Whole-genome analysis of Escherichia coli isolated from wild Amur tiger (Panthera tigris altaica) and North China leopard (Panthera pardus japonensis).

Background: Escherichia coli is an important intestinal flora, of which pathogenic E. coli is capable of causing many enteric and extra-intestinal diseases. Antibiotics are essential for the treatment of bacterial infections caused by pathogenic E. coli; however, with the widespread use of antibiotics, drug resistance in E. coli has become particularly serious, posing a global threat to human, animal, and environmental health. While the drug resistance and pathogenicity of E. coli carried by tigers and leopards in captivity have been studied intensively in recent years, there is an extreme lack of information on E. coli in these top predators in the wild environment. Methods: Whole genome sequencing data of 32 E. coli strains collected from the feces of wild Amur tiger (Panthera tigris altaica, n = 24) and North China leopard (Panthera pardus japonensis, n = 8) were analyzed in this article. The multi-locus sequence types, serotypes, virulence and resistance genotypes, plasmid replicon types, and core genomic SNPs phylogeny of these isolates were studied. Additionally, antimicrobial susceptibility testing (AST) was performed on these E. coli isolates. Results: Among the E. coli isolates studied, 18 different sequence types were identified, with ST939 (21.9%), ST10 (15.6%), and ST3246 (9.4%) being the most prevalent. A total of 111 virulence genes were detected, averaging about 54 virulence genes per sample. They contribute to invasion, adherence, immune evasion, efflux pump, toxin, motility, stress adaption, and other virulence-related functions of E. coli. Sixty-eight AMR genes and point mutations were identified. Among the detected resistance genes, those belonging to the efflux pump family were the most abundant. Thirty-two E. coli isolates showed the highest rate of resistance to tetracycline (14/32; 43.8%), followed by imipenem (4/32; 12.5%), ciprofloxacin (3/32; 9.4%), doxycycline (2/32; 6.3%), and norfloxacin (1/32; 3.1%). Conclusions: Our results suggest that E. coli isolates carried by wild Amur tigers and North China leopards have potential pathogenicity and drug resistance.

Haplotype-resolved chromosome-scale genomes of the Asian and African Savannah Elephants.

The Proboscidea, which includes modern elephants, were once the largest terrestrial animals among extant species. They suffered mass extinction during the Ice Age. As a unique branch on the evolutionary tree, the Proboscidea are of great significance for the study of living animals. In this study, we generate chromosome-scale and haplotype-resolved genome assemblies for two extant Proboscidea species (Asian Elephant, Elephas maximus and African Savannah Elephant, Loxodonta africana) using Pacbio, Hi-C, and DNBSEQ technologies. The assembled genome sizes of the Asian and African Savannah Elephant are 3.38 Gb and 3.31 Gb, with scaffold N50 values of 130 Mb and 122 Mb, respectively. Using Hi-C technology ~97% of the scaffolds are anchored to 29 pseudochromosomes. Additionally, we identify ~9 Mb Y-linked sequences for each species. The high-quality genome assemblies in this study provide a valuable resource for future research on ecology, evolution, biology and conservation of Proboscidea species.

Genome assembly and annotation of the king ratsnake, Elaphe carinata.

The king ratsnake (Elaphe carinata) of the genus Elaphe is a common large, non-venomous snake widely distributed in Southeast and East Asia. It is an economically important farmed species. As a non-venomous snake, the king ratsnake predates venomous snakes, such as cobras and pit vipers. However, the immune and digestive mechanisms of the king ratsnake remain unclear. Despite their economic and research importance, we lack genomic resources that would benefit toxicology, phylogeography, and immunogenetics studies. Here, we used single-tube long fragment read sequencing to generate the first contiguous genome of a king ratsnake from Huangshan City, Anhui province, China. The genome size is 1.56 GB with a scaffold N50 of 6.53M. The total length of the genome is approximately 621 Mb, while the repeat content is 42.26%. Additionally, we predicted 22,339 protein-coding genes, including 22,065 with functional annotations. Our genome is a potentially useful addition to those available for snakes.

Single-nucleus transcriptome inventory of giant panda reveals cellular basis for fitness optimization under low metabolism.

BACKGROUND: Energy homeostasis is essential for the adaptation of animals to their environment and some wild animals keep low metabolism adaptive to their low-nutrient dietary supply. Giant panda is such a typical low-metabolic mammal exhibiting species specialization of extremely low daily energy expenditure. It has low levels of basal metabolic rate, thyroid hormone, and physical activities, whereas the cellular bases of its low metabolic adaptation remain rarely explored. RESULTS: In this study, we generate a single-nucleus transcriptome atlas of 21 organs/tissues from a female giant panda. We focused on the central metabolic organ (liver) and dissected cellular metabolic status by cross-species comparison. Adaptive expression mode (i.e., AMPK related) was prominently displayed in the hepatocyte of giant panda. In the highest energy-consuming organ, the heart, we found a possibly optimized utilization of fatty acid. Detailed cell subtype annotation of endothelial cells showed the uterine-specific deficiency of blood vascular subclasses, indicating a potential adaptation for a low reproductive energy expenditure. CONCLUSIONS: Our findings shed light on the possible cellular basis and transcriptomic regulatory clues for the low metabolism in giant pandas and helped to understand physiological adaptation response to nutrient stress.

The genome assembly and annotation of the Oriental rat snake Ptyas mucosa.

The Oriental rat snake Ptyas mucosa is a common non-venomous snake of the colubrid family, spanning most of South and Southeast Asia. P. mucosa is widely bred for its uses in traditional medicine, scientific research, and handicrafts. Therefore, genome resources of P. mucosa could play an important role in the efficacy of traditional medicine and the analysis of the living environment of this species. Here, we present a highly continuous P. mucosa genome with a size of 1.74 Gb. Its scaffold N50 length is 9.57 Mb, and the maximal scaffold length is 78.3 Mb. Its CG content is 37.9%, and its gene integrity reaches 86.6%. Assembled using long-reads, the total length of the repeat sequences in the genome reaches 735 Mb, and its repeat content is 42.19%. Finally, 24,869 functional genes were annotated in this genome. This study may assist in understanding P. mucosa and supporting medicinal research.

The genome assembly and annotation of the many-banded krait, Bungarus multicinctus.

Snakes are a vital component of wildlife resources and are widely distributed across the globe. The many-banded krait Bungarus multicinctus is a highly venomous snake found across Southern Asia and central and southern China. Snakes are an ancient reptile group, and their genomes can provide important clues for understanding the evolutionary history of reptiles. Additionally, genomic resources play a crucial role in comprehending the evolution of all species. However, snake genomic resources are still scarce. Here, we present a highly contiguous genome of B. multicinctus with a size of 1.51 Gb. The genome contains a repeat content of 40.15%, with a total length exceeding 620 Mb. Additionally, we annotated a total of 24,869 functional genes. This research is of great significance for comprehending the evolution of B. multicinctus and provides genomic information on the genes involved in venom gland functions.

Distinct and shared endothermic strategies in the heat producing tissues of tuna and other teleosts.

Although most fishes are ectothermic, some, including tuna and billfish, achieve endothermy through specialized heat producing tissues that are modified muscles. How these heat producing tissues evolved, and whether they share convergent molecular mechanisms, remain unresolved. Here, we generated a high-quality genome from the mackerel tuna (Euthynnus affinis) and investigated the heat producing tissues of this fish by single-nucleus and bulk RNA sequencing. Compared with other teleosts, tuna-specific genetic variation is strongly associated with muscle differentiation. Single-nucleus RNA-seq revealed a high proportion of specific slow skeletal muscle cell subtypes in the heat producing tissues of tuna. Marker genes of this cell subtype are associated with the relative sliding of actin and myosin, suggesting that tuna endothermy is mainly based on shivering thermogenesis. In contrast, cross-species transcriptome analysis indicated that endothermy in billfish relies mainly on non-shivering thermogenesis. Nevertheless, the heat producing tissues of the different species do share some tissue-specific genes, including vascular-related and mitochondrial genes. Overall, although tunas and billfishes differ in their thermogenic strategies, they share similar expression patterns in some respects, highlighting the complexity of convergent evolution.

Chromosome-scale genomes reveal genomic consequences of inbreeding in the South China tiger: A comparative study with the Amur tiger.

The South China tiger (Panthera tigris amoyensis, SCT) is the most critically endangered subspecies of tiger due to functional extinction in the wild. Inbreeding depression is observed among the captive population descended from six wild ancestors, resulting in high juvenile mortality and low reproduction. We assembled and characterized the first SCT genome and an improved Amur tiger (P. t. altaica, AT) genome named AmyTig1.0 and PanTig2.0. The two genomes are the most continuous and comprehensive among any tiger genomes yet reported at the chromosomal level. By using the two genomes and resequencing data of 15 SCT and 13 AT individuals, we investigated the genomic signature of inbreeding depression of the SCT. The results indicated that the effective population size of SCT experienced three phases of decline, ~5.0-1.0 thousand years ago, 100 years ago, and since captive breeding in 1963. We found 43 long runs of homozygosity fragments that were shared by all individuals in the SCT population and covered a total length of 20.63% in the SCT genome. We also detected a large proportion of identical-by-descent segments across the genome in the SCT population, especially on ChrB4. Deleterious nonsynonymous single nucleotide polymorphic sites and loss-of-function mutations were found across genomes with extensive potential influences, despite a proportion of these loads having been purged by inbreeding depression. Our research provides an invaluable resource for the formulation of genetic management policies for the South China tiger such as developing genome-based breeding and genetic rescue strategy.

Genomic investigation of the Chinese alligator reveals wild-extinct genetic diversity and genomic consequences of their continuous decline.

Critically endangered species are usually restricted to small and isolated populations. High inbreeding without gene flow among populations further aggravates their threatened condition and reduces the likelihood of their long-term survival. Chinese alligator (Alligator sinensis) is one of the most endangered crocodiles in the world and has experienced a continuous decline over the past c. 1 million years. In order to identify the genetic status of the remaining populations and aid conservation efforts, we assembled the first high-quality chromosome-level genome of Chinese alligator and explored the genomic characteristics of three extant breeding populations. Our analyses revealed the existence of at least three genetically distinct populations, comprising two breeding populations in China (Changxing and Xuancheng) and one breeding population in an American wildlife refuge. The American population does not belong to the last two populations of its native range (Xuancheng and Changxing), thus representing genetic diversity extinct in the wild and provides future opportunities for genetic rescue. Moreover, the effective population size of these three populations has been continuously declining over the past 20 ka. Consistent with this decline, the species shows extremely low genetic diversity, a large proportion of long runs of homozygous fragments, and mutational load across the genome. Finally, to provide genomic insights for future breeding management and conservation, we assessed the feasibility of mixing extant populations based on the likelihood of introducing new deleterious alleles and signatures of local adaptation. Overall, this study provides a valuable genomic resource and important genomic insights into the ecology, evolution, and conservation of critically endangered alligators.

The chromosome-scale genome of the raccoon dog: Insights into its evolutionary characteristics.

The raccoon dog (Nyctereutes procyonoides) is an invasive canid species native to East Asia with several distinct characteristics. Here, we report a chromosome-scale genome of the raccoon dog with high contiguity, completeness, and accuracy. The intact taste receptor genes, expanded gene families, and positively selected genes related to digestion, absorption, foraging, and detoxification likely support the omnivory of raccoon dogs. Several positively selected genes and raccoon dog-specific mutations in TDRD6 and ZP3 genes may explain their high reproductivity. Enriched GO terms in energy metabolism and positively selected immune genes were speculated to be closely related to the diverse immune system of raccoon dogs. In addition, we found that several expanded gene families and positively selected genes related to lipid metabolism and insulin resistance may contribute to winter sleep of the raccoon dog. This high-quality genome provides a valuable resource for understanding the evolutionary characteristics of this species.

Chromosome-level Genome of the Muskrat (Ondatra zibethicus).

The muskrat (Ondatra zibethicus) is a semi-aquatic rodent species with ecological, economic, and medicinal importance. Here, we present an improved genome assembly, which is the first high-quality chromosome-level genome of the muskrat with high completeness and contiguity assembled using single-tube long fragment read, BGISEQ, and Hi-C sequencing technologies. The genome size of the final assembly was 2.63 Gb with 27 pseudochromosomes. The length of scaffold N50 reached 80.25 Mb with a Benchmarking Universal Single-Copy Ortholog score of 91.3%. We identified a 66.98 Mb X chromosome and a 1.14-Mb Y-linked genome region, and these sex-linked regions were validated by resequencing 32 extra male individuals. We predicted 19,396 protein-coding genes, among which 19,395 (99.99%) were functionally annotated. The expanded gene families in the muskrat genome were found to be enriched in several organic synthesis- and metabolism-related Gene Ontology terms, suggesting the likely genomic basis for the production and secretion of musk. This chromosome-level genome represents a valuable resource for improving our understanding of muskrat ecology and musk secretion.

Gene rearrangements in the mitochondrial genome of ten ascaris species and phylogenetic implications for Ascaridoidea and Heterakoidea families.

The Ascaridoidea family and Heterakoidea family are the most common and typical representative of large parasites. Although our understanding of these parasites' diversity has expanded by analyses of some mitochondrial genes, there is limited information on these species' evolutionary rates. Here we determined ten complete mitogenome sequences of five subfamilies of Ascaridoidea and one subfamily of Heterakoidea. The phylogenetic tree divided the Ascaridoidea into six monophyletic major clades, and the divergence time of Heterakoidea family and Ascaridoidea family can be placed during the early Carboniferous Period (300-360 Mya). The reconstruction of the ancestral state showed that the gene orders of all species in Ascaridoidea were conserved, and the Heterakoidea had obvious genome rearrangement. The conserved blocks between them were divided into five and the main types are tandem-duplication/random loss (TDRL). These results will help to better understand the gene rearrangements and evolutionary position of ascaris species.

Screening of cell-virus, cell-cell, gene-gene crosstalk among animal kingdom at single cell resolution.

BACKGROUND: The exact animal origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains obscure and understanding its host range is vital for preventing interspecies transmission. METHODS: Herein, we applied single-cell sequencing to multiple tissues of 20 species (30 data sets) and integrated them with public resources (45 data sets covering 26 species) to expand the virus receptor distribution investigation. While the binding affinity between virus and receptor is essential for viral infectivity, understanding the receptor distribution could predict the permissive organs and tissues when infection occurs. RESULTS: Based on the transcriptomic data, the expression profiles of receptor or associated entry factors for viruses capable of causing respiratory, blood, and brain diseases were described in detail. Conserved cellular connectomes and regulomes were also identified, revealing fundamental cell-cell and gene-gene cross-talks from reptiles to humans. CONCLUSIONS: Overall, our study provides a resource of the single-cell atlas of the animal kingdom which could help to identify the potential host range and tissue tropism of viruses and reveal the host-virus co-evolution.

Whole-genome resequencing of Chinese pangolins reveals a population structure and provides insights into their conservation.

Poaching and trafficking have a substantial negative impact on the population growth and range expansion of the Chinese pangolin (Manis pentadactyla). However, recently reported activities of Chinese pangolins in several sites of Guangdong province in China indicate a promising sign for the recovery of this threatened species. Here, we re-sequence genomes of 15 individuals and perform comprehensive population genomics analyses with previously published 22 individuals. These Chinese pangolins are found to be divided into three distinct populations. Multiple lines of evidence indicate the existence of a newly discovered population (CPA) comprises entirely of individuals from Guangdong province. The other two populations (CPB and CPC) have previously been documented. The genetic differentiation of the CPA and CPC is extremely large (FST = 0.541), which is larger than many subspecies-level differentiations. Even for the closer CPA and CPB, their differentiation (FST = 0.101) is still comparable with the population-level differentiation of many endangered species. Further analysis reveals that the CPA and CPB populations separate 2.5-4.0 thousand years ago (kya), and on the other hand, CPA and CPC diverge around 25-40 kya. The CPA population harbors more runs of homozygosity (ROHs) than the CPB and CPC populations, indicating that inbreeding is more prevalent in the CPA population. Although the CPC population has less mutational load than CPA and CPB populations, we predict that several Loss of Function (LoF) mutations will be translocated into the CPA or CPB populations by using the CPC as a donor population for genetic rescue. Our findings imply that the conservation of Chinese pangolins is challenging, and implementing genetic rescue among the three groups should be done with extreme caution.

Equus roundworms (Parascaris univalens) are undergoing rapid divergence while genes involved in metabolic as well as anthelminic resistance are under positive selection.

BACKGROUND: The evolution of parasites is often directly affected by the host's environment. Studies on the evolution of the same parasites in different hosts are of great interest and are highly relevant to our understanding of divergence. METHODS: Here we performed whole-genome sequencing of Parascaris univalens from different Equus hosts (horses, zebras and donkeys). Phylogenetic and selection analyses were performed to study the divergence and adaptability of P. univalens. RESULTS: At the genetic level, multiple lines of evidence indicate that P. univalens is mainly separated into two clades (horse-derived and zebra & donkey-derived). This divergence began 300-1000 years ago, and we found that most of the key enzymes related to glycolysis were under strong positive selection in zebra & donkey-derived roundworms, whereas the lipid-related metabolic system was under positive selection in horse-derived roundworms, indicating that the adaptive evolution of metabolism has occurred over the past few centuries. In addition, we found that some drug-related genes showed a significantly higher degree of selection in diverse populations. CONCLUSIONS: This work reports the adaptive evolution and divergence trend of P. univalens in different hosts for the first time. Its results indicate that the divergence of P. univalens is a continuous, dynamic process. Furthermore, the continuous monitoring of the effects of differences in nutritional and drug histories on the rapid evolution of roundworms is conducive to further understanding host-parasite interactions.

Comparison of genetic characteristics between captive and wild giant pandas based on 13 mitochondrial coding genes.

BACKGROUND: Research on genetic diversity based on mitochondrial DNA of giant pandas mainly focused on a single marker or a few genes. OBJECTIVE: To provide a more comprehensive assessment of the genetic diversity on giant pandas based on 13 mitochondrial protein coding genes. METHODS: We assembled 13 protein coding genes in the mitochondrial genome of the giant panda based on the whole genome sequencing data, including ND1, ND2, COX1, COX2, ATP8, ATP6, COX3, ND3, ND4L, ND4, ND5, ND6 and Cyt b. RESULTS: We successfully obtained long sequence of 11,416 base pairs with all 13 genes for 110 giant panda individual, accounting for 67.93% in length of the mitochondrial reference genome. Haplotype diversity was 0.9518 ± 0.009 and nucleotide diversity (π) was 0.00157 ± 0.00014. We detected three new haplotypes, including GPC10 and GPC21 for the CR sequence and GPB12 for the Cyt b gene. CONCLUSION: These multi-gene sequences provided more genetic variable information to compare captive and wild giant panda population.

Chromosome-Level Genome Assembly of the Green Peafowl (Pavo muticus).

The green peafowl (Pavo muticus) is facing a high risk of extinction due to the long-term and widespread threats of poaching and habitat conversion. Here, we present a high-quality chromosome-level genome assembly of the green peafowl with high contiguity and accuracy assembled by PacBio sequencing, DNBSEQ short-read sequencing, and Hi-C sequencing technologies. The final genome size was estimated to be 1.049 Gb, whereas 1.042 Gb of the genome was assigned to 27 pseudochromosomes. The scaffold N50 length was 75.5 Mb with a complete BUSCO score of 97.6%. We identified W and Z chromosomes and validated them by resequencing 14 additional individuals. Totally, 167.04 Mb repetitive elements were identified in the genome, accounting for 15.92% of the total genome size. We predicted 14,935 protein-coding genes, among which 14,931 genes were functionally annotated. This is the most comprehensive and complete de novo assembly of the Pavo genus, and it will serve as a valuable resource for future green peafowl ecology, evolution, and conservation studies.

VThunter: a database for single-cell screening of virus target cells in the animal kingdom.

Viral infectious diseases are a devastating and continuing threat to human and animal health. Receptor binding is the key step for viral entry into host cells. Therefore, recognizing viral receptors is fundamental for understanding the potential tissue tropism or host range of these pathogens. The rapid advancement of single-cell RNA sequencing (scRNA-seq) technology has paved the way for studying the expression of viral receptors in different tissues of animal species at single-cell resolution, resulting in huge scRNA-seq datasets. However, effectively integrating or sharing these datasets among the research community is challenging, especially for laboratory scientists. In this study, we manually curated up-to-date datasets generated in animal scRNA-seq studies, analyzed them using a unified processing pipeline, and comprehensively annotated 107 viral receptors in 142 viruses and obtained accurate expression signatures in 2 100 962 cells from 47 animal species. Thus, the VThunter database provides a user-friendly interface for the research community to explore the expression signatures of viral receptors. VThunter offers an informative and convenient resource for scientists to better understand the interactions between viral receptors and animal viruses and to assess viral pathogenesis and transmission in species. Database URL: https://db.cngb.org/VThunter/.

Genomic and transcriptomic analysis unveils population evolution and development of pesticide resistance in fall armyworm Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a destructive pest native to America and has recently become an invasive insect pest in China. Because of its rapid spread and great risks in China, understanding of FAW genetic background and pesticide resistance is urgent and essential to develop effective management strategies. Here, we assembled a chromosome-level genome of a male FAW (SFynMstLFR) and compared re-sequencing results of the populations from America, Africa, and China. Strain identification of 163 individuals collected from America, Africa and China showed that both C and R strains were found in the American populations, while only C strain was found in the Chinese and African populations. Moreover, population genomics analysis showed that populations from Africa and China have close relationship with significantly genetic differentiation from American populations. Taken together, FAWs invaded into China were most likely originated from Africa. Comparative genomics analysis displayed that the cytochrome p450 gene family is extremely expanded to 425 members in FAW, of which 283 genes are specific to FAW. Treatments of Chinese populations with twenty-three pesticides showed the variant patterns of transcriptome profiles, and several detoxification genes such as AOX, UGT and GST specially responded to the pesticides. These findings will be useful in developing effective strategies for management of FAW in China and other invaded areas.