3.3.6 Fleas processing and identification
Fleas were processed in the laboratory following standard procedure in (Hastriter and Whiting, 2003) cited in (Berrizbeitia et al ., 2017), involving passing fleas through series of reagents to make their feature clear enough to be identified with dichotomous key. After processing, fleas were examined under digital stereo microscope OPTA-TECH® and identified to genus and specie level following conventional dichotomous key as described in (Harimalalaet al ., 2021; Friggens et al ., 2020). Fleas were then mounted on a microscope glass slides in accordance with conventional procedures for fleas processing before identification.
Flea processing/clearing were involving puncturing of fleas at the region between abdominal sterna II and III using thin-fine needle after placing them on a wax-block. Punctured fleas were soaked in potassium hydroxide (10%) for 24 h to decolorize their dark color and make their features visible for identification. After this process fleas were immersed in distilled water for 30 min to wash excess potassium hydroxide and stop decolonization process. After 30 min fleas were removed and gently compressed on their abdomen to expel marinated soft tissues throughout the punctured hole followed with dehydration process in a series of ethanol solutions (70%, 80%, 95% and absolute) for 30 min in each step.
For clarification of flea’s exoskeleton, fleas were immersed in methyl salicylate for 15–20 min and then transferred to xylene for a minimum of 1 h. After then, they were mounted on a microscope glass slide in a Canada balsam (Campbell et al ., 2018) which finalized the process and fleas were ready for identification. Fleas voucher specimen of each species identified were reserved at college of veterinary medicine and biomedical sciences at Sokoine University of Agriculture (SUA), Morogoro Tanzania.