Fleas (Order Siphonaptera) are small, wingless insects with laterally compressed bodies undergo holometaboliam and parasitize mammals and birds (1, 2). Over, 2500 species of fleas have been identified, with approximately 200 species capable of harboring epidemic bacteria (3, 4). These serve as vectors for a range of pathogens, including Rickettsia, Bartonella, bubonic plague, and Tularemia, thereby functioning as both disease-causing vectors and reservoir hosts (5, 6). As a result of changes in the environment and human behavior, the vector-host ecology has changed, increasing human exposure to flea vectors and the pathogens they transmit, and flea-borne diseases may re-emerge as epidemics (7). Fleas are an early warning indicator of the plague epidemic, which has important significance in medicine and veterinary medicine (8). Fleas and flea-borne diseases are increasingly threatening human and animal health and causing serious economic losses, so flea identification is of great practical importance for flea-borne disease prevention and control.

Both Frontopsylla spadix and Neopsylla specialis are found in the Siphonaptera order, belonging to the families Leptopsyllidae and Ctenophthalmidae, to the Amphipsyllinae and Neopsyllinae, and Frontopsylla and Neopsylla, respectively. Species F. spadix are parasitic on wild rodents, specifically Apodemus chevrieri and Rattus flavipectus, found in regions such as Yunnan, Gansu, and Tibet in China, and extending into Nepal. This geographic distribution is considered a conduit for the transference of plague from wild to domestic rodents (9). Species N. specialis, parasitizing mammals like Apodemus chevrieri, Apodemus draco, and Apodemus latronum, is in China and acts as a principal vector of the plague in Yunnan's natural foci, akin to species F. spadix (10). Morphological characteristics of F. spadix and N. specialis have been described previously, the identification resolution of traditional flea classification methods is low, which may have certain limitations (2).

Mitochondria are placed in cells that produce energy and have a separate set of genetic material called mitochondrial DNA (11). Mitochondrial DNA is one of the most commonly used molecular markers in systematics and is widely used in phylogenetic studies of different organisms because of its simple structure, maternal inheritance, and rapid evolutionary rate (12). The analysis of mitochondrial genome structure and sequence is helpful to clarify the classification, genetic evolution, and phylogenetic relationship of fleas more clearly (13). However, at present, the mitochondrial genome data of fleas is very limited, resulting in a huge obstacle to fleas and flea-borne diseases. Therefore, we need to continuously increase and improve the flea mitochondrial gene database to lay the foundation for flea taxonomy, population genetics, and phylogeny.

In this study, we provide the first complete description of the mitochondrial genomes of F. spadix and N. specialis, analyze mitogenome structures to address the lack of mitochondrial gene resources in fleas, and construct the phylogenetic relationships of known mitochondrial genomes in the order Siphonaptera, while providing molecular information for flea prevention and control.

The endeavor to taxonomically identify and comprehend the ecological proclivities of fleas stands as an integral foundation in the mitigation and management of a plethora of naturally transpiring diseases. Fleas, being a medically salient insect group, partake substantially in perpetuating major plague epidemics and preserving natural epidemiological origin (22). Their abundance and widespread dissemination render them instrumental in the animal-mediated propagation of diseases during epidemic occurrences. Species F. spadix and N. specialis, as principal flea vectors, are posited to serve as conduits for the transmission of wild rodent plagues into domestic rats, thereby instigating epidemics.

In an examination of mitogenomes of F. spadix and N. specialis a pronounced predilection for AT bases was discerned, with an AT content surpassing that of GC, a feature congruent with arthropods (23). An anomalous stop codon was detected in N. specialis, subsequently rectified to TAA by PolyA complementation to terminate translation (24). The control region, harboring initiation sites that govern the replication and transcription of the mitochondrial genome, evolves at a rate three to five times that of other regions, accounting for its employment in population genetics and origin evolution studies (25, 26). Variability in the number and location of non-coding regions is evidenced across species, with singular, dual, and triple D-Loops manifesting in different species. Within this study's purview, species F. spadix was devoid of D-Loop, while species N. specialis harbored two, spanning 1,536 base pairs.

The six families are split into two large clades, as shown by phylogenetic clustering, except the family Pulicidae, where the remaining five families cluster in the other clade, with Paradoxopsyllus custodis located in the Superfamily Ceratophylloidae as a member of the family Leptopsyllidae, which is a discovery that also indicates that expanded sequencing of mitochondrial genomic data is beneficial for more intensive phylogenetic studies of the species. However, individual mitochondrial genes are less informative than the whole mitogenome which may bias the reflection of phylogenetic relationships (27), and in order to make the phylogenetic relationships of the flea more convincing, we need to sequence the whole mitochondrial genome of the flea more frequently (11).

Accurate differentiation and identification of flea species are essential in the diagnosis of disease and basic and applied research on these important ectoparasites. The mitochondrial genome is frequently used in phylogenetic and phylogenetic studies of different ectoparasites at various taxonomic levels due to its matrilineal inheritance, lack of recombination, and rapid rate of evolution. The in-depth analysis of the mitochondrial genomes of F. spadix and N. specialis augments the data corpus, fortifying further phylogenetic inquiry within the Siphonaptera order. This enhances both the resolution at the family echelon and the informativeness of the phylogenetic tree. The entire mitochondrial genome sequence has also been demonstrated to proffer elevated phylogenetic precision, rendering it an apt molecular marker for elucidating the evolutionary interconnections amongst flea species. Nevertheless, the sequencing of additional flea mitochondrial genomes is requisite to facilitate a more systematized and encompassing analysis of flea evolutionary relationships.

In this study, the mitochondrial genomes of F. spadix and N. specialis were successfully sequenced based on the combination of long-range PCR technology and next-generation sequencing technology. In both F. spadix and N. specialis, the mitochondrial genomes are circular with the same genetic composition and arrangement as other fleas, which provides the basis for further understanding of the molecular evolution, and phylogeny of fleas, as well as providing useful molecular markers for studying the taxonomy and systematics of the flea species.

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 approved the Laboratory Animal Management Committee of Dali University and First Affiliated Hospital of Chengdu Medical College. The study was conducted in accordance with the local legislation and institutional requirements.

YL conceived the study and wrote the manuscript. BC, XL, SL, and DJ collected specimens and participated in experimental operations. XW, LY, and RL analyzed the experimental data. QZ, LW, and XY are 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.