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.