Application of Molecular Techniques for Identification of Insects Blood meal

Abstract

Abstract Malaria and leishmaniasis are endemic diseases in Sudan and they represent major health problems. Malaria is caused by Plasmodium spp and transmitted by mosquitoes while leishmaniasis is caused by Leishmania parasites and transmitted by sand flies. The life cycle of the two parasites includes many wild and domestic reservoir hosts. Identification of the insect vector, its preferred habitats and different habits represent a milestone in designing control programmes. The objectives of this study were to evaluate the effect of two different DNA extraction methods (Collin’s method and Steiner’s method), in the quantity and quality of extracted host DNA from the blood meal of the insect vector, and to apply a RFLP- PCR protocol to identify the source of the blood meal which can be used as an indicator of the host-preferences of the vector. Wild sandfly specimens were collected from a leishmaniasis endemic site in Gedarif State, Southeastern Sudan, while blood fed female Anopheles arabiensis mosquitoes were obtained from a laboratory colony reared at the Department of Medical Entomology, Ministry of Health. The quantity and quality of extracted DNA was measured using Nanodrop spectrophotometer and the efficiency of each of the extraction methods used was estimated by comparing the quality and quantity of the extracted DNA using statistical analysis. All the extracted blood meal samples were screened twice, one time for the presence of human blood and another time for the presence of animal blood. Positive and negative controls were run simultaneously with the samples to ensure no contamination occurs during the extraction steps, and only DNA from extracted blood meal is amplified not the DNA from insect itself. The two extraction methods showed significant differences (p<0.05) in the quantity and purity of the extracted DNA with Steiner’s method was found to be suitable for DNA extraction from sandflies. The two extraction method didn’t show any significance difference (p>0.05) in the DNA concentration for mosquito samples. However, a significant difference (p<0.05) was observed when sandfly samples were used, with Steiner method produced much DNA concentration than Collin’s one. On the other hand, Steiner method showed high ii ratio of protein contamination in both mosquito and sandfly specimens. While Collin’s method showed a higher ratio of other contaminants for mosquito specimens. No PCR products were obtained when the primers amplifying human blood were used for sandfly samples. It couldn't be confirmed that no amplification were obtained due to the low human DNA concentration in the blood meal or because there was no human blood in the extracted samples. Or may be humans were not preferred hosts for the collected sand flies. Concerning mosquito samples, no human DNA was amplified because the used specimens were from a colony which has been fed by a cow blood. Only 9 sand flies and 3 mosquitoes gave bands when amplified using the animal primer pairs. This indicates that animals were available at the collection sites and they were preferred hosts. When the amplified bands were digested using Hae III restriction enzyme which digests the amplified cyt b fragment from the vertebrates host blood; no bands were observed. This may be due to the low concentration of the amplified fragment or the inefficiency of the enzyme in recognizing the restriction sites. Large number of freshly collected samples, inclusion of a DNA purification step and the use of more sensitive molecular approach were recommended.