to organochlorine (OC) and pyrethroid (PY) insecticides. RESULTS: Mortality rates of field strain of An. stephensi were 91.3 +/- 0.14 and 90 +/- 0.47% to DDT and dieldrin, respectively at one hour exposure time but was susceptible to all pyrethroids tested. The average number of take-offs per min per adult was 2.09 +/- 0.13 for DDT, 0.581 +/- 0.05 for dieldrin, 1.85 +/- 0.08 for permethrin, 1.87 +/- 0.21 for lambda-cyhalothrin, 1.53 +/- 0.13 for cyfluthrin, and 1.23 +/- 0.1 for deltamethrin. INTERPRETATION & CONCLUSION: Currently, deltamethrin is being used for indoor residual spraying against malaria vectors in the endemic areas of Iran. The findings revealed that the main malaria species is susceptible to all pyrethroids including deltamethrin, permethrin, cyfluthrin and lambda-cyhalothrin but was tolerant to DDT and dieldrin. This report and the finding are coincided with results of previous studies carried out during 1957-61 in the same area. Irritability tests to OC and PY insecticides revealed the moderate level of irritability to DDT compared to pyrethroids and dieldrin. Monitoring for possible cross-resistance between OC and PY insecticides should come into consideration for malaria control programme.

Effect of washing on the bioefficacy of insecticide-treated nets (ITNs) and long-lasting insecticidal nets (LLINs) against main malaria vector Anopheles stephensi by three bioassay methods.

BACKGROUND & OBJECTIVES: The use of pyrethoid impregnated bednets is one of the main malaria vector control strategies worldwide. The objective of the present study was to evaluate the bioefficacy of bednets impregnated with various pyrethroids after repeated washings. METHODS: The effectiveness of bednets impregnated with permethrin, deltamethrin, bifenthrin, etofenprox and long-lasting bednets like OlysetNet and PermaNet which were provided by WHOPES was evaluated. The tests were carried out according to the WHO-recommended methods. Malaria vector, Anopheles stephensi was exposed to impregnated bednets for 3 min and the mortality was measured after 24 h recovery period. Knockdown was measured as well. RESULTS: Results of three methods of bioassay tests showed that between two LLINs, PermaNet was more efficient than OlysetNet. Results of ITNs exhibited that deltamethrin and permethrin were more effective than etofenprox and bifenthrin as impregnants. INTERPRETATION & CONCLUSION: Findings of this study will be useful for WHO, local authorities and people who wish to use different pyrethroid-impregnated bednets for malaria vector control.

Morphological method for sexing anopheline larvae.

BACKGROUND & OBJECTIVES: Most of autocidal control of malaria vectors relies on the rearing and release of large numbers of sterile male into a wild population and it would be crucial to separate the males from females before release. This could result in enormous economic benefits in the mass rearing and raise the efficiency of the field operations. The development of genetic sexing of mosquitoes, enabling the release of males only, but impairing the overall fitness of the released insect has been considered greatly. Here we report on a morphological sexing method for the preferential diagnosis and separation of males in late III and IV instar larvae for the mosquitoes Anopheles stephensi Liston and An. culicifacies s.l. (Diptera: Culicidae), the principal vectors of human malaria in Asia and Indian subcontinent. METHODS: Male mosquitoes are identified by their tube like organ at the 9th abdomen segment which originates from segment parallel to the spiracles. Length and width of this organ is measured as 66.66 +/- 9.5 and 14.3 +/- 1.5 microm respectively. The whole length of the organ is 201.63 +/- 23.4 microm. Two fried eggs in the anterior portion of the segment are apparent in males. The length of tube in female is shorter than the male (almost half of the length--37.95 +/- 4.0 microm), its width is slightly stout and wider than the male (16.72 +/- 1.4 microm). Two fried eggs in the anterior portion of the segment are absent. After separation of live male larvae by those characteristics, they were transferred into the trays and emerged adults were identified to ascertain correct identification of sex. RESULTS: All the larvae with male organs developed into male adults with hairy antennae and club shaped palpi, whereas all the female larvae developed into adult females. INTERPRETATION & CONCLUSION: The sex separation at the larval stage will provide a clue for embryonic origin of sex organs, insecticide selection at the larval stage, sex related genes, male sterility and other measures.

Monitoring of insecticides resistance in main malaria vectors in a malarious area of Kahnooj district, Kerman province, southeastern Iran.

BACKGROUND AND OBJECTIVES: Kahnooj district in south of Iran is an endemic area for malaria where Anopheles stephensi (Liston) is a main malaria vector and An. dthali (Patton) a secondary vector. According to the national strategy plan on monitoring of insecticides resistance, this study was performed on susceptibility and irritability levels of An. stephensi and An. dthali to different insecticides in the district. METHODS: The susceptibility and irritability levels of field strains of An. stephensi and An. dthali at the adult and larval stages to discriminative dose of different imagicides was determined as recommended by WHO. RESULTS: Using discriminative dose and WHO criteria it was found that An. stephensi is resistant to DDT and dieldrin with 36.1 +/- 2.3 and 62.2 +/- 1.95 mortality rates, respectively; but susceptible to other insecticides. An. dthali was found to susceptible to all tested insecticides. The larvae of An. stephensi, exhibited 100% mortality for temephos and malathion, but 44 +/- 4.32 for discriminative dose of fenitrothion. The results of irritability level for DDT and pyrethroids showed that permethrin had the most irritancy effect on An. stephensi and An. dthali. DDT and deltamethrin showed the least irritancy effect against An. stephensi with 0.42 +/- 0.08 and 0.77 +/- 0.12 take-offs/min/adult, respectively, however, lambdacyhalothrin had the least irritancy effect against An. dthali with 0.096 +/- 0.02 take-offs/min/ adult. The mean number of take-offs/min/adult with permethrin showed significant difference to DDT, lambdacyhalothrin, cyfluthrin and deltamethrin. INTERPRETATION AND CONCLUSION: Pyrethroid insecticides are being used as indoor residual sprays in Iran. Based on our results, the main malaria vectors in the region are still susceptible to pyrethroid insecticides. Therefore, we propose the use of pyrethroids with low irritancy effect in rotation with carbamate insecticides in two interval seasonal peaks of malaria transmission. Biological control including larvivorous fishes, using of local made Bacillus thuringiensis and larvicides such as chlorpyrifos-methyl are the main larval control in the region. Result on larval test exhibited the susceptibility of main vectors to some larvicides, although the 100% mortality was not obtained using fenitrothion and this is postulated the use of this insecticide in agriculture pest control. Monitoring and evaluation of insecticides resistance in malaria vectors in the region could provide an essential clue for judicious use of insecticides.

Larval habitats of main malaria vectors in Hormozgan province and their susceptibility to different larvicides.

Hormozgan Province is located in the south of Iran bordering the Persian Gulf. In this area, malaria is still considered a major health problem. In the province, eleven species of Anopheles were identified: An. pulcherrimus, An. sergenti, An. apoci, An. multicolor, An. subpictus, An. turkhudi, An. fluviatilis, An. stephensi, An. d'thali, An. superpictus, and An. culicifacies, among which only the last five species play an important role in malaria transmission. Larval habitats of malaria vectors varied from clean to brackish water, as well as sewage water, under palm trees, marshland, pools, pounds, drainage, irrigation canals, wells, and water containers. Malathion, fenitrothion, chlorpyrifos, and temephos were tested against An. stephensi, An. fluviatilis and An. d' thali larvae at diagnostic doses recommended by WHO. The results showed susceptibility to malathion, chlorpyrifos and temephos, but fenitrothion was not able to yield 100% mortality.

rDNA-ITS2 based species-diagnostic polymerase chain reaction assay for identification of sibling species of Anopheles fluviatilis in Iran.

A species-specific polymerase chain reaction (PCR) assay using primers already designed, based on differences in the nucleotides of the second internal transcribed spacer (ITS2), was used to identify the species composition of the Anopheles fluviatilis complex in Iran. All the amplified DNA samples obtained from specimens collected from different areas using different collection methods yielded to a fragment of 450 bp size, a PCR product corresponding to the species denoted as Y. Some 21 ITS2 region of Iranian specimens were sequenced and compared with the already published sequence data of species Y from India. The sequence data of the Iranian specimens were 100% identical to that of the Indian specimens, and hence confirmed the PCR assay results. Species Y is presumably species T in India, which has no role in the transmission of malaria, whereas mosquitos of An. fluviatilis are known as a secondary vector in Iran. This conflict will remain to be solved by further biological and molecular studies.

Factors involved in the re-emergence of malaria in borderline of Iran, Armenia, Azerbaijan and Turkey.

The borderline of Iran with Azerbaijan, Armenia and Turkey had been considered a malaria free region. However, in 1991, after the independence of the southern countries of the former Soviet Union, a new threat of malaria importation emerged from those countries into Iran, which was affected by serious epidemics of Plasmodium vivax malaria. Various factors can affect malaria resurgence in this region, such as socioeconomic conditions, especially the displacement of massive populations from war-stricken zones in the Republic of Azerbaijan. Accordingly, in some parts of West-Azerbaijan, East-Azerbaijan, Ardebile and Gilan provinces of Iran, several malaria foci were observed. Construction of dams, people traveling from neighboring countries to Iran, urbanization, irrigation projects, lack of malaria vector control, shortage of drug supplies are also major factors in malaria outbreaks in the region. An investigation was carried out on the bionomics of the main malaria vectors in the region. The result showed that Anopheles sacharovi plays an important role in malaria transmission and An. maculipennis and An. superpictus can be secondary vectors. Larvae were found in slow flowing water and channels with water plants. They were more abundant in June. The parity rate of blood-fed females was high in May. An. sacharovi is active from May to October with two peaks of activity, which occur in August and October. The population of this species is higher in animal shelters with a zoophicity of 95%. About 90% of bites took place in the second half of the night. A CDC light trap can also catch this species. Susceptibility testing using the WHO-recommended diagnostic doses of insecticides, revealed that this species is resistant to DDT and dieldrin, but susceptible to malathion, fenitrothion, propoxur, bendiocarb, lambdacyhalothrin, permethrin, cyfluthrin, etofenprox and deltamethrin.