Evaluation of ecotourism suitability based on AHP-GIS: Taking Xiaoxiangling area of the Giant Panda National Park and the surrounding communities as an example.

The primary goal of national parks is to protect ecological environment, but also with the functions of scientific research, education, and recreation. Aiming for the realization of universal sharing, we used the analytic hierarchy process (AHP) to construct an ecotourism suitability evaluation system by selecting four factors, including landscape resources, ecological environment carrying capacity, recreation utilization capacity and social condition, taking Xiaoxiangling area of Giant Panda National Park and the surrounding communities as an example. We evaluated the ecotourism suitability based on GIS, and conducted a questionnaire survey of tourists, to propose suggestions on the functional zoning in terms of ecotourism suitability and subjective choice preferences of tourists. The results showed that the ecotourism suitability of the evaluation area could be classified into five levels. The most suitable areas were located nearby the natural landscape resources and far away from the core conservation area, and the least suitable areas distributed at the edge of the core conservation area. According to the results of suitability analysis, the evaluation area was divided into suitable development area, moderate development area, and restricted development area. Combined with the tourist preferences, we divided the recreational activities in the evaluation area into seven activities, namely, ecotourism, eco-camping, science education, leisure vacation, agricultural and animal husbandry culture experience, eco-education, and mountain adventure. These findings could help provide suitable services for different tourists and offer reference for the ecotourism developmental planning of the Xiaoxiangling area of the Giant Panda National Park.

Comprehensive assessment of river ecological environment in nature reserve based on TOPSIS combined with grey correlation method.

The main driving factors of river ecological environment were analyzed to reveal the response mechanism of river ecosystem to ecological environmental factors. The results showed that the driving factors of river water quality were resistivity, COD and reoxidation potential, the driving factors of soil environment along river banks were total phosphorus, total nitrogen and pH, and the driving factors of plant nutrition along river banks were total potassium and total nitrogen. The contribution rates of water quality, soil and plant to river ecological environment health were 43, 51 and 70%, respectively. The comprehensive ecological environment of Menghuo River, Gongyihai, Arulendi River, Yiniu River, Dahonggou River and Nanya River is superior to each other, with the comprehensive proximity index of 0.6258, 0.5908, 0.5524, 0.5265, 0.5195 and 0.3889, respectively. Each detection index can accurately and truly invert the ecological environment health status of rivers in the protected areas.

Beyond bacteria: Reconstructing microorganism connections and deciphering the predicted mutualisms in mammalian gut metagenomes.

Numerous gut microbial studies have focused on bacteria. However, archaea, viruses, fungi, protists, and nematodes are also regular residents of the gut ecosystem. Little is known about the composition and potential interactions among these six kingdoms in the same samples. Here, we unraveled the complex connection among them using approximately 123 gut metagenomes from 42 mammalian species (including carnivores, omnivores, and herbivores). We observed high variation in bacterial and fungal families and relatively low variation in archaea, viruses, protists, and nematodes. We found that some fungi in the mammalian intestine might come from environmental sources (e.g., soil and dietary plants), and some might be native to the intestine (e.g., the occurrence of Neocallimastigomycetes). The Methanobacteriaceae and Plasmodiidae families (archaea and protozoa, respectively) were predominant in these metagenomes, whereas Onchocercidae and Trichuridae were the two most common nematodes, and Siphoviridae and Myoviridae the two most common virus families in these mammalian gut metagenomes. Interestingly, most of the pairwise co-occurrence patterns were significantly positive among these six kingdoms, and significantly negative networks mainly occurred between fungi and prokaryotes (both bacteria and archaea). Our study revealed some inconvenient characteristics in the mammalian gut microorganism ecosystem: (1) the community formed by members of the analyzed kingdoms reflects the life history of the host and the potential threat posed by pathogenic protists and nematodes in mammals; and (2) the networks suggest the existence of predicted mutualism among members of these six kingdoms and of the predicted competition, mainly among fungi and other kingdoms.

Fly-over phylogeny across invertebrate to vertebrate: The giant panda and insects share a highly similar gut microbiota.

Many studies highlight that host phylogeny and diet are the two main factors influencing the animal gut microbiota. However, the internal mechanisms driving the evolution of animal gut microbiota may be more complex and complicated than we previously realized. Here, based on a large-scale meta-analysis of animal gut microbiota (16 s RNA gene data from approximately 1,800 samples; 108 metagenomes) across a wide taxonomic range of hosts, from invertebrate to vertebrate, we found high similarity in the gut microbial community (high proportion of Gammaproteobacteria (Pseudomonas)) of invertebrate insects and vertebrate bamboo-eating pandas (giant panda and red panda), which might be associated their plant-eating behavior and the presence of oxygen in the intestinal tract. A Pseudomonas strain-level analysis using 108 metagenomes further revealed that the response to either host niches or selection by the host might further lead to host-specific strains (or sub-strains) among the different hosts congruent with their evolutionary history. In this study, we uncovered new insights into the current understanding of the evolution of animals and their gut microbiota.

Host Bias in Diet-Source Microbiome Transmission in Wild Cohabitating Herbivores: New Knowledge for the Evolution of Herbivory and Plant Defense.

It is commonly understood that dietary nutrition will influence the composition and function of the animal gut microbiome. However, the transmission of organisms from the diet-source microbiome to the animal gut microbiome in the natural environment remains poorly understood, and elucidating this process may help in understanding the evolution of herbivores and plant defenses. Here, we investigated diet-source microbiome transmission across a range of herbivores (insects and mammals) living in both captive and wild environments. We discovered a host bias among cohabitating herbivores (leaf-eating insects and deer), where a significant portion of the herbivorous insect gut microbiome may originate from the diet, while in deer, only a tiny fraction of the gut microbiome is of dietary origin. We speculated that the putative difference in the oxygenation level in the host digestion systems would lead to these host biases in plant-source (diet) microbiome transmission due to the oxygenation living condition of the dietary plant's symbiotic microbiome. IMPORTANCE We discovered a host bias among cohabitating herbivores (leaf-eating insects and deer), where a significant portion of the herbivorous insect gut microbiome may originate from the diet, while in deer, only a tiny fraction of the gut microbiome is of dietary origin. We speculated that the putative difference in the oxygenation level in the host digestion systems would lead to these host biases in plant-source (diet) microbiome transmission due to the oxygenation living condition of the dietary plant's symbiotic microbiome. This study shed new light on the coevolution of herbivory and plant defense.

Social behavior of musk deer during the mating season potentially influences the diversity of their gut microbiome.

An increasing body of research has revealed that social behavior shapes the animal gut microbiome community and leads to the similarity among the same social group. However, some additional factors (e.g., diet and habitat within each social group) may also contribute to this similarity within the social group and dissimilarity between social groups. Here, we investigated the potential correlation between social behavior and the gut microbiome community in 179 musk deer from four breeding regions in the Maerkang Captive Center, Sichuan. The dominant gut microbiome phyla in the musk deer in this study were Firmicutes, Bacteroidetes, and Proteobacteria. We found significant effects on the alpha and beta diversity of the gut microbiome due to the breeding regions. The similarity within breeding regions was higher than that between the breeding regions. Due to their solitary lifestyle, captive musk deer are raised in single cages with no direct social contact most of the time. Deer in all of the breeding regions have the same diet and similar living conditions. However, during each mating season from November to January, in each region, one adult male and about six adult females will be put together into a large cage. Social behavior happens during cohabitation, including mating behavior, grooming within the same sex or between different sexes, and other social contact. Therefore, we speculated that high similarity within the breeding region might be associated with the social behavior during the mating season. This was a simple and straightforward example of the relationship between animal social behavior and the gut microbiome.

Genome-wide signatures of mammalian skin covering evolution.

Animal body coverings provide protection and allow for adaptation to environmental pressures such as heat, ultraviolet radiation, water loss, and mechanical forces. Here, using a comparative genomics analysis of 39 mammal species spanning three skin covering types (hairless, scaly and spiny), we found some genes (e.g., UVRAG, POLH, and XPC) involved in skin inflammation, skin innate immunity, and ultraviolet radiation damage repair were under selection in hairless ocean mammals (e.g., whales and manatees). These signatures might be associated with a high risk of skin diseases from pathogens and ultraviolet radiation. Moreover, the genomes from three spiny mammal species shared convergent genomic regions (EPHB2, EPHA4, and NIN) and unique positively selected genes (FZD6, INVS, and CDC42) involved in skin cell polarity, which might be related to the development of spines. In scaly mammals, the shared convergent genomic regions (e.g., FREM2) were associated with the integrity of the skin epithelium and epidermal adhesion. This study identifies potential convergent genomic features among distantly related mammals with the same skin covering type.

Geographic pattern of antibiotic resistance genes in the metagenomes of the giant panda.

The rise in infections by antibiotic-resistant bacteria poses a serious public health problem worldwide. The gut microbiome of animals is a reservoir for antibiotic resistance genes (ARGs). However, the correlation between the gut microbiome of wild animals and ARGs remains controversial. Here, based on the metagenomes of giant pandas (including three wild populations from the Qinling, Qionglai and Xiaoxiangling Mountains, and two major captive populations from Yaan and Chengdu), we investigated the potential correlation between the constitution of the gut microbiome and the composition of ARGs across the different geographic locations and living environments. We found that the types of ARGs were correlated with gut microbiome composition. The NMDS cluster analysis using Jaccard distance of the ARGs composition of the gut microbiome of wild giant pandas displayed a difference based on geographic location. Captivity also had an effect on the differences in ARGs composition. Furthermore, we found that the Qinling population exhibited profound dissimilarities of both gut microbiome composition and ARGs (the highest proportion of Clostridium and vancomycin resistance genes) when compared to the other wild and captive populations studies, which was supported by previous giant panda whole-genome sequencing analysis. In this study, we provide an example of a potential consensus pattern regarding host population genetics, symbiotic gut microbiome and ARGs. We revealed that habitat isolation impacts the ARG structure in the gut microbiome of mammals. Therefore, the difference in ARG composition between giant panda populations will provide some basic information for their conservation and management, especially for captive populations.

Immunogenicity and protection efficacy of a Salmonella enterica serovar Typhimurium fnr, arcA and fliC mutant.

Salmonella enterica serovar Typhimurium is a major food-borne pathogen that can cause self-limited gastroenteritis or life-threatening invasive diseases in humans. There is no licensed S. Typhimurium vaccine for humans to date. In this study, we attempted to construct a live attenuated vaccine strain of S. Typhimurium based on three genes, namely, the two global regulator genes fnr and arcA and the flagellin subunit gene fliC. The S. Typhimurium three-gene mutant, named SLT39 (ΔfnrΔarcAΔfliC), exhibited a high level of attenuation with a colonization defect in mouse tissues and approximately 104-fold decreased virulence compared with that of the wild-type strain. To evaluate the immunogenicity and protection efficacy of STL39, mice were inoculated twice with a dose of 107 CFU or 108 CFU at a 28-day interval, and the immunized mice were challenged with a lethal dose of the wild-type S. Typhimurium strain one month post second immunization. Compared with mock immunization, SLT39 immunization with either dose elicited significant serum total IgG, IgG1 and IgG2a and faecal IgA responses against inactivated S. Typhimurium antigens at a comparable level post second immunization, whereas the 108 CFU group induced higher levels of duodenal and caecal IgA than the 107 CFU group. Furthermore, the bacterial loads in mouse tissues, including Peyer's patches, spleen and liver, significantly decreased in the two SLT39 immunization groups compared to those in the control group post challenge. Additionally, all mice in the SLT39 (108 CFU) group and 80% of the mice in the SLT39 (107 CFU) group survived the lethal challenge, suggesting full protection and 80% protection efficacy, respectively. Thus, the S. Typhimurium fnr, arcA and fliC mutant proved to be a potential attenuated live vaccine candidate for prevention of homologous infection.

Genetic Diversity and Prediction Analysis of Small Isolated Giant Panda Populations After Release of Individuals.

Release of individuals is an effective conservation approach to protect endangered species. To save this small isolated giant panda population in Liziping Nature Reserve, a few giant pandas have been released to this population. Here we assess genetic diversity and future changes in the population using noninvasive genetic sampling after releasing giant pandas. In this study, a total of 28 giant pandas (including 4 released individuals) were identified in the Liziping, China. Compared with other giant panda populations, this population has medium-level genetic diversity; however, a Bayesian-coalescent method clearly detected, quantified, and dated a recent decrease in population size. The predictions for genetic diversity and survival of the population in the next 100 years indicate that this population has a high risk of extinction. We show that released giant pandas can preserve genetic diversity and improve the probability of survival in this small isolated giant panda population. To promote the recovery of this population, we suggest that panda release should be continued and this population will need to release 10 males and 20 females in the future.

Regulated delayed attenuation enhances the immunogenicity and protection provided by recombinant Salmonellaenterica serovar Typhimurium vaccines expressing serovar Choleraesuis O-polysaccharides.

Regulated delayed attenuation is a well-studied strategy for retaining the immunogenicity of Salmonella-vectored vaccines. In this study, this strategy was used to optimize two previously constructed recombinant Salmonella enterica serovar Typhimurium vaccines expressing S. Choleraesuis O-polysaccharides (OPS). The novel vaccine strains SLT31 (Δasd ΔrmlB-rfbP ΔPcrp::T araC PBAD) and SLT33 (Δasd ΔrfbP ΔpagL::T araC PBADrfbP ΔPcrp::T araC PBAD) were constructed by replacement of the native crp promoter with the arabinose-dependent araC PBAD promoter. As controls, two vaccine strains with direct crp mutations were also constructed, namely, SLT30 (Δasd ΔrmlB-rfbP Δcrp) and SLT32 (Δasd ΔrfbP ΔpagL::T araC PBADrfbP Δcrp). Then, the ability to deliver the heterologous S. Choleraesuis OPS on the Asd+ plasmid pCZ1 to the mouse immune system was evaluated in the strains with or without regulated delayed attenuation. The SLT30 (pCZ1) and SLT31 (pCZ1) strains expressed only the heterologous OPS, while the SLT32 (pCZ1) and SLT33 (pCZ1) strains co-expressed the homologous and heterologous OPS. The strain SLT31 (pCZ1) or SLT33 (pCZ1), which exhibited regulated delayed attenuation, colonized mouse tissues significantly better and stimulated stronger antibody responses against S. Choleraesuis LPS post immunization than the SLT30 (pCZ1) or SLT32 (pCZ1) strain. Immunization with SLT31 (pCZ1) or SLT33 (pCZ1) resulted in a significant reduction in bacterial loads in mouse tissues and a greater degree of protection against a lethal S. Choleraesuis dose compared with the effects observed after SLT30 (pCZ1) or SLT32 (pCZ1) immunization (100% vs. 80% or 70% vs. 50%, respectively). In addition, all four vaccines conferred complete protection against S. Typhimurium challenge. Overall, our study demonstrates that regulated delayed attenuation via an araC PBAD-regulated crp gene can enhance the cross-protection by Salmonella-vectored vaccines expressing heterologous OPS, and strain SLT31 (pCZ1) is a good candidate vaccine for preventing both S. Typhimurium and S. Choleraesuis infections.

Two Novel Salmonella Bivalent Vaccines Confer Dual Protection against Two Salmonella Serovars in Mice.

Non-typhoidal Salmonella includes thousands of serovars that are leading causes of foodborne diarrheal illness worldwide. In this study, we constructed three bivalent vaccines for preventing both Salmonella Typhimurium and Salmonella Newport infections by using the aspartate semialdehyde dehydrogenase (Asd)-based balanced-lethal vector-host system. The constructed Asd+ plasmid pCZ11 carrying a subset of the Salmonella Newport O-antigen gene cluster including the wzx-wbaR-wbaL-wbaQ-wzy-wbaW-wbaZ genes was introduced into three Salmonella Typhimurium mutants: SLT19 (Δasd) with a smooth LPS phenotype, SLT20 (Δasd ΔrfbN) with a rough LPS phenotype, and SLT22 (Δasd ΔrfbN ΔpagL::T araC PBADrfbN) with a smooth LPS phenotype when grown with arabinose. Immunoblotting demonstrated that SLT19 harboring pCZ11 [termed SLT19 (pCZ11)] co-expressed the homologous and heterologous O-antigens; SLT20 (pCZ11) exclusively expressed the heterologous O-antigen; and when arabinose was available, SLT22 (pCZ11) expressed both types of O-antigens, while in the absence of arabinose, SLT22 (pCZ11) expressed only the heterologous O-antigen. Exclusive expression of the heterologous O-antigen in Salmonella Typhimurium decreased the swimming ability of the bacterium and its susceptibility to polymyxin B. Next, the crp gene was deleted from the three recombinant strains for attenuation purposes, generating the three bivalent vaccine strains SLT25 (pCZ11), SLT26 (pCZ11), and SLT27 (pCZ11), respectively. Groups of BALB/c mice (12 mice/group) were orally immunized with 109 CFU of each vaccine strain twice at an interval of 4 weeks. Compared with a mock immunization, immunization with all three vaccine strains induced significant serum IgG responses against both Salmonella Typhimurium and Salmonella Newport LPS. The bacterial loads in the mouse tissues were significantly lower in the three vaccine-strain-immunized groups than in the mock group after either Salmonella Typhimurium or Salmonella Newport lethal challenge. All of the mice in the three vaccine-immunized groups survived the lethal Salmonella Typhimurium challenge. In contrast, SLT26 (pCZ11) and SLT27 (pCZ11) conferred full protection against lethal Salmonella Newport challenge, but SLT25 (pCZ11) provided only 50% heterologous protection. Thus, we developed two novel Salmonella bivalent vaccines, SLT26 (pCZ11) and SLT27 (pCZ11), suggesting that the delivery of a heterologous O-antigen in attenuated Salmonella strains is a prospective approach for developing Salmonella vaccines with broad serovar coverage.

Recombinant attenuated Salmonella Typhimurium with heterologous expression of the Salmonella Choleraesuis O-polysaccharide: high immunogenicity and protection.

Non-typhoidal Salmonella are associated with gastrointestinal disease worldwide and invasive disease in Africa. We constructed novel bivalent vaccines through the recombinant expression of heterologous O-antigens from Salmonella Choleraesuis in Salmonella Typhimurium. A recombinant Asd+ plasmid pCZ1 with the cloned Salmonella Choleraesuis O-antigen gene cluster was introduced into three constructed Salmonella Typhimurium Δasd mutants: SLT11 (ΔrfbP), SLT12 (ΔrmlB-rfbP) and SLT16 (ΔrfbP ∆pagL::TT araCPBAD rfbP). Immunoblotting demonstrated that SLT11 (pCZ1) and SLT12 (pCZ1) efficiently expressed the heterologous O-antigen. In the presence of arabinose, SLT16 (pCZ1) expressed both the homologous and heterologous O-antigens, whereas in the absence of arabinose, SLT16 (pCZ1) mainly expressed the heterologous O-antigen. We deleted the crp/cya genes in SLT12 (pCZ1) and SLT16 (pCZ1) for attenuation purposes, generating the recombinant vaccine strains SLT17 (pCZ1) and SLT18 (pCZ1). Immunization with either SLT17 (pCZ1) or SLT18 (pCZ1) induced specific IgG against the heterologous O-antigen, which mediated significant killing of Salmonella Choleraesuis and provided full protection against a lethal homologous challenge in mice. Furthermore, SLT17 (pCZ1) or SLT18 (pCZ1) immunization resulted in 83% or 50% heterologous protection against Salmonella Choleraesuis challenge, respectively. Our study demonstrates that heterologous O-antigen expression is a promising strategy for the development of multivalent Salmonella vaccines.