Population genetic structure and phylogenetic analysis of Anopheles hyrcanus (Diptera: Culicidae) inferred from DNA sequences of nuclear ITS2 and the mitochondrial COI gene in the northern part of Iran.

BACKGROUND: The Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide. METHODS: Mosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software. RESULTS: In this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity. CONCLUSIONS: An. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon.

Molecular Monitoring of Knockdown Resistance in Head Louse (Phthiraptera: Pediculidae) Populations in Iran.

Knockdown resistance (kdr) is a common mechanism of insecticide resistance in head lice to the conventionally used pyrethroid pediculosis and can be the result of various amino acid substitutions within the voltage-sensitive sodium channel (VSSC). In this study, 54 sequences from varied specimens were investigated to monitor well-known resistance mutations and probable new mutations. The Pediculus humanus capitis de Geer specimens were collected from 13 provinces in Iran. The specimens were stored in 70% ethanol until DNA extraction and PCR amplification of ~900-bp fragment of VSSC. The sequences were analyzed using different bioinformatics software for the detection of well-known kdr substitutions and additional mutations potentially associated with kdr resistance in head lice. There were six new and an old (haplotype I) kdr haplotypes within the Iranian head louse population. K794E, F815I, and N818D amino acid substitutions were reported for the first time. The P813H mutation was the most prevalent amino acid substitution in eight provinces. Among 53 sequences, 26 (49%) were homozygous susceptible, and 27 (51%) were heterozygotes. Thus, 51% of the head lice collected in Iran harbored only the P813H allele. The exact test for the Hardy-Weinberg (H-W) equilibrium showed that genotype frequencies differed significantly from the expectation in East-Azerbaijan and Tehran provinces. Moreover, these populations had an inbreeding coefficient (Fis) <0, indicating the excess of heterozygotes. This observation suggests that the populations of head lice from Iran are currently under active selective pressure. For the rest of the populations, H-W equilibrium and the expectations were significantly in harmony. The results of the current study highlight molecular techniques in the accurate detection of resistance genotypes before their establishment within the head louse population. Accurate detection of resistant genotypes seems to be helpful in decision-making on lice control programs and resistance monitoring and management.

Resistance Status of Anopheles maculipennis and Anopheles superpictus to the Conventional Insecticides in Northeastern Caspian Littoral, Iran.

BACKGROUND: Malaria resurgence has occurred in the northern half parts of Iran. The resurgence of malaria in the prone area could arise from various factors, e.g. wide use of pesticides in the agriculture sector and factors such as habitual patterns of movement of local people from problematic southeastern foci in Iran toward the Caspian Littoral. There are no new data on the resistance status of main malaria vectors in the Caspian Littoral, and this study was aimed at renewal data on conventional insecticides. METHODS: The field strain of adult Anopheles superpictus and Anopheles maculipennis were collected using the hand catch method and transferred to the laboratory. The susceptibility tests were carried out against DDT 4%, Malathion 5%, Permethrin 0.75%, Deltamethrin 0.05%, and Lambda-cyhalothrin 0.05%, followed by the WHO's procedure. RESULTS: The primary malaria vector in Caspian Littoral is An. maculipennis, revealed to be still resistant to DDT and mortality rate, LT50 and LT90 of female mosquitoes were 75.0%, 54.2, minutes and 111.3 minutes. The under 'verification required' status of An. maculipennis was also revealed to Lambda-cyhalothrin based on recent WHO's criteria. The malaria vector An. superpictus is also considered the second malaria vectors in the west parts of the studied area, which showed to be susceptible to all insecticides tested. CONCLUSION: DDT resistance is persisted in An. maculipennis despite stopping residual spraying with DDT since 1978 in the Caspian Littoral, but the occurrence of pyrethroid under 'verification required' status is a progressive threat to the possible development of cross-resistance in the future.

Prevalence of Leishmania major Yakimoff and Schokhor (Kinetoplastida: Trypanosomatidae) in Sandflies in Iran: A Systematic Review and Meta-Analysis.

Cutaneous leishmaniasis is an important parasitic and vector-borne disease in Iran and can be transmitted to humans and animals through bites of infected female sand flies. The present study aimed to determine the pooled prevalence of Leishmania major in sand flies in Iran. A systematic review and meta-analysis was performed in the present study, so that databases, namely PubMed, Web of Sciences, Science Direct, ProQuest, Embase, Cochrane Reviews, and Scopus as well as IranDoc, SID, Elmnet, and Magiran were searched for finding Persian articles. Inclusion criteria of study contained the study in Iran and investigation of L. major parasite in sand flies. The quality of studies was examined by Joanna Briggs Institute Critical Appraisal Checklist for studies that reported prevalence data; and the data were analyzed by Stata 14 software. In total, 42 studies were evaluated, all of which had investigated the infection of Phlebotomus papatasi Scopoli (Diptera: Psychodidae). The infection of Phlebotomus caucasicus group was investigated in 13 studies, Phlebotomus ansarii Lewis in four studies, Phlebotomus salehi Mesghali and Rashti in five studies, and the infection of other species of sandflies in seven studies. According to the meta-analysis, the pooled prevalence of Ph. papatasi, Ph. caucasicus group, Ph. salehi, and Ph. ansarii were equal to 2.4, 6.2, 1.6, and 9.2%, respectively, with 95% confidence interval. According to reports of different rates of sand fly infection in different regions, all studies should have certain and standard forms and formats to determine the pooled prevalence of sand fly infection.

A spatio-temporal agent-based approach for modeling the spread of zoonotic cutaneous leishmaniasis in northeast Iran.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is a neglected tropical disease worldwide, especially the Middle East. Although previous works attempt to model the ZCL spread using various environmental factors, the interactions between vectors (Phlebotomus papatasi), reservoir hosts, humans, and the environment can affect its spread. Considering all of these aspects is not a trivial task. METHODS: An agent-based model (ABM) is a relatively new approach that provides a framework for analyzing the heterogeneity of the interactions, along with biological and environmental factors in such complex systems. The objective of this research is to design and develop an ABM that uses Geospatial Information System (GIS) capabilities, biological behaviors of vectors and reservoir hosts, and an improved Susceptible-Exposed-Infected-Recovered (SEIR) epidemic model to explore the spread of ZCL. Various scenarios were implemented to analyze the future ZCL spreads in different parts of Maraveh Tappeh County, in the northeast region of Golestan Province in northeastern Iran, with alternative socio-ecological conditions. RESULTS: The results confirmed that the spread of the disease arises principally in the desert, low altitude areas, and riverside population centers. The outcomes also showed that the restricting movement of humans reduces the severity of the transmission. Moreover, the spread of ZCL has a particular temporal pattern, since the most prevalent cases occurred in the fall. The evaluation test also showed the similarity between the results and the reported spatiotemporal trends. CONCLUSIONS: This study demonstrates the capability and efficiency of ABM to model and predict the spread of ZCL. The results of the presented approach can be considered as a guide for public health management and controlling the vector population .

Relationship Between the Distribution and Biodiversity of Sand Flies (Diptera: Psychodidae) With the Incidence of Zoonotic Cutaneous Leishmaniasis in Endemic Foci of Golestan Province, Iran.

Zoonotic cutaneous leishmaniasis (ZCL) is prevalent in Golestan Province, Iran. The current study determined the relationship between the distribution and biodiversity of sand flies with cutaneous leishmaniasis at 14 villages in plain and hillsides areas. In each village from July to September 2017, 60 sticky traps and 2 CDC light traps were laid. Spearman and Mann-Whitney tests were used to determine the relationship between the incidence of ZCL and the abundance of different species of sand flies. Simpson, Shannon-Wiener, Evenness, and Margalef indices were calculated to estimate the diversity of species. A total of 5,295 phlebotomine sand flies were collected, comprising 10 species of the genus Phlebotomus (3,947 flies) and 7 species of genus Sergentomyia (1,248 flies). The abundance of sand flies and incidence of ZCL in plain areas were greater than that of hillsides areas (P = 0.013, P = 0.002). There was a significant correlation between the incidence of ZCL and the abundance of Phlebotomus papatasi (r = 0.72, P = 0.004) and P. caucasicus groups (P = 0.006; 0.022). In the Shannon-Wiener index, the rest of the biodiversity indices were reduced in higher-altitude areas. Increasing Shannon-Wiener index showed higher diversity of sand flies in higher-altitude areas. Data of the reported cases of leishmaniasis in plain areas can reveal the relationship between less diversity index (Shannon-Wiener), higher dominant diversity index (Simpson), and incidence of leishmaniasis in these areas.

Distribution of Leishmania Infection in Humans, Animal Reservoir Hosts and Sandflies in Golestan Province, Northeastern Iran: A Systematic Review and Meta-Analysis.

BACKGROUND: Leishmaniasis is one of the most important parasitic diseases in the Golestan Province, northeastern Iran. In this study, we summarize the results of studies on the disease and its causative agent Leishmania in humans, vectors and reservoirs in the Golestan Province, Iran. METHODS: We retrieved all articles related to leishmaniasis in the Golestan Province, northern Iran from 1994 to 2018 in various databases including PubMed, Scopus and Web of Science. Articles in Persian were retrieved from the IranMedex, SID and Magiran. Reference lists of relevant articles were also hand-searched. Local active researchers in the field of leishmaniasis were contacted to avoid missing any relevant articles. Overall, 54 papers were extracted, later evaluated by two research team members based on inclusion criteria. All analyses were performed using the Stata 14 software. Pooled prevalence was calculated using the metaprop command and a random-effect model. The I2 statistic was used for measuring heterogeneity of studies. RESULTS: Ten articles in the province were related to detection of Leishmania species in patients with suspected cutaneous or visceral leishmaniasis. Fifteen articles were dedicated to identification of Leishmania species in disease reservoirs and five articles were about detection of the parasite in sand flies. The pooled prevalence of L. major in patients with acute ulcer, wild rodents and sandflies was 83%, 29% and 11%, respectively. CONCLUSION: Our findings highlight the need for implementation of control measures among the reservoirs of both cutaneous and visceral leishmaniasis in the Golestan Province, Iran.

Fauna and Larval Habitats Characteristics of Mosquitoes (Diptera: Culicidae) in Golestan Province, Northeast of Iran, 2014-2015.

BACKGROUND: Mosquitoes (Diptera: Culicidae) is one of the most medically important families of Diptera. The aims of this study were to determine fauna and larval habitat characteristics of mosquitoes in Golestan Province, during 2014-15. METHODS: This study was conducted in larval habitats of mosquitoes and installed ovitraps in 14 districts of Golestan Province, Northern Iran in 2015. Samples were collected with a scoop by ladle handling for entomology. The collected larvae were transferred to Laboratory of Medical Entomology in lactophenol solution. Then microscopic slides were prepared using de Faure's formula. Species of each sample was recognized using diagnostic criteria to identify the Culicidae species. Characteristics of larval breeding places were studied based on the habitat type (Permanent or temporary), water conditions (Clear or turbid, stagnant or running), vegetation (out, in, underwater vegetation or without vegetation), sunlight exposure (Full or partial sunlight) and so on. Data were analyzed using SPSS. RESULTS: Overall, 5661 third- and fourth- instars larvae of mosquitoes were collected and 5 genera and 14 species were identified: Anopheles hyrcanus, An. maculipennis, An. pseudopictus, An. superpictus, Culex hortensis, Cx. mimiticus, Cx. perexiguus, Cx. pipiens, Cx. pusillus, Cx. theileri, Cx. tritaeniohynchus, Culiseta longiareolata, Ochlerotatus caspius, Uranotaenia unguiculata. Culex pipiens was recognized as predominant species of the family. Among the detected species, Cx. pusillus reported for the first time from Golestan Province. CONCLUSION: Due to the high species diversity of Culicidae, ecology of medical important species such as Cx. pipiens and Cx. tritaeniorhynchus needs more investigations.

Machine learning approaches in GIS-based ecological modeling of the sand fly Phlebotomus papatasi, a vector of zoonotic cutaneous leishmaniasis in Golestan province, Iran.

The distribution and abundance of Phlebotomus papatasi, the primary vector of zoonotic cutaneous leishmaniasis in most semi-/arid countries, is a major public health challenge. This study compares several approaches to model the spatial distribution of the species in an endemic region of the disease in Golestan province, northeast of Iran. The intent is to assist decision makers for targeted interventions. We developed a geo-database of the collected Phlebotominae sand flies from different parts of the study region. Sticky paper traps coated with castor oil were used to collect sand flies. In 44 out of 142 sampling sites, Ph. papatasi was present. We also gathered and prepared data on related environmental factors including topography, weather variables, distance to main rivers and remotely sensed data such as normalized difference vegetation cover and land surface temperature (LST) in a GIS framework. Applicability of three classifiers: (vanilla) logistic regression, random forest and support vector machine (SVM) were compared for predicting presence/absence of the vector. Predictive performances were compared using an independent dataset to generate area under the ROC curve (AUC) and Kappa statistics. All three models successfully predicted the presence/absence of the vector, however, the SVM classifier (Accuracy = 0.906, AUC = 0.974, Kappa = 0.876) outperformed the other classifiers on predicting accuracy. Moreover, this classifier was the most sensitive (85%), and the most specific (93%) model. Sensitivity analysis of the most accurate model (i.e. SVM) revealed that slope, nighttime LST in October and mean temperature of the wettest quarter were among the most important predictors. The findings suggest that machine learning techniques, especially the SVM classifier, when coupled with GIS and remote sensing data can be a useful and cost-effective way for identifying habitat suitability of the species.

Larval Habitats Characteristics of Mosquitoes (Diptera: Culicidae) in North-East of Iran.

BACKGROUND: There are unorganized, published documents about the ecology of mosquitoes (Diptera: Culicidae) in northeastern part of Iran. The purpose of this study was to determine the distribution and characteristics of larval habitats of Culicidae in Kalaleh County. METHODS: Larvae were collected using dipping method and adults by human landing catch technique during April-October, 2012. Larval habitat characteristics were recorded such as vegetation status, and sunlight, water situation. Lacto-phenol and de Faure's media were used for conserving and mounting samples. Data were analyzed using SPSS statistical software, version 11.5. RESULTS: Out of the 395 larvae collected, 332 were adult mosquitoes comprising; Culiseta, Culex, Anopheles and Ochlerotatus genera and 14 species including An. superpictus, An. maculipennis s.l., An. hyrcanus, An. psudopictus, An. claviger, Culex pipiens, Cx. theileri, Cx. perexiguus, Culiseta longiareolata, Cs. subochrea, Ochlerotatus caspius, Oc. echinus and Oc. geniculatus. Culex pipiens larvae were predominant (27.6%) and Cs. subochrea (1%) was found as the lowest species in terms of number. In the adult form, Cx. pipiens (28.9%) was predominant whereas, Cs. subochrea and Cx. perexiguus were reported to have had the lowest frequency. CONCLUSION: The larvae of An. superpictus and An. maculipennis species as the main vectors of malaria in north of Iran were reported in permanent habitats with clear water and vegetation, full and partial sunlight situations and muddy as well as sandy substrates that are important in larvicide application programs. Exclusive studies are necessary to diagnose An. maculipennis species complex using molecular and morphological analysis in the future.

Faunistic Study of the Aquatic Arthropods in a Tourism Area in Northern Iran.

BACKGROUND: Aquatic insects are very abundant and divers groups of insects that are associated with an aquatic or semiaquatic environment in one or more of their life stages. These insects have been, in some cases, well studied because they are vectors of several diseases. This is the first comprehensive faunistic study of aquatic insects from Babol County. The results may provide basic data for further taxonomic and ecological studies of aquatic insects as biological control agent or classification of water quality for the country. METHODS: The specimens were collected using different methods including: D-frame net collector, standard mosquito dipper (350ml), Sweep-Netting and plastic pipette. Sampling carried out in different part of breading places in several times. RESULTS: During this study a total of 196 aquatic specimens were collected from different habitats and were morphologically identified including 18 families classified in 6 orders: Diptera, Trichoptera, Ephemeroptera, Plecoptera, Hemiptera and Odonata. Babol and Amol district in Mazandaran Province are located in humid climate regions with suitable ecological factors of humidity, moderate temperature and the variety of plant species. There are different species of aquatic insects in different habitats. CONCLUSION: The results will provide information for biodeveristy, species richness, their role for biological control as well as calcification of rivers based on abundance of aquatic insects. Therefore the understanding of ecological specifications of aquatic insects could provide a clue for further Arthropod-borne disease control. Additionally aquatic insect could be used for classification of water bodies.

Predicting the Distribution of Phlebotomus papatasi (Diptera: Psychodidae), the Primary Vector of Zoonotic Cutaneous Leishmaniasis, in Golestan Province of Iran Using Ecological Niche Modeling: Comparison of MaxEnt and GARP Models.

Zoonotic cutaneous leishmaniasis (ZCL) is a prevalent vector-borne disease in the Golestan province of Iran, with Phlebotomus papatasi (Scopoli, 1786) serving as the main vector. The aim of this study was to model the probability of presence of this species in the study area, and to determine the underlying factors affecting its distribution. Three villages were selected from each county of the province and visited monthly for investigating ZCL. Sticky paper traps were used for collecting the sand flies to determine the species present. The presence of Ph. papatasi was modeled using genetic algorithm for rule-set production (GARP) and maximum entropy (MaxEnt) techniques. Both models showed the central and northern parts of the province with lowland areas were more vulnerable to Ph. papatasi propagation, in comparison with the southern parts with mountainous and forest areas. The area under curve (AUC) of MaxEnt model for the training points was calculated as 0.90, indicating excellent performance of the model in predicting Ph. papatasi distribution. Jackknife test showed that the factors with the greatest influence in vector distribution were slope, vegetation cover, annual mean temperature, and altitude. By using ecological niche models, it is possible to identify areas with higher probability of presence of Ph. papatasi, which guides public health policy makers for planning better vector control interventions.

Spatial Analyses of the Relation between Rodent's Active Burrows and Incidence of Zoonotic Cutaneous Leishmaniasis in Golestan Province, Northeastern of Iran.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is one of the most important vector-borne diseases in Iran. Wild Rodents play as a reservoir. The main aim of this study was to determine spatial analyses of the relationship between rodent's active burrows and Incidence of ZCL in Golestan Province, north east of Iran. METHODS: The cross-sectional study was conducted in 59 rural districts in Golestan Province. Spatial distribution of rodent's active burrows, human cases of ZCL and Incidence of disease were collected, using Geographical Information Systems (GIS). The relationship of them were analyzed by Sperman test, SPSS software version No.13. RESULTS: The most number of rodents' active burrows, human positive cases (100 persons) and high Incidence of disease (35/1000) were observed in Korand rural district of Gonbad-e Kavoos County. There was significant correlation between the number of rodents active burrows with Incidence rate of disease (0.470, P< 0.001) as well as the number of cases in each districts (0.465, P< 0.001), There is high correlation between higher Incidence rate and human positive cases in districts with number of rodents' active burrows. CONCLUSION: Vicinity of wild rodents' burrows to villages plays an important role in transmission of ZCL to humans.

Hard Tick Species of Livestock and their Bioecology in Golestan Province, North of Iran.

BACKGROUND: A survey on tick species composition was carried out in Golestan Province Iran during year 2010-2011.The aim was to determine tick species parasitizing domestic ruminants and their seasonal population dynamics. METHODS: A total of 124 sheep, 92 goats, 84 cattle, 74 camels and 12 horses in several villages were inspected for tick infestation. The collected ticks preserved in 70% alcohol and then were identified. RESULTS: The overall 1059 ticks (453 female, 606 male) were collected. The ticks occur on sheep, goats, cattle, camels and horses as 72.1%, 77.3%, 75.8%, 69.3%, and 50% respectively. The frequency of ticks in spring was more than other seasons and the least was observed in winter. In the spring and summer, infestation rate in domestic ruminants were calculated as 100%. Six genus and fourteen hard and soft tick species were identified including Rhipicephalus sanguineus, R. bursa, Ixodes ricinus, Haemaphysalis punctata, H. sulcata, H. erinacei, H. inermis, Hyalomma marginatum, Hy. asiaticum, Hy. dromedarii, Hy. excavatum, Hy. anatolicum, Hy. detritum, Boophilus annulatus and Argas persicus. Rhipicephalus sanguineus was the most abundant species in the study area. The largest number of ticks was collected from animal ears and tails. Haemaphysalis, Hyalomma, Rhipicephalus and Boophilus occurred in mountainous, forest and plateau areas of Golestan Province but Ixodes occurred only in mountainous and forest areas, whereas Rhipicephalus and Hyalomma were present in coastal areas of Golestan Province. CONCLUSION: The result of this study is a survey on tick species from domestic animals in Iran and implication of possible prevention measures for diseases transmitted by ticks.