Leishmania macropodum, DNA

Panahi, Elina, Shivas, Martin, Hall-Mendelin, Sonja, Kurucz, Nina, Rudd, Penny A., Araujo, Rachel De, Skinner, Eloise B., Melville, Lorna & Herrero, Lara J., 2020, Utilising a novel surveillance system to investigate species of Forcipomyia (Lasiohelea) (Diptera: Ceratopogonidae) as the suspected vectors of Leishmania macropodum (Kinetoplastida: Trypanosomatidae) in the Darwin region of Australia, International Journal for Parasitology: Parasites and Wildlife 12, pp. 192-198 : 194

2.5. Insect identification and L. macropodum DNA extraction

For the purpose of this study only field-collected F. (Lasiohelea), the suspected vectors of L. macropodum , were identified and examined for parasite DNA. Forcipomyia (Lasiohelea) species were distinguished from other Ceratopogonid biting insects by their plain wing pattern covered with suberect macrotrichia, a well-developed empodium and claws. To identify F. (Lasiohelea) to species level, a representative 10% subset of F. (Lasiohelea) were mounted on slides in Hoyer's medium, consisting of acacia gum, chloral hydrate, glycerol, and distilled water. Slides were stored at 37 ̊C for six weeks followed by morphological identification using a taxonomic key for Australasian F. (Lasiohelea) ( Debenham, 1983).

Field-collected F. (Lasiohelea) kept in containers holding an FTAṜ card, were randomly selected for parasite examination. In order to assess whether F. (Lasiohelea) could support L. macropodum development beyond blood meal stage, insects were selected between 1 and 8 days from day of collection. Forcipomyia (Lasiohelea) were either examined for L. macropodum DNA individually or in pools (n = 2–15) and were grouped into five categories according to the number of insects in that respective pool (1, ≤ 2, 3–4, 5–6, or ≥ 7). Leishmania macropodum DNA extractions were performed using the Qiagen DNeasyṜ Blood & Tissue Kit according to manufacturer's instructions. Insects were homogenised in the manufacturer's lysis buffer containing 20 mg /mL proteinase K, followed by Qiagen's protocol for purification of total DNA from insects, with a final elution volume of 200 μL AE buffer.

Moreover, to determine species of F. (Lasiohelea) carrying L. macropodum , a subset of field-collected specimens (n = 50) was processed using a non-destructive DNA extraction method ( Dougall et al., 2011). Individually F. (Lasiohelea) were digested in the Qiagen's lysis buffer/ Proteinase K buffer without homogenisation. After a 3-h incubation at 56 ̊C, insects were removed from the lysis solution for morphological identification. Purification of total DNA was hereafter performed as describe above.