Complete mitogenomes characterization and phylogenetic analyses of Ceratophyllus anisus and Leptopsylla segnis

Fleas are one of the most common ectoparasites in warm-blooded mammals and an important vector of zoonotic diseases with serious medical implications. We sequenced the complete mitochondrial genomes of Ceratophyllus anisus and Leptopsylla segnis for the first time using high-throughput sequencing and constructed phylogenetic relationships. We obtained double-stranded circular molecules of lengths 15,875 and 15,785 bp, respectively, consisting of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and two control regions. AT-skew was negative in both C. anisus (−0.022) and L. segnis (−0.231), while GC-skew was positive in both (0.024/0.248), which produced significant differences in codon usage and amino acid composition. Thirteen PCGs encoding 3,617 and 3,711 codons, respectively, isoleucine and phenylalanine were used most frequently. The tRNA genes all form a typical secondary structure. Construction of phylogenetic trees based on Bayesian inference (BI) and maximum likelihood (ML) methods for PCGs. The results of this study provide new information for the mitochondrial genome database of fleas and support further taxonomic studies and population genetics of fleas.

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 fleaborne 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.

Sample collection and DNA isolation
The C. anisus samples (two females and one male) used in this study were collected in June 2022 from wild R. tanezumi in Laojun Mountain, Lijiang City, Yunnan Province of China (26°53′N, 99°58′E). The adult fleas L. segnis specimens were collected in August 2022 in Jianchuan, Yunnan Province from R. norvegicus (26°57′N, 99°90′E) (one female and one male). Preliminary identification of the collected flea specimens based on morphological diagnostic features (14). One specimen each was selected for subsequent DNA extraction and mitochondrial genome sequencing, while the others were placed in the Museum of Parasitology, Dali University, under voucher numbers DLUP2206 and DLUP2208, respectively. Specimens for experiments were rinsed in 0.9% saline, fixed in 96% alcohol and stored at −80°C until used for DNA extraction (11). DNA extraction was performed on C. anisus and L. segnis samples using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) and following the manufacturer's instructions.

Gene annotation and data analysis
Sequencing on the Illumina NovaSeq platform using AdapterRemoval software to eliminate low-quality data, assembly by software IDBA. The A5-miseq v20150522 program was used to assemble the complete mitochondrial genome and the MITOS WebServer 1 was used for genome annotation (15). MITOZ tool for mitochondrial genome prediction and online site tRNAscan-SE 2 for secondary structure prediction of transfer RNA (tRNA) (16,17). Mitochondrial genome circle mapping with CGView Server. 3 The software DNAStar V7.1 was used for nucleotide composition analysis and the program CodonW was used to calculate the relative synonymous codon usages (RSCU). The formulas GC-skew = [G -C]/[G + C] and AT-skew = [A -T]/[A + T] were used to measure the relative base content skewness. The total mitogenome informations of C. anisus and L. segnis have been deposited in NCBI.

Phylogenetic analysis
Mitochondrial lineages from 15 fleas were determined by phylogeny based on the concatenated datasets of 13 PCGs (Table 1).

Structure analysis of mitochondrial genome
The complete mitochondrial genomics of C. anisus and L. segnis were uploaded to Genbank in TBL format under the accession number OQ366407 and OQ023576. The C. anisus and L. segnis genomes are circular molecules of 15,875 bp and 15,785 bp in length, respectively, consisting of 13 protein-coding genes, 22 tRNAs, two rRNAs, and two D-loop ( Figure 1). Fourteen tRNA genes and nine PCGs are located in the forward strand (+), and the remaining 14 genes are encoded in the reverse strand (−) ( Table 2). The average AT content of C. anisus and L. segnis complete mitochondrial genome is 78.54% (78.89%) and GC content is 21.46% (21.11%), including A = 38.41% (40.37%), T = 40.14% (38.51%), G = 8.25% (13.17%) and C = 13.21% (7.94%; Table 3). The mitochondrial genome of C. anisus has 18 intergenic regions of 929 bp, accounting for 5.85% of the total length, and 13 overlapping regions totaling 28 bp. The genome of L. segnis has 19 spacer areas and 8 overlapping regions with a total of 894 bp and 21 bp ( Table 2).

Protein-coding genes
The mitochondrial genomes of C. anisus and L. segnis consist of 13 protein-coding genes, with a total length of 11,014 bp and 11,134 bp, accounting for 69.4% and 70.5% of the total length, respectively. The PCGs of C. anisus use the standard ATN as the initiation codon, and stop codons are TAA except for nad5 and nad4 (TTA). Amino acid utilization and RSCU were calculated for the PCGs of the mitochondrial genomic of C. anisus and L. segnis, encoding 3,617 and 3,711 amino acids, and the most abundant amino acid was found to be Isoleucine and Phenylalanine, accounting for 9.95% and 9.75%, respectively ( Figure 2). The Circular map and organization of the mitochondrial genome of Ceratophyllus anisus (A) and Leptopsylla segnis (B).
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Transfer RNAs and ribosomal RNAs
The mitochondrial genomes of C. anisus and L. segnis have 22 tRNA genes and two rRNA genes. The length of 22 tRNAs ranged from 61 bp for tRNA Cys (59 bp for tRNA Gly ) to 70 bp for tRNA Lys (70 bp for tRNA Cys ), with a total length of 1,433 bp (1,397 bp). The tRNA genes of both samples can form a complete typical canonical cloverleaf structure. There is an overlap between ATP8 and ATP6 with a length of 7 bp, which is typical of arthropods (21). The 16S rRNA and 12S rRNA genes of the C. anisus and L. segnis were separated by Valine,  (Table 2), a structure consistent with that reported in the mitogenome of other flea species (22).

Phylogenetic analysis
To further analyze the phylogenetic relationships of fleas, we added the mitochondrial genomes of C. anisus and L. segnis to the analysis. Phylogenetic trees were constructed using the BI and ML methods for the concatenated nucleotide sequences of 13 PCGs of the mitochondrial genome of 15 fleas and Casmara patrona as an outgroup, and the topologies of the two methods were consistent. According to the topology analysis, C. anisus and Ceratophyllus wui are clustered in a branch with high statistical support and L. segnis is alone in a branch, forming a sister group with other families of fleas ( Figure 3). The families Ceratophyllidae, Leptopsyllidae, Vermipsyllidae, Hystrichopsyllidae, and Pulicidae form monophyletic branches, which is consistent with the previous findings (23).

Discussion
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 Frontiers in Veterinary Science 07 frontiersin.org 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.

Conclusion
In this study, the complete mitochondrial genomes of C. anisus and L. segnis are sequenced and annotated for the first time by longrange 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.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories FIGURE 3 Phylogenetic analysis based on the nucleotide sequences of the 13 PCGs in the mitogenome. Each genus is represented by different colors. The solid black triangle represents the species in this study.
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Ethics statement
The animal study was reviewed and approved by Laboratory Animal Management Committee of Dali University and First Affiliated Hospital of Chengdu Medical College.