Molecular characterization of the oriental cat flea, Ctenocephalides orientis, parasitizing goats based on nuclear and mitochondrial protein-coding genes

Abstract

Fleas are wingless ectoparasites that feed on the blood of warm-blooded animals and play a significant role as vectors of several medically and veterinary-relevant diseases. The oriental cat flea, Ctenocephalides orientis, is endemic to Asia and infests dogs more frequently than cats. However, its presence in small ruminants remains largely unexplored. Between January 2017 and October 2023, flea surveys were conducted on goat farms across seven different provinces in Thailand. Initially, flea specimens were identified using morphological keys and, subsequently, confirmed through molecular analysis of the mitochondrial genes cytochrome c oxidase subunit 1 (cox1, 450 bp) and cytochrome c oxidase subunit 2 (cox2, 678 bp), the nuclear ribosomal internal transcribed spacer 1 (ITS1, 828 bp) and the elongation factor-1 alpha (EF-1α, 904 bp) gene. In addition to characterizing these markers, the mitochondrial genome, including all protein-coding genes (PCGs), was amplified, analyzed, and subjected to comparative analyses. Among 500 goats examined, 33 (6.6%) were infested with fleas, which belonged to only one species, C. orientis. Pairwise genetic distance analysis and phylogenetic reconstruction strongly supported the placement of C. orientis within a distinct clade, consistent with the reference sequences. Of the four genetic markers analyzed, EF-1α exhibited the highest diversity. The partial mitochondrial genome of C. orientalis (14,315 bp) encoding 34 genes, including 13 PCGs, 19 transfer RNA genes, and two ribosomal RNA genes, was sequenced. Phylogenetic and genetic distance analyses based on multiple molecular markers and the mitochondrial genome revealed a close evolutionary relationship between C. orientis and C. canis. These findings confirmed that C. orientis is not only restricted to companion animals but also infests goats, suggesting its potential role in disease transmission to other animals. Furthermore, the study findings provide a dataset of both nuclear and mitochondrial molecular markers, which would facilitate future research on the taxonomy, phylogeny, and evolutionary relationships of fleas.

Keywords: Ctenocephlides orientis; EF-1α; Flea; Genetic diversity; Mitochondrial genome; cox1; cox2.

Graphical abstract

Fig. 1

A satellite-based map of Thailand depicting the collection sites of goat fleas surveyed across seven provinces. The map was obtained using Google Earth Pro v7.3.6.9345 and edited further in Microsoft PowerPoint v.2203.

Fig. 2

A Phylogenetic relationships between Ctenocephalides orientis concatenated cox1 and cox2 haplotypes obtained in this study (highlighted in red) and those previously identified in companion animals. B A heatmap illustrating pairwise genetic distances based on cox1 and cox2 sequences, including 30 sequences with a total length of 1108 bp from this study (n = 9), along with reference sequences (n = 21). The phylogenetic tree was constructed using the maximum likelihood method (K3Pu+F+I model) with 1000 bootstrap replicates, while the heatmap was generated using the Kimura 2-parameter (K80) model. Pulex irritans was used as the outgroup.

Fig. 3

A Phylogenetic relationships among Ctenocephalides orientis concatenated EF-1α and ITS1 sequences obtained in this study (highlighted in red) and those previously identified in companion animals. B A heatmap illustrating pairwise genetic distances based on concatenated EF-1α and ITS1 sequences, including 20 sequences with a total length of 1229 bp from this study (n = 17; highlighted in red), along with reference sequences (n = 3). The phylogenetic tree was constructed using the maximum likelihood method (TIM3e+I model) with 1000 bootstrap replicates, while the heatmap was generated using the Kimura 2-parameter (K80) model. Pulex irritans was used as the outgroup.

Fig. 4

Linear representation of the circular mitochondrial genome of Ctenocephalides orientis obtained in this study. Horizontal bars on the mitochondrial genome map indicate the positions of protein-coding genes, labeled using standard nomenclature, as well as ribosomal RNA genes. Dark blue bars with annotated labels denote the positions of transfer RNA (tRNA) genes. Mitochondrial genome features are color-coded, as indicated in the figure legend. The genome contains two tRNA genes for leucine (L1 for codons CUN and L2 for UUR) and two tRNA genes for serine (S1 for codons AGN and S2 for UCN). Protein-coding and ribosomal RNA genes located above the blue line are encoded on the plus strand, while those below the line are encoded on the minus strand.

Fig. 5

The relative synonymous codon usage (RSCU) in Ctenocephalides orientis observed in this study. The box below the bar chart displays all codons encoding each amino acid, while the height of the columns above represents the total RSCU values for each set of codons.

Fig. 6

Phylogenetic relationships of Ctenocephalides orientis obtained in this study and 23 other Siphonaptera species, inferred from BI analysis using 10,989 bp from all 13 mitochondrial protein-coding genes under the GTR+G+I model. Boreus elegans (GenBank: HQ696579) was used as the outgroup. The Ctenocephalides orientis sequence from this study is highlighted in red. Bayesian posterior probabilities (Bpp) are indicated at the nodes.