Resistance Mechanisms of Anopheles stephensi (Diptera: Culicidae) to Temephos.

BACKGROUND: Anopheles stephensi is a sub-tropical species and has been considered as one of the most important vector of human malaria throughout the Middle East and South Asian region including the malarious areas of southern Iran. Current reports confirmed An. stephensi resistance to temephos in Oman and India. However, there is no comprehensive research on mechanisms of temephos resistance in An. stephensi in the literature. This study was designed in order to clarify the enzymatic and molecular mechanisms of temephos resistance in this species. METHODS: Profile activities of α- and ß-esterases, mixed function oxidase (MFO), glutathione-S-transferase (GST), insensitive acetylcholinesterase, and para-nitrophenyl acetate (PNPA)-esterase enzymes were tested for An. stephensi strain with resistance ratio of 15.82 to temephos in comparison with susceptible strain. RESULTS: Results showed that the mean activity of α-EST, GST and AChE enzymes were classified as altered indicating metabolic mechanisms have considerable role in resistance of An. stephensi to temephos. Molecular study using PCR-RFLP method to trace the G119S mutation in ACE-1 gene showed lack of the mutation responsible for organophosphate insecticide resistance in the temephos-selected strain of An. stephensi. CONCLUSION: This study showed that the altered enzymes but not targets site insensitivity of ACE-1 are responsible for temephos resistance in An. stephensi in south of Iran.

Phylogenetic analysis of the oriental-Palearctic-Afrotropical members of Anopheles (Culicidae: Diptera) based on nuclear rDNA and mitochondrial DNA characteristics.

The phylogenetic relationships of Anopheles spp. at the junction of Oriental, Palearctic, and Afrotropical regions in the Iranian plateau were investigated using molecular markers. A 711-bp mitochondrial DNA cytochrome oxidase C subunit I (COI) fragment and the entire second internal transcribed spacer (ITS2) region (286-576 bp) of the nuclear ribosomal DNA (rDNA-ITS2) were sequenced from 14 and 28 taxa, respectively. The analyses included 12 species within Anopheles and 4 within the Myzorhynchus Series of the subgenus Anopheles, 8 within Neocellia, 6 within Myzomyia, 3 within Paramyzomyia, and 1 within the Pyretophorus Series of the subgenus Cellia. The congruent tree topologies of both molecular markers strongly supported monophyly of subgenera Anopheles and Cellia. Phylogenetic trees constructed on the basis of ITS2 sequences could accurately categorize all of the series according to the classical taxonomy but could not distinguish Pyretophorus (Anopheles subpictus) from Paramyzomyia Series. Although sequence data of the COI region were available for only 14 species, the inferred trees revealed good classification among the series but could not show the monophyletic relationship of Cellia spp. Except for a few cases, the tree inferred from ITS2 sequences revealed the best classification for the species studied. The molecular data could significantly improve our understanding of the phylogenetic position of the taxa.

Discrimination of relapsing fever Borrelia persica and Borrelia microtti by diagnostic species-specific primers and polymerase chain reaction-restriction fragment length polymorphism.

Tick-borne relapsing fever (TBRF) is endemic in Africa and Eurasia and attributed to different Borrelia species. In the Central Asia and Middle Eastern countries, TBRF is caused mainly by Borrelia persica; however, other Borrelia species such B. microtti, B. latyschewii, B. baltazardi, and B. caucasica have also been described. The classic taxonomy of Borrelia spp. is based on the cospeciation concept that is very complex and rather confusing. In this study, we report two DNA-based methods to discriminate B. persica and B. microtti, the two main prevalent species in the region. Molecular typing of the species was performed using (i) restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified fragments of either 16S-rRNA or glpQ genes, and (ii) species-specific PCR of glpQ gene. Sequence analyses of the data obtained in this study indicate that the glpQ gene is more variable than 16S-rRNA (6.9% vs. 1.2%); thus glpQ is a more useful marker for discrimination of B. persica from B. microtti. The 16S-rRNA fragment comprises only one useful species-specific restriction site (TaqI), whereas the glpQ fragment includes several species-specific restriction sites and its digestion by DraI, TaqI, EcoRV, HinfI, or SspI results in distinctively different PCR-restriction fragment length polymorphism patterns for the two species. Further, the species-specific primers amplified a 253-bp fragment for B. persica and a 451-bp one for B. microtti. Phylogenetic analysis of the data revealed that B. microtti and B. persica are associated to the African and new world RF agents, respectively. This study demonstrates that both typing methods are simple, sensitive, and fast, and that they allow one to differentiate between B. persica and B. microtti. This could prove that both methods are important and useful in monitoring of TBRF disease in endemic areas.

Vector incrimination of sand flies in the most important visceral leishmaniasis focus in Iran.

The prevalence, host preference, and rate of Leishmania spp. infection of sand fly species are important parameters for incrimination of parasite vectors. We applied polymerase chain reaction (PCR)-based and enzyme-linked immunosorbent assay (ELISA) methods to detect Leishmania spp. parasites and blood meals within individual sand flies in the most important visceral leishmaniasis (VL) focus in northwestern Iran. Leishmania spp. minicircles (kinetoplast DNA) were found in 14 (0.9%) of 1,569 female specimens. Sequence analysis of 650 basepairs of an internal transcribed spacer ribosomal DNA gene identified L. infantum/L. donovani in 12 specimens and L. adleri-like parasites in 2 specimens. Nine (64.3%) of 14 of the Leishmania spp.-positive sand flies were Phlebotomus perfeliewi transcaucasicus. Blood meal identification of host DNA within sand flies by PCR-based and ELISA methods showed that 30% and 28%, respectively, were positive for human blood. Results of this study showed that P. perfeliewi transcaucasicus is the most prevalent, infected, and anthropophagic sand fly and plays a major role in VL transmission in the region studied.

Phlebotomus perfiliewi transcaucasicus is circulating both Leishmania donovani and L. infantum in northwest Iran.

Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of the parasite isolated from dogs; this parasite was considered to predominate in the all foci of IVL in Iran. However, based on PCR detection and sequencing of parasite Cysteine Protease B (CPB), only one out of seven sandfly infections in Phlebotomus perfiliewi transcaucasicus was found to be L. infantum in the current investigation. The six other infections were haplotypes of Leishmania donovani, the causative agent of anthroponotic visceral leishmaniasis (AVL) in West Africa and India. The deduced amino acid of the L. donovani haplotype was found to be novel and the shortest CPB protein reported within the Leishmania spp. Circulation of both L. donovani and L. infantum by P. perfiliewi transcaucasicus, in addition to previous data indicating its ability to circulate L. tropica, suggests that this species, like other vectors of VL, is a permissive vector. Finding L. donovani infecting P. perfiliewi transcaucasicus in the area demands extensive and intensive typing of natural Leishmania infections in epidemiological investigations in Iran and the Mediterranean Basin in general.

Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L.infantum/L.donovani discrimination.

Discrimination of Leishmania infantum and L. donovani, the members of the L. (L.) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA (PCR-RFLP) and a PCR that are capable to discriminate L. donovani from L. infantum. Typing of the complex was performed by a simple PCR of cysteine protease B (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L. infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3' end of cpb gene of L. donovani whereas it does not generate an amplicon for L. infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci.