Background The head louse, Pediculus humanus capitis, is the most important ectoparasite causing many health problems. Several linkages are presented for this parasite, each representing a particular geographical distribution, prevalence rate, vector competence, susceptibility to pediculicides, and infestation rate. Determining the genetic nature of these linkages is necessary to identify the population structure and also to develop and monitor control programmes against head lice. This study was designed to analyse cox1 and cytb genes and determine the mitochondrial clades in head lice populations in the northwest of Iran. Methods Adult head lice were collected from infested females of Ardabil, East and West Azerbaijan, and Zanjan Provinces from 2016 to 2018. Partial fragments of the mitochondrial genes cox1 and cytb were amplified by PCR and some of the amplicons were sequenced. All confirmed sequences were analysed, and the frequency of each mitochondrial clade was determined in the studied areas. Results A total of 6410 females were clinically examined, and 897 adult head lice were collected from 562 infested cases. Genomic DNA was extracted from 417 samples, and fragments of cox1 and cytb genes were amplified in 348 individuals. Analysis of the 116 sequences showed the 632-bp and 495-bp fragments for cox1 and cytb genes, respectively. The nucleotide and haplotype diversities of cytb and cox1 genes were 0.02261 and 0.589 and 0.01443 and 0.424, respectively. Sequence analysis indicated 6 haplotypes clustered in two clades, A and B. The relative prevalence of clade B was 73% for cytb and 82% for cox1 gene. Haplotypes of clade B were found in all the studied areas, while those of clade A were observed only in rural and suburban areas. Conclusions To our knowledge, this is the first study investigated deeply the field populations of Pediculus and documented two clades in the Middle East. The considerable prevalence of pediculosis in the studied areas requires authorities’ attention to establish effective control and preventive measures. Given the role of cytb in monitoring population groups, application of this marker is suggested for future epigenetic studies to evaluate the factors affecting the abundance of these clades.

The human flea is an important ectoparasite causing serious public health problems worldwide. Planning and monitoring the control programs against this vector require the knowledge of population structure and vector competence. This study was carried out to identify molecular structure of internal transcribed spacer 1 (ITS1) of ribosomal gene and its capability in the survey of Pulex irritans populations as well as to investigate Rickettsia infection in these populations. Flea samples were collected via human baits from animal farms in two districts of Zanjan Province, northwest of Iran. The ITS1 region and the partial Rickettsia gltA gene were amplified from the samples of human flea, and 30 amplicons were sequenced. The 1136 collected fleas consisted of 1079 (94.98%) P. irritans, 36 (3.17%) Ctenocephalides canis, and 21 (1.85%) Ctenocephalides felis. Molecular investigation of 182 human fleas detected the infection of Rickettsia sp. in 4.9%. The ITS1 region covered 957 bp and contained three tandem units of 98-99 bp, starting at positions 145, 245, and 331. Multiple alignments of ITS1 sequences showed single-nucleotide polymorphism at position 798, which caused the substitution of cytosine for adenine in the novel haplotype. High frequency of P. irritans and its Rickettsia infection requires the application of vector control measures, and full characterization of Rickettsia sp. and its potential to cause disease in humans. Regarding the consistency of ITS1 region and its ability to differentiate insect communities, further investigations are recommended to identify the role of selective factors in maintenance of this spacer.

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The present study has been designed in order to verify the species composition within Anopheles maculipennis complex in North West of Iran. We determined ribosomal DNA sequences of the second internal transcribed spacer (ITS2) region from samples of Anopheles maculipennis Complex in Zanjan province. A total of 1536 specimens within the Complex were tested by Multiplex PCR, only An. maculipennis was found in this area. One clone out of four different individual mosquitoes of each field was generated with ITS2 PCR and half of them (192 samples) selected randomly for RFLPs. PCR-RFLP assay identified 2 haplotypes; haplotype I (99%) and haplotype II (1%). Twenty five sequences were generated comprising the 5.8S gene, the ITS2 and the 28S ribosomal gene. The alignment was 422 in length and percentage of GC content was 50.3% (26.07% A, 23.59% T, 26.78% C, 23.7% G). The ITS2 was 290 bp in length and two haplotypes were revealed varying by a single base (T<-->C) at site 378. An. maculipennis is the dominant species anopheline of the province. ITS2 analysis revealed evidence of a slightly interaspecific variation among populations. However, further investigations on the genetic polymorphism among An. maculipennis populations and in particular within those belonging to the continental haplotype are required to support any hypothesis on differences in behavior across the distribution range for this potential malaria vector.

Background: Anopheles maculipennis complex, the historic vector of malaria, causes serious medical problems worldwide and exhibits different behaviours. Studying the odorant-binding proteins (OBPs), which influence the chemosensory system and behavioural responses, is essential to understanding the population structure and developing effective control measures against this vector. The present study was designed to identify and analyse the obp1 gene in An. maculipennis. Methods: Adults of An. maculipennis sensu stricto were collected in Zanjan Province, northwest of Iran, and gDNAs of female mosquitoes were extracted. Fragments of An. maculipennis obp1 (Amacobp1) gene were amplified using degenerate and specific primers, and some of amplicons were selected for sequencing. Results: Analysis of amplified products identified that the sequence of Amacobp1 gene was 1341 bp long. This gene contains three exons (5′, internal, and 3′of 160, 256, and 18 bp, respectively) and encodes 144 amino acids. The sizes of introns I and II in deduced gene are 268 and 358 nucleotides, respectively. The amino acid sequence in the C-terminal of AmacOBP1 is similar to that of major malaria vector Anopheles species. However, its N-terminal has a specific signal peptide with 19 amino acids. This peptide is conserved in different studied populations, and its sequence of amino acids shows the most variation among anopheline species. Conclusions: Degenerate primers in this study are suggested for studying obp1 gene in Anopheles species. Ama-cobp1 gene is proposed as a molecular marker for the detection of intraspecific ecotypes and diagnosis of different species within Maculipennis Group. Moreover, the N-terminal of AmacOBP1 peptide is recommended as a molecular marker to identify the Amacobp1 expression patterns in different chemosensory organs for assessing the molecular mechanisms and developing novel behavioural disturbance agents to control An. maculipennis.

Background: Androctonus crassicauda is the most medically relevant animal and understanding its morphological characteristics is essential in the production of antiscorpion sera. Methods: Adults of A. crassicauda were collected from different areas of Zanjan Province and the morphometric pa­rameters and the cuticular fluorescence patterns of samples were studied. The crude venom of samples was extracted by electric stimulation, and their biochemical properties were analyzed by the SDS-PAGE method. Results: Values of the morphometric parameters depended on sex and altitude of the area. Except for values of the pec­tinal organ, these parameters in females were higher than in males. No significant difference was in the number, shape, and intensity of cuticular fluorescence patterns. The body length of males in high and lowlands was 72.53±1.53 and 77.33±2.70mm, respectively. Females' body lengths in that area were 81.66±2.19 and 86.55±2.33mm, respectively. Analysis of toxin proteins showed two isotypes that the 12, 13, 15, 16, 18, and 19kDa proteins were in all areas. How­ever, the 41 and 74kDa proteins, and 46 and 63kDa proteins were detected in low and highlands, respectively. Conclusion: Black fat-tailed scorpion has a considerable dominancy and developing preventive programs and providing treatment facilities in studied areas are necessary. Values of the morphological parameters and venom electrophoresis patterns depended on the geographical location. Therefore, pool crude toxin is suggested for the production of effective antivenoms. Moreover, additional field complementary works in the geographic information system based niche model­ing and mass fingerprinting of scorpion venoms are suggested for screening effective isotypes.

Background The tropical bed bug, Cimex hemipterus, is an important ectoparasite causing various health problems. This species is mainly confined to tropical regions; however, insecticide resistance, global warming, and globalization have changed its distribution map. Molecular information on pyrethroid resistance, which is essential for the development of control programs, is unknown for C. hemipterus in expanded areas. The present study was designed to determine the permethrin resistance status, characterize the pyrethroid receptor sites in voltage-gated sodium channel (vgsc) gene, and identify the resistance-related mutations in the populations of tropical bed bug in Iran. Methods Live bed bugs were collected, and adults of C. hemipterus were selected for bioassay and molecular surveys. Bioassay was performed by tarsal contact with permethrin 0.75% in mixed-sex of samples. Conventional and quantitative TaqMan and SYBR Green real-time PCR assays were conducted to characterize the vgsc gene and genotypes of studied populations. Results In the bioassay tests, the mortality rates were in the range of 30.7–38.7% and 56.2–77.4% in 24 and 48 h, respectively. The knockdown rates of studied populations were in the range of 32.2–46.6% and 61.5–83.8% in the first and second days, respectively. The KT50 and KT90 values in the Cimex lectularius Kh1 strain were presented as 5.39 and 15.55 h, respectively. These values in the selected populations of C. hemipterus varied from 27.9 to 29.5 and from 82.8 to 104.4 h, respectively. Knockdown time ratios (KR50 and KR90) in these populations varied from 5.17 to 6.17-fold compared with those of the C. lectularius Kh1 strain. Fragments of vgsc gene with 355 bp and 812 bp were amplified. Analysis of sequences revealed the A468T substitution, kdr-associated D953G, and super-kdr M918I and L1014F mutations in all populations. Conclusions The specific/sensitive, safe, and rapid diagnostic assays developed in this study are recommended for detection of kdr/super-kdr mutations and frequency of mutant alleles. The presence of super-kdr mutations and high resistance to permethrin in all the populations necessitate the reconsideration of control approaches against C. hemipterus. Graphical Abstract

Background: Androctonus crassicauda is the most medically relevant scorpion and understanding its genetic forms is essential for improvement of anti-venom sera, and risk management of scorpionism. Present study was designed to identify the variations of mitochondrial genes in different populations of A. crassicauda. Methods: Adults of A. crassicauda were collected from Zanjan Province during 2016–2017. Genomic DNA of samples was extracted and fragments of mitochondrial 16S, COI and ND1 genes were amplified and some of the amplicons were sequenced. Haplotype of samples were identified by multiple alignment of sequences, then phylogenetic trees of haplo­types were constructed. Results: Fragments of 352bp, 618bp and 680bp were amplified from 16S, COI and ND1 genes respectively. Nucleotide sequence in COI fragments was conserved, however, five haplotypes with some specific polymorphic sites were detect­ed in 16S and ND1 fragments. Haplotype I was dominant and found in all areas. Other haplotypes were rare and limited to specific regions. Analysis of the phylogenetic trees inferred from 16S and COI genes, confirmed a strong positive correlation between geographic and genetic distance. Conclusion: Mitochondrial COI, 16S and ND1 genes were detected suitable for identifying the population structure. Five genotypes were found using 16S and ND1 genes. To prepare and improve the anti-venoms quality, additional stud­ies are necessary to identify the toxin electrophoretic profile and geographical/ecological niche models of these geno­types in future.