Ticks rank second in the world as vectors of disease. Tick infestation is one of the factors threatening the health and survival of giant pandas. Here, we describe the mitogenomes of Ixodes acutitarsus and Ixodes ovatus parasitizing giant pandas, and perform comparative and phylogenetic genomic analyses on the newly sequenced and other available mitogenomes of hard ticks. All six newly determined mitogenomes contain a typical gene component and share an ancient Arthropoda gene arrangement pattern. Our study suggests that I. ovatus is a species complex with high genetic divergence, indicating that different clades of I. ovatus represent distinct species. Comparative mitogenomic analyses show that the average A + T content of Ixodidae mitogenomes is 78.08%, their GC-skews are strongly negative, while AT-skews fluctuate around 0. A large number of microsatellites are detected in Ixodidae mitogenomes, and the main microsatellite motifs are mononucleotide A and trinucleotide AAT. We summarize five gene arrangement types, and identify the trnY-COX1-trnS1-COX2-trnK-ATP8-ATP6-COX3-trnG fragment is the most conserved region, whereas the region near the control region is the rearrangement hotspot in Ixodidae mitogenomes. The phylogenetic trees based on 15 genes provide a very convincing relationship (Ixodes + (Robertsicus + ((Bothriocroton + Haemaphysalis) + (Amblyomma + (Dermacentor + (Rhipicentor + (Hyalomma + Rhipicephalus))))))) with very strong supports. Remarkably, Archaeocroton sphenodonti is embedded in the Haemaphysalis clade with strong supports, resulting in paraphyly of the Haemaphysalis genus, so in-depth morphological and molecular studies are essential to determine the taxonomic status of A. sphenodonti and its closely related species. Our results provide new insights into the molecular phylogeny and evolution of hard ticks, as well as basic data for population genetics assessment and efficient surveillance and control for the giant panda-infesting ticks.

Cystic echinococcosis, a parasitic zoonosis that causes significant economic losses and poses a threat to public health, is caused by larvae of the tapeworm Echinococcus granulosus. Infection causes infertile cysts in intermediate hosts that cannot produce protoscoleces (PSCs) or complete the life cycle. Herein, we cloned, expressed, and characterised mitochondrial fission protein 1 (Eg-Fis1) and programmed cell death protein 6 (Eg-PDCD6) from E. granulosus, and explored their functions related to infertile cysts. Eg-Fis1 and Eg-PDCD6 encode putative 157 and 174 residue proteins, respectively, and Western blotting indicated good reactogenicity for both. Eg-Fis1 and Eg-PDCD6 were ubiquitously distributed in all stages of E. granulosus. Furthermore, mRNAs of Eg-Fis1 and Eg-PDCD6 were upregulated following H₂O₂ treatment which induced apoptosis in PSCs. To investigate the regulation of apoptosis in response to oxidative stress, RNA interference (RNAi) and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays were performed. The apoptotic rate of the Eg-Fis1 RNAi group was significantly lower than non-interference group, but there was no such difference for Eg-PDCD6. In conclusion, Eg-Fis1 promotes apoptosis induced by oxidative stress, whereas Eg-PDCD6 does not appear to be a key regulator of apoptosis.

The poor reproductive capacity of giant pandas significantly hinders the development of captive populations, with 80.88% of adult individuals being unable to successfully become pregnant and deliver offspring. The disturbance of vaginal microbiota has been proven to potentially lead to miscarriage, abortion, and stillbirth in mammals. To elucidate the potential relationship between the vaginal microbiota and the reproductive capacity of giant pandas, we performed high-throughput sequencing of vaginal microbiota at the time of fertilization and conducted comparative analyses based on different pregnancy outcomes. We found that the microbial diversity in the delivery (D) group exceeded that in the non-delivery (ND) group and the vaginal microbial community structure was statistically different between the two groups. The vaginal microbiota in the delivery pandas consisted of unclassified Pseudomonadaceae which was gradually replaced by the Escherichia-Shigella type of vaginal microbiota in the ND group. A function predictions analysis showed that infectious disease, glycan biosynthesis, and metabolism were significantly enriched in the ND group. Additionally, an analysis of the microbial community phenotypic categories indicated that the ND group exhibited a significantly higher abundance of Gram-negative bacteria, facultative anaerobes, potential pathogens, and stress-tolerant species compared to the D group, predominantly driven by the elevated abundance of Escherichia-Shigella. Escherichia-Shigella can be used within LDA and ROC analyses to diagnostically distinguish the vaginal microflora associated with bad pregnancy outcomes during estrus. Our results will help to identify potential pathogens causing reproductive tract diseases, reduce the number of reproductive tract disease infections in pandas, and increase the birth rate of giant pandas in conservation breeding programs.

As apex predators, felids (Felidae) face unresolved phylogenetic controversies due to their recent rapid speciation and remarkable morphological conservatism. Previous studies, often relying on a limited number of genetic markers, were constrained by insufficient data and conflicting phylogenetic signals, leaving these disputes unresolved. Therefore, establishing a robust phylogenetic framework based on larger-scale genomic data is crucial. This study integrated complete mitogenomes from 37 species representing all major felid genera to characterize genomic diversity, selection pressures, and phylogenetic relationships. Results revealed conserved gene content and arrangement patterns but significant intergenic variation in nucleotide composition, with the light-strand encoded ND6 exhibiting pronounced strand-specific bias. Nucleotide diversity was highest in ND4L (Pi = 0.132) and ATP6 (Pi = 0.131), suggesting their utility as novel markers for species delimitation and population studies. Selection pressure analysis indicated strong purifying selection on cytochrome oxidase subunits (e.g., COX1 Ka/Ks = 0.00327) but relaxed constraints on ATP8 (Ka/Ks = 0.12304). Phylogenies reconstructed from the complete 13PCGs + 2rRNAs dataset (showing high congruence between maximum likelihood and Bayesian methods) clearly delineated Felidae into two primary clades (Pantherinae and Felinae), confirming monophyly of all genera and positioning Neofelis nebulosa as the basal lineage within Pantherinae. Crucially, exclusion of ND6 (12PCGs + 2rRNAs) yielded topologies congruent with the complete 13PCGs + 2rRNAs dataset, whereas single-gene or limited multi-gene datasets produced inconsistent trees (particularly at genus-level nodes). This demonstrates that near-complete mitogenomic data (≥12PCGs + 2rRNAs) are essential for reconstructing robust felid phylogenetic frameworks. Our study provides insights into carnivoran mitogenome evolution.

Managing endangered wildlife in pre-release or semi-wild training programs requires an understanding of how animals balance strict energetic constraints against environmental health risks. We investigated the behavioral and spatial coping mechanisms of giant pandas (Ailuropoda melanoleuca) during semi-wild adaptation training exposed to intense seasonal tick (Ixodes ovatus) parasitism. Integrating high-resolution 3D-GPS telemetry, fine-scale microhabitat architecture mapping, and longitudinal daily tick counts, we evaluated the relative efficacy of behavioral hypoactivity versus microhabitat selection using Generalized Estimating Equations (GEE). During the high-risk summer season, individuals exhibited pronounced baseline behavioral suppression. However, GEE models revealed that fine-scale microhabitat selection—rather than localized movement reduction—was the primary determinant of parasite avoidance. Actively shifting to higher-elevation microhabitats with minimal leaf litter and dense bamboo coverage significantly minimized daily tick burdens. Conversely, remaining sedentary within low-elevation, thick-litter zones yielded the highest predicted parasite loads. These findings demonstrate that physical stillness alone is insufficient to mitigate ectoparasite exposure without access to structural habitat refugia. For conservation and reintroduction programs, providing structurally complex, topographically heterogeneous enclosures is essential. Ensuring access to natural altitudinal gradients empowers managed herbivores to autonomously regulate parasitological risks, thereby enhancing pre-release animal welfare and health.

Seasonal environmental variation is a widespread stressor for wildlife and can induce plastic changes in DNA methylation. Blood DNA methylation is an integrative molecular indicator of overall physiological status, yet its seasonal dynamics in the giant panda remain unclear. This study aimed to profile genome-wide blood methylation patterns in giant pandas under natural field conditions and to reveal the epigenetic strategies underlying their seasonal adaptation. Twelve blood samples were collected from three wild-trained giant pandas in the Daxiangling Nature Reserve during winter (n = 6) and summer (n = 6) between 2023 and 2025 and were subjected to whole-genome bisulfite sequencing. The genome-wide mC/C ratio (4.18–4.98%) was dominated by the CG context. Winter–summer comparison identified 7113 differentially methylated regions and 3063 differentially methylated genes. These genes were enriched in transcriptional regulation, cell surface receptor signaling, miRNA metabolism, and MAPK, Ras, and T/B cell receptor signaling pathways. Winter-hypermethylated genes were enriched in both immune defense and energy-consuming processes, whereas winter-hypomethylated genes showed limited enrichment, indicating that hypermethylation-mediated suppression dominates seasonal adaptation. This study reveals, for the first time, a seasonal methylation fingerprint in giant panda blood characterized by a “summer immune enhancement, winter energy conservation” pattern. As sampling occurred in natural habitats, these differences reflect integrated epigenetic responses to temperature, photoperiod, food availability, and pathogen pressure rather than single-factor effects. These findings provide molecular evidence for seasonal adaptation in endangered mammals and candidate markers for captive panda management and wild-release assessment.