Phlebotomus neglectus (Diptera: Psychodidae): New Insights on Its Presence in Iran Based on Three Independent Genetic Loci.

The idea of the existence of Phlebotomus (Larroussius) neglectus (Diptera: Psychodidae) Tonnoir, 1921 in Iran and the skepticism about the existence of Phlebotomus major s.str. Annandale, 1910 had been grown recently in the country. This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of P. major s.l.Annandale, 1910, specimens collected from different parts of Iran. Two different morphotypes were found among the collected samples based on the shape of the aedeagus, ventrally located hairs of the coxite, and parameral sheets. One morphotype seemed similar to P. neglectus Tonnoir 1921 or P. major krimensis Perfiliv1966 (called here MI.N.K.); the other one was similar to P. neglectus and to some extent to P. notus Artemiev & Neronov 1984 (here called MII.N.NO). Cytochrome B, elongation factor 1-alpha, and internal transcribed spacer II loci were amplified, sequenced, and characterized. High sequence homology (98-100%) was observed between P. neglectus and these morphotypes, and phylogenetic analysis was also concordant. Phlebotomus neglectus sequences available in GenBank are located as the sister group of sequences here, particularly near to morphotype MII.N.NO. Moreover, ITS2 locus provides the maximum resolution for differentiation of two morphotypes. Based on achieving results, although a strong support for the presence of P. neglectus was provided, but it is too early to say that P. major s.str. does/does not exist in Iran. This question could be resolved by studying more samples and, most importantly, by comparing the topotypes of P. neglectus and P. major s. str. if possible in the future.

The molecular and morphological variations of Culex pipiens complex (Diptera: Culicidae) in Iran.

BACKGROUND & OBJECTIVES: Taxonomic status of Culex pipiens is well-known as many years with such a wide variety of morphological and biological characteristics. These changes have been the subject of extensive investigation by many researchers. There are a little information about the morphology and molecular data of Cx. pipiens complex in Iran. The taxonomic status of the complex is very important because of medical and veterinary importance and wide distribution in the country. METHODS: This study was carried out in 11 areas in Iran using dipping technique from April 2009 to October 2010. Molecular study was carried out using primers F1457 as forward and B1256 as reverse, which amplified Ace.2 gene and performed PCR-RFLP using ScaI restriction enzyme. RESULTS: Culex quinquefasciatus found in south to central areas of Iran and reported as sympatric with Cx. pipiens in the central regions. Culex pipiens distributed in many areas of the country. Sequencing alignment of Ace.2 gene of Cx. quinquefasciatus and Cx. pipiens showed 6.5% variation in 46bp, especially in intron locus of gene. Culex pipiens complex from Iran are located in two separate clades with sister branches using phylogenetic sequencing tree. INTERPRETATION & CONCLUSION: The male genitalia found as the most reliable diagnostic characters for identification of Cx. pipiens complex in Iran that confirmed by amplify the Ace.2 gene in the samples but we recommended the use of sequencing PCR products of microsatellite loci and COI gene in future study.

The Morphological Variations of Culex pipiens Larvae (Diptera: Culicidae) in Yazd Province, Central Iran.

BACKGROUND: Culex pipiens complex shows variations in morphological and biological characters including different biological forms and has medical and veterinary importance. Because of having morphological variations, sometimes it is not easy to separate this species from Cx. quinquefasciatus and Cx. torrentium. The aim of this study was to identify the Culex pipiens complex species in order to use in control programs in the future. METHODS: This study was carried out in two randomly selected rural villages in Yazd County, eastern Iran using dipping technique from April to October 2009. The data were analyzed using SPSS software version 16. RESULTS: Average of siphon index in fourth-instrar larvae was 3.86±0.03, the minimum and maximum were calculated 2.43 and 5.14, respectively. Siphon/Saddle index was measured as average, minimum and maximum 3.2±0.2, 2.78, and 4.42 respectively. In our study, only 4 specimens had single seta 1 on segments III and VI (2.5%) and the remaining beard double seta (97.5%). The maximum 3-6 branches seta 1a-S and 1b-S (95%) were observed on siphon. CONCLUSION: More populations of Culex pipiens from different areas of Iran need to be studied to gain complete information about the taxonomy and ecology of the species in the country.

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.

Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays.

BACKGROUND: We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to identify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method. METHODS: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006-2007. For PCR-RFLP a set of conserved vertebrate primers were used to amplify a part of the host mitochondrial cytochrome b (cyt b) gene followed by digestion of the PCR products by Hae III enzyme. RESULTS: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on humans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%), human (10%), dog (4%), human and cow (21%), dog and cow (14%), and human and dog (3%). CONCLUSION: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advantages and limitations of PCR and ELISA assays are discussed.

The use of a nested PCR-RFLP technique, based on the parasite's 18S ribosomal RNA, to characterise Cryptosporidium isolates from HIV/AIDS patients.

Since there have been few studies on human cryptosporidiosis in Iran, attempts were made to identify Cryptosporidium isolates from HIV-positive Iranians, to genotype level. A nested PCR (based on a fragment of the parasite's 18S ribosomal-RNA gene) was first used to see if faecal samples from 35 HIV-positive patients (of whom 17 had apparently been found smear-positive for Cryptosporidium oocysts) contained Cryptosporidium. Twenty-one of the samples (including all 17 of those that appeared smear-positive) were found PCR-positive. Each of these 21 samples was then investigated further, by RFLP analysis in which the amplicons from the secondary PCR were digested with VspI. Curiously, although HIV-infected individuals are known to be susceptible to infection with a wide range of Cryptosporidium genotypes, all the Iranian subjects of the present study were found to be infected with C. hominis (71%) or C. parvum (29%).