Kdr genotyping and the first report of V410L and V1016I kdr mutations in voltage-gated sodium channel gene in Aedes aegypti (Diptera: Culicidae) from Iran.

BACKGROUND: Aedes aegypti is the main vector of arboviral diseases worldwide. The species invaded and became established in southern Iran in 2020. Insecticide-based interventions are primarily used for its control. With insecticide resistance widespread, knowledge of resistance mechanisms is vital for informed deployment of insecticidal interventions, but information from Iranian Ae. aegypti is lacking. METHODS: Fifty-six Ae. aegypti specimens were collected from the port city of Bandar Lengeh in Hormozgan Province in the South of Iran in 2020 and screened for kdr mutations. The most common kdr mutations in Latin America and Asia (V410L, S989P, V1016G/I and F1534C), especially when present in combinations, are highly predictive of DDT and pyrethroid resistance were detected. Phylogenetic analyses based on the diversity of S989P and V1016G/I mutations were undertaken to assess the phylogeography of these kdr mutations. RESULTS: Genotyping all four kdr positions of V410L, S989P, V1016G/I and F1534C revealed that only 16 out of the 56 (28.57%) specimens were homozygous wild type for all kdr mutation sites. Six haplotypes including VSVF (0.537), VSVC (0.107), LSVF (0.016), LSIF (0.071), VPGC (0.257) and LPGC (0.011) were detected in this study. For the first time, 11 specimens harbouring the V410L mutation, and 8 samples with V1016I mutation were found. V410L and V1016I were coincided in 8 specimens. Also, six specimens contained 1016G/I double mutation which was not reported before. CONCLUSIONS: The relatively high frequency of these kdr mutations in Iranian Ae. aegypti indicates a population exhibiting substantial resistance to pyrethroid insecticides, which are used widely in control operations and household formulations. The detection of the 410L/1016I kdr mutant haplotype in Iranian Ae. aegypti suggests possible convergence of invasive populations from West Africa or Latin America. However, as Iran has very limited maritime/air connections with those African countries, a Latin American origin for the invasive Ae. aegypti in Iran is more plausible.

Assessment of Knowledge, Attitude and Practice of Inmates, Prison Staff and Soldiers Regarding Cimex lectularius (Hemiptera: Cimicidae) in Mazandaran Prisons.

Background: Bed bugs (Cimex lectularius) are blood-sucking insects that bite humans usually at night and cause nuisance, psychological and social problems. These insects are considered a health hazard in densely populated places such as prisons. The aim of this study was to evaluate the knowledge, attitude and practice of inmates, soldiers, and prison staff regarding bed bugs in Mazandaran prisons. Methods: This is a descriptive-analytical study in which 450 inmates and 210 staff (105 personnel and 105 soldiers) from prisons of Mazandaran Province were included in the study. Data on knowledge, attitude, and practice of the participants regarding bed bugs were collected using a structured questionnaire. To determine the relative content validity coefficient, the questionnaire was assessed by 10 specialists in terms of difficulty, inappropriateness, and ambiguity of the phrases. The reliability of the questionnaire was measured using Cronbach's Alpha and then the data were analyzed using SPSS software version 20. Results: The maximum mean scores of knowledges, attitude, and practice between the three target groups were 16.42%, 44.07% and 71.21% respectively. Prison staff had the highest knowledge and practice, and inmates showed the highest attitude regarding bed bugs. There was no significant difference between the knowledge and attitude of inmates, prison staff and soldiers (P> 0.05) but there was a significant difference in their practice (P< 0.05). Conclusion: Results of this study confirmed that all three groups of people in the prisons need appropriate education regarding all aspects of bed bug including its control.

Knowledge, attitude, and practice of healthcare workers regarding dengue fever in Mazandaran Province, northern Iran.

Background: Dengue fever is a rapidly emerging infection worldwide with a high public health burden. Adequate training of healthcare workers is essential to warrant the timely provision of health services to improve the outcome of dengue management. Methods: This is an analytical cross-sectional study, conducted to assess the knowledge, attitudes and practice (KAP) of healthcare workers regarding dengue from April 2021 to March 2022 in Mazandaran Province, northern Iran. Data was collected using a researcher-made structured questionnaire, prepared as Google Forms, and sent to target groups through social media and email. Data analysis was performed by SPSS 22 software using descriptive and inferential statistics (Chi-square) at a significant level of 5%. Results: Most of the respondents had heard about dengue (83.8%); media (32.7%) and academic education (25%) were the main sources of information. Respondents had less knowledge associated with dengue symptoms (52%) than prevention and control (69%), transmission (72.2%) and clinical management (81%). Based on the 70% cut-off point, the majority of the participants had a good attitude (81%) and practice (73%). However, only 49.6% of the respondents showed good practice regarding dengue local transmission. A significant difference was observed between participants knowledge on clinical management with occupation; attitude with gender and occupation; and practice with gender (p < 0.05). Conclusion: The results of this study revealed gaps in some dimensions of KAP in healthcare workers, therefore, a greater focus should be placed on future training programs to raise knowledge and attitude leading to sound practice and behavior for adequate management of dengue.

Entomological surveillance of invasive Aedes mosquitoes in Mazandaran Province, northern Iran from 2014 to 2020.

Mosquitoes are the most important vectors of serious infectious diseases in the world. Dengue, Zika, chikungunya and yellow fever are emerging and re-emerging infectious diseases, associated with the distribution of two key vectors i.e. Aedes aegypti and Aedes albopictus throughout the world including countries neighbouring Iran. Entomological surveillance was planned and performed monthly from May to December during 2014-2020 in selected counties of Mazandaran Province, North of Iran, by ovitrap, larval collection, hand catch and human baited trap. Overall, 4410 Aedes specimens including 2376 larvae (53.9%) and 2034 (46.1%) adults belonging to six species, namely Aedes vexans, Aedes geniculatus, Aedes caspius, Aedes echinus, Aedes pulcritarsis and Aedes flavescence were collected and morphologically identified. Over the seven years of surveillance, Ae. aegypti and Ae. albopictus were not found by any sampling method. Aedes vexans and Ae. geniculatus were the most abundant species, their populations peaked in October and November and was positively correlated with precipitation and relative humidity. Aedes flavescence was a new species record for the province. A flowchart for planning and implementation of invasive mosquito surveillance for provincial health authorities in the country is proposed. These surveillance efforts provide basic and timely information for the health system to act promptly on integrated and intensified surveillance and control programs should Ae. aegypti and Ae. albopictus detected in the province.

Entomological Surveillance of the Invasive Aedes Species at Higher-Priority Entry Points in Northern Iran: Exploratory Report on a Field Study.

BACKGROUND: Arboviral diseases such as dengue, Zika, and chikungunya are transmitted by Aedes aegypti and Ae albopictus and are emerging global public health concerns. OBJECTIVE: This study aimed to provide up-to-date data on the occurrence of the invasive Aedes species in a given area as this is essential for planning and implementing timely control strategies. METHODS: Entomological surveillance was planned and carried out monthly from May 2018 to December 2019 at higher-priority entry points in Guilan Province, Northern Iran, using ovitraps, larval collection, and human-baited traps. Species richness (R), Simpson (D), evenness (E), and Shannon-Wiener indexes (H̕) were measured to better understand the diversity of the Aedes species. The Spearman correlation coefficient and regression models were used for data analysis. RESULTS: We collected a total of 3964 mosquito samples including 17.20% (682/3964) belonging to the Aedes species, from 3 genera and 13 species, and morphologically identified them from May 2018 to December 2019. Ae vexans and Ae geniculatus, which showed a peak in activity levels and population in October (226/564, 40.07% and 26/103, 25.2%), were the eudominant species (D=75.7%; D=21.2%) with constant (C=100) and frequent (C=66.7%) distributions, respectively. The population of Ae vexans had a significant positive correlation with precipitation (r=0.521; P=.009) and relative humidity (r=0.510; P=.01), whereas it was inversely associated with temperature (r=-0.432; P=.04). The Shannon-Wiener Index was up to 0.84 and 1.04 in the city of Rasht and in July, respectively. The rarefaction curve showed sufficiency in sampling efforts by reaching the asymptotic line at all spatial and temporal scales, except in Rasht and in October. CONCLUSIONS: Although no specimens of the Ae aegypti and Ae albopictus species were collected, this surveillance provides a better understanding of the native Aedes species in the northern regions of Iran. These data will assist the health system in future arbovirus research, and in the implementation of effective vector control and prevention strategies, should Ae aegypti and Ae albopictus be found in the province.

Determination of susceptibility levels of three different cockroach species including hospitals German cockroach, Blattella germanica L. (Blattodea: Blattellidae), to common insecticides, cypermethrin, propoxur and fenitrothion.

Objective: German cockroach is highly adapted to different environments like hospitals. This pest is an important carrier of pathogenic agents and allergic compounds. Hence, it is important to German cockroaches always be monitored and controlled. This study investigated the toxicity and resistance levels of technical-grade of cypermethrin, propoxur, and fenitrothion against adult males of four strains of the German cockroach. Methods: Four German cockroaches' strains including laboratory-reared susceptible strain (S) and three hospital-collected strains (B, F, and Z) were tested in this study. Adult male cockroaches were treated topically with three technical grade insecticides on the first abdominal segment of the insects using a hand micro-applicator. Results: Using topical application methods, fenitrothion showed the most toxicity to all four strains. The LD50 values of fenitrothion in the susceptible strain (S) and the hospital-collected strains B, F, and Z were 1.89, 21.48, 25.73, and 31.55 µg/grBW, respectively. All hospital-collected strains showed different resistant levels to all insecticides. The field-collected Z strain was the most resistant strain to cypermethrin, propoxur, and fenitrothion. The resistance ratios of strain Z to cypermethrin, propoxur, and fenitrothion were 10.9, 10.47 and 16.67, respectively. Conclusion: Based on the susceptibility and resistance ratios for each insecticide, we conclude that there are high rates of insecticide resistance in Blattella germanica from different hospitals of Sari County which can be caused by different insecticides treatment histories.

Global water quality changes posing threat of increasing infectious diseases, a case study on malaria vector Anopheles stephensi coping with the water pollutants using age-stage, two-sex life table method.

BACKGROUND: Water pollution due to uncontrolled release of chemical pollutants is an important global problem. Its effect on medically important insects, especially mosquitoes, is a critical issue in the epidemiology of mosquito-borne diseases. METHODS: In order to understand the effect of water pollutants on the demography of Anopheles stephensi, colonies were reared in clean, moderately and highly polluted water for three consecutive generations at 27 °C, 75% RH, and a photoperiod of 12:12 h (L:D). The demographic data of the 4th generation of An. stephensi were collected and analysed using the age-stage, two-sex life table. RESULTS: The intrinsic rate of increase (r), finite rate of increase (λ), mean fecundity (F) and net reproductive rate (R0) of An. stephensi in clean water were 0.2568 d-1, 1.2927 d-1, 251.72 eggs, and 109.08 offspring, respectively. These values were significantly higher than those obtained in moderately polluted water (r = 0.2302 d-1, λ = 1.2589 d-1, 196.04 eggs, and R0 = 65.35 offspring) and highly polluted water (r = 0.2282 d-1, λ = 1.2564 d-1, 182.45 eggs, and R0 = 62.03 offspring). Female adult longevity in moderately polluted (9.38 days) and highly polluted water (9.88 days) were significantly shorter than those reared in clean water (12.43 days), while no significant difference in the male adult longevity was observed among treatments. CONCLUSIONS: The results of this study showed that An. stephensi can partially adapt to water pollution and this may be sufficient to extend the range of mosquito-borne diseases.

Toxicity and Anti-feeding effect of Aesculushippocastanum, Thymus daenensis, Artemisia abrotanum, and Chrozophora tinctoria on Sitophillus oryzae.

Background and aim: The approach of food storage, especially cereals and basic human food sources, has long been considered as a problem to combat storage pests. the use of plant compounds has been suggested by researchers as an alternative to phosphine in the fight against pests.In this study, four plants such as Aesculushippocastanum, Thymus daenensis, Artemisia abrotanum and Chrozophoratinctoria were evaluated based on their deadly and repellent properties of pests and insectsconsidering Iranian medicine sources. Materials and methods: First, plant extracts were prepared by ethanol solvent method and then, their lethal and Anti-feeding experiments were performed on rice insects (Sitophylusoryzae) in standard laboratory conditions. Results: Extracts of Thymus daenensis, Aesculushippocastanum, Artemisia abrotanum and Chrozophoratinctoria were estimated to be 553.2, 753.6, 878.3 and 987.3 mg / ml effective dose, respectively.The results showed that Thymus daenensis extract with 90.33% with lower concentration compared to Aesculushippocastanum, Artemisia abrotanum and Chrozophoratinctoria extracts has the highest lethal effect against S. oryzae. Also in Anti-feedingtest, Thymus daenensis extract showed the highest Anti-feedingeffect in comparison with Aesculushippocastanum, Artemisia abrotanum and Chrozophoratinctoria extracts, respectively. Conclusions: The use of plants in the control against storage pests such as rice weevils will have favorable environmental results, human health and less storage is dangerous.

Bioecological Study on the Sand Flies (Diptera: Psychodidae, Phlebotominae) in Sari County, North of Iran.

Background: Phlebotomine sand flies are vectors of Leishmania species, the causative agents of leishmaniasis in the world. Present study aimed to evaluate the bioecological aspects of sand flies in different ecotopes in Sari County, north of Iran. Methods: Sand flies were collected from four villages in mountainous, forest, plain and peri-urban areas monthly using sticky traps in May-October 2016. Mounted specimens were identified using valid identification keys under optical microscope. The Arc GIS 10.5 software was applied for showing the distribution of sand flies. Shannon-Weiner, Simpson and Evenness species diversity indices were calculated. Results: Generally, 334 specimens were captured and identified, namelly Phlebotomus kandelakii, Ph. papatasi, Ph. major, Ph. sergenti, Ph. longiductus, Ph. halepensis, Ph. tobbi, Sergentomyia dentata, Se. theodori, Se. sintoni, Se. antennata and Se. sumbarica. The most common species was Ph. kandelakii (n= 128, 38.32 %). The highest Simpson index (0.81) and abundance (N= 141) were recorded in the mountaineous area. Shannon diversity index was higher in the forest (H'= 1.53) and the highest evenness index was in the plain area (J'= 0.93). The highest richness (S= 9) and Shannon indices (H'= 1.57) were observed in June. Conclusions: Phlebotomus kandelakii, Ph. sergenti, Ph. tobbi, Ph. longiductus, Se. theodori, Se. antennata and Se. sumbarica were recorded for the first time in the study area. Since some species are incriminated for leishmaniasis transmission, further studies are required in the northern regions of Iran to timely control measures planning.

Preparation, characterisation and comparative toxicity of nanopermethrin against Anopheles stephensi and Culex pipiens.

OBJECTIVES: To assess the effectiveness of nanopermethrin as a potential new formulation for pest and vector control. METHODS: Permethrin nanoparticles were prepared by the ionic gelation method and its structure and the formulations were designed using Box-Behnken statistical technique. The effect of independent variables (Chitosan/Permethrin ratio, tripolyphosphate quantity, sonication time) on the properties of nanoparticles was investigated to determine the optimal formulation. RESULTS: The size of the nanoparticles ranged from 135.27 ± 5.88 to 539.5 ± 24.01 nm and the insecticide entrapment efficiency per cent (EE%) ranged from 7.72 ± 1.36 to 63.59 ± 3.17%. Anopheles stephensi larvae were then bioassayed with the nanopermethrin and compared with the results of the bioassay with the mother molecule of permethrin using a standard WHO-recommended mosquito larval bioassay kit. LC50 with permethrin and nanopermethrin on larvae of An. stephensi were 0.125 and 0.026 ppm showing a 4.8 times difference. The LC50 for permethrin and nanopermethrin on Culex pipiens were 0.003 and 0.00032 ppm, respectively, showing a 9.4-fold difference. CONCLUSION: Nanopermethrin is much more potent than its mother molecule against larvae of An. stephensi and Cx. pipiens.

Immunological interactions of Chilo suppressalis Walker (Lepidoptera: Crambidae) with the native entomopathogenic fungi.

Entomopathogenic fungi can attack many insect hosts and have been applied as the eco-friendly alternatives to synthetic chemicals for the control of pests. Insects have developed different defense systems encountering entomopathogens including humoral and cellular immune responses. In the present study, injection of some native entomopathogenic fungi to the Chilo suppressalis Walker larvae resulted in an enhancement of the cellular and antimicrobial defenses. The numbers of total and differential hemocytes increased rapidly in the first 3 and 6 h but those gradually reduced 12 and 24 h post-injections. The nodule formation and phenoloxidase activity increased at the time intervals after fungal infection. A similar trend was found in the transcription of antimicrobial peptides including attacin1 and 2, cecropin1 and 2, gallerimycin, defensin, lysozyme, and prophenoloxidase-activating proteinase-3 during infection fungi. In all cases, the target gene transcription was upper in the larvae injected by the fungi than that of control larvae. These results may elucidate better knowledge on the interaction of the fungi present in agroecosystems with the target insect pest.

Screening and Virulence of the Entomopathogenic Fungi Associated with Chilo suppressalis Walker.

The current study aimed to explore the entomopathogenic fungi associated with the larvae of Chilo suppressalis Walker, a serious pest of rice, in northern Iran. The collected specimens were cultured and identified through morphological and molecular methods. The 38 specimens were identified by microscopic examination and genetic sequencing of the ITS region as follows: twenty-one isolates of Beauveria bassiana, five isolates of Akanthomyces lecanii, four isolates of Akanthomyces muscarious, three isolates of Metarhizium anisopliae, two isolates of Hirsutella subulata, two isolates of Trichoderma sp. and one isolate of Aspergillus sp. All the identified isolates were treated on the larvae through bioassay, evaluating the amount of hydrophobin and the activities of proteases, chitinases and lipase to find their virulence. Moreover, the percentage of thermotolerant and cold activity of the isolates were tested to determine their environmental persistence. The overall results revealed the isolates of B. bassiana, including BBRR1, BBAL1 and BBLN1 as the most virulent and environmental adaptive isolates among the fungi associated with C. suppressalis.

Population Fluctuations and Abundance Indices of Mosquitoes (Diptera: Culicid), as the Potential Bridge Vectors of Pathogens to Humans and Animals in Mazandaran Province, Northern Iran.

BACKGROUND: Seasonal activity patterns of mosquitoes are essential as baseline knowledge to understand the transmission dynamics of vector-borne diseases. This study was conducted to evaluate the monthly dynamics of the mosquito populations and their relation to meteorological factors in Mazandaran Province, north of Iran. METHODS: Mosquito adults and larvae were collected from 16 counties of Mazandaran Province using different sampling techniques, once a month from May to December 2014. "Index of Species Abundance" (ISA) along with "Standardized ISA" (SISA) was used for assessing the most abundant species of mosquitoes based on the explanations of Robert and Hsi. Pearson's correlation coefficient (R) was used to assess the relationships between the monthly population fluctuations and meteorological variables. RESULTS: Overall, 23750 mosquitoes belonging to four genera and nineteen species were collected and identified. The highest population density of mosquitoes was in July and the lowest in May. The ISA/SISA indices for Culex pipiens were both 1 for larvae and 1.25/0.973 for adults in total catch performed in human dwellings. For Cx. tritaeniorhynchus, the ISA/SISA were 1.68/0.938 in pit shelter method. A significant positive correlation was observed between population fluctuations of Cx. tritaeniorhynchus and mean temperature (R: 0.766, P< 0.027). CONCLUSION: The results indicated that the mosquitoes are more active in July, and Cx. pipiens and Cx. tritaeniorhynchus were the most abundant species. Considering the potential of these species as vectors of numerous pathogens, control programs can be planed based on their monthly activity pattern in the area.

Zika; a continuous global threat to public health.

Zika virus is transmitted by Aedes mosquitoes, especially, Ae. aegypti and Ae. albopictus. About 80% of the cases do not manifest any symptoms, and it is a self-limiting, mild viral infection. In 20% of the cases and only in fraction of those who do show the symptoms, important complications including Guillaine Barre'syndrome and microcephaly may occur. The emergence of Zika in 2016 in Brazil spreading to about 70 other countries prompted the WHO officials to declare the disease a Public Health Emergency with International Concern (PHEIC). This has led to increased concerns in health authorities of almost all countries making them embark on the strengthened human and vector surveillance, vector control and clinical management of the disease. Although the main vectors of the disease have not yet been able to establish in Iran, because of their occurrence in neighboring countries as well as increased global travel and trade, the country established a national advisory committee for capacity building, vector and human surveillance and case management of Aedes-borne diseases. This study aims at performing a literature review about global situation of Zika and Aedes mosquitoes, their distribution, biology and ecology from the past to present and the threat posed to Iran. Aedes aegypti was historically present in the checklist of Iranian mosquitoes and Ae. albopictus has recently been collected from Southern Iran, however, the species has apparently failed to establish in the country as comprehensive follow up entomological surveillance could not reproduce the findings. Although Zika was not detected in Iran, considering the expansion in tourism, travel and trade to and from Zika infected and Aedes infested countries, suitable climate and favorable prediction for establishment of Aedes vectors, Iran may well be at risk of invasion of Aedes vector species and the diseases they carry. Therefore, this review is of value particularly to health authorities in Iran and other WHO Eastern Mediterranean countries for sustained vigilance and preparedness for early detection and response, including vector control.

First Report of Biochemical Mechanisms of Insecticide Resistance in the Field Population of Culex pipiens (Diptera: Culicidae) from Sari, Mazandaran, North of Iran.

BACKGROUND: Culex pipiens play an important role in transmission of infectious diseases. Vector control by chemical pesticides, leads inevitably to resistance development. Understanding the underlying resistance mechanisms can help improve the control programmes and insecticide resistance management. METHODS: The total contents of cytochrome p450s and the activities of glutathione S-transferases, alpha- and beta-esterases and inhibition rates of acetylcholine esterase (by propoxur) were measured in the field population of Cx. pipiens collected from Sari County, North of Iran, in 2016 and the results were compared with those of the laboratory susceptible strain according to the biochemical assay methods of WHO for adult mosquitoes. Independent sample t-test was used to compare the mean values of enzyme activities/contents between filed and laboratory susceptible populations. RESULTS: The enzyme ratio of cytochrome p450s, alpha- and beta-esterases in the field population was 2.07, 3.72 and 1.36 respectively when compared with the results of the laboratory population. Although not statistically significant, the mean GSTs activities in the field population was marginally less than the laboratory population (ER=0.92). Acetylcholinesterase was insensitive to propoxur in 62.82% of the individuals of the tested field population. There was a significant difference (P< 0.05) between all values of the activities/contents of the enzyme in the field population except for GSTs compared with the laboratory susceptible strain. The highest enzyme activity was related to alpha esterase. CONCLUSION: The present study showed a range of metabolic mechanisms, comprising p450s and esterases combined with target site insensitivity of AChE, contributing to organophosphate, carbamate and pyrethroid resistance in the field population of Cx. pipiens.

Fauna, Ecological Characteristics, and Checklist of the Mosquitoes in Mazandaran Province, Northern Iran.

Mosquitoes are important vectors of human and animal diseases. This study updates current knowledge on fauna, dominance, and distribution of mosquitoes in Mazandaran Province, Northern Iran, to inform disease control effort. Larval collections, using standard dippers or droppers, and adult collections, using total catches, shelter pits, CDC light traps, and human landing catches, were performed monthly in 30 villages across 16 counties, from May to December 2014. Ovitraps, baited with hay infusion as oviposition attractants or stimulants for Aedes (Stegomyia) mosquitoes, were installed in each village and inspected weekly for eggs. Lactophenol and Berlese media were used for preserving and mounting specimens. Overall, 36,024 mosquito specimens (19,840 larvae and 16,184 adults) belonging to 4 genera and 20 species were morphologically identified. The dominance and distribution indices showed that Culex pipiens s.s. was the eudominant species with a constant distribution of larvae (D = 69.07%, C = 100%) and adults (D = 31.86%, C = 100%), followed by Cx tritaeniorhynchus (D = 38.14%, C = 100%) and Anopheles maculipennis s.l. (D = 11.05%, C = 100%) as adults. Aedes vexans was the dominant (7.85%) species, but it had a sporadic (20%) distribution. Culex torrentium and Culiseta morsitans were added as the new species to the checklist of mosquitoes in Mazandaran Province. Due to the potential role, Cx. pipiens s.s. as a vector of various pathogens, further ecological studies are recommended.

Correlation between mosquito larval density and their habitat physicochemical characteristics in Mazandaran Province, northern Iran.

Characteristics of mosquito larval habitats are important in determining whether they can survive and successfully complete their developmental stages. Therefore, data on the ecological factors affecting mosquito density and abundance especially the physicochemical properties of water of their breeding sites, can possibly be helpful in implementing larval management programs. Mosquito larvae were collected using a standard 350 ml dipper from fixed habitats including: artificial pool, river edge, creek and etc, in 30 villages of 16 counties from May-December 2014. Water samples were collected during larval collection and temperature (°C), acidity (pH), turbidity (NTU), electrical conductivity (µS/cm), alkalinity (mg/l CaCO3), total hardness (mg/l), nitrate (mg/l), chloride (mg/l), phosphate (mg/l) and sulphate (mg/l) were measured using standard methods. Spearman correlation coefficient, Kruskal-Wallis test of nonparametric analysis, Chi-square (χ2) analysis, regression analysis and C8 interspecific correlation coefficient were used for data analysis. A total of 7,566 mosquito larvae belonging to 15 species representing three genera were collected from fixed larval breeding places. Culex pipiens was the dominant species except in four villages where An. maculipennis s.l. and Cx. torrentium were predominant. There was a significant positive correlation between the density of Cx. pipiens and electrical conductivity, alkalinity, total hardness and chloride, whereas no significant negative correlation was observed between physicochemical factors and larval density. The highest interspecific association of up to 0.596 was observed between An. maculipennis s.l/An. pseudopictus followed by up to 0.435 between An. maculipennis s.l/An. hyrcanus and An. hyrcanus/An. pseudopictus. The correlations observed between physicochemical factors and larval density, can possibly confirm the effect of these parameters on the breeding activities of mosquitoes, and may be indicative of the presence of certain mosquito fauna in a given region.

Evidence of metabolic mechanisms playing a role in multiple insecticides resistance in Anopheles stephensi populations from Afghanistan.

BACKGROUND: Malaria is endemic in most parts of Afghanistan and insecticide-based vector control measures are central in controlling the disease. Insecticide resistance in the main malaria vector Anopheles stephensi from Afghanistan is increasing and attempts should be made to determine the underlying resistance mechanisms for its adequate management. METHODS: The contents of cytochrome P450s, esterases, glutathione S-transferases (GSTs) and acetylcholine esterase (AChE) activities were measured in the Kunar and Nangarhar populations of An. stephensi from Afghanistan and the results were compared with those of the susceptible Beech strain using the World Health Organization approved biochemical assay methods for adult mosquitoes. RESULTS: The cytochrome P450s enzyme ratios were 2.23- and 2.54-fold in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The enzyme ratios for esterases with alpha-naphthyl acetate were 1.45 and 2.11 and with beta-naphthyl acetate were 1.62 and 1.85 in the Kunar and Nangarhar populations respectively compared with the susceptible Beech strain. Esterase ratios with para-nitrophenyl acetate (pNPA) were 1.61 and 1.75 in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The GSTs enzyme ratios were 1.33 and 1.8 in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The inhibition of AChE was 70.9 in the susceptible Beech strain, and 56.7 and 51.5 in the Kunar and Nangarhar populations. The differences between all values of the enzymes activities/contents and AChE inhibition rates in the Kunar and Nangarhar populations were statistically significant when compared with those of the susceptible Beech strain. CONCLUSIONS: Based on the results, the reported resistance to pyrethroid and organophosphate insecticides, and tolerance to bendiocarb in the Kunar and Nangarhar populations of An. stephensi from Afghanistan are likely to be caused by a range of metabolic mechanisms, including esterases, P450s and GSTs combined with target site insensitivity in AChE.

Pyrethroid resistance in Iranian field populations of Rhipicephalus (Boophilus) annulatus.

Resistance to acaricides in ticks is becoming increasingly widespread throughout the world; therefore, tick control requires resistance monitoring for each tick species. The aims of this study were to monitor the susceptibility status of the cattle tick, Rhipicephalus (Boophilus) annulatus (Acari: Ixodidae), against pyrethroid acaricides from Mazandaran Province, northern Iran, and where resistance was evident, and establish the possible underlying mechanisms. Fully engorged adult R. (B.) annulatus females collected on cattle from Mazandaran Province. Twenty-nine tick populations produced 10-18days old larvae and bioassayed with cypermethrin and λ-cyhalothrin by larval packet test and the levels of detoxification enzymes were measured. Population AM-29 had a maximum resistance ratio (RR99) of 20.21 to cypermethrin and 53.57% of the tick populations were resistant at LC99 level. With λ-cyhalothrin, 17.86% of the tick populations were resistant and AM-29 was the most resistant population with RR99=4.54. AM-29 also showed significant elevation of cytochrome P450 monooxygenase (P450) and glutathione S-transferase (GST) (2.76- and 2.39-fold, respectively) (P<0.001). Rhipicephalus (Boophilus) annulatus showed resistance to pyrethroid insecticides with elevated levels of P450, GST and para-nitrophenyl acetate (p-NPA) in resistant populations. Operational failure was noted in controlling R. (B.) annulatus by pyrethroid insecticides, therefore alternative pest management measures should be adopted in Iran. For the first time, a new estimate of insecticide resistance based on effective dose recommended by the pesticide manufacturer termed Operational Dose Ratio (ODR) is defined and discussed.

First report of pyrethroid resistance in Rhipicephalus (Boophilus) annulatus larvae (Say, 1821) from Iran.

Rhipicephalus (Boophilus) annulatus is one of the most important hard ticks parasitizing cattle in northern Iran. The aim of this study was to evaluate pyrethroid resistance levels of this species from Nur County, northern Iran. The hard ticks were collected through a multistage cluster randomized sampling method from the study area and fully engorged female R. (B.) annulatus were reared in a controlled insectary until they produced larvae for bioassay. Seventeen populations of the hard ticks were bioassayed with cypermethrin and 12 populations with lambda-cyhalothrin using a modified larval packet test (LPT). Biochemical assays to measure the contents/activity of different enzyme groups including mixed function oxidases (MFOs), glutathione S-transferases (GSTs) and general esterases were performed. Population 75 showed a resistance ratio of 4.05 with cypermethrin when compared with the most susceptible field population 66 at the LC50 level. With lambda-cyhalothrin the resistance ratio based on LC50 was 3.67 when compared with the susceptible population. The results of biochemical assays demonstrated significantly elevated levels of GSTs and esterases in populations tested compared with the heterozygous susceptible filed population and a correlation coefficient of these enzymes was found in association to lambda-cyhalothrin resistance. Based on the results, pyrethroid acaricides may operationally fail to control R. (B.) annulatus in North of Iran. This study is the first document of pyrethroid resistance in R. (B.) annulatus populations from Iran.