Fleas are the most notorious blood-sucking ectoparasites that transmit a variety of zoonotic pathogens to infect domestic and wild animals (1). Fleas can host a wide range of species, including human, domestic pets, rodents, and ungulates, resulting in a shortened host lifespan, while ecologically related hosts might share flea species, which increases the public health impact of fleas and causes serious economic losses (2). Fleas play a significant role in maintaining the plague’s natural foci (3). Several important pathogens have been identified in fleas, including plague, epidemic hemorrhagic fever, tularemia, leptospirosis, and murine typhus (4). Due to the significant medical, veterinary, and economic value of fleas, increasing attention has been placed on diseases transmitted by fleas, which currently cost global more than $15 billion annually (5). However, to date, phylogenetic relationships and clear classification among fleas have remained ambiguous, which has greatly hindered the control of fleas.

Ceratophyllus anisus belonging to the Siphonaptera order, Ceratophyllidae family, Ceratophyllus genus, is the main vector of plague. C. anisus can parasitize Rattus norvegecus, Rattus tanezumi, Apodemus agrarius, and Crocidura attenuata, and occasionally human. C. anisus has been found in the former Soviet Union, Korea, and Japan, and is most widely distributed in Yunnan Province, China (6). Leptopsylla segnis, in the genus Leptopsylla of the family Leptopsyllidae, has been reported in Libya and Cyprus and is common in numerous provinces of China, where wild rodents are most often infested by it (7, 8). Due to the large number of pathogens carried by fleas and the low resolution of traditional taxonomic features for fleas, the choice of a rapid and accurate identification method is necessary for the control of fleas and flea-borne diseases (9).

With the development of molecular and phylogenetic studies, the mitochondrial genome has been used in ectoparasites taxonomy, systematics, and population genetics, which to some extent make up for the limitations of traditional morphology (10, 11). The mitochondrion, the organelles of eukaryotic cells that maintain the structure of life, possess their genetic material and can replicate autonomously outside the nucleus. Mitochondrial DNA (mtDNA) has the advantages of simple structure, little recombination, fast evolution rate, and maintains inheritance in the process of evolution, which makes it an effective tool for studying species identification, relatedness, and phylogeny (12, 13). However, the mitochondrial genome data of fleas is still extremely scarce, and the phylogenetic relationship has not been established, which is a major obstacle to the prevention and control of fleas and flea-borne diseases.

This study is the first to sequence and analyze the mitochondrial genomes of C. anisus and L. segnis, with the aim of contributing to their correct identification and classification, enriching the mitochondrial genome database of fleas, facilitating the prevention and control of diseases caused by them to minimize the risk to hosts and humans, and providing new and useful markers for further species identification and molecular epidemiological studies. Genetic markers for further species identification and molecular epidemiological studies. We also studied the phylogenetic relationships with the gene sequences of other flea species in NCBI, which provides an important basis for population genetic, phylogenetic and evolutionary analysis.

As a temporary host and vector of some important human infectious diseases such as plague and endemic typhus, fleas are early warning indicators for judging the prevalence of plague and other human-animal infectious diseases (24). In recent years, plague has rebounded in some regions, with an increasing trend in incidence, which has always been a persistent and difficult problem worldwide. C. anisus and L. segnis are common species of fleas that play an important role in the transmission of zoonotic diseases.

The D-loop region has low evolutionary pressure, a large number of gene rearrangements, and rapid base substitutions, making it an effective molecular marker for population genetic studies. The frequency and location of the D-loop vary from species to species and tissue to tissue, and its length is influenced by the number of tandem repeat copies, which in turn affects the length of the entire mitogenome (25). One D-loop region was found for X. cheopis, P. irritans, and C. wui, and two D-loops existed for Ctenocephalides felis, C. anisus, and L. segnis. The two control regions were also found in some ticks and sea cucumbers, and the mtDNA was replicated more efficiently, so it is speculated that the two D-loop regions acted synergistically during the evolutionary process (26). The mtDNAs of C. anisus and L. segnis have the same gene composition and arrangement as that of most flea species. Bases mismatch appears in most tRNA genes, and G-U wobble base pairs conform to the oscillating pairing principle, which is very important for maintaining the stability of the tRNA secondary structure (27).

The phylogenetic tree derived using all flea mitochondrial genomic data in the NCBI gene bank shows that the five families are divided into two distinct branches, with Ceratphyllidae, Leptosyllidae, Vermipsyllidae, and Hystrichopsyllidae clustered into one, and the Pulicidae family as the other branch. The branch where L. segnis is located and the C. anisus and C. wui branches form a sister group with high node support. The same species of fleas from different hosts and different geographical locations are clustered together in this phylogenetic tree with posterior probabilities and bootstrap values of 1 and 100, respectively, with a high degree of confidence.

The mtDNA is a valuable marker for population biology, species identification classification, and phylogenetic studies, especially for assessing genetic diversity and identifying cryptic species as well as population structure. There are still gaps in molecular data for C. anisus and L. segnis, which is a major obstacle to the development of flea species. In this study, we obtained the complete mitochondrial genome, which provides more accurate evidence for the phylogenetic relationships of flea species. To better understand the phylogenetic relationship among fleas, the mitochondrial genome study within the Siphonaptera order must be expanded. We expect that the complete mitogenomes of C. anisus and L. segnis will provide important genome information for molecular phylogenetic studies and contribute to clarifying the phylogeny and evolution of Siphonaptera.

In this study, the complete mitochondrial genomes of C. anisus and L. segnis are sequenced and annotated for the first time by long-range PCR combined with Illumina sequencing technology which will be helpful for future research on fleas. The results of this study contributed to the fleas, filling the flea mitochondrial genome database resources and laying the foundation for further understanding the phylogenetic relationships of the fleas. With the development of molecular biology, sequencing techniques using mitochondrial genomes as molecular markers have effectively bridged the morphological gap and have been widely used in species identification, kinship, and evolutionary studies.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

The animal study was reviewed and approved by Laboratory Animal Management Committee of Dali University and First Affiliated Hospital of Chengdu Medical College.

YL conceived the study and wrote the manuscript. BC, XL, and DJ carried out the experiments and analysed the data. TW and LG contributed to the collection of C. anisus and L. segnis and discussions. QZ and XY is responsible for the interpretation of experimental data, critical revision of important knowledge content, and final approval of the version to be published. All authors contributed to the article and approved the submitted version.

This work was supported by the Yunnan Natural Science Foundation (2017FD139) and Scientific Research Fund of Yunnan Education Department (2022J0687).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.