Zoological Research

1−12

Authors: Yong-Gang Niu, Deng-Bang Wei, Xue-Jing Zhang, Ti-Sen Xu, Xiang-Yong Li, Hai-Ying Zhang, Zhi-Fang An, Kenneth B. Storey, Qiang Chen

Changes in protein abundance and reversible protein phosphorylation (RPP) play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes. To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation, we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog, Nanorana parkeri, living on the Qinghai-Xizang Plateau. In total, 5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified. Based on proteomic analysis, 674 differentially expressed proteins (438 up-regulated, 236 down-regulated) were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas lower expressed proteins were mainly involved in metabolic processes. A total of 4251 modified sites (4147 up-regulated, 104 down-regulated) belonging to 1638 phosphoproteins (1555 up-regulated, 83 down-regulated) were significantly changed in the liver. During hibernation, RPP regulated a diverse array of proteins involved in multiple functions, including metabolic enzymatic activity, ion transport, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and inducing metabolic depression. Moreover, the activities of phosphofructokinase, glutamate dehydrogenase, and ATPase were all significantly lower in winter compared to summer. In conclusion, our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

25−35

Authors: Xian-Hui Ning, Bing Han, Ye Peng, Shao-Wu Yin

Long non-coding RNAs (lncRNAs) function as key modulators in mammalian immunity, particularly due to their involvement in lncRNA-mediated competitive endogenous RNA (ceRNA) crosstalk. Despite their recognized significance in mammals, research on lncRNAs in lower vertebrates remains limited. In the present study, we characterized the first immune-related lncRNA (pol-lnc78) in the teleost Japanese flounder (Paralichthys olivaceus). Results indicated that pol-lnc78 acted as a ceRNA for pol-miR-n199-3p to target the sterile alpha and armadillo motif-containing protein (SARM), the fifth discovered member of the Toll/interleukin 1 (IL-1) receptor (TIR) adaptor family. This ceRNA network regulated the antibacterial responses of flounder via the Toll-like receptor (TLR) signaling pathway. Specifically, SARM acted as a negative regulator and exacerbated bacterial infection by inhibiting the expression of inflammatory cytokines IL-1β and tumor necrosis factor-α (TNF-α). Pol-miR-n199-3p reduced SARM expression by specifically interacting with the 3’ untranslated region (UTR), thereby promoting SARM-dependent inflammatory cytokine expression and protecting the host against bacterial dissemination. Furthermore, pol-lnc78 sponged pol-miR-n199-3p to ameliorate the inhibition of SARM expression. During infection, the negative regulators pol-lnc78 and SARM were significantly down-regulated, while pol-miR-n199-3p was significantly up-regulated, thus favoring host antibacterial defense. These findings provide novel insights into the mechanisms underlying fish immunity and open new horizons to better understand ceRNA crosstalk in lower vertebrates.

39−54

Authors: Lan Zhao, Sheng-Nan Ji, Xiao-Bing Du, Jia-Hui Liu, Bo-Lun Zhang, Pei-Hua Li, Yi-Jun Yang, Bao-Guo Li, Yan-Qing Guo, Xiao-Guang Qi

The dynamics of animal social structures are heavily influenced by environmental patterns of competition and cooperation. In folivorous colobine primates, prevailing theories suggest that larger group sizes should be favored in rainforests with a year-round abundance of food, thereby reducing feeding competition. Yet, paradoxically, larger groups are frequently found in high-altitude or high-latitude montane ecosystems characterized by a seasonal scarcity of leaves. This contradiction is posited to arise from cooperative benefits in heterogeneous environments. To investigate this hypothesis, we carried out a six-year field study on two neighboring groups of golden snub-nosed monkey (Rhinopithecus roxellana), a species representing the northernmost distribution of colobine primates. Results showed that the groups adjusted their movement and habitat selection in response to fluctuating climates and spatiotemporal variability of resources, indicative of a dynamic foraging strategy. Notably, during the cold, resource-scarce conditions in winter, the large group occupied food-rich habitats but did not exhibit significantly longer daily travel distances than the smaller neighboring group. Subsequently, we compiled an eco-behavioral dataset of 52 colobine species to explore their evolutionary trajectories. Analysis of this dataset suggested that the increase in group size may have evolved via home range expansion in response to the cold and heterogeneous climates found at higher altitudes or latitudes. Hence, we developed a multi-benefits framework to interpret the formation of larger groups by integrating environmental heterogeneity. In cold and diverse environments, even smaller groups require larger home ranges to meet their dynamic survival needs. The spatiotemporal distribution of high-quality resources within these expanded home ranges facilitates more frequent interactions between groups, thereby encouraging social aggregation into larger groups. This process enhances the benefits of collaborative actions and reproductive opportunities, while simultaneously optimizing travel costs through a dynamic foraging strategy.

69−78

Authors: Shun Wang, Peng Zhang, Fan Fei, Tianyang Tong, Xiujuan Zhou, Yajie Zhou, Jing Zhang, Mengke Wei, Yanqi Zhang, Lei Zhang, Yulong Huang, Lin Zhang, Xin Zhang, Tiantian Cai, Can Xie

Birds exhibit extraordinary mobility and remarkable navigational skills, obtaining guidance cues from the Earth’s magnetic field for orientation and long-distance movement. Bird species also show tremendous diversity in navigation strategies, with considerable differences even within the same taxa and among individuals from the same population. The highly conserved iron and iron-sulfur cluster binding magnetoreceptor (MagR) protein is suggested to enable animals, including birds, to detect the geomagnetic field and navigate accordingly. Notably, MagR is also implicated in other functions, such as electron transfer and biogenesis of iron-sulfur clusters, raising the question of whether variability exists in its biochemical and biophysical features among species, particularly birds. In the current study, we conducted a comparative analysis of MagR from two different bird species, including the migratory European robin (Erithacus rubecula) and the homing pigeon (Columba livia) . Sequence alignment revealed an extremely high degree of similarity between the MagRs of these species, with only three sequence variations. Nevertheless, two of these variations underpinned significant differences in metal binding capacity, oligomeric state, and magnetic properties. These findings offer compelling evidence for the marked differences in MagR between the two avian species, potentially explaining how a highly conserved protein can mediate such diverse functions.

95−107

Authors: Hai-Yang Wang, Lan-Xiang Liu, Xue-Yi Chen, Yang-Dong Zhang, Wen-Xia Li, Wen-Wen Li, Lian Wang,  Xiao-Long Mo, Hong Wei, Ping Ji, Peng Xie

The gut microbiome interacts with the host to maintain body homeostasis, with gut microbial dysbiosis implicated in many diseases. However, the underlying mechanisms of gut microbe regulation of host behavior and brain functions remain unclear. This study aimed to elucidate the influence of gut microbiota on brain functions via post-translational modification mechanisms in the presence or absence of bacteria without any stimulation. We conducted succinylome analysis of hippocampal proteins in germ-free (GF) and specific pathogen-free (SPF) mice and metagenomic analysis of feces from SPF mice. These results were integrated with previously reported hippocampal acetylome and phosphorylome data from the same batch of mice. Subsequent bioinformatics analyses revealed 584 succinylation sites on 455 proteins, including 54 up-regulated succinylation sites on 91 proteins and 99 down-regulated sites on 51 proteins in the GF mice compared to the SPF mice. We constructed a panoramic map of gut microbiota-regulated succinylation, acetylation, and phosphorylation, and identified cross-talk and relative independence between the different types of post-translational modifications in modulating complicated intracellular pathways. Pearson correlation analysis indicated that 13 taxa, predominantly belonging to the Bacteroidetes phylum, were correlated with the biological functions of post-translational modifications. Positive correlations between these taxa and succinylation and negative correlations between these taxa and acetylation were identified in the modulation of intracellular pathways. This study highlights the hippocampal physiological changes induced by the absence of gut microbiota, and proteomic quantification of succinylation, phosphorylation, and acetylation, contributing to our understanding of the role of the gut microbiome in brain function and behavioral phenotypes.

125−135

Authors: Jian-Chao Liang, Zhi-Feng Ding, Chun-Lin Li, Yi-Ming Hu, Zhi-Xin Zhou, Gan-Wen Lie, Xiao-Nan Niu, Wen-Bin Huang, Hui-Jian Hu, Xing-Feng Si

Geographical background and dispersal ability may strongly influence assemblage dissimilarity; however, these aspects have generally been overlooked in previous large-scale beta diversity studies. Here, we examined whether the patterns and drivers of taxonomic beta diversity (TBD) and phylogenetic beta diversity (PBD) of breeding birds in China vary across (1) regions on both sides of the Hu Line, which demarcates China’s topographical, climatic, economic, and social patterns, and (2) species with different dispersal ability. TBD and PBD were calculated and partitioned into turnover and nestedness components using a moving window approach. Variables representing climate, habitat heterogeneity, and habitat quality were employed to evaluate the effects of environmental filtering. Spatial distance was considered to assess the impact of dispersal limitation. Variance partitioning analysis was applied to assess the relative roles of these variables. In general, the values of TBD and PBD were high in mountainous areas and were largely determined by environmental filtering. However, different dominant environmental filters on either side of the Hu Line led to divergent beta diversity patterns. Specifically, climate-driven species turnover and habitat heterogeneity-related species nestedness dominated the regions east and west of the line, respectively. Additionally, bird species with stronger dispersal ability were more susceptible to environmental filtering, resulting in more homogeneous assemblages. Our results indicated that regions with distinctive geographical backgrounds may present different ecological factors that lead to divergent assemblage dissimilarity patterns, and dispersal ability determines the response of assemblages to these ecological factors. Identifying a single universal explanation for the observed pattern without considering these aspects may lead to simplistic or incomplete conclusions. Consequently, a comprehensive understanding of large-scale beta diversity patterns and effective planning of conservation strategies necessitate the consideration of both geographical background and species dispersal ability.

138−151

Authors: Tao Jiang, Zhi-Min Zhou, Zi-Qi Ling, Qing Zhang, Zhong-Zi Wu, Jia-Wen Yang, Si-Yu Yang, Bin Yang Lu-Sheng Huang

Regulatory sequences and transposable elements (TEs) account for a large proportion of the genomic sequences of species; however, their roles in gene transcription, especially tissue-specific expression, remain largely unknown. Pigs serve as an excellent animal model for studying genomic sequence biology due to the extensive diversity among their wild and domesticated populations. Here, we conducted an integrated analysis using H3K27ac ChIP-seq, H3K4me3 ChIP-seq, and RNA-seq data from 10 different tissues of seven fetuses and eight closely related adult pigs. We aimed to annotate the regulatory elements and TEs to elucidate their associations with histone modifications and mRNA expression across different tissues and developmental stages. Based on correlation analysis between mRNA expression and H3K27ac and H3K4me3 peak activity, results indicated that H3K27ac exhibited stronger associations with gene expression than H3K4me3. Furthermore, 1.45% of TEs overlapped with either the H3K27ac or H3K4me3 peaks, with the majority displaying tissue-specific activity. Notably, a TE subfamily (LTR4C_SS), containing binding motifs for SIX1 and SIX4, showed specific enrichment in the H3K27ac peaks of the adult and fetal ovaries. RNA-seq analysis also revealed widespread expression of TEs in the exons or promoters of genes, including 4 688 TE-containing transcripts with distinct development stage-specific and tissue-specific expression. Of note, 1 967 TE-containing transcripts were enriched in the testes. We identified a long terminal repeat (LTR), MLT1F1, acting as a testis-specific alternative promoter in SRPK2 (a cell cycle-related protein kinase) in our pig dataset. This element was also conserved in humans and mice, suggesting either an ancient integration of TEs in genes specifically expressed in the testes or parallel evolutionary patterns. Collectively, our findings demonstrate that TEs are deeply embedded in the genome and exhibit important tissue-specific biological functions, particularly in the reproductive organs.

160−175

Authors: Yu-Ran Li, Zheng-Wei Wang, Richard T. Corlett, Wen-Bin Yu

The insect mitogenome is typically a compact circular molecule with highly conserved gene contents. Nonetheless, mitogenome structural variations have been reported in specific taxa, and gene rearrangements, usually the tRNAs, occur in different lineages. Because synapomorphies of mitogenome organizations can provide information for phylogenetic inferences, comparative analyses of mitogenomes have been given increasing attention. However, most studies use a very few species to represent the whole genus, tribe, family, or even order, overlooking potential variations at lower taxonomic levels, which might lead to some incorrect inferences. To provide new insights into mitogenome organizations and their implications for phylogenetic inference, this study conducted comparative analyses for mitogenomes of three social bee tribes (Meliponini, Bombini, and Apini) based on the phylogenetic framework with denser taxonomic sampling at the species and population levels. Comparative analyses revealed that mitogenomes of Apini and Bombini are the typical type, while those of Meliponini show diverse variations in mitogenome sizes and organizations. Large inverted repeats (IRs) cause significant gene rearrangements of protein coding genes (PCGs) and rRNAs in Indo-Malay/Australian stingless bee species. Molecular evolution analyses showed that the lineage with IRs have lower dN/dS ratios for PCGs than lineages without IRs, indicating potential effects of IRs on the evolution of mitochondrial genes. The finding of IRs and different patterns of gene rearrangements suggested that Meliponini is a hotspot in mitogenome evolution. Unlike conserved PCGs and rRNAs whose rearrangements were found only in the mentioned lineages within Meliponini, tRNA rearrangements are common across all three tribes of social bees, and are significant even at the species level, indicating that comprehensive sampling is needed to fully understand the patterns of tRNA rearrangements, and their implications for phylogenetic inference.

189−200

Authors: Cui-Hong Tong, Zhi-Peng Huo, Lu Diao, Dan-Yu Xiao, Ruo-Nan Zhao, Zhen-Ling Zeng, Wen-Guang Xiong

Monitoring the prevalence of antimicrobial resistance genes (ARGs) is vital for addressing the global crisis of antibiotic-resistant bacterial infections. Despite its importance, the characterization of ARGs and microbiome structures, as well as the identification of indicators for routine ARG monitoring in pig farms, are still lacking, particularly concerning variations in antimicrobial exposure in different countries or regions. Here, metagenomics and random forest machine learning were used to elucidate the ARG profiles, microbiome structures, and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe. Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs (P<0.05). ANT(6)-Ib, APH(3')-IIIa, and tet(40) were identified as shared core ARGs between the two pig populations. Furthermore, the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions. Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs, respectively. Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100% and 98.7%, respectively. Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy (r=0.72–0.88). Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs. The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

215−225

Authors: Zhan-Fei Wei, Kai-Wen Ta, Nan-Nan Zhang, Shan-Shan Liu, Liang Meng, Kai-Qiang Liu, Chong-Yang Cai, Xiao-Tong Peng, Chang-Wei Shao

A total of 10 specimens of Alcyonacea corals were collected at depths ranging from 905 m to 1 633 m by the manned submersible Shenhai Yongshi during two cruises in the South China Sea (SCS). Based on mitochondrial genomic characteristics, morphological examination, and sclerite scanning electron microscopy, the samples were categorized into four suborders (Calcaxonia, Holaxonia, Scleraxonia, and Stolonifera), and identified as 9 possible new cold-water coral species. Assessments of GC-skew dissimilarity, phylogenetic distance, and average nucleotide identity (ANI) revealed a slow evolutionary rate for the octocoral mitochondrial sequences. The nonsynonymous (Ka) to synonymous (Ks) substitution ratio (Ka/Ks) suggested that the 14 protein-coding genes (PCGs) were under purifying selection, likely due to specific deep-sea environmental pressures. Correlation analysis of the median Ka/Ks values of five gene families and environmental factors indicated that the genes encoding cytochrome b (cyt b) and DNA mismatch repair protein (mutS) may be influenced by environmental factors in the context of deep-sea species formation. This study highlights the slow evolutionary pace and adaptive mechanisms of deep-sea corals.

230−232

Authors: Wenhui Lee, Ren Lai