Complete Mitochondrial Genome and Phylogenetic Analysis of Tarsiger indicus (Aves: Passeriformes: Muscicapidae).

Tarsiger indicus (Vieillot, 1817), the White-browed Bush Robin, is a small passerine bird widely distributed in Asian countries. Here, we successfully sequenced its mitogenome using the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA) for PE 2 × 150 bp sequencing. Combined with other published mitogenomes, we conducted the first comprehensive comparative mitogenome analysis of Muscicapidae birds and reconstructed the phylogenetic relationships between Muscicapidae and related groups. The T. indicus mitogenome was 16,723 bp in size, and it possessed the typical avian mitogenome structure and organization. Most PCGs of T. indicus were initiated strictly with the typical start codon ATG, while COX1 and ND2 were started with GTG. RSCU statistics showed that CUA, CGA, and GCC were relatively high frequency in the T. indicus mitogenome. T. cyanurus and T. indicus shared very similar mitogenomic features. All 13 PCGs of Muscicapidae mitogenomes had experienced purifying selection. Specifically, ATP8 had the highest rate of evolution (0.13296), whereas COX1 had the lowest (0.01373). The monophylies of Muscicapidae, Turdidae, and Paradoxornithidae were strongly supported. The clade of ((Muscicapidae + Turdidae) + Sturnidae) in Passeriformes was supported by both Bayesian Inference and Maximum likelihood analyses. The latest taxonomic status of many passerine birds with complex taxonomic histories were also supported. For example, Monticola gularis, T. indicus, and T. cyanurus were allocated to Turdidae in other literature; our phylogenetic topologies clearly supported their membership in Muscicapidae; Paradoxornis heudei, Suthora webbiana, S. nipalensis, and S. fulvifrons were formerly classified into Muscicapidae; we supported their membership in Paradoxornithidae; Culicicapa ceylonensis was originally classified as a member of Muscicapidae; our results are consistent with a position in Stenostiridae. Our study enriches the genetic data of T. indicus and provides new insights into the molecular phylogeny and evolution of passerine birds.

Hematological and biochemical parameters of giant pandas (Ailuropoda melanoleuca) in captive and semi-natural environments.

Physiological indexes like blood parameters have been widely used to monitor the health of free-roaming animals. Attempts to reintroduce one of China's most endangered species, the giant panda (Ailuropoda melanoleuca), have been hampered by a lack of data on its ecology and physiology. We examined three giant pandas' hematological and blood chemistry parameters in a soft release program and 30 captive giant pandas as controls and determined the reference intervals (RIs) for those blood parameters in the captive animals. Elevation, captivity status and the interaction of those factors were statistically significant for hematologic measures. Release pandas had significantly higher hemoglobin and hematocrit values after they moved to high elevation locations. We also found significant difference in the enzyme parameters between high and low elevation pandas such as higher aspartate aminotransferase, alanine aminotransferase, creatinine kinase, amylase and lower lactate dehydrogenase and alkaline phosphatase. Release pandas also had higher nutrition parameter values such as higher albumin, globulin and creatinine. The RI for blood parameters in our study provides a baseline to monitor the health of captive animals and forms the basis for assessing the health of free-roaming giant pandas in future reintroduction efforts.

Complete Mitogenomes of Ticks Ixodes acutitarsus and Ixodes ovatus Parasitizing Giant Panda: Deep Insights into the Comparative Mitogenomic and Phylogenetic Relationship of Ixodidae Species.

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.

Viral metagenomics unveiled extensive communications of viruses within giant pandas and their associated organisms in the same ecosystem.

Cross-species transmission events were commonplace, with numerous cases of host-switching during the viral evolutionary history, but relatively little evidence for onward transmission in different species living in the same ecosystem. For understanding the communications of viruses in giant pandas (Ailuropoda melanoleuca) and their associated organisms, based on a large size of samples (N = 2305) collected between 2015 and 2020 from giant panda (N = 776) and other four giant panda-associated organisms in the same ecosystem, red pandas (N = 700), stray cats (N = 32), wild rats (N = 42), and mosquitoes (N = 755), viromics was used for the virus identification and subsequent virus traceability. The results showed that a feline panleukopenia virus (FPV) was found in giant pandas with clinical signs of vomiting and mild diarrhea. Meanwhile, the same FPV strain was also prevalent in the healthy red panda (Ailurus fulgens) population. From the viromes of the five different organisms, 250 virus genomes were determined. Our data revealed that besides FPV, other putative pathogenic viruses, such as red panda amdoparvoviruses (RPAVs) and Getah viruses (GETVs) were responsible for previous disease or death of some red pandas. We also demonstrated that a number of viruses were involved in potential interspecies jumping events between giant pandas and their associated species. Collectively, our results shed light on the genetic diversity and relationship of diverse viral pathogens in 'Giant pandas-Associated animals-Arthropods' and report some cases of possible viral host-switching among these host species living in the same ecosystem.

Virome of Giant Panda-Infesting Ticks Reveals Novel Bunyaviruses and Other Viruses That Are Genetically Close to Those from Giant Pandas.

Tick infestations have been reported as one of the factors threatening the health of giant pandas, but studies of viral pathogens carried by ticks feeding on the blood of giant pandas are limited. To assess whether blood-sucking ticks of giant pandas can carry viral pathogens and if so, whether the viruses in ticks are associated with those previously detected in giant panda hosts, we determined the viromes of ticks detached from giant pandas in a field stocking area in Sichuan Province, southwest China. Using viral metagenomics we identified 32 viral species in ticks, half of which (including anellovirus [n = 9], circovirus [n = 3], and gemycircularvirus [n = 4]) showed homology to viruses carried by giant pandas and their associated host species (such as red pandas and mosquitoes) in the same living domain. Remarkably, several viruses in this study phylogenetically assigned as bunyavirus, hepe-like virus, and circovirus were detected with relatively high abundance, but whether these newly identified tick-associated viruses can replicate in ticks and then transmit to host animals during a blood meal will require further investigation. These findings further expand our understanding of the role of giant panda-infesting ticks in the local ecosystem, especially related to viral acquisition and transmission, and lay a foundation to assess the risk for giant panda exposure to tick-borne viruses. IMPORTANCE Ticks rank only second to mosquitoes as blood-feeding arthropods, capable of spreading pathogens (including viruses, bacteria, and parasites) to hosts during a blood meal. To better understand the relationship between viruses carried by ticks and viruses that have been reported in giant pandas, it is necessary to analyze the viromes of giant panda-parasitic blood-sucking ticks. This study collected 421 ticks on the body surface of giant pandas in Sichuan Province, China. We characterized the extensive genetic diversity of viruses harbored by these ticks and reported frequent communication of viruses between giant pandas and their ticks. While most of the virome discovered here are nonpathogenic viruses from giant pandas and potentially tick-specific viruses, we revealed some possible tick-borne viruses, represented by novel bunyaviruses. This research contributes to the literature because currently there are few studies on the virome of giant panda-infesting ticks.

Symbiotic microbiota and odor ensure mating in time for giant pandas.

To achieve reproduction, male solitary mammals need to locate females using chemical communication with high levels of precision. In the case of giant pandas, the total estrus period of females was usually 15 days each year, however, successful mating activity is finished within 3 days from respective home range. The mating pattern of giant pandas, where multiple males compete for each female requires females employ efficient systems to communicate their estrus phases. To verifying whether the scent secretions of giant pandas changes by gender and estrus progression, the microbiota and compounds in 29 anogenital gland samples from 14 individuals during estrus were analyzed by 16S rRNA sequencing and GC-MS. We show that the microbiota communities covary by gender with 4 particular compounds of scent secretions. Among 597 genera, 34 were identified as biomarkers that could be used to distinguish between different estrus phases. By bacterial-compounds co-analysis, 3 fatty ester acids and squalene compounds covaried with the development of estrus in the bacterial communities of female giant pandas. This study helps clarify how a large, solitary mammal expresses accurate information to improve the likelihood of successful reproduction by changing the composition of microbiota and odor compounds of anogenital glands during estrus.