Colonization pattern and thermal needs of immature phases of Sarcophaga argyrostoma (Diptera: Sarcophagidae): Significance for estimating postmortem interval.

Members of Sarcophagidae are necrophagous and are commonly found on decaying carcasses; and their developmental forms are important indicators for the approximation of lowest postmortem interval (PMImin). This work describes the biological characteristics of Sarcophaga argyrostoma from Tehran, Iran. Various temperature regimes were applied to estimate the thermal summation constant (k) and thermal requirements for development of S. argyrostoma. Five growth proceedings, containing 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion, were investigated under eight fixed temperature regimes (6-30 °C). The effects of fly age, freshness, and availability of oviposition substrate on oviparity and viviparity was studied. At 6 °C, no development occurs, and at 8 °C only the first ecdysis occurs. At temperatures between 10 and 30 °C, all immature stages proceeded to the adult stage, and thus immature development was analyzed at these six remaining temperatures. The development phases needed minimum (Dz ± SE) 5.4(0.4), 8.5(0.26), 5.3(0.44), 3.8(0.1), and 6.6 (0.6)°C to attain one of the succeeding developmental occasion, correspondingly. The approximated K for those five occasions were 15.04 ± 1.12, 12.62 ± 0.65, 140.36 ± 4.35, 14.59 ± 0.6, and 222.8 ± 4.18°-day (DD) accordingly. When the food substrate is available and fresh, the female prefers to lay eggs, no matter how old she is. However, the chance of ovoviviparity increased when no oviposition substrate was available. The truth that S. argyrostoma able to either larviposit or to lay eggs encompasses serious consequences for the precise estimation of the PMImin, as the existence of larvae resulting from eggs laid on the carcass could add hours (based on ambient temperature) to the PMImin.

Association Between Wolbachia Infection and Susceptibility to Deltamethrin Insecticide in Phlebotomus papatasi (Diptera: Psychodidae), the Main Vector of Zoonotic Cutaneous Leishmaniasis.

Background: Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis. Wolbachia is a symbiotic alphaproteobacteria of arthropods that can be involved in susceptibility or resistance. This study aimed to investigate the relationship between Wolbachia and Deltamethrin susceptibility/resistance in Ph. papatasi. Deltamethrin filter papers (0.00002%) were used to test sand fly field collected from southern Iran. After the test, PCR amplification of the Wolbachia surface protein gene (wsp) was used to measure Wolbachia infection rate in the killed, surviving, and control groups. Result: The rates of infection by Wolbachia strain (wPap, super group A) differed between killed (susceptible) and surviving (resistant) Ph. papatasi specimens. The rate of Wolbachia infection in susceptible individuals was more than twice (2.3) (39% vs. 17%) in resistant individuals with the same genetic background. This difference was highly significant (p < 0.001), indicating a positive association between Wolbachia infection and susceptibility to Deltamethrin. In addition, the results showed that Deltamethrin can act as a PCR inhibitor during detection of Wolbachia in Ph. papatasi. Conclusion: Results of this study show that Wolbachia is associated with Deltamethrin susceptibility level in Ph. papatasi. Also, as Deltamethrin has been identified as a PCR inhibitor, great care must be taken in interpreting Wolbachia infection status in infected populations. The results of this study may provide information for a better understanding of the host-symbiont relationship, as well as application of host symbiosis in pest management.

Development of a degree-day model to predict the growth of Anopheles stephensi (Diptera: Culicidae): implication for vector control management.

Anopheles stephensi is an efficient vector of malaria parasites in Iran. Despite its importance in malaria transmission, there is a scarcity of accurate predictive models of its rates of development at different temperatures. A laboratory colony of An. stephensi, collected from Bandar Abbas County, southern Iran, was established, and all its developmental stages were maintained in temperature-controlled incubators so that the water temperature set at 5, 8, 10, 12.5, 14, 28, 38, 39.5, 42, and 45(±0.2) °C for different treatments until subsequent adult emergence. The Lower and Upper Developmental Temperatures (LDT and UDT) and the growth degree-day (GDD) were calculated for each development stage. A 12-mo population dynamics survey of the larvae and adults of An. stephensi was performed in 3 malaria-endemic villages (Geno, Hormoodar, and Sarkhoon) of Bandar Abbas County, and the obtained data were matched with the constructed GDD model. Based on the field meteorological and dynamics data, the model was verified in the field and used to determine the appropriate date to start spraying. The LDT was determined to be 8.19, 9.74, 8.42, 5.6, 13.57, and 10.03 °C for egg hatching, first, second, and third ecdysis, pupation, and eclosion events, respectively. The UDT was 38 °C for all developmental stages. The thermal requirement for the development of all immature stages of An stephensi was determined to be 187.7 (±56.3) GDD above the LDT. Therefore, the appropriate date to start residual spraying is when the region's GDD reaches 187.7 (±56.3). Given the climatic conditions in Bandar Abbas County, it is expected that the first activity peak of adult An. stephensi would be in March. Field observations showed that An. stephensi activity starts in February and peaks in March. The GDD model can provide a good estimate for peak An. stephensi activity and indicate the optimal deployment time of residual spraying operations against the multiplication and development of malaria parasites inside the vector.

Rodents as vehicle for delivery of transgenic bacteria to make paratransgenic sand fly vectors of cutaneous leishmaniasis in field condition.

Vector-borne diseases, among them leishmaniasis, cause more than 700,000 deaths annually. The lack of an effective vaccination and the increasing resistance of sand flies to insecticides require the urgent development of innovative approaches to contain the disease. The use of engineered bacteria that express anti-parasite molecules (paratransgenesis) shows much promise. However, a challenge for implementation of this strategy is to devise means to introduce modified bacteria into sand flies in the field. In this study, we use rodent food bait as a delivery strategy to introduce two mCherry-fluorescent bacteria, Serratia AS1 and Enterobacter cloacae, into adult sand flies in field settings. Bacteria-infected food was provided to Rhombomys opimus rodents. These bacteria transiently pass through the rodent alimentary tract and are delivered to larval habitats with the rodent feces. The feces are ingested by sand fly larvae and, in the case of Serratia AS1, are trans-stadially transmitted to adults. This is the first report of targeting delivery of Serratia AS1 in a paratransgenic system to control transmission of leishmaniasis under field condition. This novel strategy shows promise for delivering transgenic bacteria to Leishmania vectors in the field.

Molecular Characterization of Paederus Spp (Coleoptera: Staphylinidae, Paederinae) the Agent of Human Linear Dermatitis in the Caspian Sea Coast, North of Iran.

Background: A combined morphological and molecular survey was performed to determine the agent of human linear dermatitis Paederus Fabricius, 1775 (Coleoptera: Staphylinidae, Paederinae) species composition in Mazandaran Province in the Caspian Sea coast in northern Iran, where most of linear dermatitis cases of the country occurred. Methods: Altogether, 397 Paederus specimens were collected from May to August 2021 and classified using morphological characters and ITS2-rDNA sequence analysis. Results: Morphological investigation revealed that all the specimens were Paederus fuscipes. ITS2 polymerase chain reaction (PCR) direct-sequences and the profiles of restriction fragment length polymorphism (RFLP) derived from digestion of PCR products by HinfI, HpaII, and SalI enzymes were identical confirming the morphological results, implying that all specimens belonged to a single taxon. Conclusion: Paederus fuscipes (Fabricius, 1775) is considered the dominant taxon and responsible for linear dermatitis in Mazandaran Province. To our knowledge, we have provided the first molecular typing of Paederus beetles at the species level, suggesting that ITS2-rDNA characterization is an alternative tool for species discrimination of Paederus spp.

Efficiency of mitochondrial genes and nuclear Alu elements in detecting human DNA in blood meals of Anopheles stephensi mosquitoes: a time-course study.

BACKGROUND: The time required for PCR detection of DNA in human blood meals in vector mosquitoes may vary, depending on the molecular markers used, based on the size and copy number of the amplicons. Detailed knowledge of the blood-feeding behavior of mosquito populations in nature is an essential component for evaluating their vectorial capacity and for assessing the roles of individual vertebrates as potential hosts involved in the transmission of vector-borne diseases. METHODS: Laboratory experiments were conducted to compare the time course of PCR detection of DNA in human blood meals from individual blood-fed Anopheles stephensi mosquitoes, using loci with different characteristics, including two mitochondrial DNA (mtDNA) genes, cytB (228 bp) and 16S ribosomal RNA (rRNA) (157 bp) and nuclear Alu-repeat elements (226 bp) at different time points after the blood meal. RESULTS: Human DNA was detectable up to 84-120 h post-blood-feeding, depending on the length and copy number of the loci. Our results suggest that 16S rRNA and Alu-repeat markers can be successfully recovered from human DNA up to 5 days post-blood-meal. The 16S rDNA and Alu-repeat loci have a significantly (P = 0.008) slower decline rate than the cytB locus. Median detection periods (T50) for the amplicons were 117, 113 and 86.4 h for Alu-repeat, 16S rDNA and cytB, respectively, suggesting an inverse linear relationship between amplicon size/copy number and digestion time. CONCLUSION: This comparative study shows that the Alu-repeat locus is the most efficient marker for time-course identification of human DNA from blood meals in female mosquitoes. It is also a promising tool for determining the anthropophilic index (AI) or human blood index (HBI), i.e. the proportion of blood meals from humans, which is often reported as a relative measure of anthropophagy of different mosquito vectors, and hence a measure of the vector competence of mosquito species collected in the field.

Wolbachia infection in native populations of Blattella germanica and Periplaneta americana.

Cockroaches are significant pests worldwide, being important in medical, veterinary, and public health fields. Control of cockroaches is difficult because they have robust reproductive ability and high adaptability and are resistant to many insecticides. Wolbachia is an endosymbiont bacterium that infects the reproductive organs of approximately 70% of insect species and has become a promising biological agent for controlling insect pests. However, limited data on the presence or strain typing of Wolbachia in cockroaches are available. PCR amplification and sequencing of the wsp and gltA genes were used to study the presence, prevalence and molecular typing of Wolbachia in two main cockroach species, Blattella germanica (German cockroach) and Periplaneta americana (American cockroach), from different geographical locations of Iran. The Wolbachia endosymbiont was found only in 20.6% of German cockroaches while it was absent in American cockroach samples. Blast search and phylogenetic analysis revealed that the Wolbachia strain found in the German cockroach belongs to Wolbachia supergroup F. Further studies should investigate the symbiotic role of Wolbachia in cockroaches and determine whether lack of Wolbachia infection may increase this insect's ability to tolerate or acquire various pathogens. Results of our study provide a foundation for continued work on interactions between cockroaches, bacterial endosymbionts, and pathogens.

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.

Comparative analysis of the gut microbiota of sand fly vectors of zoonotic visceral leishmaniasis (ZVL) in Iran; host-environment interplay shapes diversity.

The development of Leishmania parasites within sand fly vectors occurs entirely in the insect gut lumen, in the presence of symbiotic and commensal bacteria. The impacts of host species and environment on the gut microbiome are currently poorly understood. We employed MiSeq sequencing of the V3-16S rRNA gene amplicons to characterize and compare the gut microbiota of field-collected populations of Phlebotomus kandelakii, P. perfiliewi, P. alexandri, and P. major, the primary or secondary vectors of zoonotic visceral leishmaniasis (ZVL) in three distinct regions of Iran where ZVL is endemic. In total, 160,550 quality-filtered reads of the V3 region yielded a total of 72 operational taxonomic units (OTUs), belonging to 23 phyla, 47 classes, 91 orders, 131 families, and 335 genera. More than 50% of the bacteria identified were Proteobacteria, followed by Firmicutes (22%), Deinococcus-Thermus (9%), Actinobacteria (6%), and Bacteroidetes (5%). The core microbiome was dominated by eight genera: Acinetobacter, Streptococcus, Enterococcus, Staphylococcus, Bacillus, Propionibacterium, Kocuria, and Corynebacterium. Wolbachia were found in P. alexandri and P. perfiliewi, while Asaia sp. was reported in P. perfiliewi. Substantial variations in the gut bacterial composition were found between geographically distinct populations of the same sand fly species, as well as between different species at the same location, suggesting that sand fly gut microbiota is shaped by both the host species and geographical location. Phlebotomus kandelakii and P. perfiliewi in the northwest, and P. alexandri in the south, the major ZVL vectors, harbor the highest bacterial diversity, suggesting a possible relationship between microbiome diversity and the capacity for parasite transmission. In addition, large numbers of gram-positive human or animal pathogens were found, suggesting that sand fly vectors of ZVL could pose a potential additional threat to livestock and humans in the region studied. The presence of Bacillus subtilis, Enterobacter cloacae, and Asaia sp suggests that these bacteria could be promising candidates for a paratransgenesis approach to the fight against Leishmaniasis.

Molecular and Biochemical Detection of Insecticide Resistance in the Leishmania Vector, Phlebotomus papatasi (Diptera: Psychodidae) to Dichlorodiphenyltrichloroethane and Pyrethroids, in Central Iran.

The aim of the present study was to explore resistance markers and possible biochemical resistance mechanisms in the Phlebotomine sand fly Phlebotomus papatasi in Esfahan Province, central Iran. Homogenous resistant strains of sand flies were obtained by exposing P. papatasi collected from Esfahan to a single diagnostic dose of DDT. The adults from the colony were tested with papers impregnated with four pyrethroid insecticides: Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15%, and Lambdacyhalothrin 0.05% to determine levels of cross-resistance. To discover the presence of mutations, a 440 base pair fragment of the voltage gated sodium channel (VGSC) gene was amplified and sequenced in both directions for the susceptible and resistant colonies. We also assayed the amount of four enzymes that play a key role in insecticide detoxification in the resistant colonies. A resistance ratio (RR) of 2.52 folds was achieved during the selection of resistant strains. Sequence analysis revealed no knockdown resistance (kdr) mutations in the VGSC gene. Enzyme activity ratio of the resistant candidate and susceptible colonies were calculated for α-esterases (3.78), ß-esterases (3.72), mixed function oxidases (MFO) (3.21), and glutathione-S-transferases (GST) (1.59). No cross-resistance to the four pyrethroids insecticides was observed in the DDT resistant colony. The absence of kdr mutations in the VGSC gene suggests that alterations in esterase and MFO enzymes are responsible for the resistant of P. papatasi to DDT in central Iran. This information could have significant predictive utility in managing insecticide resistant in this Leishmania vector.

Evaluation of anti-malaria potency of wild and genetically modified Enterobacter cloacae expressing effector proteins in Anopheles stephensi.

BACKGROUND: Malaria is one of the most lethal infectious diseases in tropical and subtropical areas of the world. Paratransgenesis using symbiotic bacteria offers a sustainable and environmentally friendly strategy to combat this disease. In the study reported here, we evaluated the disruption of malaria transmission in the Anopheles stephensi-Plasmodium berghei assemblage using the wild-type (WT) and three modified strains of the insect gut bacterium, Enterobacter cloacae. METHODS: The assay was carried out using the E. cloacae dissolvens WT and three engineered strains (expressing green fluorescent protein-defensin (GFP-D), scorpine-HasA (S-HasA) and HasA only, respectively). Cotton wool soaked in a solution of 5% (wt/vol) fructose + red dye (1/50 ml) laced with one of the bacterial strains (1 × 109cells/ml) was placed overnight in cages containing female An. stephensi mosquitoes (age: 3-5 days). Each group of sugar-fed mosquitoes was then starved for 4-6 h, following which time they were allowed to blood-feed on P. berghei-infected mice for 20 min in the dark at 17-20 °C. The blood-fed mosquitoes were kept at 19 ± 1 °C and 80 ± 5% relative humidity, and parasite infection was measured by midgut dissection and oocyst counting 10 days post-infection (dpi). RESULTS: Exposure to both WT and genetically modified E. cloacae dissolvens strains significantly (P < 0.0001) disrupted P. berghei development in the midgut of An. stephensi, in comparison with the control group. The mean parasite inhibition of E. cloacaeWT, E. cloacaeHasA, E. cloacaeS-HasA and E. cloacaeGFP-D was measured as 72, 86, 92.5 and 92.8 respectively. CONCLUSIONS: The WT and modified strains of E. cloacae have the potential to abolish oocyst development by providing a physical barrier or through the excretion of intrinsic effector molecules. These findings reinforce the case for the use of either WT or genetically modified strains of E. cloacae bacteria as a powerful tool to combat malaria.

The different aspects of attractive toxic baits containing fipronil for control of the German cockroach (Blattella germanica).

The use of Attractive Toxic Baits (ATBs) is considered to be a low-risk practical method for controlling cockroaches. This study evaluates the attractiveness of a lab-made, fipronil-containing bait, and its effect on the food consumption and mortality of the German cockroach Blattella germanica, under field and laboratory conditions. Different developmental stages of the cockroach were used to determine their preferred carbohydrate/protein rich foods and examine the effectiveness of lab-made baits. The analysis of variance (ANOVA) with Tukey's Test was determined using SAS 9.1 and GraphPad Prism software programs. The significance level was considered at p<0.05. The most-consumed foods were carbohydrate-rich foods (biscuit and banana powder) and food consumption rate was highest in non-gravid females. The most attractive bait ingredients were 20% roasted peanut butter, 50% biscuit and 30% banana powder. The highest lethality was recorded from the baits containing 0.02% fipronil under laboratory conditions, while infested houses baited with the lab-made bait showed 76.5% and 100% decline, respectively, in cockroach numbers in the first- and fourth-week post-baiting. In conclusion, palatable foods with a pleasant odour, like biscuit powder, banana powder and peanut butter were the most effective ingredients for the ATBs. The ATBs impregnated with 0.02% fipronil provide a promising approach for control of the German cockroach. However, there is a need to evaluate the potentials of the lab-made baits, under laboratory and field conditions, in the control of other health-important cockroaches.

Study on Hard and Soft Ticks of Domestic and Wild Animals in Western Iran.

Background: Ticks are blood-sucking ectoparasites of many vertebrates and act as vectors of a wide range of vector-borne diseases. Alongside pathogens transmission, ticks also cause economic losses in animal industry such as production loss, physical damage, anemia, and poisoning. This study aimed to determine the fauna, geographical distribution and seasonal activity of ticks collected from animals in Lorestan Province, west of Iran. Methods: Ticks were collected from domestic animals including cattle, sheep, goats, chickens, turkeys, pigeons, as well as wild animals such as jackals in 2017-2018. Then, they were identified based on morphological characteristics using valid identification keys. Results: Out of a total of 706 ticks, 433 (61.33%), 104 (14.73%), 33 (4.67%) and 136 (19.26%) ticks were collected in spring, summer, autumn and winter, respectively. In terms of hard ticks, 4 genera and 6 species were identified: Hyalomma asiaticum (22.80%), Hyalomma anatolicum (3.68%), Hyalomma marginatum (2.40%), Rhipicephalus sanguineus (0.84%), Dermacentor marginatus (1.13%), and Haemaphysalis sulcata (0.64%). Additionally, two genera and four species fell into soft ticks: Argas persicus (60.48%), Argas reflexus (6.65%), Ornithodoros canstrini (0.70%) and Ornithodoros erraticus (0.42%). There was significant variation in the seasonal activity and abundance of ticks in different seasons but in the tick abundancy among different regions. Conclusion: The present study provides a perspective of the distribution status of ticks in Lorestan Province, their seasonal activity and the likelihood of emergence of related diseases.

Hyalomma spp. ticks and associated Anaplasma spp. and Ehrlichia spp. on the Iran-Pakistan border.

BACKGROUND: Anaplasmosis and ehrlichiosis are tick-borne diseases affecting humans and livestock, particularly in tropical and subtropical regions. Animal husbandry is the main activity of people on the borders of Iran and Pakistan, with thousands of cattle crossing the border each week. METHODS: PCR and sequencing of the 16S rRNA gene was used to determine the percentage and geographical distribution of the pathogens carried by Hyalomma spp. (n = 306) collected from 126 goats, cattle and camels in the region between November 2017 and late March 2018. RESULTS: In total, 1124 hard ticks including 1020 Hyalomma spp. ticks belonging to six species (Hyalomma anatolicum, Hyalomma asiaticum, Hyalomma marginatum, Hyalomma dromedarii, Hyalomma schulzei, and Hyalomma detritum) were found on the borders of Iran and Pakistan, with H. anatolicum being the most prevalent tick species. Anaplasma spp. and/or Ehrlichia spp. DNA was found in 68.3% of the engorged tick specimens (n = 256). Sequencing of a subset (12.6%) of PCR-positive samples revealed Anaplasma ovis, Anaplasma marginale, and Ehrlichia ewingii DNA in 81.8%, 9.1%, and 9.1% of the ticks, respectively. To our knowledge, this is the first report of E. ewingii, an important human pathogen, in Iran. CONCLUSIONS: Based on molecular analysis, three pathogenic Anaplasmataceae were detected in six Hyalomma spp. parasitizing cattle, goats and camels, confirming the presence of these pathogens along the Iran-Pakistan border.

Utility of Complete Mitochondrial Genomes in Phylogenetic Classification of the Species of Anopheles (Culicidae: Anophelinae).

BACKGROUND: Among the blood-sucking insects, Anopheles mosquitoes have a very special position, because they transmit parasites of the genus Plasmodium, which cause malaria as one of the main vector-borne disease worldwide. The aim of this review study was to evaluate utility of complete mitochondrial genomes in phylogenetic classification of the species of Anopheles. METHODS: The complete mitochondrial genome sequences belonging to 28 species of the genus Anopheles (n=32) were downloaded from NCBI. The phylogenetic trees were constructed using the ML, NJ, ME, and Bayesian inference methods. RESULTS: In general, the results of the present survey revealed that the complete mitochondrial genomes act very accurately in recognition of the taxonomic and phylogenetic status of these species and provide a higher level of support than those based on individual or partial mitochondrial genes so that by using them, we can meticulously reconstruct and modify Anopheles classification. CONCLUSION: Understanding the taxonomic position of Anopheles, can be a very effective step in better planning for controlling these malaria vectors in the world and will improve our knowledge of their evolutionary biology.

Biochemical Mechanism of Insecticide Resistance in Malaria Vector, Anopheles gambiae s.l in Nigeria.

BACKGROUND: Malaria is a parasitic vector-borne disease endemic in the tropical and subtropical countries of the world. The aim of this study was to investigate the current activities of the detoxification enzymes in resistant and susceptible Anopheles gambiae s.l. in northern Nigeria. METHODS: Anopheles larvae were collected from northeast and northwestern Nigeria between Aug and Nov 2018. Biochemical analyses was carried out on the mosquitoes exposed to various insecticides (deltamethrin, DDT, bendiocarb, malathion) to measure and compare the enzymatic activities of the major detoxification enzymes (P450, GSTs, Esterase). RESULTS: High levels of resistance was observed; DDT 37%-53% (95%, CI: 29-61), bendiocarb 44%-55% (CI: 39-60) and deltamethrin 74%-82% (CI: 70-86). However, these mosquitoes were found to be susceptible to malathion 99%-100% (CI: 98-100). The P450 and GSTs enzymes were found to be elevated in the resistant mosquitoes exposed to deltamethrin (1.0240±0.1902); (1.3088±1.2478), DDT (1.7703±1.4528); (1.7462±0.9418) and bendiocarb (1.1814±0.0918); (1.4479±1.0083) compared to the Kisumu strain (0.764±0.4226); (0.6508±0.6542), (0.3875±0.3482); (0.4072±0.4916) and (0.6672±0.3949); (0.7126±0.7259) at P<0.05. Similarly, the resistant mosquitoes expressed increased activity to esterase (0.7606±1.1477), (0.3269±1.1957) and (2.8203±0.6488) compared to their susceptible counterpart (0.6841±0.7597), (0.7032±0.5380) and (0.6398±0.4159) at P<0.05. The enzyme ratio was found to be: P450 (1.341, 4.568 and 1.77); GSTs (2.011, 4.288 and 2.031); Esterases (1.111, 0.469 and 4.408). One way Anova and single sample t-test were also conducted to determine the effect of the enzymes on the resistant and susceptible strains. CONCLUSION: High level of insecticide resistance was observed with significant elevation of detoxification enzymes activities in the resistant mosquitoes.

An integrated overview of the bacterial flora composition of Hyalomma anatolicum, the main vector of CCHF.

The microbial flora associated with Hyalomma anatolicum ticks was investigated using culture-dependent (CD) and independent (next generation sequencing, NGS) methods. The bacterial profiles of different organs, development stages, sexes, and of host cattle skins were analyzed using the CD method. The egg and female gut microbiota were investigated using NGS. Fourteen distinct bacterial strains were identified using the CD method, of which Bacillus subtilis predominated in eggs, larval guts and in adult female and male guts, suggesting probable transovarial transmission. Bacillus velezensis and B. subtilis were identified in cattle skin and tick samples, suggesting that skin is the origin of tick bacteria. H.anatolicum males harbour lower bacterial diversity and composition than females. The NGS analysis revealed five different bacterial phyla across all samples, Proteobacteria contributing to >95% of the bacteria. In all, 56611sequences were generated representing 6,023 OTUs per female gut and 421 OTUs per egg. Francisellaceae family and Francisella make up the vast majority of the OTUs. Our findings are consistent with interference between Francisella and Rickettsia. The CD method identified bacteria, such B. subtilis that are candidates for vector control intervention approaches such paratransgenesis whereas NGS revealed high Francisella spp. prevalence, indicating that integrated methods are more accurate to characterize microbial community and diversity.

Thermal requirements of immature stages of Chrysomya albiceps (Diptera: Calliphoridae) as a common forensically important fly.

Entomological material may be used to estimate the time since death occurred (postmortem interval, PMI) in forensically obscure cases. The method that is commonly used to calculate minimum post-mortem interval (mPMI, i.e., the least amount of time since one can be confident death occurred) is based on the relationship between insect development and ambient temerature. Isomegalen and isomorphen diagrams are among methods allowing to calculate the age of necorphagous insects, yet thermal summation models provide the most precise and acurate estimations. The digrams are prepared based on the length or the developmental stages of the larvae as a function of time and mean ambient temperature. A knowledge of thermal requirements, in particular lower temperature threshold (Dz) at which development of a species terminates, is of essential importance to calculate ADD (Accumulated Degree Days). In this study different temperature regimes were used to construct the isomorphen diagram, examinate changes in larval body length at different ambient temperatures and to estimate the thermal requirements for developemnt of Chrysomya albiceps, the most common dipteran species reported on human and animal cadavers in Iran. Six development events including hatching, 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion were studied under eleven constant temperature regims (17-37 0C). The development rate of Ch. albiceps increased as temperature increased. The larval length peaked at the end of third stage and then decreased at wandering stage. The maximum larval length occurred at 72 h post oviposition at either 31, 33, or 35 °C. At 17 °C, larvae did not hatch from eggs and at 37 °C wandering larvae did not proceed to pupariation, and thus larval development were analysed at the nine left over temperatures. The development stages required at least (Dz ± SE) 13.04 ± 0.37, 14.29 ± 0.45, 15.69 ± 0.56, 15.18 ± 0.56, 14.94 ± 0.48, and 11.23 ± 0.41 °C to reach one of the successive developmentl events, respectively. The estimated thermal summation constant (k) for those the six events were 10.43 ± 0.27, 19.31 ± 0.32, 27.87 ± 1.3, 55.94 ± 1.82, 66.69 ± 3.5, and 143.52 ± 5.61 ADD accordingly.

Relationship between Wolbachia infection in Culex quinquefasciatus and its resistance to insecticide.

Many studies have been done on the various factors affecting resistance to insecticides. The relationship between Wolbachia bacteria and resistance to insecticides is one of the factors that has attracted a lot of attentions. Wolbachia are obligatory intracellular endosymbionts that naturally occur in a wide range of arthropods and nematodes, including the mosquito Culex quinquefasciatus. Initially, the presence of bacteria was proved by molecular assays. Then the resistance level of this species was evaluated in adults against DDT 4.0% and deltamethrin 0.05% using the standard WHO guideline. After elimination of Wolbachia by tetracycline and its proof by molecular assays, the susceptibility tests were conducted again on uninfected line. Finally, the two lines were compared in terms of responding to insecticides. The findings indicated that there is no significant correlation between susceptibility of two lines in response to DDT 4.0% while they represented a significant correlation for deltamethrin (P =0.00). We propose that Wolbachia bacteria increase the susceptibility to deltamethrin but they show neutral effect on DDT susceptibility in Cx. quinquefasciatus. However, more studies on other vectors and insecticides still need to be done.

An Eco-Epidemiological Study on Zoonotic Cutaneous Leishmaniasis in Central Iran.

BACKGROUND: Leishmaniasis is an expanding neglected tropical disease in the world reporting from 98 countries including Iran. This study focused on eco-epidemiological determinants of the disease following a rapid and unexpected increase of leishmaniasis incidence in a strategic residential district in North-East of Isfahan County, Iran. METHODS: This study was accomplished from Apr 2012 to Jan 2014 in a strategic residential zone in North-East of Isfahan County, Esfahan, Iran. Monthly activity, parity, Leishmania infection and susceptibility tests, were determined on sand flies. Some portion of inhabitants and school children were surveyed to find active or passive cases of leishmaniasis and also wild rodents were collected to determine reservoir host. RESULTS: Totally 5223 sand flies belonging to Phlebotomus and Sergentomyia genus were collected and identified; Ph. papa-tasi was the dominant species and started to appear in May and disappeared in Oct. The majority of living dissected sand flies were unfed and parous. Ph. papatasi showed 4.6% Leishmania infection through direct examination and 39.54% by nested-PCR respectively. Phlebotomus papatasi was susceptible against deltametrin 0.05%. Totally 2149 people were surveyed and incidence and prevalence of zoonotic cutaneous leishmaniasis estimated as 45.39 and 314.40 per 1000 population. Rodents showed 73.91% and 80% Leishmania infection by direct examination and nested-PCR respectively. CONCLUSION: Cutaneous leishmaniasis due to L. major has been established in this area. Rodent control operation and personal protection are highly recommended to control the disease in this focus.