Genetic structure of the malaria vector Anopheles superpictus in Iran using mitochondrial cytochrome oxidase (COI and COII) and morphologic markers: a new species complex?

Anopheles superpictus has been implicated as the most widespread malaria vector in Iran. We collected adult specimens from eight provinces across the country and subjected them to a morphological investigation as well as molecular analysis of mitochondrial DNA COI-COII region, using PCR-RFLP and analysis of DNA sequence alignment for 708bp of the COI locus. Two distinct morphological forms (A and B) of the species were found sympatric in all areas of study. PCR-RFLP using AluI separated the specimens into at least three genotypes (X, Y, and Z), and alignment of DNA sequences revealed a 12.3% variation in the COI region between the genotypes. However, the sequence variation does not correspond to the morphological forms. Our observations suggest that A. superpictus in Iran is likely a group species. However, further ecological, molecular, cytological, and epidemiological studies are necessary to clear the status of the taxon and the potential role of each putative species in the transmission of malaria.

Pattern of mitochondrial DNA variation between and within Anopheles stephensi (Diptera: Culicidae) biological forms suggests extensive gene flow.

Anopheles stephensi Liston is the most prevalent anopheline species and plays an important role in malaria transmission in Indian subcontinent and Middle East including southern parts of Iran. It exists as three biological forms; "type", "intermediate", and variety mysorensis. The type form is reported to be an efficient vector of urban malaria, whereas mysorensis and intermediate are considered to be rural species and poor vectors. Moreover, differences in cuticular hydrocarbon and chromosomal characters have been described between urban and rural forms. However, the genetic structure of the biological forms remains unclear. This study was conducted to determine the genetic structure of the An. stephensi biological forms in south of Iran where all three forms are present. Live specimens were collected from the field and transferred to insectaries, reared, and identified based on egg morphological characters. Genetic structure of the biological forms was studied using PCR-RFLP of 1512 bp of mitochondrial DNA (mtDNA) cytochrome oxidase subunit I and II (COI-COII) and sequence of about 712 bp of COI and 562 bp of COII genes. Sequence analysis showed that except for a few substitutions in COII, all three forms and populations were nearly identical. The high homology of COI and COII sequence of An. stephensi forms indicates extensive gene flow between populations and forms in the region. This data will serve as first report on the sequence of mDNA COI-COII of biological forms of An. stephensi, which could be used as a diagnostic tool to identify vector/non-vector, gene flow, and geographical exchanges.

Bionomics of Anopheles stephensi Liston in the malarious area of Hormozgan province, southern Iran, 2002.

Anopheles stephensi Liston is an important malaria vector in Hormozgan province, where it is the most prevalent anopheline mosquito. It shows two annual activity peaks, one in spring and another in the autumn. In mountainous areas the second peak starts earlier than in coastal regions. Adults are endophilic and endophagic, but in the hot season when people sleep outside buildings they frequently bite outdoors. Larvae are found in a wide-range of habitats, both natural and man-made. All three biological forms of the species, occur in the province, i.e. An. stephensi stephensi (type form), An. stephensi mysorensis, and the intermediate form. An. stephensi mysorensis is found only in rural-mountainous areas, whereas the type and intermediate forms occur in urban-coastal regions and the rural plains, with the type form predominant. The presence of the type form in urban areas and mysorensis in rural areas is consistent with the available epidemiological data for malaria in the region and with the finding in India that the type form is an efficient malaria vector inhabiting urban areas whereas mysorensis is rural and has a lower vectorial capacity. Insecticide susceptibility tests on field collected adult mysorensis and adults from laboratory strains of the type and intermediate forms were carried out according to WHO standard methods. These showed that all three forms are susceptible to bendiocarb, propoxur, malathion, fenitrothion, deltamethrin, permethrin, cyfluthrin, and lambdacyhalothrin, but are resistant to DDT and show low level of tolerance to dieldrin. Examination of the larvicidal activity of malathion, fenitrothion, temephos and chlorpyrifos at diagnostic doses showed that these stephensi forms are susceptible to all larvicides except fenitrothion. Irritability tests to pyrethroid insecticides showed high levels of irritability to permethrin and lambdacyhalothrin, but low irritability to cyfluthrin and deltamethrin. The importance of these findings for the epidemiology and control of malaria in the region are discussed.

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.