Unique myoglobin adaptation to endothermy and flight since the origin of birds.

Myoglobin (Mb) mediates oxygen diffusion and storage in muscle tissue and thus is important for the energy utilization and activity of animals. Birds generally have a high body temperature, and most species also possess the capability of powered flight. Both of these require high levels of aerobic metabolism. Within endothermic mammals, bats also independently evolved flight. Although the functional evolution of myoglobins in deep-diving amniote vertebrates has been well-studied, the functional evolution of myoglobin since the origins of both birds and bats is unclear. Here, with Mb-coding sequences from >200 extant amniote species, we reconstructed ancestral sequences to estimate the functional properties of myoglobin through amniote evolution. A dramatic change in net surface charge on myoglobin occurred during the origin of Aves, which might have been driven by positively selected amino acid substitutions that occurred on the lineage leading to all birds. However, in bats, no change in net surface charge occurred and instead, the Mb genes show evidence of strong purifying selection. The increased net surface charge on bird myoglobins implies an adaptation to flight-related endothermic and higher body temperatures, possibly by reducing harmful protein aggregations. Different from the findings of net surface charge, myoglobins of extant birds show lower stability compared with other amniotes, which probably accelerates the rate of oxygen utilization in muscles. In bats and other mammals, higher stability of Mb may be an alternative pathway for adaptation to endothermy, indicating divergent evolution of myoglobin in birds and bats.

A Nonsynonymous Substitution of Lhx3 Leads to Changes in Body Size in Dogs and Mice.

Lhx3 is a LIM-homeodomain transcription factor that affects body size in mammals by regulating the secretion of pituitary hormones. Akita, Shiba Inu, and Mame Shiba Inu dogs are Japanese native dog breeds that have different body sizes. To determine whether Lhx3 plays a role in the differing body sizes of these three dog breeds, we sequenced the Lhx3 gene in the three breeds, which led to the identification of an SNP in codon 280 (S280N) associated with body size. The allele frequency at this SNP differed significantly between the large Akita and the two kinds of smaller Shiba dogs. To validate the function of this SNP on body size, we introduced this change into the Lhx3 gene of mice. Homozygous mutant mice (S279N+/+) were found to have significantly increased body lengths and weights compared to heterozygous mutant (S279N+/-) and wild-type (S279N-/-) mice several weeks after weaning. These results demonstrate that a nonsynonymous substitution in Lhx3 plays an important role in regulating body size in mammals.

Research Note: SOCS2 contributes to reduction of the third digit during development of the chicken forelimb.

The development of the avian wing pattern has been the subject of heated debate due to its special shape. The Suppressor of cytokine signaling 2 (SOCS2) gene encodes a negative regulator of growth hormone (GH) signaling and bone growth and is known to be strongly expressed in the third digit of chicken forelimbs. These observations suggest that SOCS2 might regulate the morphology of the avian wing, however, the function of SOCS2 in avian limb development remains unknown. Here, we reexamined SOCS2 expression in successive developmental stages of chicken limb development by in situ hybridization (ISH) and describe extended expression from the posterior of the stypolod to the third digit of the forelimbs. We used the RCAS avian retrovirus to overexpress SOCS2 in the developing chicken limb buds, which resulted in reduced or malformed chicken wings while hindlimbs developed normally. Transcriptome sequencing (mRNA-Seq) revealed changes in expression of genes known to be associated with growth and development in forelimbs with overexpressed SOCS2. This study highlights a pivotal role for SOCS2 during the development of the wing in the chicken and provides new insight into molecular mechanisms regulating avian limb development.

CD206 modulates the role of M2 macrophages in the origin of metastatic tumors.

Tumor metastasis is a key factor affecting the life of patients with malignant tumors. For the past hundred years, scientists have focused on how to kill cancer cells and inhibit their metastasis in vivo, but few breakthroughs have been made. Here we hypothesized a novel mode for cancer metastasis. We show that the phagocytosis of apoptotic tumor cells by macrophages leads to their polarization into the M2 phenotype, and that the expression of stem cell related as well as drug resistance related genes was induced. Therefore, it appears that M2 macrophages have "defected" and have been transformed into the initial "metastatic cancer cells", and thus are the source, at least in part, of the distal tissue tumor metastasis. This assumption is supported by the presence of fused cells with characteristics of both macrophage and tumor cell observed in the peripheral blood and ascites of patients with ovarian cancer. By eliminating the expression of CD206 in M2 macrophages using siRNA, we show that the growth and metastasis of tumors was suppressed using both in vitro cell line and with experimental in vivo mouse models. In summary, we show that M2 macrophages in the blood circulation underwent a "change of loyalty" to become "cancer cells" that transformed into distal tissue metastasis, which could be suppressed by the knockdown of CD206 expression.

Evolution and expression patterns of the neo-sex chromosomes of the crested ibis.

Bird sex chromosomes play a unique role in sex-determination, and affect the sexual morphology and behavior of bird species. Core waterbirds, a major clade of birds, share the common characteristics of being sexually monomorphic and having lower levels of inter-sexual conflict, yet their sex chromosome evolution remains poorly understood. Here, by we analyse of a chromosome-level assembly of a female crested ibis (Nipponia nippon), a typical core waterbird. We identify neo-sex chromosomes resulting from fusion of microchromosomes with ancient sex chromosomes. These fusion events likely occurred following the divergence of Threskiornithidae and Ardeidae. The neo-W chromosome of the crested ibis exhibits the characteristics of slow degradation, which is reflected in its retention of abundant gametologous genes. Neo-W chromosome genes display an apparent ovary-biased gene expression, which is largely driven by genes that are retained on the crested ibis W chromosome but lost in other bird species. These results provide new insights into the evolutionary history and expression patterns for the sex chromosomes of bird species.

Genomic evolution of island birds from the view of the Swinhoe's pheasant (Lophura swinhoii).

Island endemic birds account for the majority of extinct vertebrates in the past few centuries. To date, the evolutionary characteristics of island endemic bird's is poorly known. In this research, we de novo assembled a high-quality chromosome-level reference genome for the Swinhoe's pheasant, which is a typical endemic island bird. Results of collinearity tests suggest rapid ancient chromosome rearrangement that may have contributed to the initial species radiation within Phasianidae, and a role for the insertions of CR1 transposable elements in rearranging chromosomes in Phasianidae. During the evolution of the Swinhoe's pheasant, natural selection positively selected genes involved in fecundity and body size functions, at both the species and population levels, which reflect genetic variation associated with island adaptation. We further tested for variation in population genomic traits between the Swinhoe's pheasant and its phylogenetically closely related mainland relative the silver pheasant, and found higher levels of genetic drift and inbreeding in the Swinhoe's pheasant genome. Divergent demographic histories of insular and mainland bird species during the last glacial period may reflect the differing impact of insular and continental climates on the evolution of species. Our research interprets the natural history and population genetic characteristics of the insular endemic bird the Swinhoe's pheasant, at a genome-wide scale, provides a broader perspective on insular speciation, and adaptive evolution and contributes to the genetic conservation of island endemic birds.

Succinylation proteomic analysis identified differentially expressed succinylation sites affecting porcine muscle fiber type function.

Muscle fiber type is a major factor in pork meat quality, however, the role of post-translational protein modifications, especially succinylation, in the regulation of muscle fiber type is not fully understood. Here we performed protein succinylation profiles of fast-type biceps femoris (BF) and slow-type soleus (SOL) muscles. A total of 4,221 succinylation sites were identified from these samples, of which 294 sites were differentially expressed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these succinylated proteins were mainly involved in glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Succinylation modification of the CRAT and RAB10 proteins was verified by co-immunoprecipitation. Protein-protein interaction (PPI) network analysis unveiled the interactions of these succinylated proteins that regulate pig myofiber type conversion. This investigation offers fresh perspectives into the molecular roles of protein succinylation in the regulation of pig myofiber type transformation and meat quality.

The genome evolution of Marek's disease viruses in chickens and turkeys in China.

The virus that causes Marek's disease (MD) is globally ubiquitous in chickens, continuously evolving, and poses a significant threat to the poultry industry. Although vaccines are extensively used, MD still occurs frequently and the virus has evolved increased virulence in China. Here, we report an outbreak of MD in vaccinated chickens and unvaccinated turkeys in a backyard farm in Guangdong province, China, in 2018. Phylogenetic analysis revealed two lineages of MDVs at this farm, with one lineage, containing isolates from two turkeys and five chickens, clustering with virulent Chinese strains and displays a relatively high genetic divergence from the vaccine strains. These new isolates appear to have broken through vaccine immunity, yielding this outbreak of MD in chickens and turkeys. The second lineage included four chicken isolates that clustered with the CVI988 and 814 vaccine strains. The large diversity of MDVs in this single outbreak reveals a complex circulation of MDVs in China. Poor breeding conditions and the weak application of disease prevention and control measures make backyard farms a hotbed for the evolution of viruses that cause infectious diseases. This is especially important in MDV as the MD vaccines do not provide sterilizing immunity, which allows the replication and shedding of virulent field viruses by vaccinated individuals and supporting the continuous evolution of MDVs. Hence, constant monitoring of the evolution of MDVs is necessary to understand the evolution of these field viruses and potential expansions of their host range.

Discovery and identification of a novel canine coronavirus causing a diarrhea outbreak in Vulpes.

Cross-species transmission of viruses from wildlife animal reservoirs, such as bats, poses a threat to human and domestic animal health. Previous studies have shown that domestic animals have important roles as intermediate hosts, enabling the transmission of genetically diverse coronaviruses from natural hosts to humans. Here, we report the identification and characterization of a novel canine coronavirus (VuCCoV), which caused an epidemic of acute diarrhea in Vulpes (foxes) in Shenyang, China. The epidemic started on November 8, 2019, and caused more than 39,600 deaths by January 1, 2022. Full-length viral genomic sequences were obtained from 15 foxes with diarrhea at the early stage of this outbreak. The VuCCoV genome shared more than 90% nucleotide identity with canine coronavirus (CCoV) for three of the four structural genes, with the S gene showing a larger amount of divergence. In addition, 67% (10/15) of the VuCCoV genomes contained an open reading frame (ORF3) gene, which was previously only detected in CCoV-I genomes. Notably, VuCCoV had only two to three amino acid differences at the partial RNA-dependent RNA polymerase (RdRp) level to bat CoV, suggesting a close genetic relationship. Therefore, these novel VuCCoV genomes represent a previously unsampled lineage of CCoVs. We also show that the VuCCoV spike protein binds to canine and fox aminopeptidase N (APN), which may allow this protein to serve as an entry receptor. In addition, cell lines were identified that are sensitive to VuCCoV using a pseudovirus system. These data highlight the importance of identifying the diversity and distribution of coronaviruses in domestic animals, which could mitigate future outbreaks that could threaten livestock, public health, and economic growth.

Early-life prophylactic antibiotic treatment disturbs the stability of the gut microbiota and increases susceptibility to H9N2 AIV in chicks.

BACKGROUND: Antibiotics are widely used for prophylactic therapy and for improving the growth performance of chicken. The problem of bacterial drug resistance caused by antibiotic abuse has previously attracted extensive attention; however, the influence of early-day use of prophylactic antibiotics on the gut microflora and on the disease resistance ability in chicks has not been explored. Here, we comprehensively evaluate the growth performance, gut microbial dynamics, level of antibiotic resistance genes (ARGs) in the gut microbial community, and resistance to H9N2 avian influenza virus (AIV) in chickens following long-term and short-term early-day prophylactic antibiotic treatment. RESULTS: Unexpectedly, long-term prophylactic enrofloxacin treatment slowed the growth rate of chickens, whereas short-term antibiotics treatments were found to increase the growth rate, but these changes were not statistically significant. Strikingly, expansions of Escherichia-Shigella populations were observed in early-life prophylactic antibiotics-treated groups of chickens, which is in contrast to the general perception that antibiotics should control their pathogenicity in chicks. The gut microbiota composition of chickens treated long term with antibiotics or received early-day antibiotics treatment tend to be more dramatically disturbed compared to the gut microbiome of chickens treated with antibiotics for a short term at a later date, especially after H9N2 AIV infection. CONCLUSIONS: Our data provide evidence that early-day and long-term antibiotic treatments have a more adverse effect on the intestinal microbiome of chickens, compared to short-term late age antibiotic treatment. Furthermore, our metagenomic data reveal that both long-term and short-term antibiotic treatment increase the relative abundance of ARGs. Our findings highlight the adverse effects of prophylactic antibiotic treatment and provide a theoretical basis for the cautious administration of antibiotics in food-producing animal management. Video Abstract.

Hybrid origin of a primate, the gray snub-nosed monkey.

Hybridization is widely recognized as promoting both species and phenotypic diversity. However, its role in mammalian evolution is rarely examined. We report historical hybridization among a group of snub-nosed monkeys (Rhinopithecus) that resulted in the origin of a hybrid species. The geographically isolated gray snub-nosed monkey Rhinopithecus brelichi shows a stable mixed genomic ancestry derived from the golden snub-nosed monkey (Rhinopithecus roxellana) and the ancestor of black-white (Rhinopithecus bieti) and black snub-nosed monkeys (Rhinopithecus strykeri). We further identified key genes derived from the parental lineages, respectively, that may have contributed to the mosaic coat coloration of R. brelichi, which likely promoted premating reproductive isolation of the hybrid from parental lineages. Our study highlights the underappreciated role of hybridization in generating species and phenotypic diversity in mammals.

Hologenomic insights into mammalian adaptations to myrmecophagy.

Highly specialized myrmecophagy (ant- and termite-eating) has independently evolved multiple times in species of various mammalian orders and represents a textbook example of phenotypic evolutionary convergence. We explored the mechanisms involved in this unique dietary adaptation and convergence through multi-omic analyses, including analyses of host genomes and transcriptomes, as well as gut metagenomes, in combination with validating assays of key enzymes' activities, in the species of three mammalian orders (anteaters, echidnas and pangolins of the orders Xenarthra, Monotremata and Pholidota, respectively) and their relatives. We demonstrate the complex and diverse interactions between hosts and their symbiotic microbiota that have provided adaptive solutions for nutritional and detoxification challenges associated with high levels of protein and lipid metabolisms, trehalose degradation, and toxic substance detoxification. Interestingly, we also reveal their spatially complementary cooperation involved in degradation of ants' and termites' chitin exoskeletons. This study contributes new insights into the dietary evolution of mammals and the mechanisms involved in the coordination of physiological functions by animal hosts and their gut commensals.

Pathogenicity, tissue tropism and potential vertical transmission of SARSr-CoV-2 in Malayan pangolins.

Malayan pangolin SARS-CoV-2-related coronavirus (SARSr-CoV-2) is closely related to SARS-CoV-2. However, little is known about its pathogenicity in pangolins. Using CT scans we show that SARSr-CoV-2 positive Malayan pangolins are characterized by bilateral ground-glass opacities in lungs in a similar manner to COVID-19 patients. Histological examination and blood gas tests are indicative of dyspnea. SARSr-CoV-2 infected multiple organs in pangolins, with the lungs the major target, and histological expression data revealed that ACE2 and TMPRSS2 were co-expressed with viral RNA. Transcriptome analysis indicated that virus-positive pangolins were likely to have inadequate interferon responses, with relative greater cytokine and chemokine activity in the lung and spleen. Notably, both viral RNA and viral proteins were detected in three pangolin fetuses, providing initial evidence for vertical virus transmission. In sum, our study outlines the biological framework of SARSr-CoV-2 in pangolins, revealing striking similarities to COVID-19 in humans.

Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals.

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Diversified Mammalian Visuasl Adaptations to Bright- or Dim-Light Environments.

Photic niche shifts of mammals are associated with changing visual capabilities, primarily mediated by three visual pigments, two (SWS1 and M/LWS) of them for color vision and rhodopsin (RH1) for dim-light vision. To further elucidate molecular mechanisms of mammalian visual adaptations to different light environments, a systematic study incorporating evolutionary analyses across diverse groups and in vitro assays have been carried out. Here, we collected gene sequences for the three opsins from 220 species covering all major mammalian clades. After screening for cone opsin gene losses, we estimated selective pressures on each of the three genes and compared the levels of selection experienced by species living in bright- and dim-light environments. SWS1 pigment is shown to experience accelerated evolution in species living in bright-light environments as has RH1 in aquatic cetaceans, indicating potential shifts for ecological adaptations. To further elucidate the functional mechanisms for these two pigments, we then carried out site-directed mutagenesis in representative taxa. For SWS1, violet and ultraviolet sensitivities in the pika and mouse are mainly affected by substitutions at the critical sites 86 and 93, which have strong epistatic interaction. For RH1, the phenotypic difference between the sperm whale and bovine sequences is largely contributed by a substitution at site 195, which could be critical for dim-light sensation for deep-diving species. Different evolutionary patterns for the visual pigments have been identified in mammals, which correspond to photic niches, although additional phenotypic assays are still required to fully explain the functional mechanisms.

Multi-omics Investigation of Freeze Tolerance in the Amur Sleeper, an Aquatic Ectothermic Vertebrate.

Freeze tolerance, the ability of an organism to survive internal ice formation, is a striking survival strategy employed by some ectotherms living in cold environments. However, the genetic bases of this remarkable adaptation are largely unknown. The Amur sleeper (Perccottus glenii), the only known freeze-tolerant fish species, can overwinter with its entire body frozen in ice. Here, we sequenced the chromosome-level genome of the Amur sleeper and performed comparative genomic, transcriptomic, and metabolomic analyses to investigate its strategies for surviving freezing. Evolutionary analysis suggested that the Amur sleeper diverged from its closest non-cold-hardy relative about 15.07 million years ago and has experienced a high rate of protein evolution. Transcriptomic and metabolomic data identified a coordinated and tissue-specific regulation of genes and metabolites involved in hypometabolism, cellular stress response, and cryoprotectant accumulation involved in freezing and thawing. Several genes show evidence of accelerated protein sequence evolution or family size expansion were found as adaptive responses to freezing-induced stresses. Specifically, genetic changes associated with cytoskeleton stability, cryoprotectant synthesis, transmembrane transport, and neuroprotective adaptations were identified as potentially key innovations that aid in freezing survival. Our work provides valuable resources and opportunities to unveil the molecular adaptations supporting freeze tolerance in ectothermic vertebrates.

Comparative Proteomic Analysis of Glycolytic and Oxidative Muscle in Pigs.

The quality of meat is highly correlated with muscle fiber type. However, the mechanisms via which proteins regulate muscle fiber types in pigs are not entirely understood. In the current study, we have performed proteomic profiling of fast/glycolytic biceps femoris (BF) and slow/oxidative soleus (SOL) muscles and identified several candidate differential proteins among these. We performed proteomic analyses based on tandem mass tags (TMTs) and identified a total of 26,228 peptides corresponding to 2667 proteins among the BF and SOL muscle samples. Among these, we found 204 differentially expressed proteins (DEPs) between BF and SOL muscle, with 56 up-regulated and 148 down-regulated DEPs in SOL muscle samples. KEGG and GO enrichment analyses of the DEPs revealed that the DEPs are involved in some GO terms (e.g., actin cytoskeleton, myosin complex, and cytoskeletal parts) and signaling pathways (PI3K-Akt and NF-kappa B signaling pathways) that influence muscle fiber type. A regulatory network of protein-protein interaction (PPI) between these DEPs that regulates muscle fiber types was constructed, which demonstrates how three down-regulated DEPs, including PFKM, GAPDH, and PKM, interact with other proteins to potentially control the glycolytic process. This study offers a new understanding of the molecular mechanisms in glycolytic and oxidative muscles as well as a novel approach for enhancing meat quality by transforming the type of muscle fibers in pigs.

Comparison of embryonic development, from HH21 to HH40, between ostrich (Struthio camelus) and chicken (Gallus gallus).

BACKGROUND: The chicken has been a representative model organism to study embryonic development in birds, however important differences exist among this class of species. As a representative of one of oldest existing clades of birds, the African ostrich (Struthio camelus), has the largest body among birds, and has two toes. Our purpose is to establish the corresponding stages in ostrich embryo development that match the well-established HH system of the chicken to facilitate comparative studies between the ostrich and other birds to better understand differences in development. RESULTS: Here we describe in detail the middle period of embryonic development using microscopic images and skeletal staining. We found that clear morphological differentiation between the ostrich and the chicken begins at stage 26. Bird limb cartilage first form in stage 25, while the development of the limb skeletons differs after stage 31. Calcification of limb skeletons in the chicken was completed faster. The first and second toes of the ostrich disappear at stages 36 and 38, respectively. CONCLUSIONS: This study should greatly aid ostrich-related developmental and morphological research and provide a reference for studying the development and evolution of avian limb skeletons, including molecular research. Questions that can now be addressed include studies into the fusion of tarsometatarsal skeleton, ossification, and digit loss.

Embryonic and skeletal development of the albino African clawed frog (Xenopus laevis).

The normal stages of embryonic development for wild-type Xenopus laevis were established by Nieuwkoop and Faber in 1956, a milestone in the history of understanding embryonic development. However, this work lacked photographic images and staining for skeleton structures from the corresponding stages. Here, we provide high-quality images of embryonic morphology and skeleton development to facilitate studies on amphibian development. On the basis of the classical work, we selected the albino mutant of X. laevis as the observation material to restudy embryonic development in this species. The lower level of pigmentation makes it easier to interpret histochemical experiments. At 23°C, albino embryos develop at the same rate as wild-type embryos, which can be divided into 66 stages as they develop into adults in about 58 days. We described the complete embryonic development system for X. laevis, supplemented with pictures of limb and skeleton development that are missing from previous studies, and summarized the characteristics and laws of limb and skeleton development. Our study should aid research into the development of X. laevis and the evolution of amphibians.