Insights for the Captive Management of South China Tigers Based on a Large-Scale Genetic Survey.

There is an urgent need to find a way to improve the genetic diversity of captive South China tiger (SCT, Panthera tigris amoyensis), the most critically endangered taxon of living tigers, facing inbreeding depression. The genomes showed that 13 hybrid SCTs from Meihuashan were divided into two groups; one group included three individuals who had a closer relationship with pureblood SCTs than another group. The three individuals shared more that 40% of their genome with pureblood SCTs and might be potential individuals for genetic rescuing in SCTs. A large-scale genetic survey based on 319 pureblood SCTs showed that the mean microsatellite inbreeding coefficient of pureblood SCTs decreased significantly from 0.1789 to 0.0600 (p = 0.000009) and the ratio of heterozygous loci increased significantly from 38.5% to 43.2% (p = 0.02) after one individual of the Chongqing line joined the Suzhou line and began to breed in the mid-1980s, which is a reason why the current SCTs keep a moderate level of microsatellite heterozygosity and nucleotide diversity. However, it is important to establish a back-up population based on the three individuals through introducing one pureblood SCT into the back-up population every year. The back-up population should be an important reserve in case the pureblood SCTs are in danger in the future.

Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation.

BACKGROUND: Among six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers. RESULTS: In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious mutations, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records. CONCLUSIONS: The identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos.

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.

Comparative analysis of the fecal microbiome and metabolomics of healthy versus captive South China tigers with mild diarrhea.

Diarrhea-predominant irritable bowel syndrome (IBS-D) is common among the captive South China tigers in zoos. 16S rRNA gene sequencing was performed to demonstrate the compositions and structures of the gastrointestinal microbiota of this species with IBS-D. Their healthy (F1) and mushy (F2) feces were allocated into two groups. A total of 21 and 31 fecal bacterial communities of major phyla and genera were detected, respectively. The F1 and F2 groups had five common microbiotas at the phylum level (Firmicutes, Proteobacteria, Fusobacteria, Actinobacteria, and Bacteroidetes). Among the five phyla, the abundance of Bacteroidetes in the F2 group was significantly lower than that in the F1 group. The diversity level of fecal microbiota within the mild-diarrhea stool was also significantly lower than that of the healthy counterpart. Thirty-two metabolites were correlated to four genus-level bacteria (Bacteroides, Pseudoclavibacter, Streptococcus, and Ruminococcaceae-UCG-005). Due to its normal role in protein degradation and metabolism, we hypothesized that the lower abundance of Bacteroides within the F2 group could be associated with the IBS-D symptoms. Therefore, this work implied that ameliorating the daily diet with a supplement of probiotics, such as Bacteroides, could improve the gut health of this species.

Antibody Response of an H9 Subtype Avian Influenza Poultry Vaccine on Three Kinds of Wild Birds in Shanghai Zoo.

A semiannual immunization with a commercial inactivated H9 subtype avian influenza virus (AIV) vaccine developed for poultry has been used to prevent and control the avian influenza (AI) infections among captured wild birds in Shanghai Zoo. However, the overall safety and effectiveness of the poultry vaccine for housed birds in the zoo remain unclear. To verify the safety and efficacy of the commercial inactivated H9 AI vaccine on zoo birds and to explore a more reasonable and effective immunization procedure, 48 zoo birds, including 11 Oriental white storks, 25 peafowl, and 12 silver pheasants, were administered the AI vaccine developed for poultry use. Then, the clinical signs of the immunized birds were observed for 2 weeks, and the antibodies against H9 AI were determined via the hemagglutination inhibition test. Results showed that no harmful effects related to the vaccination were observed, and the antibody titers of the Oriental white stork, peafowl, and silver pheasants were all higher than 7 log 2 at 21 days, 30 days, 60 days, 120 days, and 180 days postimmunization. For further study, the H9 AIV titers of 11 peafowls and 6 Oriental storks, which were raised in the nursing ground, were continuously monitored for 15 months. All of their antibody titers were above the national standards of China (5 log 2; GB/T18936-2003), even at 12 months and 15 months postimmunization. We concluded that the commercial inactivated H9 AI vaccine used at the present time in Shanghai Zoo can induce high and prolonged immune responses in vaccinated birds.

Artículo regular­Respuesta de anticuerpos de una vacuna contra influenza aviar subtipo H9 en tres tipos de aves silvestres en el zoológico de Shanghai. Se ha utilizado una inmunización semestral con una vacuna comercial inactivada contra el virus de la influenza aviar del subtipo H9 (AIV) desarrollada para la avicultura comercial para prevenir y controlar las infecciones por influenza aviar (IA) entre aves silvestres cautivas en el zoológico de Shanghai. Sin embargo, la seguridad y eficacia generales de la vacuna avícola para las aves alojadas en el zoológico siguen sin estar completamente determinadas. Para verificar la seguridad y eficacia de la vacuna comercial contra la influenza aviar H9 inactivada en las aves de zoológico y para explorar un procedimiento de inmunización más razonable y eficaz, una vacuna contra la influenza desarrollada para su uso en avicultura comercial se administró a 48 aves de zoológico, incluyendo 11 cigüeñas blancas orientales, 25 pavos reales y 12 faisanes plateados. Posteriormente, se observaron los signos clínicos de las aves inmunizadas durante 2 semanas y se determinaron los anticuerpos contra el subtipo H9 del virus de influenza aviar mediante la prueba de inhibición de la hemaglutinación. Los resultados mostraron que no se desarrollaron efectos nocivos relacionados con la vacunación, y los títulos de anticuerpos de la cigüeña blanca oriental, el pavo real y los faisanes plateados fueron todos superiores a 7 log2 a los 21 días, 30 días, 60 días, 120 días y 180 días posinmunización. Para un estudio adicional, los títulos contra el subtipo H9 de 11 pavos reales y 6 cigüeñas orientales que se criaron en el área de recría, se evaluaron continuamente durante 15 meses. Todos sus títulos de anticuerpos estaban por encima de los estándares nacionales de China (5 log2; GB/T18936-2003), incluso a los 12 y 15 meses posteriores a la inmunización. Se concluye que la vacuna comercial de influenza aviar inactivada con el subtipo H9 que se utiliza actualmente en el zoológico de Shanghai puede inducir respuestas inmunitarias elevadas y prolongadas en las aves vacunadas.

The complete mitochondrial genome of De Brazza's monkey (Cercopithecus neglectus).

The complete mitochondrial genome of Cercopithecus neglectus was described. The mitogenome is 16,490 bp in length and consists 13 protein-coding genes (PCGs), 22 transfer-RNA genes, two ribosomal-RNA genes, and one non-coding region. All the 13 PCGs were 11,398 bp in length with most common start codon of ATG and termination codon of TAA. The overall GC content was 42.5%. The result of phylogenetic analysis showed that the relationship of C. neglectus was close to C. mona, C. pogonias, C. wolfi and C. denti.

Comparison of fecal microbiota composition of blue sheep fed Lolium perenne versus Sorghum sudanense.

Lolium perenne L. and Sorghum sudanense (Piper) Stapf. are 2 common forages fed to blue sheep (Pseudois nayaur Hodgson, 1833) in captivity. However, the effect of these 2 forages on the gastrointestinal microbiota is largely unknown. We analyzed the diversity of the microbiota in the feces of captive blue sheep fed with L. perenne (group F1) and S. sudanense (group F2) by 16S rRNA sequencing. A total of 20 major phyla and 29 genera fecal bacterial communities were detected in the 2 groups. The F1 and F2 groups shared common microbiota at the phylum level, which mainly consisted of Firmicutes and Bacteroidetes. Ruminococcaceae_UCG-005, Eubacterium_coprostanoligenes_group, Ruminococcaceae_UCG-013, and Ruminococcaceae_UCG-010 were the top 4 dominant taxa at the genus level. The percentage of Ruminococcaceae_UCG-010 was significantly higher in the F2 group (∼2.75-fold) than in F1 group. The diversity and abundance of the microbial community in F2 was higher than that in F1. Although both of L. perenne and S. sudanense affect the metabolism of the gastrointestinal microbiota of blue sheep, the S. sudanense improves more aspects of metabolism and biogenesis. In summary, our results demonstrated that L. perenne and S. sudanense affect blue sheep gastrointestinal microbiota in different ways. But S. sudanense efficiently improved the gastrointestinal microbiota of blue sheep.

Sorting Out the Genetic Background of the Last Surviving South China Tigers.

The South China tiger (Panthera tigris amoyensis) is endemic to China and also the most critically endangered subspecies of living tigers. It is considered extinct in the wild and only about 150 individuals survive in captivity to date, whose genetic heritage, however, is ambiguous and controversial. Here, we conducted an explicit genetic assessment of 92 studbook-registered South China tigers from 14 captive facilities using a subspecies-diagnostic system in the context of comparison with other voucher specimens to evaluate the genetic ancestry and level of distinctiveness of the last surviving P. t. amoyensis. Three mtDNA haplotypes were identified from South China tigers sampled in this study, including a unique P. t. amoyensis AMO1 haplotype not found in other subspecies, a COR1 haplotype that is widespread in Indochinese tigers (P. t. corbetti), and an ALT haplotype that is characteristic of Amur tigers (P. t. altaica). Bayesian STRUCTURE analysis and parentage verification confirmed the verified subspecies ancestry (VSA) as the South China tiger in 74 individuals. Genetic introgression from other tigers was detected in 18 tigers, and subsequent exclusion of these and their offspring from the breeding program is recommended. Both STRUCTURE clustering and microsatellite-based phylogenetic analyses demonstrated a close genetic association of the VSA South China tigers to Indochinese tigers, an issue that could only be elucidated by analysis of historical South China tiger specimens with wild origin. Our results also indicated a moderate level of genetic diversity in the captive South China tiger population, suggesting a potential for genetic restoration.

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes.

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P. t. amoyensis mitochondrial genomes were also highly similar to those of P. uncia. Additionally, the rate of point mutation was only 0.3% and a total of 59 variable sites between P25 and P27 were found. Out of the 59 variable sites, 6 were located in 6 different tRNA genes, 6 in the 2 rRNA genes, 7 in non-coding regions (one located between tRNA-Asn and tRNA-Tyr and six in the D-loop), and 40 in 10 protein-coding genes. COI held the largest amount of variable sites (9 sites) and Cytb contained the highest variable rate (0.7%) in the complete sequences. Moreover, out of the 40 variable sites located in 10 protein-coding genes, 12 sites were nonsynonymous.