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Sand fly fever is caused by Naples (SFNV) and Sicilian viruses (SFSV) and the closely related Toscana virus of the Phlebovirus genus in the family Bunyaviridae, and transmitted by Phlebotomine sand flies. Phlebotomus papatasi Scopoli, 1786 is known as the main vector of the disease. This study aimed to investigate the distribution of Phlebotomine sand flies as vector of sand fly fever viruses, and the effects of some environmental variables on their potential dispersion to new areas in some provinces of Iran. Sand flies were collected during their active season in the region using CDC light traps. Ecological parameters were recorded for each collection site. Arc GIS 10.3 software was used for data analysis and mapping the distribution of sand flies. Sampling in the study areas was carried out in six different climatic zones. Seventeen sand fly species were collected including eight species of genus Phlebotomus and nine species of genus Sergentomyia. The Medium Semi-Arid climatic zone had the highest species diversity. Fourteen species of sand flies were collected at altitude between 2 and 325 m, and seven species were collected between 326 and 1380 m above sea level. There was significant correlation between sand fly density and all tested environmental variables. Phlebotominae sand flies have wide distribution in Iran and have a major public health concern in the country. P. papatasi and Sergentomyia sintoni Pringle, 1933 prefers hot summers and rainfall. This poses a threat of transmission of sand fly fever caused by SFSV and SFNV across the country.

Members of the Culex (Culex) pipiens assemblage are known vectors of deadly encephalitides, periodic filariasis, and West Nile virus throughout the world. However, members of this assemblage are morphologically indistinguishable or hard to distinguish and play distinct roles in transmission of the diseases. The current study aimed to provide further evidence on utility of the two most popular nuclear (ITS2-rDNA) and mitochondrial (COI barcode region) genetic markers to identify members of the assemblage. Culex pipiens assemblage specimens from different climate zones of Iran were collected and identified to species level based on morphological characteristics. Nucleotide sequences of the loci for the specimens plus available data in the GenBank were analyzed to find species specific genetic structures useful for diagnosis purposes. ITS2 region was highly divergent within species or populations suggesting lack of consistency as a reliable molecular marker. In contrast, sequence analysis of 710 bp of COI gene revealed three fixed haplotypes named here "C, T, H" within the assemblage which can be distinguished by HaeIII and AluI enzymes. There were a correlation between the haplotypes and the world climate regions, where the haplotypes H/T and C are present mainly in temperate and tropical regions of the world, respectively. In the New world, Australia, and Japan only haplotype H is found. In conjunction between tropical and temperate regions such Iran, China, and Turkey, a mix of C/H or C/H/T are present. Although, the haplotypes are not strictly species-specific, however, Cx. quinquefasciatus was mainly of haplotype C. Due to the lack of mating barrier and questionable taxonomic situation of the complex members, the mentioned haplotypes in combination with other morphological and molecular characters might be used to address the genetic structure of the studied populations.

In the current study, the effects of the presence of symbiotic bacteria on the activity of the enzymes involved in An. stephensi resistance to temephos are evaluated for the first time. Four different strains (I. susceptible strain, II. resistant strain, III. resistant strain + antibiotic, and IV. resistant strain + bacteria) were considered in order to determine the possible effects of the symbiotic bacteria on their hosts' resistance to temephos. The median values of all enzymes of susceptible strain were compared with those of other resistant strains. The results of this study indicated a direct relationship between the presence of bacteria in the symbiotic organs of An. stephensi and resistance to temephos. The profile of enzymatic activities in the resistant strain changed to a susceptible status after adding antibiotic. The resistance of An. stephensi to temephos could be completely broken artificially by removing their bacterial symbionts in a resistant population.

Background Anaplasmosis / Ehrlichiosis are tick-borne diseases affecting human and livestock in tropical and subtropical regions of the world. Due to infection both animals and humans and monitoring of ticks and the pathogens they carry, an extensive survey was conducted in border line of Iran-Pakistan of Sistan and Baluchistan, southeast corner of Iran in 2016-2017, where animal husbandry is the main activity of people and every week thousands of cattle cross the borders into the countries. The aim of the survey was to determine the prevalence and geographical distribution of Anaplasmosis / Ehrlichiosis agents in Hyalomma spp hard ticks. Ticks were collected, identified and processed for Anaplasma / Ehrlichia spp DNA detection. Results Six Hyalomma species were found in the region, where Hy. anatolicum was the most prevalent species collected on goats, cattle, and camel. Anaplasma / Ehrlichia genomes were found in 68.3% of the specimens. Anaplasma ovis, A.marginale, and E.ewingii DNAs prevalence were 81.82, 9.09, and 9.09% in the infected ticks respectively. DNA sequence and phylogenetic analysis of the 16SrRNA gene confirmed the detection of these three anaplasmosis agents while they had 99-100% identity with the strains previously reported in genbank from different parts of the world. Conclusion Because A.ovis and A.marginale are important livestock pathogens, and E.ewingii is an important human pathogen, both farmers and people in border line of the countries which engaged in livestock need to be made aware of the risks of tick infestation and the tick-borne disease they transmit.

This study was designed to identify populations of Rhipicephalus sanguineus collected from Iran and also to study molecular taxonomy of Rhipicephalus species using cytochrome C oxidase subunit 1 (COI) and internal transcribed spacer 2 (ITS2) sequences. Tick specimens were collected from livestock (sheep and goat) in 14 Iranian provinces. DNA of individual specimens was extracted and PCR was done on these samples. So, 62 sequencing (33 COI and 29 ITS2) were done, successfully. Morphologically, we identified four Rhipicephalus species, namely R. bursa, R. sanguineus (s.l.), R. sanguineus (s.s.), and R. turanicus based on taxonomic keys. The data obtained from the phylogenetic analyses of COI and ITS2 fragments present a possible conflict regarding the identity of R. sanguineus species. Thus, the molecular identification of R. sanguineus group might be different according to mitochondrial and nuclear DNA. The results show a phylogenic conflict based on COI and ITS2 phylogeny in a tree topology. We dealt with three genetic entities in R. sanguineus group (i.e. R. sanguineus (s.s.), R. sanguineus (s.l.), and R. turanicus) based on COI phylogeny and two genetic clades (i.e. R. sanguineus (s.s.) and R. sanguineus (s.l.)/R. turanicus) according to ITS2 phylogeny.

Background: Enterobacter cloacae subsp. dissolvens bacterium is a known commensal of the gut microflora of Phlebotomus papatasi, the main vector for zoonotic cutaneous Leishmaniasis, and nominated for paratransgenesis in sand flies. In this study, we evaluated dynamics and fitness costs of engineered E. cloacae for its potential to serve as a 'Trojan Horse' in P. papatasi. Methods: The engineered strain of E. cloacae transformed with a constantly active expressed red fluorescent protein plus defensin (EC-DR) plasmid and was fed to sand fly colonies via larval food to larvae. A wild type the bacterium (EC-WT) and intact food were used as controls. Fitness characters as well as dynamics of the EC-DR at various development stages of sand fly larvae were tested by plating homogenized specimens and counting fluorescent expressing colonies on the Tet-BHI agar medium. Results: Enterobacter cloacaeDR producing red fluorescent protein could be isolated from the larvae gut after 36 days when the bacteria were added once in larval pots. The EC-DR with multiple applications had no negative effect on emergence time of instar II larvae, pupae, and adults but increased slightly mortality rate of P. papatasi larvae. The experiment also confirmed lack or weak trans-stadial transmission of E. cloacae DR in P. papatasi. It has minimal fitness cost on P. papatasifeeding behavior and survival. Conclusion: Results of this study showed that E. cloacae DR is suitable for paratransgenesis of P. papatasi at only adult stage because it did not transmit transstadially.

Objective: To estimate the infection of ticks to Anaplasma, Ehrlichia, Babesia, Theileria, and Brucellaceae using molecular methods in borderline of Iran, Azerbaijan, and Armenia. Methods: Totally, 2 022 ticks were collected from different livestock. Then, species were diagnosed under stereomicroscope according to valid morphological keys. Tick DNA was extracted followed by PCR to detect Anaplasma, Ehrlichia, Theileria, Babesia and Brucellaceae infection in ticks. Results: A total of 498 males [24.62% (95% CI 22.76%-26.57%)], 741 females [36.64% (95% CI 34.54%-38.79%)], 782 nymphs [38.67% (95% CI 36.55%-40.84%)] and 1 larva [0.04% (95% CI 0.00%-0.28%)] were identified. Among identified samples, we found four genera including Hyalomma, Rhipicephalus, Haemaphysalis, and Dermacentor. Molecular assay revealed that the prevalence of ticks to Anaplasma or Ehrlichia, and Brucellaceae was 22.02% (95% CI 16.01%-29.06%) and 15.03% (95% CI 9.43%-22.26%), respectively. Phylogenetic analysis showed that the identified Anaplasma sp. had the most similarity with Anaplasma centrale, Anaplasma platys, Anaplasma camelii, and Anaplasma phagocytophilum, submitted in GenBank. Furthermore, the detected Ehrlichia sp. and Brucellaceae bacterium had the most similarity with Ehrlichia ruminantium and Mycoplana peli, respectively. However, no sign of the presence of Theileria and Babesia spp. was seen in the studied samples. Conclusions: Anaplasmosis, ehrlichiosis and brucellosis should be considered as important health threats in northwestern Iran and consistent monitoring on infection of ticks and livestock should be performed regularly.

Use of sterile fly larvae (maggots) of blow flies for the treatment of many different types of skin and soft tissue wounds is called Maggot debridement therapy (MDT). The larvae of blow flies secrete a broad spectrum of compounds with diverse mechanisms of action in the gut and salivary glands called excretion/secretion (ES) products which showed to have antimicrobial activities against Gram negative and positive bacteria. Cutaneous leishmaniasis (CL) which is the common form of leishmaniasis is difficult to treat. In this study, the effect of ES from 2nd and 3rd stages of L. sericata and C. vicina larvae on in vitro Leishmania major amastigote growth in macrophage was evaluated. The effect of ES on Leishmania growth was estimated by assessing the rate of macrophage infection and the number of amastigotes per infected macrophages. In addition, the anti Leishmania activities of larval and ES of L. sericata and C. vicina on the skin lesion induced by L. major infection was evaluated in susceptible BALB/c mice. The results showed that ES of both flies reduced the number of infected macrophages; 2.6 and 1.5-fold using L. sericata ES and C. vicina ES, respectively, and inhibited amastigotes growth in macrophages; 2.03 and 1.36-fold by L. sericata ES and C. vicina ES, respectively as compared to the control group. The results showed that L. sericata ES was significantly more effective than C. vicina ES to inhibit in vitro L. major amastigotes growth, The size of lesion was significantly smaller in BALB/c mice treated with L. sericata ES than treated with C. vicina ES. The results of in vivo experiments suggested that pre-treatment with ES derived from L. sericata may have some protective effects on the development of L. major lesion. Therefore, it seems that maggot ES might be considered as a possible candidate for the treatment of cutaneous leishmaniasis.

Several mosquito species are vectors of disease; however, to understand their role in disease transmission, accurate species identification is of particular importance. Morphological identification is the main method used, but molecular techniques have emerged as a tool for the identification of closely related species. In this study, mosquitoes from the West Azerbaijan Province in northwestern Iran were characterized on the basis of their rDNA-ITS2 sequences. Nine populations of 6 species of mosquitoes belonging to the genera Anopheles, Culex, Culiseta, and Ochlerotatus were studied. To the best of our knowledge, ITS2 sequences of Culiseta longiareolata and Culex hortensis have been reported for the first time. In addition, ITS2 sequences of Culex theileri and Ochlerotatus caspius have been reported for the first time in Iran. Phylogenetic analysis based on ITS2 showed that subfamilies Anophelinae and Culicinae of the family Culicidae could be differentiated successfully and subgenera Anopheles and Cellia of the genus Anopheles were separated. The analysis showed that the genera Culex, Culiseta, and Ochlerotatus have diverged separately.

Black flies are insects of medical, veterinary, and environmental significance. Historically, they have attacked humans and caused simuliotoxicosis in livestock in the Aras River Basin in northwest Iran. However, information on the species and their bionomics is limited in the region. Adult flies were collected from diverse ecotopes of the Aras River Basin. After morphological identification, representative specimens of each morphological group were subjected to mtDNA COI gene sequence analysis for species diagnosis and to infer relationships. Flies also were examined for pollinia. A total of 1880 specimens representing 12 morphotaxa in two genera (Simulium and Metacnephia) were identified: Simulium turgaicum (n=1834), S. kiritshenkoi (n=12), S. bezzii (n=7), S. brevitarse (n=7), S. pseudequinum (n=5), S. aureum species group (n=4), S. vernum species group (n=3), S. transcaspicum (n=1), three unidentified species of the subgenus Simulium (n=5), and Metacnephia possibly persica (n=2). Fifty two haplotypes were detected for the 65 COI sequences analyzed. Intraspecific genetic divergence was 0.19-8.83%, whereas the mean interspecific genetic distances among the morphotaxa were 1.41-19.58%. Molecular analyses recovered three well-supported lineages within S. turgaicum. One lineage included black flies collected from agricultural fields, a second lineage involved black flies captured from animals, and a third lineage included specimens that had visited flowers, as evidenced by presence of pollinia. The relative abundance (97%) and observations of the S. turgaicum complex biting humans are important epidemiological factors. Future studies are needed to define the potential epidemiological risk of simulids in Khoda-Afarin County of Iran.

Background: Entomotoxicology as a subset of forensic entomology can be used by analysis of carcass feeding in­sects to detecting of drugs or toxins, as well as the cause and manner of death in cases of ante-mortem drugs intoxi­cation. Morphine is one of the deacetylate metabolites of heroin. The aim of this study was to determine the presence and quantity of morphine in insects on the carcass and compare them with decomposing carcass. Methods: Field of this study was in Chalabeh District and toxicological tests were carried out at the Department of Forensic Toxicology, Legal Medicine Center, Kermanshah, Iran in 2017. Morphine was inoculated into live rabbit as experimental model at concentrations of 12.5, 25, 50mg/ml, similar to those normally encountered in human over­doses, then quality and quantity of morphine were determined in insects such as Chrysomya albiceps (as the first wave of insect succession on human cadavers) fed on carcass. Results: Quantitative assessment at larvae showed that morphine was detected in all larvae (feeding and post feeding stage) fed on tissues from carcasses administered morphine, except for post-feeding larvae from R1 which received 12.5mg/ml dosage of morphine. Conclusions: Necrophagous insects are an indicator on the scene of crime and a potential source of information about the antemortem situation. Detection of drug in insects which is actually a reflection of the cause of death is possible.

Background Leishmaniasis is caused by Leishmania parasites and is transmitted to humans through the bite of infected sand flies. Development of Leishmania to infective metacyclic promastigotes occurs within the sand fly gut where the gut microbiota influences development of the parasite. Paratransgenesis is a new control method in which symbiotic bacteria are isolated, transformed and reintroduced into the gut through their diet to express anti-parasitic molecules. In the present study, the midgut microbiota of three sand fly species from a steppe and a mountainous region of northern Iran, where zoonotic visceral leishmaniasis (ZVL) is endemic, was investigated. Methods Briefly, adult female sand flies was collected during summer 2015 and, after dissection, the bacterial composition of the guts were analyzed using a culture-dependent method. Bacterial DNA from purified colonies was extracted to amplify the 16S rRNA gene which was then sequenced. Results Three ZVL sand fly vectors including Phlebotomus major, P. kandelakii and P. halepensis were found in the highlighted regions. In total, 39 distinct aerobic bacterial species were found in the sand fly midguts. The sand fly microbiota was dominated by Proteobacteria (56.4%) and Firmicutes (43.6%). Bacterial richness was significantly higher in the steppe region than in the mountainous region (32 vs 7 species). Phlebotomus kandelakii, the most important ZVL vector in the study area, had the highest bacterial richness among the three species. Bacillus subtilis and Pantoea agglomerans were isolated from the guts of the sand flies; these are already used for the paratransgenesis of sand flies and mosquitoes, respectively. Conclusions The existence of B. subtilis and P. agglomerans in the ZVL vectors and other sand fly species studied so far suggests that these two bacterial species are potential candidates for paratransgenic approach to prevent ZVL transmission. Further research needs to test the possible relationship between the gut microbiome richness and the vector competence of the ZVL vectors.

Sand flies of Phlebotomus papatasi and P. sergenti are the main vectors of cutaneous leishmanisis (CL) in the old world. We aimed to screen Iranian P. papatasi and P. sergenti for their natural infections with Wolbachia and to determine their phylogenetic association with other species. Wolbachia surface protein (wsp) gene was PCR amplified from DNA extracted from Phlebotomus species, sequenced, and were analysed in combination with wsp sequences related to Phelebtominae and other insects. All Wolbachia-infecting Iranian sand flies of P. papatasi and P. sergenti were classified in the Supergroup A., Wolbachia isolated from P. sergenti were clustered in a new subgroup within Supergroup A so-called wSreg. The Wolbachia strains identified from the P. papatasi clustered mainly in the subgroup wPap and partly in wSerg. Multiple Wholbachia infection within a single population of P.papatasi warrants investigation on existence and intensity of cytoplasmic incompatibility between the wPap and wSerg subgroups.

Understanding how arthropod vectors acquire their bacteria is essential for implementation of paratransgenic and RNAi strategies using genetically modified bacteria to control vector-borne diseases. In this study, a genetically marked Serratia AS1 strain expressing the mCherry fluorescent protein (mCherry-Serratia) was used to test various acquisition routes in six arthropod vectors including Anopheles stephensi, Culex pipiens, Cx. quinquefaciatus, Cx. theileri, Phlebotomus papatasi, and Hyalomma dromedarii. Depending on the species, the bacteria were delivered to (i) mosquito larval breeding water, (ii) host skin, (iii) sugar bait, and (iv) males (paratransgenic). The arthropods were screened for the bacteria in their guts or other tissues. All the hematophagous arthropods were able to take the bacteria from the skin of their hosts while taking blood meal. The mosquitoes were able to take up the bacteria from the water at larval stages and to transfer them transstadially to adults and finally to transfer them to the water they laid eggs in. The mosquitoes were also able to acquire the bacteria from male sperm. The level of bacterial acquisition was influenced by blood feeding time and strategies (pool or vessel feeding), dipping in water and resting time of newly emerged adult mosquitoes, and the disseminated tissue/organ. Transstadial, vertical, and venereal bacterial acquisition would increase the sustainability of the modified bacteria in vector populations and decrease the need for supplementary release experiments whereas release of paratransgenic males that do not bite has fewer ethical issues. Furthermore, this study is required to determine if the modified bacteria can be introduced to arthropods in the same routes in nature.

Background: The abundance, diversity, distribution and ecology of mosquitoes (Diptera: Culicidae), especially ar­bovirus vectors are important indices for arthropod-borne diseases control. Methods: Larvae and adult mosquitoes were collected using the standard methods from different habitats in nine localities of three counties in the East Azerbaijan Province, Northwestern Iran during June to October 2017. In addi­tion, species richness (R), Simpson’s diversity index (D), Shannon–Wiener index (H̕) and evenness (E) as measures of diversity, were calculated. Results: Overall, 1401 mosquito specimens including 1015 adults and 386 larvae were collected in the study area. The properties of geographical larval habitats were recorded. Four genera along with 10 species were collected and identified, including Anopheles hyrcanus, An. maculipennis s.l., An. superpictus s.l., Aedes caspius, Ae. vexans, Cu­lex pipiens, Cx. theileri, Cx. perexiguus, Culiseta longiareolata and Cs. subochrea. Among the three counties, Ahar region presented the highest species richness (R: 1.5) and diversity values (D: 0.79, H’: 1.74, E: 0.73). Conclusions: This study provides important information on the diversity, distribution and ecology of ten mosquito species in the region. This information leads to a better understanding of mosquito population dynamics in relation to vector control measures.

Getting to the guts of mosquito control Malaria persistently evades our best efforts to eliminate it. Pike et al. genetically modified malaria vector mosquitoes to be more immune-resistant to infection by the parasite, which altered the composition of the mosquitoes' gut bacteria. Genetically modified male (female) mosquitoes preferentially mated with wild-type females (males). Ten generations later, the genetically modified mosquitoes constituted 90% of a caged population without losing resistance to the malaria parasite. In an alternative strategy, Wang et al. engineered mosquitoes' gut bacteria. A strain of nonpathogenic bacteria, AS1, was both sexually and transgenerationally transmitted. The strain infected a laboratory population of mosquitoes and persisted for at least three generations. AS1 engineered to inhibit malaria parasite development in the midgut could do so without handicapping the mosquitoes. Science , this issue p. 1396 , p. 1399

Objective To identify the vector(s), the parasite and the species composition of sand flies in the district during May–October 2012. Methods For reaching our objectives we used polymerase chain reaction of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism. Results Two species of Phlebotomus sergenti and Phlebotomus tobbi were the most prevalent among 8 species identified comprising 51.1% and 32.9% respectively. Among the 160 specimens of female sand flies tested by polymerase chain reaction of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphisms, only 1 out of 80 Phlebotomus tobbi (1.25%) were positive to Leishmania infantum parasites. Conclusions Our finding showed that Phlebotomus tobbi may play as a vector to circulate the parasite of Leishmania infantum among reservoir(s) and human.

Objective To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis. Methods Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer (ITS1) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Leishmania species in sand flies specimens. Results Three out of 115 females of Phlebotomus sergenti ( P. sergenti ) (2.6%) were positive to Leishmania tropica ( L. tropica ). Conclusions This is the first report on P. sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HRM analysis. This method is rapid, sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.

Recent epidemiological evidences revealed the higher prevalence of 'O' blood group in the residents of malaria-endemic areas. Also some data indicated preference of mosquitoes to 'O' group. The aim of this study was to determine ABO group ratio in the residents as well as ABO group preference of Anopheles in two malaria endemic areas in south of Iran. Agglutination method was used for ABO typing of residents. Field blood fed Anopheles specimens were tested against vertebrate DNA using mtDNA-cytB PCR-RFLP and then the human fed specimens were tested for ABO groups using multiplex allele-specific PCR. A total of 409 human blood samples were identified, of which 150(36.7%) were 'O' group followed by 113(27.6%), 109(26.7%), and 37(9.0%) of A, B, and AB groups respectively. Analyzing of 95 blood fed mosquitoes revealed that only four Anopheles stephensi had fed human blood with A(1), B(1), and AB(2) groups. Result of this study revealed high prevalence of O group in south of Iran. To our knowledge, it is the first ABO molecular typing of blood meal in mosquitoes; however, due to low number of human blood fed specimens, ABO host choice of the mosquitoes remains unknown. This study revealed that ABO blood preference of malaria vectors and other arthropod vectors deserves future research.

Anaplasmosis, a disease caused by various species of Anaplasma, poses important economic con- straints to animal breeders. Ehrlichiosis is a worldwide zoonosis illness and mostly occurs in tropical and subtropical regions that are close to the vector's distribution. Tick-borne pathogens lead to over 100,000 cases of illness in the world each year. Besides the costs of the additional ve- terinary care, anaplasmosis causes abortion in animals, reduction of milk production, body weight, and frequently leads to death. In this study, we investigated on infection of ticks to Anaplasma and Ehrlichia pathogens in Zabol and Zahak County in Sistan and Baluchestan Province where is bor- dered with Afghanistan. Totally from June 2013 to May 2014, 369 ticks were caught from goats, cows and sheep. Molecular studies on 53 of these samples which represented all specimens, showed that Ehrlichia's DNA and Anaplasma's DNA was found in 14 (26.4%) out of the 53 selected specimens. The results showed the infection of Rhipicephalus sanguineus and Hyalomma anatoli- cum with Anaplasma ovis. Also we saw infection of H. anatolicum and H. asiaticum ticks to Ehrlichia spp. This study has been intended to do a comprehensive survey of Ehrlichia and Anaplasma dis- tribution in ticks caught from east of Iran; it was designed to investigate the presence of Anaplas- ma spp. and Ehrlichia spp. in Zabol and Zahak Counties, Iran. These results show that these pa- thogens should be controlled in such regions. * Corresponding author.

Objective: To identify the biological forms, sporozoite rate and molecular characterization of the Anopheles stephensi (An. stephensi) in Hormozgan and Sistan-Baluchistan provinces, the most important malarious areas in Iran. Methods: Wild live An. stephensi samples were collected from different malarious areas in southern Iran. The biological forms were identified based on number of egg-ridges. Molecular characterization of biological forms was verified by analysis of the mitochondrial cytochrome oxidase subunit I and II (mtDNA-COI/COII). The Plasmodium infection was examined in the wild female specimens by species-specific nested–PCR method. Results: Results showed that all three biological forms including mysorensis, intermediate and type are present in the study areas. Molecular investigations revealed no genetic variation between mtDNA COI/COII sequences of the biological forms and no Plasmodium parasites was detected in the collected mosquito samples. Conclusions: Presence of three biological forms with identical sequences showed that the known biological forms belong to a single taxon and the various vectorial capacities reported for these forms are more likely corresponded to other epidemiological factors than to the morphotype of the populations. Lack of malaria parasite infection in An. stephensi, the most important vector of malaria, may be partly due to the success and achievement of ongoing active malaria control program in the region.

Objective: To evaluate the susceptibility of Anopheles stephensi (An. stephensi) Liston, the main malaria vector in southern Iran, to WHO recommended insecticides. Methods: Larvae of An. stephensi were collected from three different larval habitats in both urban and rural area of Bandar Abbas city and one rural area in Rudan county southern Iran. WHO standard method was used for evaluation of adult and larval mosquito susceptibility. Bendiocarb, permethrin, lambda-cyhalothrin, deltamethrin as insecticide and temephos and chlorpyriphos as larvicide were used at the diagnostic dosages recommended by WHO. Results: Findings of this study showed all larval populations of An. stephensi were completely susceptible to temephos and candidate for resistance to chlorpyriphos. Adult mosquitoes in rural areas of Bandar Abbas city were resistant to pyrethroid and carbamate insecticides. Conclusion: Comparison of the results of this survey with previous studies indicates that the resistance to pyrethroids and carbamates in this malaria endemic region is increasing. Wide use of pesticides in agriculture is certainly effective in increasing resistance. The inter-sectoral coordination and collaboration in health and agriculture seem to be necessary to manage insecticide resistance in malaria vectors.

Background: Leishmaniasis is one of the most important health problems in many areas of Iran. This study aimed to identify leishmania species in patients with cutaneous leishmaniasis using PCR teqnique based on sequencing ITS1 primers in villages of the central county of Qom province. Materials and Methods: This descriptive cross-sectional study was done on 169 patients who with leishmaniosis, based on clinical and laboratory confirmation treated and were followed in health center of Qomrood in 2010. This data was recorded in epidemiologic data forms. The DNA was extracted by KIAGEN kits. The extracted DNA was exploited to identify the parasite by PCR technique. The data were analyzed by SPSS-17 software. Chi square and Fisher’s exact test were used for the evaluation of the hypothesis. Results: The PCR result confirmed the parasite positive slides and obtained bands from these slides were seen in the range of 350 bp which are expected band for the leishmania major parasite. The most frequent age group was above 15 years old (71.5%). Hands and feet were the most common sites of ulcer (78.8%). 19.5 of patients had 3 ulcers and more. Highest disease prevalence was observed in months October, December and November. Conclusion: The PCR results illustrated that the prevalent CL in the central county of Qom province is Zoonotic type (ZCL), and the agent of disease is leishmania major parasite. In conclusion, identification of the disease and parasite type can help the health officials to make appropriate strategies for its prevention and control.

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.

an increasing trend. So we investigated some epidemiological aspects of the Leishmaniasis disease in order to update our information about infection routes and epidemiological characteristics of the disease in Qomrood District, Qom Province, Central Iran. Materials & Methods: In this descriptive cross-sectional study, patients population includes those have been treated and followed with the diagnosis of cutaneous Leishmaniasis in Qomrood District Health Center clinically or based on laboratory data from the beginning of April to the end of March 2009 and the related information have been documented in summarized epidemiologic data forms of cutaneous Leishmaniasis by the "disease control" personnel of the province health center. Data were analyzed using chi- square test. Results: The incidence of cutaneous Leishmaniasis in Qomrood rural areas has reached to 32.7/1000 in 2009 which used to be 9/1000 in 2008 The most common sites of skin lesions have been upper and lower extremities. The disease has been appeared in winter and autumn. Conclusions: According to the current condition, recognizing the vector and reservoir, and to arranging controlling programs in the area according to epidemiological aspects of the disease and mass education are mandatory. &nbsp

Background and Objectives: Cutaneous leishmaniasis is a major health problem in many parts of Iran and its main foci are in various parts of the country. This study was carried out with the intention of examining the species composition of the possible disease reservoirs in the cutaneous leishmaniasis focus in the selected villages of central county of Qom province. Methods: Study was done as a descriptive, cross-sectional study on rodents (possible leishmaniasis reservoirs) in the selected rural areas of Qomrood and Ghanavat located in central parts of Qom province in 2010. In this research, a total of 46 small rodents were hunted by live traps from 5 selected villages. A Smear prepared from auricle or suspicious lesions on skin, and after Giemsa staining, the smears were examined for the presence of leishman’s bodies. After extracting DNA from positive smears, PCR technique was exploited to determine the parasite species. Results: A total 46 small mammals were hunted and identified by the authentic keys. They included 31 rodents (67.4%) were Meriones libycus , 8 (17.4%) Allactaga elater , 4 (8.7%) Mus musculus , 2 (4.3%) Nesokia indica and one (2.7%) Hemiechinus auritus . The results of PCR demonstrated that 3.7% of M. libycus rodents were infected by Leishmania major. Conclusion: The results of this study showed that epidemic cutaneous leishmaniasis in the central county of Qom province was zoonotic and its reservoir was species of rodents. Therefore, determining disease type and its reservoir can help health care authorities to adopt appropriate strategies for prevention and control of the disease in this area.

Issues, like emerging insecticide resistance in Anopheles mosquitoes, have led to a breakdown in many vector control programs. In this study, a recombinant Escherichia coli with plasmid expressing a green fluorescent protein (E.coli-GFP) was used as a paratransgenesis model to determine: the possibility of E. coli-GFP trans-stadial transmission. The effect of the water microflora, of bacteria-impregnated sugar solutions, and of blood-feeding on E. coli-GFP colonization and localization within An. stephensi tissues, were studied. The results demonstrated the persistence of E. coli-GFP during molting and metamorphosis events and its trans-stadial transmission. Also the efficacy of bacteria-impregnated sugar solutions for transferring the bacteria to the adult mosquito’s midgut was shown. A blood meal dramatically increased the number of bacteria within 24–48 h post feeding. In addition to fluorescence microscope evaluation, GFP gene PCR amplification showed the presence of the bacteria in the midgut of larvae, pupae, and adults up to 13 days after eclosion. Massive colonization of bacteria was observed in the larvae and in the adult mosquito’s malpighian tubules which may play a role in retaining bacteria in adult mosquitos. The results of this study showed that E. coli could be used as a laboratory model in paratransgenesis studies for the evaluation of various effector molecules as anti-parasite agents for malaria and filariasis.

Canine monocytic ehrlichiosis is a disease which can cause death in dogs and cats by the Ehrlichia canis and transmitted by hard ticks. The main vector is Rhipicephalus sanguineus which is a common tick in Iran and common ectoparasite on dogs. Few researches has been done to show Ehrlichiosis situation in dog’s ticks. Animal husbandry in Meshkin-Shahr County from Ardebil province (North-West Iran) is the main job of people. Specimens were collected on dogs’ ears, neck, shoulder and toes and they were transferred to the Entomology Laboratory, School of Public Health, Tehran University of Medical Sciences. After DNA extraction, Ehrlichia agent was detected by nested-PCR, 16 s rRNA amplification. Determination of sequence homologies have been done in Gen-Bank. 146 ticks were identified which included 29.44% female and 47.94% male. Rhipicephalus sanguineus were the most prevalent ticks. Ehrlichia spp and Anaplasma spp were found in 43.84% of all the specimens containing Anaplasma ovis, and Ehrlichia sp. and Herlichia canis. Rhipicephalus sanguineus is widely spread and the most common ticks on dogs. As far as we know, this is the first report of E. canis in vector from Iran. Nested PCR showed that hard ticks can contain Anaplasma ovis, Ehrlichia spp and E. canis. These results warrant studying on vector competence of ticks for the Ehrlichiosis agents.

Leishmaniasis is a Neglected Tropical Disease (NTD) and emerging parasitic infection that affect mainly poor regions around the world. This study aimed to determine the baseline susceptibility of Phlebotomus papatasi to commonly used insecticides in a hyper endemic area using WHO standard procedure in central Iran. A total of 4-5 replicates containing 120-200 sand flies were used for each insecticide. Baseline susceptibility to DDT and pyrethroids was assessed on 5326 specimens collected from the study area. The LT50 and LT90 values were measured according to the World Health Organisation test using probit analysis and regression lines. The test results against males P. papatasi revealed that LT50 values to DDT 4%,Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% were 564.07, 38.08, 1.95, 0.60 and 9.78s and the figures for females were 584.44, 110.10, 11.64, 1.53 and 16.91s, respectively. Our results indicated that P. papatasi as the main cutaneous leishmaniasis vector was susceptible to Cyfluthrin 0.15%, Lambdacyhalothrin 0.05%, Permethrin 0.75% and Deltamethrin 0.05% and tolerant to DDT 4%. This study was carried out in one out of many Leishmaniasis foci in Iran. We recommend that future studies incorporate other regions and use the same procedure for monitoring and evaluating sand fly resistance. Also, WHO can provide a specific guideline and create a test kit for sand fly resistance monitoring and for applying susceptibility test because the tubes prepared for mosquitoes are not actually fit for sand flies.

Viscerotropic leishmaniasis (VTL) is a parasitic disease with non-specific manifestations caused by Leishmania tropica. Specific antigens produced by Viscerotropic leishmaniasis gene have been used for diagnosis of VTL. The aim of this study was to compare the expression level of VTL gene among the viscerotropic L. tropica isolates (n: 3) and visceral L. infantum isolates (n: 4). Also, the expression level was compared in L. tropica (n: 21) and L. major (n: 8) isolates, the main causes of cutaneous leishmaniasis in Iran by real time-RT-PCR. Results showed viscerotropic leishmaniasis gene was expressed in all 3 species; L. tropica, L. major and L. infantum. The most expression rate was in L. tropica and L. major as the cutaneous species and the lowest in visceral isolates including L. infantum and viscerotropic L. tropica strains respectively.Conclusion: Results revealed that VTL gene can play an important role in visceralization process of L. tropica although there are other mechanisms to keep parasite visceralized. According to these primary results, increased the expression level of VTL gene probably could contribute to inhibit the invasive behavior of Leishmania parasites. However, more experimental researches are needed to confirm this idea.

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 µg/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 CO 2 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 CO 2 in the attraction of An. stephensi.

Visceral leishmaniasis (VL) is an important health problem in Ardebil, where it borders Azerbaijan in the northwestern Iran. In spite of the presence of both cutaneous and visceral leishmaniasis (CL and VL) in northwestern Iran, previous researches have consistently revealed the etiologic agent of VL in the region to be Leishmania infantum. This is the first report of natural infection of Phlebotomus tobbi with L. infantum in Bilesavar district in the northern part of Ardebil province bordering Azerbaijan. Polymerase chain reaction (PCR) of kDNA, ITS1-rDNA, and CPB genes of the parasite followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses revealed presence of L. infantum in six out of 433 tested female sand fly specimens. Although sand flies of P. tobbi were infrequent, two out of 32 (6.25%) females captured in the area were found infected with the parasite. Phlebotomus perfiliewi transcaucasicus, the known vector of VL in the area, were the most dominant species but only four out of 273 (1.47%) tested were infected with L. infantum. This study showed that P. tobbi similar to P. perfiliewi transcaucasicus could play a significant role in the transmission of the L. infantum. However more investigations are needed to demonstrate that L. infantum is the only species circulating in the focus.

Bashagard is an important malaria endemic area in south of Iran. An epidemiological and entomological survey was performed during 2002-2010. The aim of study was to determine malaria situation, species composition of anopheline mosquitoes and susceptibility status of main vectors to insecticides/larvicides. A total of 13,490 malaria cases were recorded, Plasmodium vivax (99.64%), P. falciparum (0.35%) and mix cases (0.01%). The highest and lowest Annual Parasite Incidence (API) were observed in 2007 (145.72/1000) and 2009 (6.29/1000), respectively. Anopheles culicifacies, An. dthali, An. stephensi, An. superpictus, An. fluviatilis, An. moghulensis, An. turkhudi and An. apoci were collected from the area. Two peak activities occur in April and October. The first five species were confirmed as malaria vectors in Iran. No indication of sporozoite in mosquitoes using molecular method was performed. Susceptibility tests using diagnostic dose of insecticides and larvicides showed only resistance of An. stephensi to DDT. Tolerance in An. stephensi to deltamethrin and bendiocarb is reported. The same phenomenon was observed in An. culicifacies to DDT, propoxur and deltamethrin, and in An. dthali to malathion and deltamethrin. Larvae of vectors were susceptible to all larvicides, except for An. stephensi that exhibited tolerance to fenthion. In conclusion it should be emphasized that malaria transmission is a complex process in Bashagard. This event is attributed to five proven vectors with different behaviors which are active in the area. Regarding tolerance of vectors to deltamethrin, resistance management is suggested by using new insecticide with novel mode of action.

To describe the midgut microbial diversity and to find the candidate bacteria for the genetic manipulation for the generation of paratransgenic Anopheline mosquitoes refractory to transmission of malaria, the microbiota of wild larvae and adult Anopheles stephensi mosquito midgut from southern Iran was studied using a conventional cell-free culture technique and analysis of a 16S ribosomal RNA (rRNA) gene sequence library. Forty species in 12 genera including seven Gram-negative Myroides, Chryseobacterium, Aeromonas, Pseudomonas, Klebsiella, Enterobacter and Shewanella and five Gram-positive Exiguobacterium, Enterococcus, Kocuria, Microbacterium and Rhodococcus bacteria were identified in the microbiota of the larvae midgut. Analysis of the adult midgut microbiota revealed presence of 25 Gram-negative species in five genera including Pseudomonas, Alcaligenes, Bordetella, Myroides and Aeromonas. Pseudomonas and Exiguobacterium with a frequency of 51% and 14% at the larval stage and Pseudomonas and Aeromonas with a frequency of 54% and 20% at the adult stage were the most common midgut symbionts. Pseudomonas, Aeromonas and Myroides genera have been isolated from both larvae and adult stages indicating possible trans-stadial transmission from larva to adult stage. Fast growth in cheap media, Gram negative, and being dominantly found in both larvae and adult stages, and presence in other malaria vectors makes Pseudomonas as a proper candidate for paratransgenesis of An. stephensi and other malaria vectors.

Accurate identification of animal reservoirs of transmissible diseases is an absolute requirement to any epidemiological survey of zoonoses and is essential for predicting species-specific population outbreaks and therefore to develop accurate ecological control strategies. The systematic status of the great gerbil (Rhombomys opimus) remains unclear, despite the fundamental role of these rodents as the main known reservoir hosts of the protozoan parasite Leishmania major in the epidemiology of zoonotic cutaneous leishmaniasis (ZCL) in central and south Asia. In the present work, we represent molecular evidence supporting the identification of at least two major lineages (subspecies) within the species of R. opimus in Iran. Analysis of the mitochondrial cytochrome b (cyt b) gene, revealed a range of 1–10% genetic variation among populations, which were well associated with biogeographic origins and subspecies. Results of laboratory cross hybridization between the subspecies and finding sympatric haplotypes of the two subspecies suggested that no pre- or post-zygotic barriers exist between the subspecies indicating that they still belong to a single taxon. However, the amount of genetic variations between populations/subspecies is high enough to lead them to speciation in future. Implications of such findings on the eco-epidemiology of ZCL in Iran are discussed.

Malaria is still one of the most important health-problems in the world and is endemic in Iran. Since 1994, after collapse of former Soviet Union, a new threat of malaria importation emerged from those countries into the northern Iran. This work was carried out to provide further evidence on the status of anopheline species composition, the malaria parasite species, and natural infectivity of mosquitoes distributed in Pars-Abad district, on the borderline of Azerbaijan in northwestern Iran. Mosquitoes were collected from May to December 2008 in anopheline seasonal activity and were identified at the species level. The genus- and species-specific primers against Plasmodium ssrDNA gene were used for specific amplification on female mosquito head + thorax. Members of the Anoheles maculipennis complex were identified by sequence analysis of the ribosomal internal transcribed spacer II (ITS2-rDNA). Morphological character-based identification showed that out of 1455 anopheline female specimens, 1121 (77%) were of A . maculipennis s.l. and 334 (23%) were of Anoheles hyrcanus. Molecular analysis of the species complex indicated the presence of Anoheles sacharovi 984 (67.6%) and A. maculipennis 137 (9.4%) in the region. None of themosquito's head–thorax was found to be naturally infected by malaria parasite. Results of this study, particularly high dominance of A. sacharovi , suggest a potential risk of malaria epidemic in the region, and the need for a continuous epidemiological surveillance.

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.

Tick-borne relapsing fever (TBRF) is endemic in Africa and Eurasia and attributed to different Borrelia species. In the Central Asia and Middle Eastern countries, TBRF is caused mainly by Borrelia persica; however, other Borrelia species such B. microtti, B. latyschewii, B. baltazardi, and B. caucasica have also been described. The classic taxonomy of Borrelia spp. is based on the cospeciation concept that is very complex and rather confusing. In this study, we report two DNA-based methods to discriminate B. persica and B. microtti, the two main prevalent species in the region. Molecular typing of the species was performed using (i) restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified fragments of either 16S-rRNA or glpQ genes, and (ii) species-specific PCR of glpQ gene. Sequence analyses of the data obtained in this study indicate that the glpQ gene is more variable than 16S-rRNA (6.9% vs. 1.2%); thus glpQ is a more useful marker for discrimination of B. persica from B. microtti. The 16S-rRNA fragment comprises only one useful species-specific restriction site (TaqI), whereas the glpQ fragment includes several species-specific restriction sites and its digestion by DraI, TaqI, EcoRV, HinfI, or SspI results in distinctively different PCR-restriction fragment length polymorphism patterns for the two species. Further, the species-specific primers amplified a 253-bp fragment for B. persica and a 451-bp one for B. microtti. Phylogenetic analysis of the data revealed that B. microtti and B. persica are associated to the African and new world RF agents, respectively. This study demonstrates that both typing methods are simple, sensitive, and fast, and that they allow one to differentiate between B. persica and B. microtti. This could prove that both methods are important and useful in monitoring of TBRF disease in endemic areas.

An epidemiological study was carried out on the vectors and reservoirs of cutaneous leishmaniasis in rural areas of Damghan district, Semnan province, central Iran, during 2008-2009. Totally, 6110 sand flies were collected using sticky papers and were subjected to molecular methods for detection of Leishmania parasite. Phlebotomus papatasi Scopoli was the common species in outdoor and indoor resting places. Polymerase chain reaction technique showed that 24 out of 218 P. papatasi (11%) and 4 out of 62 Phlebotomus caucasicus Marzinovskyi (6.5%) were positive for parasites Leishmania major Yakimoff and Schokhor. Twenty-one rodent reservoir hosts captured using Sherman traps were identified as Rhombomys opimus Lichtenstein (95%) and Meriones libycus Lichtenstein (5%). Microscopic investigation on blood smear of the animals for amastigote parasites revealed 8 (40%) rodents infected with R. opimus. L. major infection in these animals was then confirmed by polymerase chain reaction against internal transcribed spacer ribosomal DNA (rDNA) loci of the parasite followed by restriction fragment length polymorphism. Further, sequence analysis of 297 bp of ITS1-rDNA loci revealed the presence of L. major and Leishmania turanica in P. papatasi, and L. major in R. opimus. This is the first molecular report of L. major infection in both vectors (P. papatasi and P. caucasicus) and reservoir host (R. opimus) in this region. The results indicated that P. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and P. caucasicus could be considered as a secondary vector. Further, our study showed that R. opimus is the most important host reservoir for maintenance of the parasite source in the area.

Shiraz district in south of Iran is a classical focus of Cutaneous leishmaniasis (CL) and previous research has consistently documented the etiologic agent to be Leishmania tropica and Leishmania major in urban and rural areas, respectively. However, none of the Phlebotomus sergenti, a known vector for L. tropica, of the region has been found infected. We report the first isolation of L. tropica from sandflies in urban community of southern part of Shiraz city. Parasite polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses indicate CL cases in this community were caused by either L. major or L. tropica. Sandflies of P. sergenti were infrequent, however, three out of 10 (30.0%) females captured in urban area were found infected with L. tropica. But, no human cases were found to be infected with L. tropica. Phlebotomus papatasi were found the most dominant and infected species where 41 out of 207 (20%) tested individuals harboring L. major in suburb area of the city. Patients have been lived in the suburb area of the city where people keep normally domestic animals in their houses which provide appropriate environment for completion of sandfly life cycle and expansion of CL disease in the region.

Objective To reveal subspecies composition of Rhombomy opimus ( R. opimus ) in Iran. Methods In this study, field specimens of the gerbil were collected from all its geographical rang in northeast and central parts of Iran and identified on basis of morphological characteristics. Results Results revealed presence of two subspecies of R. opimus sodalis and R. opimus sargadensis in the country. The first subspecies with brown to hazel-nut color and a size bigger than a typical R. opimus was found only in Golestan province, an isolated colony which is located in plains of north slopes of Alborz Chain Mountains of the country, connected with the Turkmenistan. The subspecies of R. opimus sargadensis with a yellowish color and similar size as a typical R. opimus was found in all other areas of the great gerbil distribution in Iran. There was a distinct topographic difference but similar ectoparasites between colonies of the two subspecies in the study area. Conclusions Further ecological and genetic investigations are required for more detailed description of the R. opimus subspecies range and structure. The R. opimus – Phlebotomus papatasi-Leishmania major association and the ZCL severity and outcome in hosts are discussed.

Mosquitoes of four Iranian islands (Abu-Musa, Hormuz, Larak and Qeshm), Hormozgan Province of southern Iran, were surveyed between 2005 and 2007. In 521 specimens collected, 14 species belonging to four genera were identified: Anopheles culicifacies Giles sensu lato (s.l.), An. dthali Patton, An. fluviatilis James s.l., An. stephensi Liston, An. turkhudi Liston, Culex bitaeniorhynchus Giles, Cx. perexiguus Theobald, Cx. pseudovishnui Colless, Cx. quinquefasciatus Say, Cx. sitiens Wiedemann, Cx. theileri Theobald, Cx. tritaeniorhynchus Giles, Culiseta longiareolata (Macquart) and Ochlerotatus (= Aedes) caspius (Pallas) s.l. This is the first record of all of these species on the islands. Cytochrome c oxidase I (COI) barcode sequences were used to confirm the taxonomic identity of An. stephensi, Cx. quinquefasciatus and Oc. caspius s.l. from the islands and mainland of Hormozgan Province, with genetic isolation noted in both Oc. caspius s.l. and Cx. quinquefasciatus samples collected in Abu-Musa Island. Mor...

Background & objectives: Olfaction is the major sensory modality involved in the resource searchingbehaviour of insects including vector mosquitoes (Diptera: Culicidae). To date, our current countrywideknowledge on the host-seeking behaviour of Iranian mosquitoes is mainly confined to hostpreference which has exclusively come from field studies. Olfactometer is a scientific tool bywhich more naive aspects of man-vector contact can be clarified under controlled and less biasedconditions.Methods: The wind tunnel and stimulus delivery system was constructed from acrylic materialsbased on previously introduced models with some modifications. Air supply and required lightwere ensured by a powerful compressor and incandescent bulbs, respectively. Desired level oftemperature was maintained by controllable heating radiators. For humidity production a uniquein-built piezo system was devised in the course of the air flow. Fine regulators facilitated thecontinuous generatation of the humidity at a preset level.Results: Titanium tetrachloride smoke plus monitoring of the wind speed revealed that the flow ofair was proper and invariable. A desired level of humidity and temperature could be set up in just10 and 15–45 min, respectively. These physical parameters varied only ±2% (humidity) and±0.15ºC (temperature) in a typical 20 min duration.Conclusion: The first sophisticated olfactometer in the field of medical entomology in Iran isreported here. Fast set up and stability of physical parameters are its salient features. It is expectedthat with the aid of this olfactometer further information on the physiological principles of thehost-seeking behaviour of mosquitoes become available soon.

Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of the parasite isolated from dogs; this parasite was considered to predominate in the all foci of IVL in Iran. However, based on PCR detection and sequencing of parasite Cysteine Protease B (CPB), only one out of seven sandfly infections in Phlebotomus perfiliewi transcaucasicus was found to be L. infantum in the current investigation. The six other infections were haplotypes of Leishmania donovani, the causative agent of anthroponotic visceral leishmaniasis (AVL) in West Africa and India. The deduced amino acid of the L. donovani haplotype was found to be novel and the shortest CPB protein reported within the Leishmania spp. Circulation of both L. donovani and L. infantum by P. perfiliewi transcaucasicus, in addition to previous data indicating its ability to circulate L. tropica, suggests that this species, like other vectors of VL, is a permissive vector. Finding L. donovani infecting P. perfiliewi transcaucasicus in the area demands extensive and intensive typing of natural Leishmania infections in epidemiological investigations in Iran and the Mediterranean Basin in general.

The prevalence, host preference, and rate of Leishmania spp. infection of sand fly species are important parameters for incrimination of parasite vectors. We applied polymerase chain reaction (PCR)�based and enzyme-linked immunosorbent assay (ELISA) methods to detect Leishmania spp. parasites and blood meals within individual sand flies in the most important visceral leishmaniasis (VL) focus in northwestern Iran. Leishmania spp. minicircles (kinetoplast DNA) were found in 14 (0.9%) of 1,569 female specimens. Sequence analysis of 650 basepairs of an internal transcribed spacer ribosomal DNA gene identified L. infantum/L. donovani in 12 specimens and L. adleri -like parasites in 2 speci- mens. Nine (64.3%) of 14 of the Leishmania spp.-positive sand flies were Phlebotomus perfeliewi transcaucasicus . Blood meal identification of host DNA within sand flies by PCR-based and ELISA methods showed that 30% and 28%, respec- tively, were positive for human blood. Results of this study showed that P. perfeliewi transcaucasicus is the most prevalent, infected, and anthropophagic sand fly and plays a major role in VL transmission in the region studied. In the Old World, cutaneous leish- maniasis (CL) is predominantly caused by L. major , L. tropica , L. aethiopica , and L. infantum , whereas L. donovani and L. infantum are responsible for visceral disease. Leishmaniasis causes a wide range of clinical forms ranging from a mere ulcer to a systemic disease that can be fatal if not treated correctly. Visceral leishmaniasis (VL) in Mediterranean region and Iran is caused by L. infantum , and it is considered a severe, often fatal disease. 4 Domestic dogs ( Canis familiaris ) are the prin- ciple reservoir host, and some sand fly species of the genus Phlebotomus subgenus Larroussius are the primary vectors of VL in the region. 5 During 1998-2006, approximately 2,056 cases of VL were reported in Iran, and Ardabil Province contained approximately 30.4% of the cases. More than 90% of VL cases are reported in infants less than 10 years of age and domes- tic dogs are primary reservoir host of the disease. 7 However, on the basis of entomologic studies, two species ( P. kandelakii and P. perfeliewi transcaucasicus ) are naturally infected with Leishmania spp. promasigotes in northwestern Iran and sus- pected as being probable vectors of VL in the region. 8

The field strain of Anopheles stephensi, the main malaria vector in south of Iran, was colonized in laboratory and selected with DDT and dieldrin in two separate lines for 3 generations to a level of 19.5- and 14-fold for DDT and dieldrin resistance, respectively. Synergist tests with chlorofenethol (DMC) and piperonyl butoxide (PBO) on the selected strains indicated that dehydrochlorination and oxidative detoxification might be the underlying mechanisms involved in the resistance to dieldrin and DDT in selected strains. DDT selection decreased susceptibility to DDT and pyrethroids including lambdacyhalothrin, permethrin deltamethrin and cyfluthrin. The result also showed that selection with dieldrin caused negative and positive cross-resistance to pyrethroid and fipronil, respectively. Based on these results, it can be concluded that besides metabolic resistance mechanisms, other factors such as mutation in γ aminobutyric acid (GABA) and voltage-gated sodium channels (Kdr) might be involved.

Insecticide resistance in the German cockroach can be mediated by a number of mechanisms, the most common being enhanced enzymatic metabolism. Seven field-collected strains of German cockroach, Blattella germanica (L.) with various levels of resistance to pyrethroids, five out of which were also cross-resistant to DDT were used in this study. The investigation of possible mechanisms responsible for permethrin resistance was carried out using the synergists PBO, DEF and DMC and biochemical assays, including general esterases, glutathione S-transeferases and monooxygenases assays, using an automated microtitre plate reader. PBO and DEF, the inhibitors of cytochrome p450 monooxygenases and general esterases, respectively, affected permethrin resistance to varying degrees depending on the strain. DDT resistance in five strains were not completely eliminated by the synergist DMC, an inhibitor of glutathione S-transferase enzymes, suggesting that a further non-metabolic resistance mechanism such as kdr-type may be present. This suggestion was further supported by GST assay data, where a little elevation in GST activity was detected in only two strains. The synergist data supported by biochemical assays implicated that cytochrome p450 monooxygenases or hydrolases are involved in permethrin resistance in some strains. However, these results implicated both enhanced oxidative and hydrolytic metabolism of permethrin as resistance mechanism in the other strains. The results of synergist and biochemical studies implicated that all the field-collected permethrin resistant strains have developed diverse mechanisms of resistance, although these strains have been collected from the same geographic area. The change in resistance ratios of some strains by using PBO or DEF is discussed. It is of interest to note that because resistance to permethrin was not completely eliminated by DEF and PBO, it is likely that one or more additional mechanisms are involved in permethrin resistance in every strain studied.

The West Nile virus (WNV) transmission cycle includes a wide range of migratory wetland birds as reservoirs, mosquitoes as biological vectors, and equines and humans as dead-end hosts. Despite the presence of potential vector species, there is no information about the existence of WNV in mosquito vectors in Iran. The Iranian West Azerbaijan Province is located in the northwestern part of Iran and has borders with Turkey, Iraq, Armenia, and the Republic of Azerbaijan. The current study was conducted to identify the wetland mosquitoes of the West Azerbaijan Province and their infection with WNV. In this study, 2143 specimens were collected, comprising 1541 adults and 602 larvae. Six species belonging to four genera were collected and identified: Anopheles maculipennis sensu lato (s.l.), Culex (Cx.) hortensis, Cx. pipiens s.l., Cx. theileri, Culiseta longiareolata, and Aedes (Ae.) (Ochlerotatus) caspius. In total, 45 pools of mosquitoes were examined. Two of the adult pools collected from the same location showed the presence of WNV in Ae. (Och.) caspius, from Sangar, Makoo County, as confirmed by PCR and sequencing. Due to the discovery of WNV in the mosquito population of the region, and the presence of wetlands and significant populations of migratory birds, the health sector should carefully monitor the factors involved in the cycle of this disease.

Although Iran has a large geographic area encompassing 13 ecoregions, its simuliid fauna remains largely unexplored. To begin redressing this faunal gap, we reviewed literature records and coupled morphological and chromosomal identifications of material newly collected from 16 sites in Iran. Twenty-three nominal species are now recognized, including new country records for Simulium crassicaulum (Rubtsov) and Simulium alajense Rubtsov, and the southernmost world record for Simulium transcaspicum Enderlein. Multiple cytoforms of the Simulium aureum group, Simulium bezzii complex, and Simulium ornatum group were found.