Evaluation of the Time Course on the Effectiveness of WHO Standard Pyrethroid and Carbamate Impregnated Test Papers against Anopheles stephensi, the Main Malaria Vector in Iran.

Background: Anopheles stephensi is a major vector of malaria in some parts of the world. A standard method for determining resistance in adult mosquito populations is the bioassay test recommended by the world health organization (WHO). The papers used in this method have an expiry date. This study aimed to determine the effectiveness of outdated susceptibility test papers for use in insecticide resistance monitoring programs. Methods: Beech and Bandar Abbas strains of An. stephensi were reared in the insectary. Permethrin 0.75%, Deltamethrin 0.05%, and Bendiocarb 0.1% impregnated test papers prepared by Universiti Sains Malaysia were used. Probit analysis was used to analyze the results and prepare time-mortality regression lines of LT50 and LT90. Results: There was a difference in the mortality of both tested strains of An. stephensi was exposed to all tested insecticides. Both expired and not expired Permethrin and Deltamethrin papers induced 100% mortality at the diagnostic time (60min), but their insecticidal properties were reduced gradually in serial times. The highest efficacy of test papers was in the first trimester after the expiry date and decreased over time. Conclusion: At the diagnostic time of 60 minutes, the mortality rate of both dated and expired papers was 100% in the pyrethroid insecticides, even three years after expiry dates, if stored in the package provided by the producer, in a refrigerator. This value was reduced to less than 100% in the expired papers of Bendiocarb comparing the dated papers that induced 100% mortality.

Sex-typing of ingested human blood meal in Anopheles stephensi mosquito based on the amelogenin gene.

Identifying the sex of human hosts of insect disease vectors, using PCR amplification of the amelogenin gene (AMEL) from the ingested blood meal is an increasingly useful technique for epidemiological studies of vector-borne diseases, as well as within the criminal justice system. Detection of DNA from ingested blood is influenced by the choice of DNA extraction method, genomic target region, type and length of PCR, and rate of degradation in the DNA samples over time. Here, we have tested two types of PCR (i.e. conventional and nested), producing differently-sized PCR products, in time-course assays targeting the human AMEL gene in Anopheles stephensi mosquitoes that were fed on human male and female blood. The fed female mosquitoes were allowed to digest at 28 °C for times ranging from 0 to 120 h. Three AMEL primer pairs were used to amplify three sequences that were 977, 539, and 106 bp for the X chromosome and 790, 355, and 112 bp for Y. We found that time since feeding had a significant negative effect on the success of PCR amplification. The shortest fragments (106 and 112 bp) were amplified for the longest time after blood feeding (up to 60 h), whereas the medium and longest loci were not amplified by conventional PCR even at 0 h. However, the nested PCR protocol, targeting the medium sequence, could detect small amounts of human DNA up to 36 h (1.5 days) after the blood meal. The shortest PCR assay standardized herein successfully detected small amounts of human DNA in female mosquitoes up to 60 h after the blood meal. This assay represents a promising tool for identifying the sex of the human host from the blood meal in field-collected female mosquitoes.

Drosophila melanogaster Laboratory Rearing for Wolbachia-Based Control Programs, a Component of Dengue Control.

Background: Drosophila melanogaster flies are smooth, low upkeep and safe model organisms, they can be effortlessly used in different fields of life sciences like genomics, biotechnology, genetics, disease model, and Wolbachia-based approaches to fight vectors and the pathogens they transmit. Methods: Fruit fly specimens were collected in 25 districts (14 provinces) of Iran and their morphological recognition was proven by molecular analysis based on sequence homology of mitochondrial COI barcode region. Essential information and specific requirements were provided for laboratory rearing of D. melanogaster. Results: Drosophila melanogaster colonies were found in 23 out of 25 districts. Also, five related species coincident with D. melanogaster were reported in this study including D. ananassae/D. parapallidosa, D. hydei, D. repleta, Zaprionus indianus (Diptera: Drosophilidae), and Megaselia scalaris (Diptera: Phoridae). The Iranian D. melanogaster molecular signature and their rearing techniques have been described here. The complete life cycle, from (egg to adult), takes approximately 8 days at 25 °C. Some biological points have been presented with highlighting capturing, rearing, culturing, and embryo collection along with primitive recognition and segregation between females and males have been presented. A recipe for culture media and the quantity of various ingredients have been provided. Conclusion: This is the first report on the D. repleta and D. ananassae/D. parapallidosa species for the country. Results of this study provide efficient and effective rearing procedures which are requirement for both small-scale for facilitating entomological research and large-scale use in justifiable vector control management such as disease model or Dengue control.

Evaluation of the Entomopathogenic Fungus Beauveria bassiana on Different Stages of Phlebotomus papatasi (Diptera: Psychodidae), Vector of Zoonotic Cutaneous Leishmaniasis in Iran.

Background: Zoonotic cutaneous leishmaniasis is a major public health problem in Iran with the main vector of Phlebotomus papatasi. The use of entomopathogenic fungi for biological control of the vector is a potential substitute for the current methods which are being used. The purpose of the current study was to assess the virulence of two local isolates of Beauveria bassiana (OZ2 and TV) against Ph. papatasi. Methods: To perform the bioassay test, fungal suspensions were applied for every stage of the sand fly life cycle. The mortality rate, longevity, and number of eggs laid were determined. Also, the probability of fungal survival on the surface of rodent's body was assessed. Results: The longevity of infected adult sand flies with both isolates of B. bassiana was significantly lower (P< 0.05) in comparison to the negative control. The estimated Lethal concentration 50 (LC50) values for adult female and male sand flies treated with OZ2 isolate were 1.4×106 and 2.2×107 conidia/ml, respectively, while they were 6.8×106 and 2.3×108 conidia/ml for TV isolate, respectively. Both isolates of B. bassiana exhibited nonsignificant mortality rates in sand fly larvae and pupae and fecundity rate (P> 0.05). According to our findings for both isolates, the fungus continued to spread throughout the surface of the rodent's body for 144 hours after spraying. Conclusion: The current study demonstrated that both isolates of B. bassiana have considerable biological control capacity against adult sand flies.

Ace-1 Target Site Status and Metabolic Detoxification Associated with Bendiocarb Resistance in the Field Populations of Main Malaria Vector, Anopheles stephensi in Iran.

Background: Anopheles stephensi is the main vector of malaria in Iran. This study aimed to determine the susceptibility of An. stephensi from the south of Iran to bendiocarb and to investigate biochemical and molecular resistance mechanisms in this species. Methods: Wild An. stephensi were collected from Hormozgan Province and reared to the adult stage. The susceptibility test was conducted according to the WHO protocols using bendiocarb impregnated papers supplied by WHO. Also, field An. Stephensi specimens were collected from south of Kerman and Sistan and Baluchistan Provinces. To determine the G119S mutation in the acetylcholinesterase (Ace1) gene, PCR-RFLP using AluI restriction enzyme and PCR direct-sequencing were performed for the three field populations and compared with the available GenBank data. Also, biochemical assays were performed to measure alpha and beta esterases, insensitive acetylcholinesterase, and oxidases in the strains. Results: The bioassay tests showed that the An. stephensi field strain was resistant to bendiocarb (mortality rate 89%). Ace1 gene analysis revealed no G119S in the three field populations. Blast search of sequences revealed 98-99% identity with the Ace1 gene from Pakistan and India respectively. Also, the results of biochemical tests revealed the high activity of non-sensitive acetylcholinesterase, alpha and beta-esterase in the resistant strain compared to the susceptible strain. No G119S was detected in this study additionally the enhanced enzyme activity of esterases and acetylcholinesterase suggesting that resistance was metabolic. Conclusion: The use of alternative malaria control methods and the implementation of resistance management strategies are suggested in the study area.

Impact of meteorological parameters on mosquito population abundance and distribution in a former malaria endemic area, central Iran.

Meteorological parameters, have been identified as an important factor involved in the transmission of vector-borne diseases. Mosquitoes are extremely sensitive to weather conditions. The aim of this study was investigate the correlation between meteorological parameters and the abundance of mosquitoes in Kashan County. Mosquitoes were collected using four different traps, including hand catch, animal baited bed net trap (usually a cow), human baited bed net trap and BG-Sentinel trap with CO2 from May to December 2019. A total number of mosquitoes collected were 1756 out of which 1621 (92.31%) were Culex, 22 (1.25%) Culiseta and 113 (6.44%) Anopheles in nine species. Most mosquitoes were collected by BG-Sentinel trap with CO2 (63.78%). Monthly distribution of the mosquitoes indicated different monthly peaks. Their high density were recorded in September and were low in December. The spearman's correlation of the mosquito abundance and the meteorological parameters shows that correlation of the number of total collected mosquitoes with relative humidity and precipitation (Rainfall) was weak negative, and there was week correlation with wind speed, and positive strong correlation with temperature. Data collected with various trap types and mosquito correlation with meteorological parameters in this study can be used for mosquito surveillance and control programs. However, meteorological parameters affect the abundance of mosquitoes, but their impact is complex and most of these variables are species specific.

Pathogenic fungi infection attributes of malarial vectors Anopheles maculipennis and Anopheles superpictus in central Iran.

BACKGROUND: Due to the effect of synthetic and commercial insecticides on non-target organisms and the resistance of mosquitoes, non-chemical and environmentally friendly methods have become prevalent in recent years. The present study was to isolate entomopathogenic fungi with toxic effects on mosquitoes in natural larval habitats. METHODS: Larvae of mosquitoes were collected from Central, Qamsar, Niasar, and Barzok Districts in Kashan County, Central Iran by standard dipping method, from April to late December 2019. Dead larvae, live larvae showing signs of infection, and larvae and pupae with a white coating of fungal mycelium on the outer surface of their bodies were isolated from the rest of the larvae and sterilized with 10% sodium hypochlorite for 2 min, then washed twice with distilled water and transferred to potato-dextrose-agar (PDA) and water-agar (WA) media and incubated at 25 ± 2 °C for 3-4 days. Larvae and fungi were identified morphologically based on identification keys. RESULTS: A total of 9789 larvae were collected from urban and rural areas in Kashan County. Thirteen species were identified which were recognized to belong to three genera, including Anopheles (7.89%), Culiseta (17.42%) and Culex (74.69%). A total of 105 larvae, including Anopheles superpictus sensu lato (s.l), Anopheles maculipennis s.l., Culex deserticola, Culex perexiguus, and Culiseta longiareolata were found to be infected by Nattrassia mangiferae, Aspergillus niger, Aspergillus fumigatus, Trichoderma spp., and Penicillium spp. Of these, Penicillium spp. was the most abundant fungus isolated and identified from the larval habitats, while An. superpictus s.l. was the most infected mosquito species. CONCLUSIONS: Based on the observations and results obtained of the study, isolated fungi had the potential efficacy for pathogenicity on mosquito larvae. It is suggested that their effects on mosquito larvae should be investigated in the laboratory. The most important point, however, is the proper way of exploiting these biocontrol agents to maximize their effect on reducing the population of vector mosquito larvae without any negative effect on non-target organisms.

Fauna and Larval Habitat Characteristics of Mosquitoes (Diptera: Culicidae) in Kashan County, Central Iran, 2019.

BACKGROUND: Mosquitoes are responsible for spreading devastating parasites and pathogens causing some important infectious diseases. The present study was done to better understand and update the fauna of Culicidae and to find out the distribution and the type of their larval habitats in Kashan County. METHODS: This study was done in four districts of Kashan County (Central, Qamasr, Niasar and Barzok). Mosquito larvae were collected from 23 active larval habitats using a standard 350ml capacity mosquito dipper from April to late December 2019. The collected larvae were transferred to containers containing lactophenol, and after two weeks individually mounted in Berlese's fluid on a microscope slide and identified to species by morphological characters and valid keys. RESULTS: In this study, a total of 9789 larvae were collected from urban and rural areas in Kashan County. The identified genera were Anopheles, Culiseta and Culex. In this study larvae of An. turkhudi, Cx. perexiguus, Cx. mimeticus, Cx. deserticola and Cs. subochrea were collected for the first time from Kashan County. CONCLUSION: The results of this study indicate the presence and activity of different mosquito species in Kashan County that some of them are vectors of arbovirus and other vector-borne diseases.

Insecticide Resistance Status of Malaria Vectors in a Malarious Area, Southeast of Iran.

Background: Malaria continues to be the main vector-borne disease in Iran. The endemic foci of malaria are in Sistan and Baluchistan Province, the borderline of Iran and Pakistan. By the year 2020 the program of the country is malaria elimination. The main vector control is using insecticide as Indoor Residual Spraying. The aim of the study was to evaluate the susceptibility of main malaria vectors to different insecticides recommended by WHO. Methods: All the insecticides papers supported by WHO and evaluation of insecticide resistance of Anopheles stephensi, Anopheles culicifacies, Anopheles superpictus to different chemical groups of imagicides including DDT 4%, malathion 5%, propoxur 01.%, lambdacyhalothrin 0.05%, deltamethrin 0.025% and permethrin 0.75% were followed by the WHO guideline. Results: Results of the susceptibility test against different insecticides revealed that An. stephensi and An. culicifacies are resistant to DDT and susceptible to other insecticides. An. superpictus is susceptible to all groups of pesticides. Conclusion: Knowledge on insecticide resistance in target species is a basic requirement to guide insecticide use in malaria control programmes in local and global scales.

Preparation of a Nanoemulsion of Essential Oil of Acroptilon repens Plant and Evaluation of Its Larvicidal Activity agianst Malaria Vector, Anopheles stephensi.

Background: Extensive use of chemical larvicides to control larvae, has led to resistance in vectors. More efforts have been conducted the use of natural products such as plant essential oils and their new formulations against disease vectors. Nanoformulation techniques are expected to reduce volatility and increase larvicidal efficacy of essential oils. In this study for the first time, a larvicide nanoemulsion from the essential oil of Acroptilon repens was developed and evaluated against Anopheles stephensi larvae under laboratory conditions. Methods: Fresh samples of A. repens plant were collected from Urmia, West Azarbaijan Province, Iran. A clevenger type apparatus was used for extracting oil. Components of A. repens essential oil (AEO) were identified by gas chromatography-mass spectrometry (GC-MS). All larvicidal bioassay tests were performed according to the method recommended by the World Health Organization under laboratory condition. Particle size and the morphologies of all prepared nanoformulations determined by DLS and TEM analysis. Results: A total of 111 compounds were identified in plant. The LC50 and LC90 values of AEO calculated as 7 ppm and 35 ppm respectively. AEO was able to kill 100% of the larvae in 4 days. Conclusion: The nanoemulsion of AEO showed a weak effect on the larvar mortality. It may therefore be suggested that this kind of nanoemulsion is not appropriate for the formulation as a larvicide. It is important to screen native plant natural products, search for new materials and prepare new formulations to develop alternative interventions with a long-lasting impact.

Investigation on Mosquitoes Fauna (Diptera: Culicidae) and Probable Vector of West Nile Virus in Lorestan Province, Western Iran.

Background: Fauna and larval habitat characteristics studies on mosquitoes are important tools to identify the breeding places of the vectors and management of the control strategies. This study was done to provide data on Culicidae fauna, larval habitat characteristics and identifying potential vectors of West Nile virus in Lorestan Province, west of Iran. Methods: Culicidae mosquitoes were collected at three counties and nine site stations from Lorestan Province, west of Iran in 2017. Adult mosquitoes were collected using human and animal bite collection methods, New Jersey and CDC light traps and pit shelters by aspirator. Larva were collected by dipping method. RT-PCR technique was employed for detection of the West Nile virus among mosquito's samples. Results: 4805 mosquitoes were collected from three counties and nine sites in Lorestan Province during June-October 2017, including 4363 adults and 442 larvae. The most abundant species collected from all counties in both adult and larval stages were Culex pipiens (49.10%), Cx. theileri (31.82%), Anopheles maculipennis (11.09%), An. superpictus (2.66%), An. stephensi (2.12%), Cx. perexiguus (1.89%), An. dthali (1.17%) and An. sacharovi (0.15%) respectively. West Nile virus was detected in none of mosquitoes examined. Conclusion: The results of this study revealed that arbovirus vectors such as Cx. pipiens along with Cx. theileri and Cx. perexiguus are well adapted to a broad range of habitats and different climatic conditions in Lorestan Province. That necessitates further routine surveillance of arboviral infections.

Evaluation of Susceptibility of Aedes caspius (Diptera: Culicidae) to Insecticides in a Potent Arboviral-Prone Area, Southern Iran.

BACKGROUND: Southern part of the country is a high risk for mosquito transmitted Arboviruses. This study was carried out to determine the base line susceptibility of the Aedini mosquitoes to the WHO-recommended insecticide. METHODS: Larval collection was carried out by dipping method and adult collection occurred by suction tube from January to December 2017. The adult susceptibility test was assessed to Bendiocarb 0.1%, DDT 4%, Deltamethrin 0.05%, Lambda-cyhalothrin 0.05%, Malathion 5% and, Permethrin 0.75% at different interval times as well as at discriminative dose recommended by WHO. The larval susceptibility test was occurred using Temephos and Bacillus thuringiensis serotype H-14, at different concentrations. The LT50, LT90 and LC50, LC90 values were calculated for plotting the regression line using Microsoft office Excel software ver. 2007. RESULTS: Aedes caspius was quite resistant to DDT, Malathion, Bendiocarb and showed susceptible or tolerant to other insecticides.The LT50 and LT90 values to DDT in this species were 157.896, and 301.006 minutes, respectively. The LC50 and LC90 values of Ae. caspius to Temephos were 0.000068, and 0.000130ppm, the figures for B. thuringiensis was 111.62 and 210.2ppm, respectively. CONCLUSION: A routine and continuous study for monitoring and evaluation of different species of Aedes to insectides is recommend at different parts of country for decision making.

Autochthonous Transmission of West Nile Virus by a New Vector in Iran, Vector-Host Interaction Modeling and Virulence Gene Determinants.

Using molecular techniques and bioinformatics tools, we studied the vector-host interactions and the molecular epidemiology of West Nile virus (WNV) in western Iran. Mosquitoes were collected during 2017 and 2018. DNA typing assays were used to study vector-host interactions. Mosquitoes were screened by RT-PCR for the genomes of five virus families. WNV-positive samples were fully sequenced and evolutionary tree and molecular architecture were constructed by Geneious software and SWISS-MODEL workspace, respectively. A total of 5028 mosquito specimens were collected and identified. The most prevalent species was Culex (Cx.) pipiens complex (57.3%). Analysis of the blood-feeding preferences of blood-fed mosquitoes revealed six mammalian and one bird species as hosts. One mosquito pool containing non-blood-fed Cx. theileri and one blood-fed Culex pipiens pipiens (Cpp.) biotype pipiens were positive for WNV. A phylogram indicated that the obtained WNV sequences belonged to lineage 2, subclade 2 g. Several amino acid substitutions suspected as virulence markers were observed in the Iranian WNV strains. The three-dimensional structural homology model of the E-protein identified hot spot domains known to facilitate virus invasion and neurotropism. The recent detection of WNV lineage 2 in mosquitoes from several regions of Iran in consecutive years suggests that the virus is established in the country.

Morphological and molecular identification of Culicidae mosquitoes (Diptera: Culicidae) in Lorestan province, Western Iran.

Culicidae mosquitoes are main vectors of arboviruses that cause arboviral diseases in humans. Studies on fauna, ecology, biology, resting behaviors of Culicidae mosquitoes are important and greatly impacts the control of arboviral diseases that are transmitted by vectors. The aim of the present study was to determine fauna of mosquitoes (Diptera: Culicidae) based on morphological and molecular (genomic) identification and their habitats in Lorestan province, Western Iran. Meanwhile mosquito samples were examined for arbovirus infection. Culicidae mosquitoes were caught in 2015 and 2016 from human homes, animal dwellings, storehouses and pit shelters in Lorestan province, Western Iran, using an oral aspirator (hand catch), total catch, human and animal bait and light trap methods. The samples were identified on the genus and species. Six species of Culex and eight species of Anopheles were caught. One complex species (Cx. pipiens complex) and a hybrid between Cx. pipiens pipiens biotype pipiens and Cx. pipiens pipiens biotype molestus were identified. Among all of the trapped mosquitoes (4211), 94.68% were from genus Culex mosquitoes (3987), which indicate that this genus is the dominant in Lorestan province, Western Iran. Anopheles comprised of 201 individuals out of the total catch. Arboviruses were not detected in these samples.

Ecology of Malaria Vectors in an Endemic Area, Southeast of Iran.

BACKGROUND: Malaria has long been regarded as one of the most important public health issues in Iran. Although the country is now in the elimination phase, some endemic foci of malaria are still present in the southeastern areas of the country. In some endemic foci, there are no data on the malaria vectors. To fill this gap, the present study was designed to provide basic entomological data on malaria vectors in the southeastern areas of Iran. METHODS: Adult and larval stages of Anopheles mosquitoes were collected by using different catch methods. Resistance of the main malaria vector in the study area to selected insecticides was evaluated using diagnostic doses advised by the World Health Organization in 2013-2014. RESULTS: A total of 3288 larvae and 1055 adult Anopheles mosquitoes were collected, and identified as: Anopheles stephensi (32.1%), Anopheles culicifacies s.l. (23.4%), Anopheles dthali (23.2%), Anopheles superpictus s.l. (12.7%), and Anopheles fluviatilis s.l. (8.6%). Anopheles stephensi was the most predominant mosquito species collected indoors at the study area, with two peaks of activity in May and November. This species was found to be resistant to DDT 4%, tolerant to malathion 5% and susceptible to other tested insecticides. CONCLUSION: All the five malaria vectors endemic to the south of Iran were collected and identified in the study area. Our findings on the ecology and resting/feeding habitats of these malaria vectors provide information useful for planning vector control program in this malarious area.

Employing Different Traps for Collection of Mosquitoes and Detection of Dengue, Chikungunya and Zika Vector, Aedes albopictus, in Borderline of Iran and Pakistan.

BACKGROUND: Southeastern Iran has been established as an area with the potential to harbor Asian tiger mosquito populations. In 2013, a few numbers of Aedes albopictus were detected in three sampling sites of this region. This field study was aimed to evaluate the efficacy of various traps on monitoring mosquitoes and status of this dengue vector, in five urban and 15 suburban/rural areas. METHODS: For this purpose, four adult mosquito traps (BG-sentinel 2, bednet, Malaise, and resting box trap) were used and their efficacy compared. In addition, large numbers of CDC ovitraps were employed, within 12 months. RESULTS: A total of 4878 adult samples including 22 species covering five genera were collected and identified from traps. It was not revealed any collection of Ae. albopictus. Statistical analysis showed no significant difference in meteorological variables between the two periods, the previous report and the current study. There were significant differences in the total number of mosquitoes collected by various traps in the region across different months. CONCLUSION: The resulting data collected here on the efficiency of the various trap types can be useful for monitoring the densities of mosquito populations, which is an important component of a vector surveillance system. While the presence of Ae. albopictus was determined in this potential risk area, there is no evidence for its establishment and further monitoring needs to be carried out.

Correction: DNA barcodes corroborating identification of mosquito species and multiplex real-time PCR differentiating Culex pipiens complex and Culex torrentium in Iran.

[This corrects the article DOI: 10.1371/journal.pone.0207308.].

Biodiversity of Mosquitoes (Diptera: Culicidae) with Emphasis on Potential Arbovirus Vectors in East Azerbaijan Province, Northwestern Iran.

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 (H') 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). CONCLUSION: 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.

Baseline Susceptibility of Culiseta longiareolata (Diptera: Culicidae) to Different Imagicides, in Eastern Azerbaijan, Iran.

BACKGROUND: Culiseta longiareolata is an important vector for many human diseases such as brucellosis, avian influenza and West Nile encephalitis. It is likely an intermediate host of avian Plasmodium that can transmit Malta fever. The aim of this study was to determine the susceptibility level of Cs. longiareolata to different classes of imagicides which are recommended by World Health Organization . METHODS: Larval stages of the Cs. longiareolata were collected from their natural habitats in Marand County at East Azerbaijan Province, northwestern of Iran in 2017. Adult susceptibility test were carried out with using impregnated papers to insecticides including DDT 4%, Cyfluthrin 0.15%, Deltamethrin 0.05%, Propoxur 0.1% and Fenitrothion 1% by standard test kits. RESULTS: Results showed that Cs. longiareolata adult is more susceptible to pyrethroid and carbamate insecticides. Among tested insecticides, Cyfluthrin was the most toxic against Cs. longiareolata with LT50 value of 11.53 minutes and Fenitrothion had the least toxic effect (LT50: 63.39 min). CONCLUSIONS: This study provided a guideline for monitoring and evaluation of insecticide susceptibility tests against Cs. longiareolata mosquitoes for further decision making.

Host-feeding patterns of Culex mosquitoes in Iran.

BACKGROUND: Different mosquito-borne pathogens are circulating in Iran including Sindbis virus, West Nile virus, filarioid worms and malaria parasites. However, the local transmission cycles of these pathogenic agents are poorly understood, especially because ecological data on vector species are scarce and there is limited knowledge about the host range; this understanding could help to direct species-specific vector control measurements or to prioritize research. METHODS: In the summers of 2015 and 2016, blood-fed mosquitoes were collected at 13 trapping sites on the coast of the Caspian Sea in northern Iran and at an additional trapping site in western Iran. Mosquitoes were generally collected with either a Biogents Sentinel trap or a Heavy Duty Encephalitis Vector Survey trap installed outside. A handheld aspirator was used at the trapping site in western Iran, in addition to a few samplings around the other trapping sites. On average, eight trapping periods were conducted per trapping site. The sources of blood meals were identified using a DNA barcoding approach targeting the cytochrome b or 16S rRNA gene fragment. RESULTS: The source of blood meals for 580 blood-fed mosquito specimens of 20 different taxa were determined, resulting in the identification of 13 different host species (9 mammals including humans, 3 birds and 1 reptile), whereby no mixed blood meals were detected. Five mosquito species represented more than 85.8% of all collected blood-fed specimens: Culex pipiens pipiens form pipiens (305 specimens, 55.7% of all mosquito specimens), Cx. theileri (60, 10.9%), Cx. sitiens (51, 9.3%), Cx. perexiguus (29, 5.3%) and Anopheles superpictus (25, 4.6%). The most commonly detected hosts of the four most abundant mosquito species were humans (Homo sapiens; 224 mosquito specimens, 40.9% of all mosquito specimens), cattle (Bos taurus; 171, 31.2%) and ducks (Anas spp.; 75, 13.7%). These four mosquito species had similar host-feeding patterns. The only exceptions were a relatively high proportion of birds for Cx. pipiens pipiens f. pipiens (23.2% of detected blood meal sources) and a high proportion of non-human mammals for Cx. theileri (73.4%). Trapping month, surrounding area, or trapping method had no statistically significant impact on the observed host-feeding patterns of Cx. pipiens pipiens f. pipiens. CONCLUSIONS: Due to the diverse and overlapping host-feeding patterns, several mosquito species must be considered as potential enzootic and bridge vectors for diverse mosquito-borne pathogens in Iran. Most species can potentially transmit pathogens between mammals as well as between mammals and birds, which might be the result of a similar host selection or a high dependence on the host availability.