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Abstract Background Malaria is a major global health challenge, and for the elimination and eradication of this disease, transmission-blocking vaccines (TBVs) are a priority. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising TBV candidate, is essential for gamete fertilization. The HAP2-GCS1 domain of this antigen as well as its cd loop could induce antibodies that partially inhibit transmission of P. falciparum. Methods In the current study, a new synthetic fusion antigen containing cd loop and HAP2-GCS1 domain (cd-HAP) of PfGCS1 was evaluated as a transmission blocking vaccine candidate. Initially, the profile of naturally acquired IgG antibodies to the cd-HAP antigen was analysed in Iranian individuals infected with P. falciparum, to confirm that this new fusion protein has the appropriate structure containing common epitopes with the native form of PfGCS1. Then, the immunogenicity of cd-HAP was evaluated in BALB/c mice, using different adjuvant systems such as CpG, MPL, QS-21, and a combination of them (CMQ). Furthermore, the blocking efficacy of polyclonal antibodies induced against these formulations was also assessed by oocyst intensity and infection prevalence in the Standard Membrane Feeding Assay (SMFA). Results The naturally acquired antibodies (dominantly IgG1 and IgG3 subclasses) induced in P. falciparum-infected individuals could recognize the cd-HAP antigen which implies that the new fusion protein has a proper conformation that mimics the native structure of PfGCS1. Concerning the immunogenicity of cd-HAP antigen, the highest IgG levels and titers, by a Th1-type immune profile, and elevated antibody avidity were induced in mice immunized with the cd-HAP antigen formulated with a combination of adjuvants (P

Abstract Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p

BackgroundsMalaria, a preventive and treatable disease, is still responsible for annual deaths reported in most tropical regions, principally in sub-Saharan Africa. Subunit recombinant transmission-blocking vaccines (TBVs) have been proposed as promising vaccines to succeed in malaria elimination and eradication. Here, a provisional study was designed to assess the immunogenicity and functional activity of alanyl aminopeptidase N (APN1) of Anopheles stephensi, as a TBV candidate, administered with MPL, CpG, and QS21 adjuvants in the murine model.Methodology/principal findingsThe mouse groups were immunized with recombinant APN1 (rAPN1) alone or formulated with CpG, MPL, QS-21, or a combination of adjuvants (CMQ), and the elicited immune responses were evaluated after the third immunization. The standard membrane feeding assay (SMFA) measured the functional activity of antibodies against bacterial-expressed APN1 protein in adjuvanted vaccine groups on transmission of P. falciparum (NF54) to An. stephensi mosquitoes. Evaluation of mice vaccinated with rAPN1 formulated with distinct adjuvants manifested a significant increase in the high-avidity level of anti-APN1 IgG and IgG subclasses; however, rAPN1 induced the highest level of high-avidity anti-APN1 IgG1, IgG2a, and IgG2b antibodies in the immunized vaccine group 5 (APN1/CMQ). In addition, vaccine group 5 (receiving APN1/CMQ), had still the highest level of anti-APN1 IgG antibodies relative to other immunized groups after six months, on day 180. The SMFA data indicates a trend towards higher transmission-reducing activity in groups 2 and 5, which received the antigen formulated with CpG or a combination of three adjuvants.Conclusions/significanceThe results have shown the capability of admixture to stimulate high-affinity and long-lasting antibodies against the target antigen to hinder Plasmodium parasite development in the mid-gut of An. stephensi. The attained results authenticated APN1/CMQ and APN1/CpG as a potent APN1-based TBV formulation which will be helpful in designing a vaccine in the future.

Objectives: Lucilia sericata (Diptera: Calliphoridae) is used in larval therapy for wound healing. Netrin-A is an enzyme secreted from the salivary glands of these larvae, and has a central role in neural regeneration and angiogenesis. This study aimed to produce the recombinant Netrin-A protein from Lucilia sericata larvae by the baculovirus expression vector system in the Sf9 insect cell line. Methods: The coding sequence of Netrin-A was cloned, amplified in the pTG19 vector, and then cloned in the pFastBac HTA vector. It was then transformed into DH10Bac, and the recombinant Bacmid was subsequently transfected into Sf9 cells. The recombinant Netrin-A was purified by Ni-NTA agarose. The evaluation was done using SDS-PAGE and western blot, respectively. Finally, its concentration was calculated with the Bradford assay. Results: The molecular weight of this protein was 52 kDa with 404 amino acids. The signal peptide was located between amino acids 24 and 25. The concentration of Netrin-A was calculated to be 48.8 μg/ml. It reaffirmed the characterized gene codes of Lucilia sericata Netrin-A in a previous study. Conclusions: The generation of recombinant Netrin-A could be used in larval therapy, and as a biomarker in certain diseases. The netrin-A of Lucilia sericata was unprecedentedly cloned and expressed in a eukaryotic cell line. Given that this larva is FDA-approved, and non-pathogenic, it conduces to research on the development of maggot therapy in future.

Abstract Background The spread of Plasmodium vivax strains resistant to chloroquine (CQ) has posed a challenge to control strategies aimed at eliminating malaria. Molecular analysis of candidate resistance markers is very important for monitoring the P. vivax resistance to CQ in different endemic regions. In the present study, the multidrug resistance 1 (pvmdr1) gene, a possible marker for CQ resistance in P. vivax, was evaluated by molecular methods. Methods A simple PCR–RFLP method was developed for mutation analysis in pvmdr1 gene. A number of 120 blood spots were obtained from patients with P. vivax mono-infection in 2021. All of the samples were collected from Pakistani patients who travelled to Iran. Results None of the samples had any mutation at codon 976 of pvmdr1, while the 1076 mutation was detected in 96.2% of the examined isolates. Only two pvmdr1 haplotypes were identified, including the single mutant (Y976/1076L) as the most prevalent haplotype (with 96.2% frequency) and the wild type (Y976/F1076; with 3.8% frequency). Conclusions In this study, the major CQ resistance-mediating mutation and multiple mutant haplotypes of the pvmdr1 gene was not detected. However, continuous monitoring of drug resistance markers and close supervision of the efficacy of CQ is essential to detect the potential emergence of CQ-resistant P. vivax isolates in Iran. This data is important for performing future epidemiological surveillance to monitor CQ resistance in this endemic area and the bordering regions.

Abstract CRISPR-mediated integration could be used to develop the recombinant CHO (rCHO) cells by knock-in into the hotspot loci. However, low HDR efficiency besides the complex donor design is the main barrier for achieving so. The recently introduced MMEJ-mediated CRISPR system (CRIS-PITCh) uses a donor with short homology arms, being linearized in the cells via two sgRNAs. In this paper, a new approach to improve CRIS-PITCh knock-in efficiency by employing small molecules was investigated. Two small molecules, B02, a Rad51 inhibitor, and Nocodazole, a G2/M cell cycle synchronizer, were used to target the S100A hotspot site using a bxb1 recombinase comprised landing pad in CHO-K1 cells. Following transfection, the CHO-K1 cells were treated with the optimum concentration of one or combination of small molecules, being determined by the cell viability or flow cytometric cell cycle assay. Stable cell lines were generated and the single-cell clones were achieved by the clonal selection procedure. The finding showed that B02 improved the PITCh-mediated integration approximately twofold. In the case of Nocodazole treatment, the improvement was even more significant, up to 2.4-fold. However, the combinatorial effects of both molecules were not substantial. Moreover, according to the copy number and out-out PCR analyses, 5 and 6 of 20 clonal cells exhibited mono-allelic integration in Nocodazole and B02 groups, respectively. The results of the present study as the first attempt to enhance the CHO platform generation by exploiting two small molecules in the CRIS-PITCh system could be used in future researches to establish rCHO clones.

Background and Objectives: Although the study on the bacteria residing in the mid-gut, salivary gland, and reproductive organs of insect vectors have drawn appeal to the host-pathogen interactions, we know comparatively less about microbiota that naturally exist in different mosquito organs within Iran. Materials and Methods: In the current investigation, PCR assay by using 16S rRNA gene amplification and DNA sequencing, in addition to the traditional culture-based approach utilized for the detection of cultivable bacterial assemblages in mid-gut and reproductive tracts of Culex quinquefasciatus. Results: The identified bacteria isolated from different tissues of 45 individuals were consisted of Achromobacter, Aeromonas, Arthrobacter, Asaia, Enterobacter, Gluconobacter, Klebsiella, Lysinibacillus, Micrococcus, Psuedomonas and Serratia. The results showed that Proteobacteria was the most prevalent phylum in both genders' mid-gut and reproductive tracts, and Asaia was the most common bacteria that originated in adult females and males’ tissues. Conclusion: These outcomes recommend that the discovered microbiome may span through Cx. quinquefasciatus populations. This data can be utilized to interfere with the transmission of pathogens and design new strategies for the control of mosquito-borne diseases.

Background: A given amino acid sequence can be encoded by a huge number of different nucleic acid sequences. These sequences, however, have proved not to be equally useful. The choice of sequence can significantly impact the expression of an encoded protein. Given the importance of protein-coding sequence and promising industrial and medicinal applications of Clostridium histolyticum collagenase, this study examined the codon optimization of the Col H gene so as to enhance collagenase expression in Escherichia coli (E. coli). Methods: This is an experimental study in which the CDS of Col H gene was optimized according to the codon usage of E. coli, using Gene Designer software (DNA 2.0). Results: The results revealed that relative frequency of codon usage in Col H gene was adapted to the most preferred triplets in E. coli in such a way that codon usage bias in E. coli was enhanced after codon optimization. The higher level of collagenase expression was more likely the result of substituting rare codons with optimal codons. Conclusion: The findings of this study suggest that codon optimization provides a theoretical improvement in Col H gene expression in E. coli. Nevertheless, experimental research is needed to confirm the improvement.

Abstract Background According to the World Health Organization reports, billions of people around the world are at risk for malaria disease and it is important to consider the preventive strategies for protecting the people that are living in high risk areas. One of the main reasons of disease survival is diversity of vectors and parasites in different malaria regions that have their specific features, behaviour and biology. Therefore, specific regional strategies are necessary for successful control of malaria. One of the tools that needs to be developed for elimination and prevention of reintroduction of malaria is a vaccine that interrupt malaria transmission (VIMTs). VIMT is a broad concept that should be adjusted to the biological characteristics of the disease in each region. One type of VIMT is a vector-based vaccine that affects the sexual stage of Plasmodium life cycle. According to recent studies, the aminopeptidase N-1 of Anopheles gambiae (AgAPN-1) is as a potent vector-based VIMT with considerable inhibition activity against the sexual stage of Plasmodium parasite. Methods Systems for rapid amplification of cDNA ends (3ʹ-RACE) and genome walking methods were used for sequence determination of apn-1 gene from Anopheles stephensi and distinct bioinformatics software were used for structural analysis. AsAPN-1 was expressed in Spodoptera frugiperda (Sf9) insect cell line using the baculovirus expression system. Recombinant AsAPN-1 was purified under the hybrid condition and its biological activity was assayed. Results Asapn-1 gene and its coded protein from An. stephensi were characterized for the first time in this study. Subsequently, the structural features and immunological properties of its coded protein were evaluated by in silico approaches. Enzymatic activity of the recombinant AsAPN-1, which was expressed in Sf9 insect cell line, was equal to 6 unit/μl. Conclusions Results of this study revealed that AsAPN-1 is very similar to its counterpart in An. gambiae. In silico evaluation and fundamental data which are necessary for its evaluation as a VIMT-based vaccine in the next steps were acquired in this study and those could be useful for research groups that study on malaria vaccine for countries that An. stephensi is the main malaria vector there.

Abstract Background The recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined. Methods The APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis. Results APL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus. Conclusions APL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa.

Abstract Background According to scientific recommendations, paratransgenesis is one of the solutions for improving the effectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, affecting the fitness and longevity of vectors or reducing the vectorial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, findings showed that RNase III mutant bacteria are more competent for this aim. Asaia spp. have been introduced as potent paratransgenesis candidates for combating malaria and, based on their specific features for this goal, could be considered as effective dsRNA production and delivery tools to Anopheles spp. Therefore, we decided to characterize the rnc gene and its related protein to provide the basic required information for creating an RNase III mutant Asaia bacterium. Methods Asaia bacteria were isolated from field-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplification of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purified recombinant protein was assayed. Furthermore, Asaia RNaseIII amino acid sequence was analyzed by in silico approaches such as homology modeling and docking to determine its structural properties. Results In this study, the structure of rnc gene and its related operon from Asaia sp. was determined. In addition, by performing superimposition and docking with specific substrate, the structural features of Asaia RNaseIII protein such as critical residues which are involved and essential for proper folding of active site, binding of magnesium ions and double stranded RNA molecule to protein and cleaving of dsRNA molecules, were determined. Conclusions In this study, the basic and essential data for creating an RNase III mutant Asaia sp. strain, which is the first step of developing an efficient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in different medically-important vectors and these data are potentially very helpful for researchers studying paratransgenesis and vector-borne diseases and are interested in applying the RNAi technology in the field.

Larval therapy with Lucilia sericata is a promising strategy in wound healing. Axon guidance molecules play vital roles during the development of the nervous system and also regulate the capacity of neuronal restoration in wound healing. Netrin-1, one of the proteins that larvae secrete, plays a useful role in cell migration and nerve tissue regeneration. The UNC-5 receptor combines with a netrin-1 signal and transmits the signal from one side of the membrane to the other side, initiating a change in cell activity. In the current study, we identified the full length of the UNC-5 receptor mRNA in L. sericata using different sets of primers, including exon junction and specific region primers. The coding sequence (CDS) of the UNC-5 receptor was sequenced and identified to include 633 base-pair nucleic acids, and BLAST analysis on its nucleotide sequence revealed 96% identity with the Lucilia cuprina netrin-1 UNC-5 receptor. The protein residue included 210 amino acids (aa) and coded for a protein with 24 kD weight. This gene lacked the signal peptide. Furthermore, the UPA domain is conserved in UNC-5. It lied at the interval of 26–131 aa. We identified the CDS of netrin-1 UNC-5 receptor in L. sericata. It could be applied to research activities implementing a new essential component design in wound healing.

Mosquitoes are vectors of viruses affecting animal and human health. In Iran, the prevalence of mosquito-borne viruses remains poorly investigated. Once infected, mosquito females remain infected for all their life making virus detections possible at early steps before infections are reported in vertebrate hosts. In this study, we used a recently developed high-throughput chip based on the BioMark Dynamic arrays system capable of detecting 37 arboviruses in a single experiment. A total of 1,212 mosquitoes collected in Mazandaran, North-Khorasan, and Fars provinces of Iran were analyzed. Eighteen species were identified, belonging to five genera; the most prevalent species were Anopheles maculipennis s.l. (42.41%), Culex pipiens (19.39%), An. superpictus (11.72%), and Cx. tritaeniorhynchus (10.64%). We detected chikungunya virus (CHIKV) of the Asian genotype in six mosquito pools collected in North Khorasan and Mazandaran provinces. To our knowledge, this is the first report of mosquitoes infected with CHIKV in Iran. Our high-throughput screening method can be proposed as a novel epidemiological surveillance tool to identify circulating arboviruses and to support preparedness to an epidemic in animals and humans.

Abstract Background In recent years, the genus Asaia (Rhodospirillales: Acetobacteraceae) has been isolated from different Anopheles species and presented as a promising tool to combat malaria. This bacterium has unique features such as presence in different organs of mosquitoes (midgut, salivary glands and reproductive organs) of female and male mosquitoes and vertical and horizontal transmission. These specifications lead to the possibility of introducing Asaia as a robust candidate for malaria vector control via paratransgenesis technology. Several studies have been performed on the microbiota of Anopheles mosquitoes (Diptera: Culicidae) in Iran and the Middle East to find a suitable candidate for controlling the malaria based on paratransgenesis approaches. The present study is the first report of isolation, biochemical and molecular characterization of the genus Asaia within five different Anopheles species which originated from different zoogeographical zones in the south, east, and north of Iran. Methods Mosquitoes originated from field-collected and laboratory-reared colonies of five Anopheles spp. Adult mosquitoes were anesthetized; their midguts were isolated by dissection, followed by grinding the midgut contents which were then cultured in enrichment broth media and later in CaCO3 agar plates separately. Morphological, biochemical and physiological characterization were carried out after the appearance of colonies. For molecular confirmation, selected colonies were cultured, their DNAs were extracted and PCR was performed on the 16S ribosomal RNA gene using specific newly designed primers. Results Morphological, biochemical, physiological and molecular results indicated that all isolates are members of the genus Asaia. Conclusions Contrary to previous opinions, our findings show that Asaia bacteria are present in both insectary-reared colonies and field-collected mosquitoes and can be isolated by simple and specific methods. Furthermore, with respect to the fact that we isolated Asaia within the different Anopheles specimens from distinct climatic and zoogeographical regions, it is promising and may be concluded that species of this genus can tolerate the complicated environmental conditions of the vector-borne diseases endemic regions. Therefore, it can be considered as a promising target in paratransgenesis and vector control programs. However, we suggest that introducing the new technologies such as next generation sequencing and robust in silico approaches may pave the way to find a unique biomarker for rapid and reliable differentiation of the Asaia species.

Background: The salivary glands of Lucilia sericata are the first organs to express specific endopeptidase enzymes. These enzymes play a central role in wound healing, and they have potential to be used therapeutically. Methods: Rapid amplification of cDNA ends and rapid amplification of genomic ends were used to identify the coding sequence of MMP-1 from L. sericata. Different segments of MMP1 gene, namely the middle part, 3′ end, and 5′ end, were cloned, sequenced, and analyzed using bioinformatics tools to determine the distinct features of MMP-1 protein. Results: Assembling the different segments revealed that the complete mRNA sequence of MMP-1 is 1932 bp long. CDS is 1212 bp long and is responsible for the production of MMP-1 of 404 amino acid residues with a predicted molecular weight of 45.1 kDa. The middle part, 3′ end, and 5′ end sequences were 933, 503, and 496 bp. In addition, it was revealed that the MMP-1 genomic sequence includes three exons and two introns. Furthermore, the three-dimensional structure of L. sericata MMP-1 protein was evaluated, and its alignment defined that it has high similarity to chain A of human MMP-2 with 100% confidence, 72% coverage, and 38% identity according to the SWISS-MODEL modeling analysis. Conclusions: MMP-1 of L. sericata has a close relationship with its homologs in invertebrates and other insects. The present study significantly contributes to understanding the function, classification, and evolution of the characterized MMP-1 from L. sericata and provides basic required information for the development of an effective medical bioproduct.

Non-invasive therapeutic methods have recently been used in medical sciences. Enzymes have shown high activity at very low concentrations in laboratories and pharmaceutical, enabling them to play crucial roles in different biological phenomena related to living organism, especially human medicine. Recently, using the therapeutic methods based on non-invasive approaches has been emphasized in medical society. Researchers have focused on producing medicines and tools reducing invasive procedures in medical. Collagenases are proteins which catalyze chemical processes and break the peptide bonds in collagen. Collagen may be generated more than the required amount or produced in unsuitable sites or may not degrade after a certain time. In such cases, using an injectable collagenase or its ointment can be helpful in collagen degradation. In both in vitro and in vivo tests, it has been revealed that collagenases have several therapeutic properties in wound healing, burns, nipple pain and some diseases including intervertebral disc herniation, keloid, cellulite, lipoma among others. This review describes the therapeutic application of collagenase in medical sciences and the process for its production using novel methods, paving the way for more effective and safe applications of collagenases.

Abstract The dual occurrence of Pseudomonas‐like and Wolbachia endobacteria has not been investigated in the Pederus beetles yet. We investigated pederin‐producing bacteria (PPB) infection in Paederus fuscipes specimens from the southern margins of the Caspian Sea by designed genus‐specific (OprF) and species‐specific (16S rRNA) primers. Wolbachia infection was studied through a nested‐PCR assay of Wolbachia surface protein (wsp) gene. Of the 125 analyzed beetles, 42 females (82.35%) and 15 males (20.27%) were positive to PPB infection; this is the first study reporting male P. fuscipes infection to PPB. Wolbachia infection was found in 45 female (88.23%) and 50 male (67.57%) analyzed beetles. Surprisingly, a number of 36 females (70.59%) and 13 males (17.57%) were found to be infected with both PPB and Wolbachia endosymbionts. In general, population infection rates to PPB and Wolbachia were determined to be 45.6% and 76%, respectively. The infection rates of female beetles to PPB and PPB‐Wolbachia were significantly higher than males. In Paederus species, only female beetles shelter PPB and the discovery of this bacterium in adult males may reflect their cannibalistic behavior on the contaminated stages. Phylogenetic analysis showed that the sequences of OprF gene were unique among Pseudomonas spp.; however, sequences of 16S rRNA gene were related to the PPB of Pederus species. The co‐occurrence and random distribution of these endobacteria may imply putative tripartite interactions among PPB, Wolbachia, and Paederus. In order to elucidate these possible tripartite interactions, further studies are required even at gender level.

Background: Some mosquito species which belong to the Culex. pipiens complex are primary vectors for West Nile virus, Sindbis, Dirofilaria immitis, and many arboviruses. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene of Cx. pipiens that is inherited, is one of the important threats for the efficacy of pyrethroids insecticides. Knockdown resistance (kdr) mutation, L1014F, is a well-defined mechanism of resistance to pyrethroids and DDT in many insect species. The aim of study was to determine the mechanisms of Insecticide resistance in this species Methods: Specimens of Cx. pipiens, the major vector of West Nile virus, were obtained in Tehran, Iran by collecting larvae from polluted wastewater in Qarchak of Tehran. In 2016 Insecticide susceptibility tests were performed according to WHO methods with deltamethrin 0.05%. We focused on determination of this point mutation in the VGSC gene of Cx. pipiens by Real-time PCR. Results: Our results revealed high levels of resistance to deltamethrin 0.05%. The lethal times i.e. LT50 and LT90 for deltamethrin were 2.1530 and 8.5117 h respectively. The result of Real-time PCR confirmed the presence of resistant genotype in all the members of tested population. This study is the first report on kdr genotyping of Cx. pipiens from Tehran and our results on the VGSC gene in position L1014F confirmed the TTA to TTT nucleotide change. Conclusion: This finding will provide a clue for management of insecticide resistance in mosquito which are vectors of arboviruses and decision for replacement of novel approach for vector control.

Background: Mosquito galactose-specific C-type lectins (mosGCTLs), such as mosGCTL-1, act as ligands to facilitate the invasion of flaviviruses like West Nile virus (WNV). WNV interacts with the mosGCTL-1 of Aedes aegypti (Culicidae) and facilitates the invasion of this virus. Nevertheless, there is no data about the role of mosGCTL-1 as a transmission-blocking vaccine candidate in Culex pipiens, the most abundant Culicinae mosquito in temperate regions. Methods: Adult female Cx. pipiens mosquitoes were experimentally infected with a WNV infectious blood meal, and the effect of rabbit anti-rmosGCTL-1 antibodies on virus replication was evaluated. Additionally, in silico studies such as the prediction of protein structure, homology modeling, and molecular interactions were carried out. Results: We showed a 30% blocking activity of Cx. pipiens mosGCTL-1 polyclonal antibodies (compared to the 10% in the control group) with a decrease in infection rates in mosquitoes at day 5 post-infection, suggesting that there may be other proteins in the midgut of Cx. pipiens that could act as cooperative-receptors for WNV. In addition, docking results revealed that WNV binds with high affinity, to the Culex mosquito lectin receptors. Conclusions: Our results do not support the idea that mosGCTL-1 of Cx. pipiens primarily interacts with WNV to promote viral infection, suggesting that other mosGCTLs may act as primary infection factors in Cx. pipiens.

Vector competence is an important parameter in evaluating whether a species plays a role in transmission of an arbovirus. Although the protocols are similar, interpretation of results is unique given the specific interactions that exist between a mosquito population and a viral genotype. Here, we assessed the infection (IR), dissemination (DR), and transmission (TR) rates of Cx. pipiens s.l., collected from Iran, for West Nile virus (WNV) lineage 1a. We showed that Cx. pipiens s.l. mosquitoes in Iran were susceptible to WNV with IR up to 89.7%, 93.6%, and 83.9% at 7, 14, and 21 days post-infection (dpi) respectively. In addition, DR and TR reached respectively 92.3% and 75.0% at 21 dpi, and the number of viral particles delivered with saliva reached up to 1.33 × 105 particles. Therefore, an unexpected high risk of WNV dissemination in the region where Cx. pipiens s.l. mosquitoes are well established should be considered carefully and surveillance measures implemented accordingly.

The midgut microbiota associated with Anopheles stephensi and Anopheles maculipennis (Diptera: Culicidae) was investigated for development of a paratransgenesis-based approach to control malaria transmission in Eastern Mediterranean Region (EMR). Here, we present the results of a polymerase chain reaction (PCR) and biochemical-based approaches to identify the female adult and larvae mosquitoe microbiota of these two major malaria vectors, originated from South Eastern and North of Iran. Plating the mosquito midgut contents from lab-reared and field-collected Anopheles spp. was used for microbiota isolation. The gram-negative and gram-positive bacterial colonies were identified by Gram staining and specific mediums. Selected colonies were identified by differential biochemical tests and 16S rRNA gene sequence analysis. A number of 10 An. stephensi and 32 An. maculipennis adult mosquitoes and 15 An. stephensi and 7 An. maculipennis larvae were analyzed and 13 sequences of 16S rRNA gene bacterial species were retrieved, that were categorized in 3 classes and 8 families. The majority of the identified bacteria were belonged to the γ-proteobacteria class, including Pseudomonas sp. and Aeromonas sp. and the others were some closely related to those found in other vector mosquitoes, including Pantoea, Acinetobacter, Brevundimonas, Bacillus, Sphingomonas, Lysinibacillus and Rahnella. The 16S rRNA sequences in the current study aligned with the reference strains available in GenBank were used for construction of the phylogenetic tree that revealed the relatedness among the bacteria identified. The presented data strongly encourage further investigations, to verify the potential role of the detected bacteria for the malaria control in Iran and neighboring countries.