Mitochondrial COI barcoding of Pulicidae fleas and ultrastructural differentiation of the cat flea by scanning electron microscopy

Fleas are widespread ectoparasites found across the globe—even in polar regions—and exhibit low host specificity, allowing them to infest both humans and animals, including birds. They feed on the blood of their hosts and serve as vectors for various infectious diseases, particularly in tropical and subtropical regions. In this study, the COI barcodes and ultrastructural characteristics using scanning electron microscopy (SEM) were performed to confirm classical morphological identification of cat flea taxonomic levels. Four species of medically important Pulicidae fleas were collected from hosts in various provinces of Thailand and identified based on their distinctive morphological characteristics: Xenopsylla cheopis, Echidnophaga gallinacea, Ctenocephalides felis and Ctenocephalides orientis. Phylogenetic analyses and calculated sequence distance based on mitochondrial COI barcodes were performed. The four species clearly formed monophyletic groups with low intraspecific distance (0 % -0.24 %) and high interspecific distance (4.60 % -21.26 %). Ctenocephalides felis and C. orientis were separated at the closely related level and separated into distinct clusters, with a sequence distance of 8.42 %. and C. orientis has shown closely genetic relationship with C. canis (4.60 %). Scanning electron microscopy (SEM) revealed ultrastructural characteristics that clearly differentiate C. felis and C. orientis, including differences in head shape and minute bristles on the dorsal end of the antennal fossa. Specifically, C. felis frons are elongated and pointed anteriorly, whereas C. orientis frons are short and rounded anteriorly. Additionally, the C. orientis female has 3–4-minute bristles at the dorsal end of the antennal fossa, while this structure is absent in the C. felis female but present and numerous (with 13–18 bristles) in all males of the genus Ctenocephalides. Fleas were identified, and their sex or ambiguous structures were determined using a stereoscope or low-power binocular microscope. DNA barcoding and ultrastructural analysis using SEM for differentiation of structures of taxonomic significance are useful for subspecies/species identification.