Biodiversity of culicid mosquitoes in rural Neka township of Mazandaran province, northern Iran.

BACKGROUND & OBJECTIVES: This paper presents the results of a study on ecological parameters including diversity indices applied on populations of Culicidae mosquitoes (Diptera: Culicidae) from three rural areas (Darvishan, Chalmardi and Khairabad) of Neka township in Mazandaran province, northern Iran. METHODS: Adult and larval stages of mosquitoes were systematically sampled to obtain data required for measuring diversity of mosquitoes in these areas. Alfa and beta indices were analyzed to compare mosquito diversity. A total of 5270 specimens belonging to four genera and 14 different species were collected and identified. RESULTS: Evaluation of dominance structure of species showed that culicid mosquitoes of Neka rangelands include one eudominant species, two dominant species, two subdominant species, two rare species and eight subrare species when Heydemann classification was used. INTERPRETATION & CONCLUSION: Data analysis revealed a higher diversity of mosquitoes in Khairabad with a low degree of interspecific dominance, while Darvishan and Chalmardi recorded the lowest observed diversity and a high degree of interspecific dominance. The cluster analysis based on Jaccard's index indicated the relationship between the categories and indicates that Darvishan and Khairabad are the closest categories in their specific composition.

Interleukin 4 (IL-4) gene promoter polymorphisms in Rhombomys opimus, the main reservoir of zoonotic cutaneous leishmaniasis.

Great gerbils (Rhombomys opimus) are the most common gerbils in center to northeast of Iran as well as central Asia and serve as reservoirs for the zoonotic agents, including Leishmania major, the principal etiologic agent of zoonotic cutaneous leishmaniasis (ZCL). The outcome of L. major infection in gerbils is not uniform. Among several immune-related factors including cytokine genes, the polymorphism in interleukin 4 (IL-4) promoter gene showed a great impact on outcome and pathological symptoms of L. major infection at least in mouse model. In this study gerbils' IL-4 promoter gene polymorphism is assessed. Specific primers were designed to develop a PCR-based assay to amplify IL-4 promoter gene to possibly define IL-4 promoter gene polymorphism in great gerbil populations with a range of Leishmania infection and symptoms collected from different foci of the central, north and northeast regions of Iran. The results showed that the designed primers amplify 689bp of the promoter gene. Sequence analysis of the promoter gene revealed five polymorphic sites assembly six haplotypes among the gerbil populations. Further studies are needed to assess whether or not the five polymorphisms cause different outcome phenotypes following infection with L. major in great gerbils. The data might be used to characterize the immune responses of R. opimus against L. major infection.

Molecular detection of Leishmania infection in sand flies in border line of Iran-Turkmenistan: restricted and permissive vectors.

A molecular study was carried out to incriminate sand fly vectors of cutaneous leishmaniasis (CL) in rural areas of Sarakhs district, Khorassane-Razavi Province, northeastern Iran, in 2011. Sand flies of Sergentomyia with three species and Phlebotomus with six species respectively comprised 73.3% and 26.7% of the specimens. Phlebotomus papatasi was the most common Phlebotomine species in outdoor and indoor resting places. Leishmania infection was found at least in 17 (22%) specimens including Ph. papatasi (n=9 pool samples), Phlebotomus caucasicus (n=6), Phlebotomus alexandri (n=1), and Sergentomyia sintoni (n=1). The parasites were found comprised Leishmania major (n=5), Leishmania turanica (n=10), and Leishmania gerbilli (n=4). Infection of Ph. papatasi with both L. major and L. turanica supporting the new suggestion indicating that it is not restricted only with L. major. Circulation of L. major by Ph. alexandri, and both L. gerbilli and L. turanica by Ph. caucasicus, in addition to previous data indicating the ability of Ph. alexandri to circulate Leishmania infantum and Leishmania donovani, and Ph. caucasicus to circulate L. major, suggests that these two species can be permissive vectors. The results suggest that Ph. papatasi and Ph. alexandri are the primary and secondary vectors of CL where circulating L. major between human and reservoirs, whereas Ph. caucasicus is circulating L. turanica and L. gerbilli between the rodents in the region.

Natural infection of wild caught Phlebotomus tobbi to Leishmania infantum in East Azerbaijan province, northwestern Iran.

BACKGROUND & OBJECTIVES: Zoonotic visceral leishmaniasis is caused by Leishmania infantum, which is transmitted to humans by bites of phlebotomine sandflies and is one of the most important public health problems in Iran. To detect and identify the Leishmania parasites and their corresponding vector(s), an investigation was carried out in Azarshahr County, a new and important focus of the disease in East Azerbaijan province in northwestern Iran during late April to late October 2010. METHODS: Sandflies were sampled using sticky papers (A4 white paper soaked in castor oil) from inside and outside of the houses and animal shelters, close to the vegetation and crevices. The head and three last abdomen segments of the specimens were removed and mounted in Puri's medium for species identification. The rest of body was subjected to molecular methods for detection of leishmanial parasites. RESULTS: Among 400 female sandflies tested by polymerase chain reaction (PCR) of kDNA, ITS1-rDNA, and CPB genes of the parasite followed by restriction fragment length polymorphism (RFLP), only 2 out of 8 Phlebotomus tobbi were positive to L. infantum parasites. CONCLUSION: The results indicated that, P. tobbi was the only species found infected by L.infantum and the principal vector of the disease agent to human.

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 2009­10 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 0.570­0.829 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.

Upwind responses of Anopheles stephensi to carbon dioxide and L-lactic acid: an olfactometer study.

Excretion of carbon dioxide and L-lactic acid through exhalation and perspiration provides olfactory signals to mosquitoes which allow them to find and bite humans; however, mosquito species differ in this regard. This study investigated upwind responses of Anopheles stephensi, mysorensis form, an important malaria vector in Asia, to carbon dioxide and L-lactic acid under laboratory conditions. While a minimal dose of carbon dioxide (90 ppm) activated the mosquitoes, 10 times this amount suppressed them. L-lactic acid alone did not produce a significant effect by itself, but addition of 6 microg/min of L-lactic acid to a range of 90 to 410 ppm carbon dioxide resulted in attraction. The results provide further support for the hypothesis that CO2 plays an important role in the host-seeking behaviour of zoophilic mosquitoes, and suggests that L-lactic acid might play a more critical role than CO2 in the attraction of An. stephensi.

First detection of Leishmania infantum in Phlebotomus kandelakii using molecular methods in north-eastern Islamic Republic of Iran.

Shirvan district in north-eastern Islamic Republic of Iran is a new focus of visceral leishmaniasis. This study aimed to identify the vector(s), the parasite and the species composition of sand flies in the district during July-September 2009 using polymerase chain reaction techniques. In all, 2088 sand flies were collected from 3 villages. Nine sand fly species were identified: Phlebotomus kandelakii (Shchurenkova), P. major (Annandale), P. halepensis (Theodor), P. papatasi (Scopoli), P. sergenti (Parrot), P. longidactus (Parrot), P. caucasicus (Marzinovsky), Sergentomyia sintoni (Pringle) and S. sumbarica (Perfil'ev). P. sergenti and P. kandelakii were the most prevalent Phlebotomus species at 31.3% and 10.0% respectively. Of 59 female P. kandelakii, 2(3.4%) were naturally infected with L. infantum. This is the first finding of natural infection of P. kandelakii by L. infantum in this region suggesting P. kandelakii may be the vector of L. infantum in the area although it is the second most prevalent phlebotomine species.

Knowledge, attitudes and practices regarding malaria control in an endemic area of southern Iran.

Iran is in the malaria pre-elimination stage. In this situation community based strategies are important for malaria control. In order to intensify elimination activities we need to have a better understanding of the knowledge, attitudes and practices (KAP) of malaria prevention in the community. We carried out a KAP study in one malaria endemic district in southern Iran in 2009 using a structured questionnaire. Five villages with indigenous malaria cases during the previous year and a high potential for anopheline mosquito breeding were selected. All the households in each village were visited and the head of the household or other responsible adult was interviewed. Data were analyzed using SPSS 11.5. A total of 494 respondents from the five villages participated. More than 33% of people in the study area were living in sheds with poor facilities. The illiteracy level of the studied population was high (44.2%) and significantly affected the knowledge and practices of the respondents about malaria control (p<0.05). Descriptive analysis showed significant differences between sex, job and history of malaria infection of the respondents and their knowledge and practices about malaria control (p<0.05). Knowledge of the respondents about the route of transmission in malaria was good; 72.1% knew mosquitoes were the vector. Most people knew at least one symptom of the disease. The practices of respondents were 59.5% for bednet use and 9.3% for screening windows. The knowledge, attitudes and practices of respondents in this survey about malaria control were good in some cases, but in general needed to be improved with educational programs.

Ecology of malaria vector Anopheles culicifacies in a malarious area of Sistan va Baluchestan province, south-east Islamic Republic of Iran.

A study was done of the bionomics, insecticide susceptibility and irritability status of Anopheles culicifacies in Sistan va Baluchestan province. Sampling was performed to determine the following parameters: species identification, seasonal activity, adult and larval susceptibility tests, irritability tests, anthropophily index and sporozoite rate. An. culicfacies adults were susceptible to all tested pyrethroid insecticides. An. culicfacies started to appear indoors in late May, showing 2 peaks in June and September. Fenitrothion, cyfluthrin and permethrin had the least irritancy effect and DDT the highest. Only 2/860 females tested were infected with Plasmodium spp. (sporozoite rate: 0.25%). ELISA testing of 250 blood meals derived from night-biting collections of female mosquitoes from humans and cows revealed that only 12.5% were human-fed.

New Data on Two Subspecies of Mesobuthus eupeus, the Most Medically Important Scorpion Species in Northwestern Iran.

Scorpions are among the most medically important arthropods in Iran, particularly northwestern areas. To date, five scorpion species, i.e. Mesobuthus eupeus, Mesobuthus caucasicus, Androctonus crassicauda, Hottentotta saulcyi, and scorpio maurus, have been identified. The family Buthidae is responsible for most cases of scorpionism in Iran. The Mesobuthus eupeus species belong to this family and is commonly distributed from Turkey to China, including Iran. Among these species, Mesobuthus eupeus is regarded as the most medically important species and responsible for most cases of envenomation in this area. Morphological differences between some species collected in the study area have been reported. The present study, thus, aimed to identify the subspecies of Mesobuthus eupeus in northwestern Iran. Scorpions were captured in the summer months from 37 localities in three northwestern provinces in Iran: West Azerbaijan, East Azerbaijan, and Ardabil. Scorpion collection was carried out using rock rolling and ultraviolet methods. A total of 376 specimens of Mesobutus eupeus (177males and 199 females) were collected and identified as Mesobuthus eupeus (98.4%) and Mesobuthus eupeus philippovitschi (1.6%). Owing to the findings of our study, M.e.philippovitschi has been added to the scorpion fauna of northwestern parts of Iran for the first time. Unlike M.e. eupeus which is widely distributed from plains to mountainous regions, M.e.philippovitschi has limited distribution and is found mostly along the borders with neighboring countries. This subspecies is the most medically important and most prevalent one in the region. The findings of the present study also provide the basis for future consideration of regional antivenom production for this medically important species.

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.

The Anopheles superpictus complex: introduction of a new malaria vector complex in Iran.

Anopheles superpictus Grassi is one of the most widespread malaria vectors in Iran. Two morphologically independent forms of this mosquito, both at larval and adult stage as well as a great diversity in its mtDNA loci have been previously described in Iran. Because of existence of mtDNA haplotypes, co-occurrence of two forms in diverse climates as well as different roles of populations played in malaria transmission, we hypothesized the possibility of emerging species (or sub-species) within the taxon. We surveyed the molecular variation in sympatric and allopatric populations of the two forms, using sequences from the ribosomal-DNA spacer region (ITS2). This analysis revealed a high degree of polymorphism (32.3%) as well as a length polymorphism (357 vs. 378 bp) in the ITS2 region among the populations but not so among morphological forms. Further examination identified three different ITS2 sequences, designated as genotypes X, Y and Z within species. Interestingly, while the sympatric Y and Z genotypes appear to be exclusive to the populations from the southeastern part of the country, genotype X is geographically separated and present in the North, the West, the South and the Central territories. The degree of divergence in ITS2 is much more than an intra-specific variation seen within the anopheline mosquitoes, and it points out the possibility of cryptic species within the taxon. Further studies are necessary to identify the species composition of the An. superpictus and their role played in the transmission of malaria in its geographical range.

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.

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.

Geographical variation in populations of Phlebotomus (Paraphlebotomus) caucasicus (Diptera: Psychodidae) in Iran.

A comparative morphological and molecular study was carried out on 11 different populations of Phlebotomus (Paraphlebotomus) caucasicus Marzinovsky 1917 caught in 7 provinces in Iran (2004-2005). Differences in the implantation level of the two distal spines of the style, the number of setae of the basal lobe of coxite, and the length of the third antennal segment, revealed the existence of two morphotypes within P. (Pa) caucasicus, a species having a confused history if we take into account an unclear synonymisation with Phlebotomus (Paraphlebotomus) grimmi Porchinsky 1874. Sequencing of mtDNA (a fragment of cytchromeB gene, tRNA for serine gene and a fragment of NADH1 gene) and Neighbour-Joining analysis showed a partial correlation between morphotypes and haplotypes. We also found a correlation between the latter and the geographical origin of the specimens. These results need further studies in order to appreciate the role of each morphotype/haplotype in the transmission of Leishmania major.

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.

Detection of Leishmania major DNA within wild caught Phlebotomus papatasi and species composition of sand flies in endemic focus of cutaneous leishmaniasis, in western Iran.

Cutaneous leishmaniasis is one of the most important public health problem in many developing countries. The present study was conducted to determine the vector(s), the parasite and the species composition of sand flies in the Dehloran County during May-November 2012. Sand flies were collected by sticky traps and mounted in Puri's medium for species identification. Polymerase chain reaction (PCR) techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism (RFLP) were used for identification of DNA of Leishmania parasites in infected sand flies. A total of 82443 specimens comprising 15 species of sand flies (5 Phlebotomus and 10 Sergentomyia) were collected and identified. The species of Phlebotomus papatasi was dominant in outdoor and indoor resting places. Among the 280 specimens of female P. papatasi tested by PCR of kDNA, ITS1-rDNA genes of the parasite followed by RFLP, only 5 of them (1.8 %) were positive to Leishmania major parasites. This is the first molecular detection of leishmania infection of P. papatasi to L. major in this region. The results indicated that, P. papatasi was only species found infected by L. major and the principal vector of disease agent to human.

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.