Analysis of complete mitogenomes and phylogenetic relationships of Frontopsylla spadix and Neopsylla specialis

Fleas represent a group of paramount medical significance, subsisting on blood and acting as vectors for an array of naturally occurring diseases. These pathogens constitute essential elements within the plague biome, exerting deleterious effects on both human and livestock health. In this study, we successfully assembled and sequenced the whole mitochondrial genome of Frontopsylla spadix and Neopsylla specialis using long-range PCR and next-generation sequencing technologies. The mitogenomes of F. spadix and N. specialis both have 37 genes with full lengths of 15,085 bp and 16,820 bp, respectively. The topology of the phylogenetic tree elucidates that species F. spadix is clustered in a branch alongside other members of the family Leptopsyllidae, whereas species N. specialis is a sister taxon to Dorcadia ioffi and Hystrichopsylla weida qinlingensis. It also suggests that Pulicidae form a monophyletic clade, Ctenopthalmidae, Hystrichopsyllidae, Vermipsyllidae form a sister group to Ceratophyllidae/Leptopsyllidae group. The mitochondrial genomes of F. spadix and N. specialis were sequenced for the first time, which will contribute to a more comprehensive phylogenetic analysis of the Siphonaptera order. The foundation for subsequent systematic studies, and molecular biology of fleas was established.


Introduction
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.

Sample collection and DNA extraction
Adult specimens of F. spadix (one female and one male) were collected in July 2020 from Luoping Mountain, eryuan City, Dali Bai Autonomous Prefecture, Yunnan Province, China (26 • 07 ′ N, 99 • 85 ′ E).Three females and one male adult specimens of N. specialis were found in June 2022 from Laojun Mountain, Lijiang City, Yunnan Province of China (26 • 53 ′ N, 99 • 58 ′ E).Species identification was conducted based on morphological characteristics with F. spadix and N. specialis samples extracted from an adult female using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) following the manufacturer's instructions.

PCR amplification
The study design included the development of two sets of overlapping long fragment PCR primers to amplify the mitochondrial genomes of F. spadix and N. specialis.This was conducted using cox1 and 12S rRNA genes of Ctenophthalmus quadratus (OQ023577) and Leptopsylla segnis (OQ023576), with primer design achieved through Primer 5.0 software, as delineated in Table 1.The PCR was performed in the 50 µl system, including 10 µl 5× PrimerSTAR GXL Buffer (Takara, Japan), 4 µl of each primer, 4 µl of dNTPs, 1 µl of PrimerSTAR GXL DNA Polymerase (Takara, Japan), 4 µl of DNA template and 23 µl of ddH 2 O under the following reaction conditions: 92 • C for 2 min for initial denaturation, followed by 35 cycles of denaturation at 92

Phylogenetic analysis
The 13 protein-coding gene sequences from 15 flea species were independently aligned using MUSCLE nucleotide mode, and datasets were manually concatenated.Positions containing gaps and incomplete data were excluded through Bioedit v7.0.5.3 software.Phylogenetic relationships were analyzed using Casmara patrona as an outgroup (Table 2), and trees were constructed with MEGA 7.0 software and Mrbayes v.3.2.7 software.The ML tree was formulated using GTR+G+I as the optimal model based on the Akaike Information Criterion (AIC) (17), employing the maximum likelihood method across 1,000 bootstrap datasets.The BI tree underwent 10,000,000 generations, sampled every 1,000 generations.The evolutionary relationships among flea species were visually depicted using the software Figtree v1.4.2.

Organization of mitochondrial genome
The mitochondrial genomes of F. spadix and N. specialis, which are typically closed double-stranded molecular structures, were uploaded to Genbank in TBL format and obtained accession numbers OQ366408 and OQ366409, respectively.The length of the mitochondrial genome was 15,085 bp and 16,820 bp, respectively (Figure 1), with differences in length mainly determined by the length of the control region.Most of the genes including 14 tRNAs and 9 PCGs are distributed on the positive strand, the same as the other fleas (18,19).Both intergenic regions and overlapping domains are present within the mitochondrial genome (Table 3).The mitogenomes of F. spadix and N. specialis had a significant AT preference with AT content of 78.83% and 77.27%, respectively, and the base content was 37.99% (38.64%)A, 40.84% (38.63%)T, 12.85 (14.22%)C, and 8.31 (8.51%) G (Table 4).

Protein-coding genes
The PCGs of F. spadix and N. specialis were 11,144 bp and 11,142 bp long, accounting for 73.87% and 66.24% of the complete mitochondrial genome length, respectively.Of the 13 proteincoding genes (PCGs) of F. spadix, which encode a total of 3713 codons, the initiation codon is the standard codon ATN, with TAA as the termination codon except for NAD3 (TAG).N. specialis encodes a total of 3714 codons, with incomplete termination codons occurring in NAD5 and NAD4, and NAD3 with TAG as a stop codon.Leucine is the dominant amino acid and cysteine is the rarest amino acid (Figure 2).The mitochondrial genomes of F. spadix and N. specialis are mostly nonpolar amino acid groups with 1851 (49.85%) and 1933 (52.05%), respectively, and the remaining polar, basic, and acidic amino acid groups are 1209 (32.51%) and 1206 (32.47%), 235 (6.33%) and 230 (6.19%), 194 (5.22%) and 157 (4.23%).

Transfer RNA genes and ribosomal RNA genes
The mitogenomes of F. spadix and N. specialis have 14 tRNAs located in the positive strand with full lengths of 1439 bp and 1408 bp, respectively.Among the 22 tRNA genes of F. spadix, the length of tRNA genes ranged from 62 bp (trnL1) to 70 bp (trnK), with the shortest amino acid of 61 bp (trnC) in N. specialis.The mitochondrial genome was conjured according to the special genetic code so that all the 22 tRNA genes could be identified.G-U oscillating bases appear as a common mismatch in most tRNA genes to maintain tRNA secondary structure (20).The relationship between base mismatch and evolution needs further consideration.With a length of 7 bp, which is typical of arthropods, ATP8 and ATP6 overlap (21).The 16S rRNA and 12S rRNA of F. spadix and N. specialis are both located in the reverse strand, separated by Valine, with AT contents of 82.98% (80.43%) and 81.42% (79.01%), respectively (Table 4).

Phylogenetic analysis
We harnessed available flea genomic data from the NCBI database pertaining to fleas and amalgamated this with our successfully sequenced F. spadix and N. specialis, thus facilitating a refined exploration of the topology of flea phylogenetic relationships.Utilizing the maximum likelihood method, we constructed a phylogenetic tree anchored on the concatenated nucleotide sequences of 13 PCG genes, thereby providing an insightful perspective into flea evolutionary trajectories.The ML and BI trees show identical topologies.According to ML and BI analysis, the families Ctenopthalmidae, Hystrichopsyllidae, Vermipsyllidae, and Pulicidae form a monophyletic clade, while the family Ceratophyllidae and Leptopsyllidae are paraphyletic.A principal clade encompasses species of the family Pulicidae, crystallizing into a definitive monophyletic clade.In juxtaposition, Ctenopthalmidae, Hystrichopsyllidae, and Vermipsyllidae form a sister aggregation to the Ceratophyllidae and Leptopsyllidae group.Notably, F. spadix and Leptopsylla segnis belonging to the Leptopsyllidae family emerge as the most phylogenetically congruent entities, bolstered by robust node support values.N. specialis resides solitarily on a branch, constituting a strongly endorsed linkage with Dorcadia ioffi and Hystrichopsylla weida qinlingensis and fostering sister group affiliations (Figure 3).

Discussion
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, ./fvets. .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.

Conclusion
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.

FIGURE
FIGUREMap of the mitochondrial genome of Frontopsylla spadix (A) and Neopsylla specialis (B).Genes in the inner circle plot are located on the reverse strand, and the rest of the genes are located on the forward strand.

FIGURE
FIGUREPhylogenetic analysis based on the nucleotide sequences of thePCGs in the mitogenome.The number beside the nodes are posterior probabilities (BI) and bootstrap (ML).The black square markers represent the species in this study.
TABLE PCR primers for sequencing the mitogenome of F. spadix and N. specialis.
TABLE Mitochondrial genome sequence information used in this paper.
TABLE Summary of the mitogenome of F. spadix and N. specialis.TABLE Composition and skewness of F. spadix and N. specialis mitogenome.