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. 2025 Dec 11;16:39. doi: 10.1038/s41598-025-28795-9

Morphological traits and mitogenome of Thaumapsylla breviceps orientalis endemic to China provide insight into the evolution of the order Siphonaptera

Ju Pu 1, Xiaoxia Lin 1, Xiaobin Huang 1, Xiaoyan Zheng 1, Xianzheng Zhang 1, Wenge Dong 1,✉
PMCID: PMC12764565  PMID: 41381548

Abstract

The Thaumapsylla breviceps orientalis is endemic to China and exhibits extreme host specificity (monoxenous). This study reports the first mitogenome sequenced for the family Ischnopsyllidae and provides comprehensive analysis of both the morphological characteristics and mitogenome of T. b. orientalis. The assembled mitogenome is 15,631 bp in length with a high AT content (78.5%), and its codon usage bias is predominantly shaped by natural selection. Evolutionary analysis based on evolutionary rates and nucleotide diversity across different families within Siphonaptera revealed that Pulicidae has the fastest evolutionary rate and the highest nucleotide diversity, a pattern likely driven by differences in their hosts and habitats. We observed that early-diverging flea lineages are predominantly polyxenous, whereas later-diverging lineages are primarily pleioxenous or monoxenous. Phylogenetic results indicate that the taxonomic status of the families Ctenophthalmidae, Vermipsyllidae, and Hystrichopsyllidae requires further study and revision. This research addresses a key knowledge gap in Ischnopsyllidae mitogenomics and clarifies the phylogenetic relationships within the order Siphonaptera.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-28795-9.

Keywords: Thaumapsylla breviceps orientalis, Ischnopsyllidae, Mitogenome, Phylogeny

Subject terms: Taxonomy, Genome, Bayesian inference

Introduction

The known family Ischnopsyllidae comprises 2 subfamilies, 5 tribes, 20 genera and 162 species, of which 6 genera and 30 species have been recorded in China1. The Ischnopsyllidae is a distinct family and morphologically closely related to the Leptopsyllidae and Ceratophyllidae. Fleas do not choose their hosts randomly, although opportunistic behavior may occur, their choice is influenced by specific requirements for conditions such as light, airflow, temperature, blood, hair, and host behavior2. Based on host specificity, fleas are classified into three primary types: (1) Polyxenous, characterized by low host specificity and the ability to parasitize a wide range of hosts; (2) Pleioxenous, restricted to hosts within a single family; (3) Monoxenous, exhibiting high host specificity, typically limited to a single host species or closely related species within the same genus. Thaumapsylla breviceps orientalis is a typical monoxenous flea species, primarily parasitizing fruit bats (mainly Rousettus leschenaultii)3. The high host specificity of T. b. orientalis may be attributed to several proposed factors: (1) the optimal body temperature of bats provides a suitable microenvironment for these fleas4; (2) the unique chemical composition of bat blood and their distinct olfactory cues may facilitate host recognition and preference5; and (3) the specific microclimatic conditions within bat caves may further influence flea survival and reproduction6. T. b. orientalis had no record for medical significance, its primary host (Rousettus leschenaultii) can carry various viruses, such as Coronavirus, Nipah virus, and Bartonella7–9, thereby serving as a critical component in a potential “virus reservoir - vector - new host” transmission chain.

Mitogenome also known as the second genetic system, was the primary genetic material outside nuclear in animals10. Owing to its strictly maternal inheritance, high mutation rate, and low recombination rate, the mitogenome serves as a powerful molecular marker and was widely used in studies of insect phylogeny, population genetics, and the identification of cryptic species11. Throughout evolutionary history, the base composition of most insect mitogenomes has remained relatively conserved. However, their structural organization can exhibit significant variation, including gene rearrangements, indels, as well as mutations in tRNA anticodons and the presence of atypical start codons12,13. Research on Siphonapteran mitogenomes has progressed more slowly compared to other major insect orders such as Diptera, Lepidoptera, and Coleoptera14–16. Notably, a complete mitogenome from the family Ischnopsyllidae has not been available until now. To address this gap and to understand the evolution and phylogeny within Siphonaptera, we combined a detailed morphological description of T. b. orientalis with the first sequencing and analysis of its mitogenome. This study provides a valuable genomic resource for understanding the evolution and phylogeny of the Ischnopsyllidae.

Materials and methods

Specimen collection, morphological identification, DNA extraction and mitogenome sequencing

In June 2023, 17 individuals (male: 7, female: 10) of T. b. orientalis were collected from Rousettus leschenaultii (Chiroptera: Pteropodidae) captured in Baoshan City, Yunnan Province. The fleas were placed in Eppendorf tubes containing 95% ethanol, and data on the habitat, host and weather were recorded. Subsequently, they were transported to the laboratory for storage in an ultra-low temperature refrigerator at − 80 °C. All specimens were collected and preserved with the approval of the Animal Ethics Committee of Dali University (approval number: MECDU-201912-20).

The collected T. b. orientalis individuals were identified primarily based on morphological characteristics described in Fauna Sinica Insecta Siphonaptera1. Specimens selected for morphological documentation were sealed and dried in a 47 °C incubator for 3–5 days. Subsequently, both male and female specimens were photographed using an X-type optical microscope. The remaining specimens were sent to Shenzhen Huitong Biotechnology Co., Ltd. (China) for DNA analysis. Following tests for DNA purity and integrity, a sequencing library with an insertion fragment of 350 bp was constructed using the NEB Next Ultra DNA Library Prep Kit for Illumina (NEB, USA). Double-end sequencing was then performed on an Illumina Novaseq 6000 platform. Raw data were processed with the fastp software (https://github.com/OpenGene/fastp) to obtain clean reads. The filtering criteria included: removal of reads containing over 5% N-bases, removal of reads with more than 50% of bases having a quality score ≤ 5, and removal of adapter-contaminated reads.

Mitochondrial sequence assembly, annotation and analysis

The mitogenome was assemble using MitoZ 2.317. The reliability of the assembled was evaluated with BWA v 0.7.1718 and Samtools v 0.1.2019. To further validate the assembly, we used Geneious Prime 11.020 with the following parameters: a minimum overlap of 50 bp and a minimum overlap homology of 100%. PCGs and rRNA genes were identified using BLAST21. The tRNA genes were predicted with tRNAscan SE22 and ARWEN23, and all gene annotations were manually verified and corrected. The annotated mitogenome sequence of T. b. orientalis was deposited in GenBank (accession number: PP973737). Subsequent analyses were performed using a suite of tools, including CodonW v.1.4.2 (https://sourceforge.net/projects/codonw/), R package24, MEGA25 and DnaSP v 526. Nucleotide composition biases were assessed by calculating the AT-skew and GC-skew using the formulas: AT-skew = (A - T) / (A + T) and GC-skew = (G - C) / (G + C).

Phylogenetic analysis

The Boreus elegans (Carpenter, 1935) was selected as the outgroup for phylogenetic tree construction. A concatenated nucleotide dataset based on 13 PCGs and two rRNA genes was analyzed using both Bayesian inference (BI)27 and Maximum likelihood (ML)28 methods. Species information is provided in Table S2. First, the sequences of the 13 PCGs and two rRNA genes were individually aligned using MAFF29 and optimized with MACSE v2.6030. The resulting PCGs alignments were then trimmed with Gblocks31, the rRNA gene alignments were trimmed with trimAl32. The processed sequences were concatenated into a supermatrix. Nucleotide substitution saturation was assessed for this dataset using DAMBE33. The results (Iss < Iss.c, p < 0.05) indicated no significant saturation, confirming its suitability for phylogenetic reconstruction. Second, the optimal nucleotide substitution model was determined using ModelFinder34 (Table S3). An ML tree was constructed with IQ-TREE35, performing 1000 bootstrap replicates. A BI tree was constructed using MrBayes 3.2.6, running four independent Markov Chain Monte Carlo (MCMC) chains for 1 million generations each and sampling every 1000 generations. Finally, FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize and finalize the phylogenetic trees for presentation.

Results

Morphological characteristics

The head of T. b. orientalis was shorter than that of most fleas (Ceratophyllidae, Hystrichopsyllidae, Pulicidae and Ctenophthalmidae). It was fracticipital, characterized by its short and wide shape, which distinguished it from most other flea species. There were 2 genal combs. Column 1 of frons bristle had 6 to 7 bristles. There was one ocular bristle, and the club was long and narrow. Males had 36 to 38 pronotal combs, while females had 34 to 37. The mesothorax was longer than both the prothorax and the metathorax. The coxal bristle on the fore leg were numerous and densely arranged. There were 3 notches on the posterior edge of the tibia. An antepygidial bristle was present in both males and females, with one bristle per individual. There was no boundary between the immovable process and the clasper, and the movable process was a wide and short oval. The hilla was round. The presence of a convex margin along the apex of the basimere was an important character distinguishing T. b. orientalis from other male subspecies (Fig. 1).

Fig. 1.

Fig. 1

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Morphological characteristics of a (♂) & b (♀) of T. b. orientalis from China. Image was acquired using a Leica DM3000 microscope.

Mitogenome structure and organization

The insect mitogenome was a double-stranded circular DNA molecule. The mitogenome of T. b. orientalis (GenBank accession number: PP973737) was 15,631 bp in size (the non-coding region was not complete and was not discussed in this paper). It contained 37 genes typical of metazoan animals, with 23 genes were located on the major chain (J-strand), including nine protein-encoding genes (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6 and cob) and fourteen transfer RNA genes (trnI, trnM, trnW, trnL2 (taa), trnK, trnD, trnG, trnA, trnR, trnN, trnS1 (tct), trnE, trnT and trnS2 (tga)). The remaining fourteen genes were located on the minor chain (N-strand), including four protein-encoding genes (nad1, nad4, nad4L and nad5), eight transfer RNA genes (trnY, trnC, trnQ, trnV, trnL1 (tag), trnP, trnH and trnF) and two rRNA genes (rrnL and rrnS) (Fig. 2). The mitogenome base composition of T. b. orientalis was A: 37.8%, T: 40.7%, C: 13.1%, G: 8.4%, with a high AT content (78.5%). The AT content at the first codon position was 71.8%, 68.7% at the second codon position, and as high as 89.9% at the third codon position, indicating a stronger AT preference at the third codon position (Table 1). There were fourteen gene overlap regions (totaling 35 bp), and the largest overlap region was 7 bp (between atp8 and atp6 / nad4 and nad4L). There were ten intergenic regions (totaling 33 bp), and the largest intergenic region was 17 bp (between trnS2 and nad1) (Table S1).

Fig. 2.

Fig. 2

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Organization of the T. b. orientalis mitogenome. tRNA genes were shown with the single-letter abbreviations of their corresponding amino acids. The two leucines are denoted by L1 (tag) and L2 (taa), and the two serines are denoted by S1 (tct) and S2 (tag).

Table 1.

Nucleotide composition and skewness of the T. b. orientalis mitogenome.

Gene A% T% C% G% AT% AT-skew GC-skew
PCGs 30.5 41.8 14.3 13.5 72.3 -0.156 -0.03
1st codon position 34.5 37.3 11.1 17.1 71.8 -0.039 0.212
2nd codon position 21.3 47.4 17.3 14.0 68.7 -0.380 -0.107
3rd codon position 43.5 46.4 5.9 4.3 89.9 -0.032 -0.164
rRNAs 39.2 40.3 6.5 13.9 79.5 -0.013 0.363
tRNAs 40.1 38.1 10.1 11.8 78.2 0.026 0.075
Whole genome 37.8 40.7 13.1 8.4 78.5 -0.037 -0.221
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Protein coding genes, codon usage bias and tRNA secondary structure analysis

All PCGs of T. b. orientalis used ATN (ATT/ATC/ATG/ATA) as start codon, and all PCGs used TAA or TAG as stop codons except for nad5 and nad4 with incomplete T– as stop codons (Table S1). Codon usage bias directly affects translational efficiency. As this process occurs at the mRNA level (where T was replaced by U), the codons consist of A, U, G, and C. Analysis of the 13 PCGs identified 27 preferred codons (RSCU > 1), including UUA, UCU, CCU, CGA, ACU, GUA and GGA. The least frequently used codons were UCG, CUG, CGC, ACG, AGC, GCG and GGC (Fig. 3a). In the ENC-plot, except for cob, nad4L, nad4, atp6 and cox2 with Nc > 35, the other eight genes had Nc < 35. Most genes were located considerably below the standard curve, indicating that codon usage bias was primarily influenced by natural selection (Fig. 3b). The neutral curve showed that the points for each gene did not fall on the diagonal, and the correlation between GC12 and GC3s was not significant (R = 0.105). This suggests that the base composition of GC12 and GC3s differed and that the genomic GC content was highly conserved, with codon usage bias being more strongly influenced by natural selection (Fig. 3c). The PR2 analysis revealed that all genes were distributed away from the central point (0.5, 0.5), with A ≠ T and G ≠ C, indicating that both selection and mutation pressures collectively shaped codon usage bias (Fig. 3d).

Fig. 3.

Fig. 3

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Preference analysis of 13 PCGs of T. b. orientalis. (a Relative Synonymous Codon Usage; b: ENC-plot; c: Neutral curve; d: PR2)

The 22 tRNA genes of T. b. orientalis ranged in length from 63 to 70 bp (Table S1). Among them, only trnS1 lacked the D-arm, while the remaining 21 tRNAs had typical cloverleaf-like structures. Most tRNAs formed typical Watson-Crick pairing, with a few mismatches (T-G: 15, T-T: 2, C-A: 1). Most of these mismatches occurred at the junction of the stem and the loop (Fig. S1).

Analysis of evolutionary differences among Siphonaptera based on evolutionary rate, nucleotide diversity and codon usage bias

Due to the availability of only a single sequenced mitogenome from the families Hystrichopsyllidae, Pygiopsyllidae, Ischnopsyllidae, and Vermipsyllidae, the average Ka/Ks values were not calculated for these groups. The average Ka/Ks values for the remaining families were ranked as follows: Pulicidae (0.7457) > Ceratophyllidae (0.5767) > Leptopsyllidae (0.5702) > Ctenophthalmidae (0.5504). Furthermore, the species with the highest evolutionary rates were Ctenocephalides felis (Bouche, 1835): 0.8423, C. f. felis (Bouche, 1835): 0.7999, C. orientis (Jordan, 1925): 0.7868, C. canis (Curtis, 1826): 0.7219, and Xenopsylla cheopis (Rothschild, 1903): 0.6873 (Table S2). Families that diverged around the Late Cretaceous (Pulicidae, Pygiopsyllidae, and Ctenophthalmidae) were predominantly characterized by polyxenous. In contrast, families that diverged during the Paleocene to Eocene (Hystrichopsyllidae, Vermipsyllidae, Leptopsyllidae, and Ceratophyllidae) were predominantly characterized by pleioxenous or monoxenous (Table 2). Additionally, the flea species parasitizing Rattus tanezumi and R. norvegicus were highly diverse. Among the 25 sequenced Siphonaptera mitogenomes in this study, 12 and 9 species were associated with R. tanezumi and R. norvegicus, respectively (Table 2).

Table 2.

Divergence times and host information for fleas.

Divergence times Species Host

Pulicidae

(Late Cretaceous)

 Ctenocephalides felis Felis catus, Canidae, Rattus tanezumi, Lepus sinensis, Tupaia belangeri, Homo sapiens(Carnivora, Rodentia, Scandentia, Primates)
Ctenocephalides felis felis Felis catus, Canidae, Rattus tanezumi, Lepus sinensis, Tupaia belangeri, Homo sapiens(Carnivora, Rodentia, Scandentia, Primates)
Ctenocephalides orientis It is mainly parasite on the family Canidae, a few mammals (Carnivora).
Ctenocephalides canis It is mainly parasitic in the family Canidae, a few mammals (Carnivora).
Pulex irritans It is parasite on 15 orders, 77 genera and 130 species.
Xenopsylla cheopis Rattus norvegicus, Rattus tanezumi, Rattus rattus, Spermophilus dauricus, Apodemus agrarius, Suncus murinus, Suncus etruscus (Rodentia, Eulipotyphla)
Hystrichopsyllidae (Paleocene to Eocene.) Hystrichopsylla weida qinlingensis Anourosorex squamipes, Sorex cylindricauda (Family Soricidae)

Vermipsyllidae

(Paleocene to Eocene.)

 Dorcadia ioffi Ovis aries, Capra hircus (Family Bovidae)

Pygiopsyllidae

(Late Cretaceous)

 Aviostivalius aklossi bispiniformis Leopoladmys edwardsi, Rattus tanezumi, Rattus nitidus, Niviventer fulvescen, Rattus rattus, Niviventer confucianus, Tupaia belangeri, Anourosorex squamipes, Berylmys bowersi, Rattus brunneusculus, Bandicota indica, Niviventer coninga, Ratufa bicolor (Rodentia, Scandentia, Eulipotyphla)

Ctenophthalmidae

(Late Cretaceous)

 Neopsylla specialis Apodemus agrarius, Apodemus chevrier, Rattus tanezumi, Niviventer confucianus, Niviventer fulvescen, Eothenomys miletus, Niviventer coxingi, Niviventer confucianus, Rattus losea, Rattus norvegicus (Mainly parasite on the family Muridae.)
Stenischia humilis Niviventer fulvescen, Apodemus agrarius, Rattus norvegicus, Rattus tanezumi, Apodemus chevrier, Eothenomys miletus, Rattus nitidus, Crocidura attenuata, Tupaia glis, Phodopus sungorus, Tscherskia triton, Spermophilus dauricus (Mainly parasite on Rodentia; a very few are parasite on Eulipotyphla and Scandentia.)
Stenischia montanis yunlongensis Apodemus draco, Apodemus chevrieri, Niviventer confucianus, Eothenomys custos, Rattus norvegicus, Anourosorex squamipes, Scaptonyx fusicaudus, Neotetracus sinensis (Rodentia, Eulipotyphla)
Ctenophthalmus yunnanus Neodon, Eothenomys fidelis, Eothenomys custos, Rattus norvegicus, Ochotona thibetana, Apodemus peninsulae (Mainly parasite on Rodentia; a very few are parasite on Lagomorpha)
Ctenophthalmus quadratus Eothenomys miletus, Eothenomys custos, Eothenomys olitor, Apodemus draco, Apodemus latronum, Rattus tanezumi, Rattus nitidus, Berylmys bowersi, Rattus rattus, Mus musculus, Dremomys pernyi, Crocidura attenuate (Mainly parasite on Rodentia; a very few are parasite on Eulipotyphla.)

Leptopsyllidae

(Paleocene to Eocene.)

 Paradoxopsyllus custodis Rattus norvegicus, Rattus tanezumi, Eothenomys custos, Rupestes forresti, Niviventer fulvescen (Family Muridae)
Frontopsylla spadix Apodemus sylvaticus, Apodemus agrarius, Niviventer fulvescen, Niviventer confucianus, Rattus losea, Rattus tanezumi, Cricetulus longicaudatus, Allactaga sibirica, Apodemus chevrier etc. (Rodentia)
Frontopsylla diqingensis Apodemus peninsulae, Ochotona thibetana, Apodemus agrarius, Rattus nitidus, Rattus tanezumi, Niviventer eha (Rodentia)
Leptopsylla segnis Mus musculus, Rattus norvegicus, Rattus rattus, Rattus tanezumi, Rattus losea, Niviventer fulvescen, Rattus nitidus, Apodemus agrarius, Apodemus sylvaticus, Tscherskia triton (Family Muridae)

Ceratophyllidae

(Paleocene to Eocene.)

 Citellophilus tesquorum Spermophilus undulatus, Spermophilus erythrogenys, Marmota baibacina, Apodemus sylvaticus, Cricetus migratorius, Apodemus sylvaticus, Felis silvestris, Rhombomys opimus, Spermophilus dauricus, Meriones unguiculatus, Meriones meridianus, Myospalax aspalax (Rodentia)
Ceratophyllus wui Aerodramus brevirostris innominate (Family Apodidae)
Nosopsyllus laeviceps Meriones meridianus, Rhombomys opimus, Meriones libycus, Marmota baibacina, Cricetus migratorius, Dipus sagitta, Mus musculus, Cricetulus barabensis, Rattus norvegicus, Meriones tamariscinus, Vulpes vulpes (Mainly parasite on Rodentia; a very few are parasite on Carnivora.)
Ceratophyllus anisus Rattus norvegicus, Rattus tanezumi, Rattus losea, Rattus nitidus, Berylmys bowersi, Apodemus agrarius, Mus musculus, Callosciurus erythraeus, Tupaia belangeri, Crocidura attenuate etc. (Mainly parasite on Rodentia; a very few are parasite on Primates and Carnivora.)
Macrostylophora euteles Dremomys lokriah, Dremomys rufigenis, Dremomys pernyi, Sciurotamias davidianus, Rupestes forresti, Tamiops swinhoei, Callosciurus erythraeus, Hylopetes alboniger, Apodemus agrarius, Tupaia belangeri (Mainly parasite on the family Sciuridae)

Ischnopsyllidae

(Paleocene to Eocene.)

 Thaumapsylla breviceps orientalis Rousettus leschenaultii (Pteropodidae)
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The divergence time of fleas is referred to Wu et al.1 and Zhu et al.36. The aforementioned host information is based on currently reported data1,37, with occasional and temporary hosts not included in this list.

Since there was only one species from the families Ischnopsyllidae, Hystrichopsyllidae, Pygiopsyllidae and Vermipsyllidae represented in the mitogenome database, an analysis of nucleotide diversity could not be performed. For the analyzable families, the nucleotide diversity (π) and the number of variable sites were as follows: Pulicidae (π = 0.3644; 6723 sites), Ctenophthalmidae (π = 0.3023; 5751 sites), Leptopsyllidae (π = 0.2390; 3733 sites), and Ceratophyllidae (π = 0.2594; 5609 sites) (Fig. 4). In addition, the nad5 and nad6 genes were characterized by greater sequence polymorphism and higher substitution rate (Fig. 4). A significant positive correlation was identified between the mitogenome AT content and evolutionary rate (Fig. 5). Statistical analysis of the Relative Synonymous Codon Usage (RSCU) across Siphonaptera species revealed that UUA was the most frequently used codon (461 times). Furthermore, most of the preferred codons for highly expressed genes ended with A or U (Fig. 6).

Fig. 4.

Fig. 4

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Nucleotide diversity analysis of 4 families of Siphonaptera.

Fig. 5.

Fig. 5

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Correlation analysis between evolutionary rate and AT content of Siphonaptera.

Fig. 6.

Fig. 6

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RSCU cluster analysis of Siphonaptera based on PCGs.

Exploring the evolutionary relationship of the siphonaptera

Using Boreus elegans as the outgroup, a phylogenetic tree was constructed based on 13 PCGs and 2 rRNA gene sequences by Bayesian method (BI) and Maximum likelihood (ML) methods (Fig. 7). The phylogenetic relationships of Vermipsyllidae, Hystrichopsyllidae and Ctenophthalmidae were inconsistent between the BI and ML analyses. In addition to the outgroup, there are two main clades. The topological structure of BI tree was (((Ceratophyllidae + Ischnopsyllidae + Leptopsyllidae) + ((Vermipsyllidae + Hystrichopsyllidae) + Ctenophthalmidae)) + (Pulicidae + Pygiopsyllidae)). The topological structure of ML tree was (((Ceratophyllidae + Ischnopsyllidae + Leptopsyllidae) + (Ctenophthalmidae + Hystrichopsyllidae + Vermipsyllidae)) + (Pulicidae + Pygiopsyllidae)). The clade of Vermipsyllidae, Ctenophthalmidae and Hystrichopsyllidae was low supported(Bpp<0.75, Bsp<61). The results showed that T. b. orientalis and Leptopsylla segnis clustered together as a branch with high support (BPP = 1, BSP = 78).

Fig. 7.

Fig. 7

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Phylogenetic relationship of Siphonaptera was reconstructed by Bayesian inference and Maximum likelihood method based on 13 PCGs. (Note:① suggests that the BI and ML topologies are inconsistent.)

Discussion

Among the currently sequenced mitogenomes of Siphonaptera, T. b. orientalis was the only parasite that infests the body surface of bats. The flight capability and specific body structure of bats have likely driven distinct morphological specializations in T. b. orientalis: (1) Adaptation to host environment: studies have shown that prolonged habitation in lightless environments leads to eye degeneration in species38. Correspondingly, the T. b. orientalis lacks pigment in its compound eyes, an adaptation consistent with the cave-dwelling and nocturnal habits of its host; (2) Attachment and Stability: the small size, flattened body, and resilient cuticle of fleas facilitate movement through the host’s pelage. We hypothesize that the well-developed pronotal comb (bearing 34–38 spines) in T. b. orientalis serves as a key adaptive trait, enabling the flea to maintain a firm grip on the host, particularly during flight. This is analogous to the rapid evolution of body size and color in feather lice in response to host plumage39; (3) Sensory Perception: the club (the third antenna segment) serves as the primary olfactory organ in fleas, playing a critical role in detecting host chemical signals and mechanical stimuli40. The pygidium, located at the abdominal terminus, is involved in tactile and chemosensory perception, aiding in host location and skin attachment41. The well-developed club and pygidium in T. b. orientalis are therefore essential for host localization and survival. In summary, these morphological characteristics collectively enhance the stability of its attachment to the bat’s body and improve its survival capacity within the unique ecological niche provided by its volant host.

To explore the molecular level of adaptive evolution in T. b. orientalis, we analyzed its mitogenome structure, codon usage bias, and evolutionary rate. The gene arrangement was consistent with that of the putative arthropod ancestor (Drosophila yakuba)42. We identified 27 high-frequency codons (predominantly A- or U-ending) in its mitogenome. As codon usage bias is a key regulator of gene expression that influences transcription, translation, and protein folding43,44. Our results indicate that natural selection plays a crucial role in shaping the molecular evolution of T. b. orientalis. Furthermore, our phylogenetic analysis revealed a general evolutionary trend: early-diverging flea lineages are predominantly polyxenous, whereas later-diverging lineages are primarily pleioxenous or monoxenous This pattern suggests a history of concerted evolution and increasing host specificity within Siphonaptera. The reasons for the high diversity of flea species parasitizing certain hosts, such as R. tanezumi and R. norvegicus, warrant further study. We also investigated factors influencing mitogenome evolutionary rates across Siphonaptera. A significant positive correlation was found between evolutionary rate and AT content, identifying base composition as a putative driver of rate variation. Notably, the subspecies Ctenocephalides felis felis exhibited a higher evolutionary rate (0.8423) than the nominate species Ctenocephalides felis (0.7999), raising the question of whether subspecies-level taxa generally display lower evolutionary rates—a hypothesis requiring future validation. Finally, previous studies have proposed nad5 and nad6 as superior markers to the traditionally used cox1 for species differentiation and population genetics in eukaryotes45. In line with this, we observed high sequence polymorphism and substitution rates in nad5 and nad6, suggesting their strong potential as molecular markers for resolving phylogenetic controversies within Siphonaptera.

Compared to most holometabolous insects, fleas exhibit a relatively complex taxonomic hierarchy that includes superfamily, family, subfamily, tribe, genus, species, and subspecies46. The topologies of the phylogenetic trees constructed using the two methods were largely congruent, differing only in the unstable positions of the families Ctenophthalmidae, Vermipsyllidae, and Hystrichopsyllidae—a finding consistent with previous studies47,48. The taxonomic status of Ctenophthalmidae remains particularly problematic, as it contains approximately one-quarter of all known flea species and has historically served as a “catch-all” category for taxa that are difficult to assign to other families46. One notable result was the clustering of Macrostylophora euteles (Ceratophyllidae) and Paradoxopsyllus custodis (Leptopsyllidae) into a highly supported clade (BPP = 1, BSP = 100). Further sequence alignment revealed an exceptionally high mitochondrial DNA sequence similarity of 98.8% between them (98.6% for cox1, 98.8% for cob, 99.5% for rrnL, and 99.8% for rrnS). This high genetic similarity suggests that the sequences deposited under these two names in GenBank may belong to the same species, indicating a potential misidentification. This hypothesis is surprising given that the two taxa are considered morphologically distinct and unlikely to be conspecific, highlighting a critical discrepancy between molecular and morphological data that warrants further investigation. As sequencing technologies continue to advance and more flea mitogenomes become available, expanding the taxonomic sampling will be crucial for significantly improving the resolution and robustness of the flea phylogenetic tree.

Conclusion

We present the first mitochondrial genome of T. b. orientalis and provide a comprehensive analysis of its morphology and genomic features. Our results indicate that natural selection is the dominant force shaping codon usage bias in this species. We observed an evolutionary trend from polyxenous early-diverging lineages toward pleioxenous or monoxenous later-diverging lineages. We propose that host specificity, ecological niche, and dispersal capacity are key factors underlying the variation in evolutionary rates among flea species. Furthermore, our phylogenetic analyses confirm the unstable taxonomic positions of the families Ctenophthalmidae, Vermipsyllidae, and Hystrichopsyllidae. This study lays a foundation for understanding the evolution and phylogeny of the family Ischnopsyllidae.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (458.9KB, pdf)

Acknowledgements

We thank the Technology Division of Shenzhen Huitong Biotechnology Co., Ltd. for its support in genomics and data analysis.

Author contributions

Ju Pu, Xiao-xia Lin and Wen-ge Dong designed and conducted the research. Xiao-bin Huang, Xian-zheng Zhang, and Xiao-yan Zheng provided the biological samples. Ju Pu and Xiao-xia Lin assembled and annotated the mitogenome. All authors read and approved the final manuscript.

Funding

We acknowledge funding support from the National Natural Science Foundation of China (NO. 32260152 to Wen-Ge Dong).

Data availability

The nucleotide sequences of the T. b. orientalis mitogenome were deposited in GenBank (https://www.ncbi.nlm.nih.gov/) under accession number PP973737.

Declarations

Competing interests

The authors declare no competing interests.

Statement

All methods were performed in accordance with the relevant guidelines and regulations.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (458.9KB, pdf)

Data Availability Statement

The nucleotide sequences of the T. b. orientalis mitogenome were deposited in GenBank (https://www.ncbi.nlm.nih.gov/) under accession number PP973737.

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