The effects of Leishmania RNA virus 2 (LRV2) on the virulence factors of L. major and pro-inflammatory biomarkers: an in vitro study on human monocyte cell line (THP-1).

BACKGROUND: Cutaneous Leishmaniasis (CL) is a parasitic disease with diverse outcomes. Clinical diversity is influenced by various factors such as Leishmania species and host genetic background. The role of Leishmania RNA virus (LRV), as an endosymbiont, is suggested to not only affect the pathogenesis of Leishmania, but also impact host immune responses. This study aimed to investigate the influence of LRV2 on the expression of a number of virulence factors (VFs) of Leishmania and pro-inflammatory biomarkers. MATERIALS AND METHODS: Sample were obtained from CL patients from Golestan province. Leishmania species were identified by PCR (LIN 4, 17), and the presence of LRV2 was checked using the semi-nested PCR (RdRp gene). Human monocyte cell line (THP-1) was treated with three isolates of L. major with LRV2 and one isolate of L. major without LRV2. The treatments with four isolates were administered for the time points: zero, 12, 24, 36, and 48 h after co-infection. The expression levels of Leishmania VFs genes including GP63, HSP83, and MPI, as well as pro-inflammatory biomarkers genes including NLRP3, IL18, and IL1ß, were measured using quantitative real-time PCR. RESULTS: The expression of GP63, HSP83, and MPI revealed up-regulation in LRV2 + isolates compared to LRV2- isolates. The expression of the pro-inflammatory biomarkers including NLRP3, IL1ß, and IL18 genes in LRV2- were higher than LRV2 + isolates. CONCLUSION: This finding suggests that LRV2 + may have a probable effect on the Leishmania VFs and pro-inflammatory biomarkers in the human macrophage model.

Effect of Leishmania RNA virus 2 on virulence factors and cytokines gene expression in a human macrophage infected with Leishmania major: A preliminary study.

Cutaneous leishmaniasis (CL) is one of the most important infectious parasitic diseases in the world caused by the Leishmania parasite. In recent decades, the presence of a virus from the Totiviridae family has been proven in some Leishmania species. Although the existence of LRV2 in the Old world Leishmania species has been confirmed, almost no studies have been done to determine the potential impact of LRV2 on the immunopathogenicity of the Leishmania parasite. In this preliminary study, we measured the expression of target genes, including Glycoprotein 63 (gp63), Heat Shock Protein 70 (hsp70), Cysteine Protease b (cpb), Interleukin 1 beta (IL-1ß), IL8 and IL-12 in LRV2 positive Leishmania major strain (LRV2+L. major) and LRV2 negative L. major strain (LRV2-L. major). We exposed THP-1, a human leukemia monocytic cell line, to promastigotes of both strains. After the initial infection, RNA was extracted at different time points, and the relative gene expression was determined using a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Findings showed that the presence of LRV2 in L. major was able to increase the expression of gp63, hsp70, and cpb genes; also, we observed lower levels of expression in cytokine genes of IL-1ß, IL-8, IL-12 in the presence of LRV2+, which are critical factors in the host's immune response against leishmaniasis. These changes could suggest that the presence of LRV2 in L. major parasite may change the outcome of the disease and increase the probability of Leishmania survival; nevertheless, further studies are needed to confirm our results.

Delivery of a Genetically Marked Serratia AS1 to Medically Important Arthropods for Use in RNAi and Paratransgenic Control Strategies.

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

Sequence analysis of mtDNA COI barcode region revealed three haplotypes within Culex pipiens assemblage.

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

A survey of Wolbachia infection in brachyceran flies from Iran.

Wolbachia is a maternally inherited intracellular bacterium that is considered to be the most plentiful endosymbiont found in arthropods. It reproductively manipulates its host to increase the chances of being transmitted to the insect progeny; and it is currently used as a means of suppressing disease vector populations or controlling vector-borne diseases. Studies of the dissemination and prevalence of Wolbachia among its arthropod hosts are important for its possible use as a biological control agent. The molecular identification of Wolbachia relies on different primers sets due to Wolbachia strain variation. Here, we screened for the presence of Wolbachia in a broad range of Brachycera fly species (Diptera), collected from different regions of Iran, using nine genetic markers (wsp, ftsZ, fbpA, gatB, CoxA, gltA, GroEL dnaA, and 16s rRNA), for detecting, assessing the sensitivity of primers for detection, and phylogeny of this bacterium. The overall incidence of Wolbachia among 22 species from six families was 27.3%. The most commonly positive fly species were Pollenia sp. and Hydrotaea armipes. However, the bacterium was not found in the most medically important flies or in potential human disease vectors, including Musca domestica, Sarcophaga spp., Calliphora vicinia, Lucilia sericata, and Chrysomya albiceps. The primer sets of 16s rRNA with 53.0% and gatB with 52.0% were the most sensitive primers for detecting Wolbachia. Blast search, phylogenetic, and MLST analysis of the different locus sequences of Wolbachia show that all the six distantly related fly species likely belonging to supergroup A. Our study showed some primer sets generated false negatives in many of the samples, emphasizing the importance of using different loci in detecting Wolbachia. The study provides the groundwork for future studies of a Wolbachia-based program for control of flies.

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.

The monsoon-associated equine South African pointy mosquito 'Aedes caballus'; the first comprehensive record from southeastern Iran with a description of ecological, morphological, and molecular aspects.

The equine South African pointy vector mosquito, Aedes caballus, poses a significant threat to human health due to its capacity for transmitting arboviruses. Despite favorable climate for its existence in southeast Iran, previous records of this species in the area have indicated very low abundance. This comprehensive field and laboratory study aimed to assess its current adult population status in this region, utilizing a combination of ecological, morphological and molecular techniques. Four distinct types of traps were strategically placed in three fixed and two variable mosquito sampling sites in the southern strip of Sistan and Baluchistan Province. Subsequently, DNA was extracted from trapped mosquitoes and subjected to PCR amplification using the molecular markers COI, ITS2, and ANT. In total, 1734 adult Ae. caballus specimens were collected from rural areas, with the majority being captured by CO2-baited bednet traps. A notable increase in the abundance of this species was observed following rainfall in February. The genetic analysis revealed multiple haplotypes based on COI and ITS2 sequences, with COI gene divergence at 0.89%, and ITS2 sequence divergence at 1.6%. This suggests that previous challenges in morphological identification may have led to misidentifications, with many adults previously classified as Ae. vexans potentially being Ae. caballus. The findings of this study hold significant implications for public health authorities, providing valuable insights for integrated and targeted vector control and disease management efforts.

Detection of Enterobius vermicularis in archived formalin-fixed paraffin-embedded (FFPE) appendectomy blocks: It's potential to compare genetic variations based on mitochondrial DNA (cox1) gene.

Acute appendicitis represents one of the most common causes of emergency abdominal surgery worldwide. Meanwhile, Enterobius vermicularis has been suggested as one of the probable causes of appendicitis. In this study, the morphological characteristics of the remnant pinworms and pathologic changes were explored in old-archived FFPE tissues of appendectomies. Moreover, we provide the first molecular identification, genetic, and haplotype variation of this nematode from the old-archived FFPE tissue section of appendectomy using the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Seventeen FFPE appendectomies with E. vermicularis infection, stored over 12-22 years, were collected from two different geographical areas of Iran. In the histopathological examination, tissue changes were observed in thirteen cases (76.4%) and inflammation in four blocks (23.5%). After DNA extraction, the cox1 gene was amplified in twelve (70.6%) cases using the nested polymerase chain reaction (PCR). Phylogenetic analysis and a median-joining network of 78 available cox1 sequences of E. vermicularis revealed 59 haplotypes. We identified five haplotypes that fell into type B. All Haplotypes are novel except for two haplotypes, Hap32 and Hap37, identical to E. vermicularis sequences from Iran, Greece, and Germany. The ranges of diversity distance and haplotype diversity within the isolates were 0-1.9% and HD:0.643-0.667, subsequently. Overall, the absence of inflammation or even tissue changes in some sections can suggest the possible non-inflammatory role of E. vermicularis in appendicitis. Although FFPE material suffers from PCR inhibition, we could successfully use nested PCR to characterize E. vermicularis in old-archived appendectomy blocks and suggest this method as a complementary diagnosis technique in pathology. While the predominant type was B in the Middle East and Europe, further studies on a larger sample size from different geographical regions could probably confirm the results obtained in the present study.

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.

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.

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.

Susceptibility of Phlebotomus papatasi (Diptera: Psychodidae) against DDT and Deltamethrin in an Endemic Focus of Zoonotic Cutaneous Leishmaniasis in Iran.

Background: Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis (ZCL) in Iran. The nonstandard use of pesticides against pests, particularly in agriculture, indirectly has caused the development of resistance and, consequently, the threat of control measures in ZCL endemic areas. Up to 2023, several reports of resistance in Ph. papatasi have been declared in the Old World. The purpose of this study was to measure the lethal time (LT50 and LT90) of Ph. papatasi sand flies in the ZCL endemic center of Esfahan to DDT and deltamethrin insecticides. Methods: Sand flies were collected in Borkhar and were tested using WHO adult mosquito test kit against DDT 4% and deltamethrin 0.0002%. The sand fly's survival was recorded during exposure time in 225, 450|, 900, 1800, and 3600-seconds' intervals for DDT and Deltamethrin and they were allowed to recover for 24 hours. Then LT50 and LT90 were analyzed using probit software. Phlebotomus papatasi were identified using morphological keys and other sand flies' species were excluded from the analysis. Results: The insecticide against female Ph. papatasi revealed hundred percent mortality when exposed to DDT 4% and deltamethrin 0.0002%. The LT50 and LT90 were 19.32 and 22.74 minutes for DDT 4% and 39.92 and 51.33 minutes for deltamethrin 0.0002% respectively. Conclusion: Results of this study revealed that Ph. papatasi is still susceptible to DDT and deltamethrin. This data provides valuable knowledge to implement effective control strategies against ZCL main vector and help to manage insecticide resistance in the region.

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.

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.

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.

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.

Molecular detection of Dirofilaria spp. and host blood-meal identification in the Simulium turgaicum complex (Diptera: Simuliidae) in the Aras River Basin, northwestern Iran.

BACKGROUND: Blackflies (Diptera: Simuliidae) are known as effective vectors of human and animal pathogens, worldwide. We have already indicated that some individuals in the Simulium turgaicum complex are annoying pests of humans and livestock in the Aras River Basin, Iran. However, there is no evidence of host preference and their possible vectorial role in the region. This study was conducted to capture the S. turgaicum (s.l.), to identify their host blood-meals, and to examine their potential involvement in the circulation of zoonotic microfilariae in the study areas. METHODS: Adult blackflies of the S. turgaicum complex were bimonthly trapped with insect net in four ecotopes (humans/animals outdoors, irrigation canals, lands along the river, as well as rice and alfalfa farms) of ten villages (Gholibaiglou, Gungormaz, Hamrahlou, Hasanlou, Khetay, Khomarlou, Larijan, Mohammad Salehlou, Parvizkhanlou and Qarloujeh) of the Aras River Basin. A highly sensitive and specific nested PCR assay was used for detection of filarial nematodes in S. turgaicum (s.l.), using nuclear 18S rDNA-ITS1 markers. The sources of blood meals of engorged specimens were determined using multiplex and conventional cytb PCR assays. RESULTS: A total of 2754 females of S. turgaicum (s.l.) were collected. The DNA of filarial parasites was detected in 6 (0.62%) of 960 randomly examined individuals. Sequence analysis of 420 base pairs of 18S rDNA-ITS1 genes identified Dirofilaria spp. including 5 D. immitis and 1 D. repens. Importantly, all filarial positive specimens have been captured from humans and animals outdoors. Cytb-PCR assays showed that in all ecotypes studied, members of the S. turgaicum complex had preferably fed on humans, dogs, bovids, and birds, respectively. CONCLUSIONS: To the best of our knowledge, this is the first report of D. immitis/D. repens detection in blackflies. Results showed that S. turgaicum (s.l.) was the most abundant (97%) and anthropophilic (45%) blackfly in all studied ecotypes/villages and that DNA of Dirofilaria spp. was detected in the flies taken from six villages. Dirofilariasis is a common zoonosis between humans and carnivores, with mosquitoes (Culicidae) as the principal vectors. Further investigations are needed to demonstrate that blackflies are actual vectors of Dirofilaria in the studied region.

Identification of Forensically Important Flesh Flies Using the Cytochrome C Oxidase Subunits I and II Genes.

Forensically important flesh flies (Diptera: Sarcophagidae) often are not morphologically distinguishable, especially at the immature stage. In addition, female flies are quite similar in general morphology, making accurate identifications difficult. DNA-based technologies, particularly mitochondrial DNA (mtDNA), have been used for species-level identification. The cytochrome oxidase subunits I and II (COI-COII) sequences of Iranian Sarcophagidae are still unavailable in GenBank. In this study as many as 648 (540 males and 106 females) fly specimens from family Sarcophagidae, representing 10 sarcophagid species, including eight forensically important species were collected from seven locations in five Iranian provinces. Of these, 150 male specimens were identified based on both morphology of male genitalia and DNA sequencing analysis. Sequence data from the COI-COII regions for 10 flesh fly species collected in Iran were generated for the first time. Digestion of COI-COII region by restriction enzymes RsaI, EcoRV, and HinfI provided distinct restriction fragment length polymorphism profiles among the species and can serve as molecular markers for species determination. Phylogenetic analysis represented that the COI-COII sequences are helpful for delimitation of sarcophagid species and implementation in forensic entomology. However, the application of the COI-COII fragment as a species identifier requires great caution and additional species and markers should be studied to ensure accurate species identification in the future.

Effect of Serratia AS1 (Enterobacteriaceae: Enterobacteriales) on the Fitness of Culex pipiens (Diptera: Culicidae) for Paratransgenic and RNAi Approaches.

The mosquito Culex pipiens is the primary vector of Rift Valley fever, West Nile, encephalitis, and Zika viruses, and periodic lymphatic filariasis. Developing insecticide resistance in mosquitoes demands the development of new approaches to fight these diseases. Paratransgenesis and RNAi approaches by using engineered bacteria have been shown to reduce mosquito vector competence. Serratia-AS1 is a bacterium found in mosquitoes and was genetically modified for expression of antimalaria effector molecules that repress development of malaria parasites in mosquitoes. The aim of this study was to determine how a genetically marked Serratia strain expressing the mCherry fluorescent protein (mCherry-Serratia) affects the colonization potential, life span, blood feeding behavior, fecundity, and fertility of Cx. pipiens. mCherry-Serratia bacteria disseminated into larvae, pupae, and newly emerged adults and dramatically increased in numbers following a blood meal. The bacterium was transmitted to progeny, showing that it can extend horizontally, transstadially, and vertically through the mosquito population. The presence of mCherry-Serratia did not affect blood feeding behavior, survival rate, fecundity, and fertility of Culex mosquitoes. This is the first study to evaluate the effects of an engineered bacteria on the fitness of Cx. pipiens. Although challenges remain, such as producing engineered bacteria to secrete anti-pathogens associated with Cx. pipiens, introducing such bacteria into mosquito populations, our findings of minimal fitness cost caused by Serratia-AS1 bode well for the development of paratransgenesis and RNAi approaches.