OdTx12/GNA, a chimeric variant of a ß excitatory toxin from Odontobuthus doriae, reveals oral toxicity towards Locusta migratoria and Tenebrio molitor.

OdTx12, a beta excitatory toxin from Odontobothus doriae had previously been identified and characterized. It had been shown that OdTx12 causes significant lethal effects on insects by injection but does not show any toxicity on mice. Due to the natural ineffectiveness of scorpion toxins to act as oral toxins, OdTx12 was fused to Galanthus nivalis agglutinin (GNA), a protein with the potential to cross the insect gut. The sequence of OdTx12/GNA gene was chemically synthesized, cloned in Escherichia coli, and expressed. The effect of the purified fusion protein (OdTx12/GNA) was assessed on the insect and mammalian cell lines, insect larvae and mice. Toxicity assay on insect cell culture (SF9 cell line) showed comparable toxicity between OdTx12 and OdTx12/GNA (LD50 of 0.0030 and 0.0048 µM, respectively). Also very similar mortality rates were observed by injecting OdTx12 and OdTx12/GNA to Locusta migratoria and Tenebrio molitor. Oral administration of OdTx12/GNA, after five days of feeding, resulted in 96.6% and 98.3% mortality of L. migratoria and T. molitor larvae with an LC50 of 0.69 and 0.43 nmol/g of insect food, respectively, while OdTx12 alone did not cause any toxic effects on the larvae orally, suggesting the role of GNA in delivering the toxin to the insect's haemolymph. No toxicity or mortality was observed after toxicity testing of OdTx12/GNA on a mammalian cell line (HEP-2) or any mortality in vivo, by testing the protein in the laboratory mouse. Herein, we demonstrated that the fusion protein OdTx12/GNA could be considered an effective toxin for the biological control of insects.

Cloning and expression of OdTx12, a ß excitatory toxin from Odontobuthus doriae, in Escherichia coli and evaluation of its bioactivity in Locusta migratoria.

The venom of Odontobuthus doriae contains several peptide toxins that interfere with the sodium channel function of cell membranes, some of which specifically act on the insect's sodium channel without affecting mammalian cells. In this study sodium channel toxins of Odontobuthus doriae were aligned to other closely related toxins by BLAST and ClustalW servers. Among these toxins, NaTx12 (OdTx12) showed more than 90% similarity to the most known beta excitatory toxin, AaHIT1; furthermore, our modeling studies confirmed high tertiary structure similarity of these proteins. OdTx12 was cloned and expressed in E.coli, using pET26-b and pET28-a expression vectors. Tris-tricine SDS-PAGE and Western blot analysis showed OdTx12 expression by pET28-a, only. After purification, bioactivity of the purified protein was analyzed by injection and oral administration to Locusta migratoria larvae, and toxicity to mammals was tested on mice. Injection of OdTx12 resulted in the killing of larvae with LD50 of 0.4 and 0.2 after 48 and 72 h respectively, but oral administration of OdTx12 had no significant effect on Locusta migratoria, nor did the injection to mice show any signs of toxicity. These results showed that OdTx12, as a novel ß excitatory toxin can be considered as a candidate for insect control purposes.

Molecular Characterization of Mosquitoes (Diptera: Culicidae) in Northwestern Iran by Using rDNA-ITS2.

Several mosquito species are vectors of disease; however, to understand their role in disease transmission, accurate species identification is of particular importance. Morphological identification is the main method used, but molecular techniques have emerged as a tool for the identification of closely related species. In this study, mosquitoes from the West Azerbaijan Province in northwestern Iran were characterized on the basis of their rDNA-ITS2 sequences. Nine populations of 6 species of mosquitoes belonging to the genera Anopheles, Culex, Culiseta, and Ochlerotatus were studied. To the best of our knowledge, ITS2 sequences of Culiseta longiareolata and Culex hortensis have been reported for the first time. In addition, ITS2 sequences of Culex theileri and Ochlerotatus caspius have been reported for the first time in Iran. Phylogenetic analysis based on ITS2 showed that subfamilies Anophelinae and Culicinae of the family Culicidae could be differentiated successfully and subgenera Anopheles and Cellia of the genus Anopheles were separated. The analysis showed that the genera Culex, Culiseta, and Ochlerotatus have diverged separately.

Fauna and Larval Habitats of Mosquitoes (Diptera: Culicidae) of West Azerbaijan Province, Northwestern Iran.

BACKGROUND: Several important diseases are transmitted by mosquitoes. Despite of the potential of the occurrence of some mosquito-borne diseases such as West Nile, dirofilariasis and malaria in the region, there is no recent study of mosquitoes in West Azerbaijan Province. The aim of this investigation was to study the fauna, composition and distribution of mosquitoes and the characteristics of their larval habitats in this province. METHODS: Larvae and adult collections were carried out from different habitats using the standard methods in twenty five localities of seven counties across West Azerbaijan Province. RESULTS: Overall, 1569 mosquitoes including 1336 larvae and 233 adults were collected from 25 localities. The details of geographical properties were recorded. Five genera along with 12 species were collected and identified including: Anopheles claviger, An. maculipennis s.l., An. superpictus, Culex pipiens, Cx. theileri, Cx. modestus, Cx. hortensis, Cx. mimeticus, Culiseta Longiareolata, Ochlerotatus caspius s.l., Oc. geniculatus and Uranotaenia unguiculata. This is the first record of Oc. geniculatus in the province. CONCLUSION: Due to the geographical location of the West Azerbaijan Province, it comprises different climatic condition which provides suitable environment for the establishment of various species of mosquitoes. The solidarity geographical, cultural and territorial exchanges complicate the situation of the province and its vectors as a threat for future and probable epidemics of mosquito-borne diseases.