==== Front Front Vet Sci Front Vet Sci Front. Vet. Sci. Frontiers in Veterinary Science 2297-1769 Frontiers Media S.A. 10.3389/fvets.2023.1218488 Veterinary Science Original Research Complete mitogenomes characterization and phylogenetic analyses of Ceratophyllus anisus and Leptopsylla segnis Liu Yafang 1 Chen Bin 1 Lu Xinyan 1 Jiang Dandan 2 Wang Tao 1 Geng Ling 1 Zhang Quanfu 3 * Yang Xing 1 * 1Integrated Laboratory of Pathogenic Biology, College of Preclinical Medicine, Dali University, Dali, China 2School of Public Health, Dali University, Dali, China 3Department of Gastroenterology, Clinical Medical College and The First Affiliated Hospital of Chengdu Medical College, Chengdu, Sichuan, China Edited by: Guillermo Tellez-Isaias, University of Arkansas, United States Reviewed by: Roberto Senas Cuesta, University of Arkansas, United States; Jesús Adonai Maguey González, National Autonomous University of Mexico, Mexico *Correspondence: Quanfu Zhang, 2622045807@qq.com Xing Yang, yang08220013@163.com 16 6 2023 2023 10 121848807 5 2023 30 5 2023 Copyright © 2023 Liu, Chen, Lu, Jiang, Wang, Geng, Zhang and Yang. 2023 Liu, Chen, Lu, Jiang, Wang, Geng, Zhang and Yang https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 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. fleas Ceratophyllus anisus Leptopsylla segnis mitogenome phylogenetic Yunnan Natural Science Foundation2017FD139 Scientific Research Fund of Yunnan Education Department2022J0687 section-at-acceptanceParasitology ==== Body pmcIntroduction 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. Materials and methods 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. PCR amplification The mitochondrial genomes of C. anisus and L. segnis were amplified using two sets of overlapping long-fragment RCR primers. Specific PCR primers were constructed from the Xenopsylla cheopis (MW310242) and Pulex irritans (NC063709) mitochondrial genomes, which correspond to the COX1 and 12S rRNA genes, using Primer Premier 5.0 software (CA1: 5′-TTC CCT ACC TGT GCT TGC AG-3′; 3′-AAG AAT TGG ATC TCC CCC GC-5′; CA2: 5′-GCT TGA AAC TTA AAG AAT TTG GCG G-3′, 3′-AAG AGC GAC GGG CAA TAT GT-5′; LS1: 5′-GCA GGA GGG GGT GAT CCT AT-3′; 3′-ATC GTC GAG GTA TTC CTG CT-5′; LS2: 5′-CTT GAA ACT TAA AGA ATT TGG CGG T-3′, 3′-TCC AGT ACA TCT ACT ATG TTA CGA C-5′). The long-range PCR was performed in a total volume of 50 μL, consisting of 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 ddH2O under the following conditions: initial denaturation at 92°C for 2 min, followed by 30 cycles of denaturation at 92°C for 10 s, annealing at 68°C for 30 s and extension at 68°C for 10 min and the final extension step was subjected to 68°C for 10 min. The PCR products were tested using 1% agarose gel electrophoresis, which was purified and sequenced by Sangon Biotech Company (Shanghai, China). 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 WebServer1 was used for genome annotation (15). MITOZ tool for mitochondrial genome prediction and online site tRNAscan-SE2 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). Phylogenetic trees were constructed using the Bayesian inference (BI) and maximum likelihood (ML) methods in the Mrbayes v.3.2.7 (18) and MEGA 7.0 software, respectively. The BI approach selected GTR + G + I as the best model, running 10,000,000 generations in total, sampling every 1,000 generations. The ML analysis was based on 1,000 bootstrapped, which assessed branch reliability and nodal robustness. The resultant tree was visualized and edited with FigTree v1.4.2 (19). Table 1 List of the 15 flea species and Casmara patrona analyzed in this paper with their GenBank numbers. Species Family Length (bp) Accession number Ceratophyllus anisus Ceratophyllidae 15,875 OQ366407.1 Leptopsylla segnis Leptopsyllidae 15,785 OQ023576.1 Xenopsylla cheopis Pulicidae 18,902 MW310242.1 Pulex irritans Pulicidae 20,337 NC063709.1 Ctenocephalides canis Pulicidae 15,609 ON109770.1 Ctenocephalides canis Pulicidae 15,609 NC063710.1 Ctenocephalides felis Pulicidae 20,873 NC049858.1 Ctenocephalides felis Pulicidae 20,911 MW420044.1 Ceratophyllus wui Ceratophyllidae 18,081 NC040301.1 Jellisonia amadoi Ceratophyllidae 17,031 NC022710.1 Jellisonia amadoi Ceratophyllidae 17,031 KF322091.1 Dorcadia ioffi Vermipsyllidae 16,785 NC036066.1 Dorcadia ioffi Vermipsyllidae 16,785 MF124314.1 Hystrichopsylla weida qinlingensis Hystrichopsyllidae 17,173 NC042380.1 Hystrichopsylla weida qinlingensis Hystrichopsyllidae 17,173 MH259703.1 Casmara patrona Oecophoridae 15,393 NC053695.1 Results 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). Figure 1 Circular map and organization of the mitochondrial genome of Ceratophyllus anisus (A) and Leptopsylla segnis (B). Table 2 Organization of the Ceratophyllus anisus and Leptopsylla segnis mitochondrial genomes. Gene Strand Position Size(bp) Initiation codon Stop codon Anticodon Intergenic nucleotide D-loop N 1-347/117-287 347/171 91/321 trnI N 439-501/609-672 63/64 GAT 7/5 trnQ J 577-509/746-678 69/69 TTG −1/41 trnM N 577-643/788-854 67/67 CAT nad2 N 644-1657/855-1868 1014/1014 ATT/ATT TAA/TAA −2/−1 trnW N 1656-1722/1868-1932 67/65 TCA 6/7 trnC J 1789-1729/2001-1940 61/62 GCA trnY J 1853-1790/2062-2002 64/61 GTA −3/0 cox1 N 1851-3386/2063-3598 1535/1536 ATC/ATC TAA/TAA 4/4 trnL2 N 3391-3454/3603-3666 64/64 TAA 1/1 cox2 N 3456-4136/3668-4348 681/681 ATG/ATG TAA/TAA 2/2 trnK N 4139-4208/4351-4420 70/70 CTT −1/0 trnD N 4208-4272/4421-4488 65/68 GTC 9/0 atp8 N 4282-4443/4489-4650 162/162 ATA/ATA TAA/TAA −7/−7 atp6 N 4437-5111/4644-5318 675/675 ATG/ATG TAA/TAA −1/−1 cox3 N 5111-5893/5318-6100 783/783 ATG/ATA TAA/TAA trnG N 5894-5956/6101-6159 63/59 TCC −3/0 nad3 N 5957-6307/6160-6513 350/354 ATA/ATA TAA/TAA 2/−2 trnA N 6310-6373/6512-6575 64/64 TGC −1/−1 trnR N 6373-6435/6575-6637 63/63 TCG −3/4 D-loop N 0/6642-6679 /38 0/3 trnN N 6433-6497/6683-6747 65/65 GTT trnS1 N 6498-6565/6748-6815 68/68 TCT trnE N 6566-6630/6816-6878 65/63 TTC −3/0 trnF J 6694-6628/6944-6879 67/66 GAA −2/2 nad5 J 8410-6693/8660-6947 1717/1714 ATT/ATT TTA/TTA 61/1 trnH J 8474-8412/8724-8662 63/63 GTG −1/0 nad4 J 9810-8474/10063-8725 1337/1339 ATG/ATG TTA/TTA 59/−7 nad4l J 10097-9804/10350-10057 294/294 ATG/ATG TAA/TAA 2/2 trnT N 10100-10165/10353-10416 66/64 TGT trnP J 10228-10166/10480-10417 63/64 TGG 11/2 nad6 N 10240-10746/10483-10992 507/510 ATT/ATT TAA/TAA 0/−1 cob N 10747-11886/10992-12131 1140/1140 ATG/ATG TAA/TAA 2/2 trnS2 N 11889-11952/12134-12196 64/63 TGA 19/19 nad1 J 12910-11972/13151-12216 938/936 ATT/ATT TAA/TAA 7/1 trnL1 J 12980-12918/13214-13153 63/62 TAG 0/1 rrnL J 14198-12981/14489-13216 1218/1274 79/30 trnV J 14346-14278/14585-14520 69/66 TAC −1/−1 rrnS J 15124-14346/15363-14585 779/780 519/420 D-loop N 15643-15875/0 233/0 48/0 Table 3 Composition and skewness of Ceratophyllus anisus and Leptopsylla segnis mitogenome. Region A% C% G% T% A + T% G + C% AT skew GC skew Whole genome 38.41/40.37 13.21/7.94 8.25/13.17 40.14/38.51 78.54/78.89 21.46/21.11 −0.022/0.024 −0.231/0.248 nad2 34.91/35.80 11.14/10.65 7.99/7.00 45.96/46.55 80.87/82.35 19.13/17.65 −0.137/−0.131 −0.165/−0.207 cox1 29.30/29.49 16.47/16.21 13.93/13.15 40.30/41.15 69.60/70.64 30.40/29.36 −0.158/−0.165 −0.084/−0.104 cox2 34.65/33.19 15.42/15.57 10.57/10.72 39.35/40.53 74.01/73.72 25.99/26.28 −0.099/−0.100 −0.187/−0.185 atp8 43.83/40.12 6.17/11.73 4.32/3.70 45.68/44.44 89.51/84.57 10.49/15.43 −0.021/−0.051 −0.176/−0.521 atp6 30.96/33.04 15.26/14.52 9.78/9.04 44.00/43.41 74.96/76.44 25.04/23.56 −0.174/−0.136 −0.219/−0.233 cox3 27.97/29.63 17.11/16.09 13.03/12.77 41.89/41.51 69.86/71.14 30.14/28.86 −0.199/−0.167 −0.135/−0.115 nad3 31.91/31.92 13.11/12.43 7.12/7.34 47.86/48.31 79.77/80.23 20.23/19.77 −0.200/−0.204 −0.199/−0.257 nad5 34.69/34.89 7.16/6.65 13.80/13.54 44.35/44.92 79.05/79.81 20.95/20.19 −0.122/−0.126 0.317/0.341 nad4 33.43/33.76 7.85/7.24 13.84/12.85 44.88/46.15 78.31/79.91 21.69/20.09 −0.146/−0.155 0.276/0.279 nad4l 36.05/34.35 2.72/3.40 13.61/13.61 47.62/48.64 83.67/82.99 16.33/17.01 −0.138/−0.172 0.667/0.600 nad6 35.90/35.10 10.26/11.57 5.72/4.90 48.13/48.43 84.02/83.53 15.98/16.47 −0.146/−0.160 −0.284/−0.405 cob 31.50/31.58 15.35/15.96 11.40/10.53 41.67/41.93 73.25/73.51 26.75/26.49 −0.139/−0.141 −0.148/−0.205 nad1 30.78/33.01 7.14/7.26 15.76/15.49 46.33/44.23 77.10/77.24 22.90/22.76 −0.202/−0.145 0.176/0.362 rrnl 40.48/41.52 6.16/5.73 12.89/12.32 40.48/40.42 80.95/81.95 19.05/18.05 0/0.013 0.353/0.365 rrns 41.98/40.90 6.55/6.41 12.45/12.31 39.02/40.38 81.00/81.28 19.00/18.72 0.037/0.006 0.311/0.315 trnI 41.27/40.62 7.94/7.81 12.70/12.50 38.10/39.06 79.37/79.69 20.63/20.31 0.040/0.020 0.230/0.231 trnQ 40.58/40.58 4.35/4.35 11.59/11.59 43.48/43.48 84.06/84.06 15.94/15.94 −0.034/−0.034 0.454/0.454 trnM 38.81/37.31 17.91/19.40 10.45/13.43 32.84/29.85 71.64/67.16 28.36/32.84 0.083/0.111 −0.263/−0.182 trnW 41.79/43.08 13.43/10.77 7.46/9.23 37.31/36.92 79.10/80.00 20.90/20.00 0.057/0.077 −0.286/−0.077 trnC 44.26/38.71 4.92/8.06 13.11/16.13 37.70/37.10 81.97/75.81 18.03/24.19 0.080/0.021 0.454/0.334 trnY 39.06/42.62 10.94/8.20 18.75/13.11 31.25/36.07 70.31/78.69 29.69/21.31 0.111/0.083 0.263/0.230 trnL2 34.38/32.81 12.50/12.50 14.06/14.06 39.06/40.62 73.44/73.44 26.56/26.56 −0.064/−0.106 0.059/0.059 trnK 32.86/32.86 15.71/15.71 17.14/15.71 34.29/35.71 67.14/68.57 32.86/31.43 −0.021/−0.042 0.044/0 trnD 47.69/50.00 7.69/5.88 9.23/7.35 35.38/36.76 83.08/86.76 16.92/13.24 0.148/0.153 0.091/0.111 trnG 44.44/42.37 6.35/8.47 7.94/11.86 41.17/37.29 85.71/79.66 14.29/20.34 0.038/0.064 0.111/0.167 trnA 37.50/40.32 6.25/9.68 9.38/9.68 46.88/40.32 84.38/80.65 15.62/19.35 −0.111/0 0.200/0 trnR 39.68/34.92 11.11/11.11 11.11/9.52 38.10/44.44 77.78/79.37 22.22/20.63 0.020/−0.120 0/−0.077 trnN 44.62/44.62 7.69/7.69 9.23/9.23 38.46/38.46 83.08/83.08 16.92/16.92 0.364/0.364 0.091/0.091 trnS1 42.65/39.71 10.29/10.29 10.29/10.29 36.76/39.71 79.41/79.41 20.59/20.59 0.281/0 0/0 trnE 41.54/39.68 7.69/9.52 4.62/6.35 46.15/44.44 87.69/84.13 12.31/15.87 −0.053/−0.057 −0.250/−0.120 trnF 35.82/40.91 10.45/6.06 14.93/13.64 38.81/39.39 74.63/80.30 25.37/19.70 −0.040/0.019 0.177/0.385 trnH 42.86/36.51 3.17/3.17 12.70/15.87 41.27/44.44 84.13/80.95 15.87/19.05 0.019/−0.219 0.600/0.667 trnT 42.42/39.06 4.55/6.25 9.09/9.38 43.94/45.31 86.36/84.38 13.64/15.62 −0.018/−0.074 0.333/0.200 trnP 38.10/42.19 4.76/3.12 15.87/14.06 41.27/40.62 79.37/82.81 20.63/17.19 −0.040/0.019 0.539/0.636 trnS2 42.19/42.86 6.25/7.94 12.50/12.70 39.06/36.51 81.25/79.37 18.75/20.63 0.039/0.080 0.333/0.230 trnL1 39.68/38.71 6.35/6.45 12.70/14.52 41.27/40.32 80.95/79.03 19.05/20.97 −0.020/−0.020 0.333/0.385 trnV 43.48/40.91 5.80/7.58 7.25/7.58 43.48/43.94 86.96/84.85 13.04/15.15 0/−0.039 0.111/0 OH 44.31/44.50 2.41/3.35 2.41/11.48 50.86/40.67 95.17/85.17 4.83/14.83 −0.069/0.045 0/0.548 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 AT-skew and GC-skew of the PCGs of the C. anisus range from −0.202 (for NAD1) to −0.021 (for ATP8) and from −0.296 (for NAD3) to 0.320 (for NAD5), respectively. Among the 13 protein-coding genes, both C. anisus and L. segnis had nine genes in the forward chain (NAD2, COX1, COX2, ATP8, ATP6, COX3, NAD3, NAD6, Cob) and four genes in the reverse chain (NAD1, NAD5, NAD4, NAD4L), which are consistent with most metazoans such as fleas and ticks (5, 20). Figure 2 Relative synonymous codon usage (RSCU) of codons. (A) Ceratophyllus anisus and (B) Leptopsylla segnis. The horizontal coordinates represent all codons encoding each amino acid, and the vertical coordinates represent the sum of all RECU values. 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 tRNACys (59 bp for tRNAGly) to 70 bp for tRNALys (70 bp for tRNACys), 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, and were 1,218 bp (1,274 bp) and 779 bp (780 bp) in length, respectively (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). 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. 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 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 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. Data availability statement 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. 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. Author contributions 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. Funding This work was supported by the Yunnan Natural Science Foundation (2017FD139) and Scientific Research Fund of Yunnan Education Department (2022J0687). Conflict of interest 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. Publisher’s note 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. 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