Ame-miR-1-3p of bee venom reduced cell viability through the AZIN1/OAZ1-ODC1-polyamines pathway and enhanced the defense ability of honeybee (Apis mellifera L.).

Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3' untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.

Resistin up-regulates LPL expression through the PPARγ-dependent PI3K/AKT signaling pathway impacting lipid accumulation in RAW264.7 macrophages.

Resistin is a cysteine-rich cytokine, which has been indicated as a mediator of insulin resistance and inflammation. Previous studies demonstrated that lipoprotein lipase (LPL) was an important enzyme that could mediate lipid accumulation in macrophages. Additionally, the intracellular molecules phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase (AKT)/peroxisome proliferator-activated receptor (PPARγ) were supposed to be involved in the lipid accumulation process in cells. However, it remains unclear whether resistin was correlated with the dysregulation of lipid metabolism in macrophages. The present study investigated that resistin could up-regulate the expression of LPL and increase the contents of intracellular triglyceride (TG) and total cholesterol (TC) in RAW264.7 macrophages. In addition, intracellular molecules PI3K, AKT and PPARγ were significantly up-regulated and activated in resitin-stimulated RAW264.7 macrophages (P < 0.05). In contrast, the effects of resistin on RAW264.7 macrophages could be abrogated by specific inhibitors for LPL (LPL-siRNA) and PI3K/AKT signaling pathway (LY294002). All together, this study demonstrated that resistin could up-regulate the expression of LPL and induce lipid accumulation in RAW264.7 macrophages. More importantly, the PPARγ-dependent PI3K/AKT signaling pathway was relevant to the lipid accumulation process in resistin-stimulated macrophages.

Activation of Porcine Alveolar Macrophages by Actinobacillus pleuropneumoniae Lipopolysaccharide via the Toll-Like Receptor 4/NF-κB-Mediated Pathway.

Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia. Overproduction of proinflammatory cytokines, like interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha, and resistin, in the lung is an important feature of A. pleuropneumoniae infection. These proinflammatory cytokines enhance inflammatory and immunological responses. However, the mechanism that leads to cytokine production remains unclear. As a major virulence factor of A. pleuropneumoniae, lipopolysaccharide (LPS) may act as a potent stimulator of Toll-like receptor 4 (TLR4), triggering a number of intracellular signaling pathways that lead to the synthesis of proinflammatory cytokines. Porcine alveolar macrophages (PAMs) are the first line of defense against pathogenic microbes during pathogen invasion. The results of the present study demonstrate that A. pleuropneumoniae LPS induces PAMs to produce inflammatory cytokines in time- and dose-dependent manners. Moreover, PAMs were activated by A. pleuropneumoniae LPS, resulting in upregulation of signaling molecules, including TLR4, MyD88, TRIF-related adaptor molecule, and NF-κB. In contrast, the activation effects of A. pleuropneumoniae LPS on PAMs could be suppressed by specific inhibitors, like small interfering RNA and Bay11-7082. Taken together, our data indicate that A. pleuropneumoniae LPS can induce PAMs to produce proinflammatory cytokines via the TLR4/NF-κB-mediated pathway. These findings partially reveal the mechanism of the overproduction of proinflammatory cytokines in the lungs of swine with A. pleuropneumoniae infection and may provide targets for the prevention of A. pleuropneumoniae-induced pneumonia. All the data could be used as a reference for the pathogenesis of respiratory infection.

Activation of the porcine alveolar macrophages via toll-like receptor 4/NF-κB mediated pathway provides a mechanism of resistin leading to inflammation.

Resistin, a previously discovered cysteine-rich adipokine known to regulate glucose metabolism, has been emerged as a mediator in inflammation and immunity. Its level was supposed to be related to the expression of indicators, such as interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) in inflammation. Toll-like receptor 4 (TLR4) was reported to be a receptor for resistin in cells, like leukocytes and peripheral blood mononuclear cells (PBMC). However, the pro-inflammatory role of resistin and its intracellular mechanisms in alveolar macrophages have not been thoroughly validated. Here we found that the pro-inflammatory cytokine expression in porcine alveolar macrophages (PAMs) was positively correlated with resistin. Our results also showed that resistin induced the expression of TLR4, intracellular molecules myeloid differentiation primary response protein 88 (MyD88), TRIF-related adaptor molecule (TRAM) and nuclear factor κB (NF-κB) in PAMs. In contrast, inhibition of TLR4, MyD88, TRAM and NF-κB abrogated the pro-inflammatory effect of resistin on PAMs. Additionally, the associations among TLR4, MyD88/TRAM and NF-κB were investigated by introducing TLR4-siRNA, MyD88-siRNA and TRAM-siRNA respectively into PAMs prior to the treatment with resistin. Taken together, our findings demonstrated that resistin promoted the production of pro-inflammatory cytokine in PAMs via TLR4/NF-κB-mediated pathway (TLR4/MyD88/TRAM/NF-κB).

Diversity of methanogens in the hindgut of captive white rhinoceroses, Ceratotherium simum.

BACKGROUND: The white rhinoceros is on the verge of extinction with less than 20,200 animals remaining in the wild. In order to better protect these endangered animals, it is necessary to better understand their digestive physiology and nutritional requirements. The gut microbiota is nutritionally important for herbivorous animals. However, little is known about the microbial diversity in the gastrointestinal tract (GIT) of the white rhinoceros. Methanogen diversity in the GIT may be host species-specific and, or, function-dependent. To assess methanogen diversity in the hindgut of white rhinoceroses, an archaeal 16S rRNA gene clone library was constructed from pooled PCR products obtained from the feces of seven adult animals. RESULTS: Sequence analysis of 153 archaeal 16S rRNA sequences revealed 47 unique phylotypes, which were assigned to seven operational taxonomic units (OTUs 1 to 7). Sequences assigned to OTU-7 (64 out of 153 total sequencs - 42%) and OTU-5 (18%, 27/153) had 96.2% and 95.5% identity to Methanocorpusculum labreanum, respectively, making Methanocorpusculum labreanum the predominant phylotype in these white rhynoceroses. Sequences belonging to OTU-6 (27%, 42/153) were related (97.6%) to Methanobrevibacter smithii. Only 4% of the total sequences (6/153) were assigned to Methanosphaera stadtmanae (OTU-1). Sequences belonging to OTU-2 (4%, 6/153), OTU-3 (3%, 5/153) and OTU-4 (2%, 3/153) were distantly related (87.5 to 88,4%) to Methanomassiliicoccus luminyensis and were considered to be novel species or strains that have yet-to-be cultivated and characterized. CONCLUSION: Phylogenetic analysis indicated that the methanogen species in the hindgut of white rhinoceroses were more similar to those in the hindgut of horses. Our findings may help develop studies on improving the digestibility of forage for sustainable management and better health of these endangered animals.

The pheromones of laying workers in two honeybee sister species: Apis cerana and Apis mellifera.

When a honeybee colony loses its queen, workers activate their ovaries and begin to lay eggs. This is accompanied by a shift in their pheromonal bouquet, which becomes more queen like. Workers of the Asian hive bee Apis cerana show unusually high levels of ovary activation and this can be interpreted as evidence for a recent evolutionary arms race between queens and workers over worker reproduction in this species. To further explore this, we compared the rate of pheromonal bouquet change between two honeybee sister species of Apis cerana and Apis mellifera under queenright and queenless conditions. We show that in both species, the pheromonal components HOB, 9-ODA, HVA, 9-HDA, 10-HDAA and 10-HDA have significantly higher amounts in laying workers than in non-laying workers. In the queenright colonies of A. mellifera and A. cerana, the ratios (9-ODA)/(9-ODA + 9-HDA + 10-HDAA + 10-HDA) are not significantly different between the two species, but in queenless A. cerana colonies the ratio is significant higher than in A. mellifera, suggesting that in A. cerana, the workers' pheromonal bouquet is dominated by the queen compound, 9-ODA. The amount of 9-ODA in laying A. cerana workers increased by over 585% compared with the non-laying workers, that is 6.75 times higher than in A. mellifera where laying workers only had 86% more 9-ODA compared with non-laying workers.

De Novo Genome Assembly of Chinese Plateau Honeybee Unravels Intraspecies Genetic Diversity in the Eastern Honeybee, Apis cerana.

Apis cerana abansis, widely distributed in the southeastern margin of the Qinghai-Tibet Plateau, is considered an excellent model to study the phenotype and genetic variation for highland adaptation of Asian honeybee. Herein, we assembled and annotated the chromosome-scale assembly genome of A. cerana abansis with the help of PacBio, Illumina and Hi-C sequencing technologies in order to identify the genome differences between the A. cerana abansis and the published genomes of different A. cerana strains. The sequencing methods, assembly and annotation strategies of A. cerana abansis were more comprehensive than previously published A. cerana genomes. Then, the intraspecific genetic diversity of A. cerana was revealed at the genomic level. We re-identified the repeat content in the genome of A. cerana abansis, as well as the other three A. cerana strains. The chemosensory and immune-related proteins in different A. cerana strains were carefully re-identified, so that 132 odorant receptor subfamilies, 12 gustatory receptor subfamilies and 22 immune-related pathways were found. We also discovered that, compared with other published genomes, the A. ceranaabansis lost the largest number of chemoreceptors compared to other strains, and hypothesized that gene loss/gain might help different A. cerana strains to adapt to their respective environments. Our work contains more complete and precise assembly and annotation results for the A. cerana genome, thus providing a resource for subsequent in-depth related studies.

The Dominating Role of Genetic Background in Shaping Gut Microbiota of Honeybee Queen Over Environmental Factors.

A balanced, diverse gut microbiota is vital for animal health. The microbial population is shaped by multiple factors including genetic background and environment, but other determinants remain controversial. Numerous studies suggest that the dominant factor is genetic background while others emphasize the environmental factors. Here, we bred asexual hybridization queens (AHQs) of honeybees through nutritional crossbreeding (laid in Apis mellifera colony but bred in Apis cerana colony), sequenced their gut microbiome, and compared it with normally bred sister queens to determine the primary factor shaping the gut microbiota. Our results showed that the dominant genera in the gut microbiota of AHQs were Brevundimonas, Bombella, and Lactobacillus, and its microbial community was more related to A. mellifera queens. The AHQs had a moderate number of different bacterial species and diversity, but total bacterial numbers were low. There were more significant taxa identified in the comparison between AHQ and A. cerana queen according to LEfSe analysis results. The only genetic-specific taxon we figured out was Brevundimonas. The growth of core bacterial abundance showed different characteristics among different queen groups in the first week after emerging. Collectively, this study suggested that the genetic background played a more dominant role than environmental factors in shaping the gut microbiota of honeybee queen and the microbiota of midgut was more sensitive than that of rectum to this impact.

Efficiency and mechanism of a vermicompost additive in enhancing composting of swine manure.

Vermicompost was used as an additive in swine manure composting to investigate the expression of bacterial functional genes on nutrients biotransformation. Three treatments with vermicompost compositions of 10%, 20%, and 30% in swine manure were set up. Raw manure was used as the control. The thermophilic period increased to 12 days, the NH4+ -N/NO3- -N ratio decreased to 0.85, and the germination index (GI) increased to 166% after vermicompost addition. Furthermore, higher relative abundances of Firmicutes were observed in the substrate during the initial stages of experiment. The abundance of the dominant phylum Proteobacteria and its related pathogenic genera Acinetobacter and Stenotrophomonas decreased in the thermophilic stage while the potentially beneficial genera Actinomadura and Chryseolinea increased. The expression of primary functional genes associated with the metabolism of carbohydrates, amino acids, xenobiotics, and fatty acids was enhanced during the thermophilic phase. Besides, most dominant genera showed strengthened correlations with NO3--N and GI, which were the strongest environmental factors for bacterial communities. Network analysis revealed a new metabolic pathway associated with dominant genera Pseudomonas, Acinetobacter, Stenotrophomonas, and Oceanobacter, whose abundance increased with vermicompost addition. Collectively, the results of this study indicate that vermicompost can promote composting efficiency by increasing the potentially beneficial bacteria, decreasing pathogenic bacteria, and enhancing the metabolic capacity of bacterial communities.

Protective effects of sodium butyrate on rotavirus inducing endoplasmic reticulum stress-mediated apoptosis via PERK-eIF2α signaling pathway in IPEC-J2 cells.

BACKGROUND: Rotavirus (RV) is a major pathogen that causes severe gastroenteritis in infants and young animals. Endoplasmic reticulum (ER) stress and subsequent apoptosis play pivotal role in virus infection. However, the protective mechanisms of intestinal damage caused by RV are poorly defined, especially the molecular pathways related to enterocytes apoptosis. Thus, the aim of this study was to investigate the protective effect and mechanism of sodium butyrate (SB) on RV-induced apoptosis of IPEC-J2 cells. RESULTS: The RV infection led to significant cell apoptosis, increased the expression levels of ER stress (ERS) markers, phosphorylated protein kinase-like ER kinase (PERK), eukaryotic initiation factor 2 alpha (eIF2α), caspase9, and caspase3. Blocking PERK pathway using specific inhibitor GSK subsequently reversed RV-induced cell apoptosis. The SB treatment significantly inhibited RV-induced ERS by decreasing the expression of glucose regulated protein 78 (GRP78), PERK, and eIF2α. In addition, SB treatment restrained the ERS-mediated apoptotic pathway, as indicated by downregulation of C/EBP homologous protein (CHOP) mRNA level, as well as decreased cleaved caspase9 and caspase3 protein levels. Furthermore, siRNA-induced GPR109a knockdown significantly suppressed the protective effect of SB on RV-induced cell apoptosis. CONCLUSIONS: These results indicate that SB exerts protective effects against RV-induced cell apoptosis through inhibiting ERS mediated apoptosis by regulating PERK-eIF2α signaling pathway via GPR109a, which provide new ideas for the prevention and control of RV.

Comb construction in mixed-species colonies of honeybees, Apis cerana and Apis mellifera.

Comb building in mixed-species colonies of Apis cerana and Apis mellifera was studied. Two types of cell-size foundation were made from the waxes of these species and inserted into mixed colonies headed either by an A. cerana or an A. mellifera queen. The colonies did not discriminate between the waxes but the A. cerana cell-size foundation was modified during comb building by the workers of both species. In pure A. cerana colonies workers did not accept any foundation but secreted wax and built on foundation in mixed colonies. Comb building is performed by small groups of workers through a mechanism of self-organisation. The two species cooperate in comb building and construct nearly normal combs but they contain many irregular cells. In pure A. mellifera colonies, the A. cerana cell size was modified and the queens were reluctant to lay eggs on such combs. In pure A. cerana colonies, the A. mellifera cell size was built without any modification but these cells were used either for drone brood rearing or for food storing. The principal elements of comb-building behaviour are common to both species, which indicates that they evolved prior to and were conserved after speciation.

Responses of queenright and queenless workers of Apis cerana to 9-keto-2(E)-decenoic acid, a pheromonal constituent of the mandibular gland.

In dequeened honeybee colonies ovarian activation occurs in some workers, and the pheromonal bouquets of these laying workers become more queen-like. In the Asiatic honeybee, Apis cerana, we compared the amount of 9-keto-2(E)-decenoic acid (9-ODA), a mandibular gland pheromone component, between non-laying workers from queenright colonies and laying workers from queenless colonies, and further, applied synthetic 9-ODA to workers to determine whether they discriminate workers with activated ovaries based on the level of this compound. Levels of 9-ODA were higher in laying workers from dequeened colonies than in non-laying workers from queenright colonies. In both queenright and queenless colonies, workers attacked more workers treated with 9-ODA than control-treated workers. These results suggest that detection of pseudoqueens in A. cerana is mediated by changes in 9-ODA.

Removal of phosphate by aluminum-modified clay in a heavily polluted lake, Southwest China: Effectiveness and ecological risks.

Lake eutrophication is a main water environmental problem. When the extraneous nutrients are effectively controlled, nutrients in water mainly source from endogenous release from the sediment. In situ passivation is an important pollution control technique for endogenous pollution in lakes or reservoirs. This study focused on Qianling Lake, a cascade-channel lake in Southwest China polluted by phosphorus (P), which was selected for an in situ passivation project using a novel Al-based passivator on the polluted water area. Accordingly, the release of endogenous nutrients from the sediment can be controlled to remediate the polluted water, and the remediation effect and ecological risk of the passivation were evaluated. The results showed that after 12 months of passivation, the release of P from the sediments can be effectively inhibited. Concentrations of total P (TP) and Chl-a within water of the passivation area were reduced by approximately 80% and 70%. Meanwhile, water transparency and the content of dissolved oxygen were remarkably enhanced. The application of the passivator remarkably improved the water quality. P in water and at the sediment-water interface was fixed on the surface of sediment in the form of Al-combined state. This passivator exhibits favorable P-controlling performance in the restoration of lakes and reservoirs polluted by endogenous P. This aluminum-modified clay is an effective passivator for remediation of internal P pollution in potentially similar lakes and reservoirs.

[Resistin promotes the production of inflammatory factors in cultured bovine alveolar macrophages and its mechanism].

Objective To explore the relationship between resistin and PPARγ and to investigate the pro-inflammatory functions of bovine resistin. Methods Bovine alveolar macrophages (BAMs) were incubated with 100 ng/mL bovine-resistin for 0, 1.5, 3, 6, 12, 24 hours, respectively. The mRNA expression levels of PPARγ, NF-κB, and resistin were tested by qRT-PCR, the protein expression levels of PPARγ and NF-κB were analyzed by Western blot analysis, and the levels of pro-inflammatory cytokines (IL-1ß and TNF-α) in the supernatant were measured by ELISA. Results After BAMs were treated with resistin for 1.5 hours, the level of PPARγ of BAMs was significantly reduced and there was a time dependent effect. Meanwhile, the mRNA levels of NF-κB and resistin in BAMs increased significantly since induced by bovine resistin for 6 hours and to peak at 12 hours. The protein expression of PPARγ in BAMs decreased significantly after incubating with bovine-resistin for 12 hours, while the expression of NF-κB increased significantly at 12 hours. Both IL-1ß and TNF-α increased in a time-dependent manner after 1.5 hours. Conclusion Bovine-resistin might induce BAMs producing pro-inflammatory cytokines such as IL-1ß and TNF-α via inhibiting PPARγ whereas activating the NF-κB mediated pathway.

The near complete mitochondrial genome of white-lipped Treefrog, Polypedates braueri (Anura, Rhacophoridae).

We newly sequenced the near complete mitochondrial genome of Polypedates braueri. The total length of the P. braueri partial mitogenome is 13 144 bp, with GenBank accession no. KT921226. It consists of 11 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNA), and 18 transfer RNA genes (tRNA). Most of the genes are encoded on the H-strand, except for seven tRNA and ND6, which are encoded on the L-strand. Our mitogenomic phylogenetic tree shows that the relationships between the genera Rhacophorus and Polypedates were well supported.

First near complete mitochondrial genome of large toothed toad, Oreolalax major (Anura: Megophryidae) from southwest China.

In this study, the near complete mitogenome sequence (15,469 bp) of Oreolalax major was determined using polymerase chain reaction (PCR). It includes 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes and 19 transfer RNA (tRNA) genes (GenBank accession number KU310894). The features of O. major have one more tRNA gene (tRNAMet ) behind the original one before ND2 which is similar to Leptobrachium boringii. Phylogenetic analyses were based on the concatenated sequences of the 13 protein-encoding genes of O. major and other related species.

The complete mitochondrial genome sequence of Red knobby newt Tylototriton shanjing (Amphibia: Caudata).

The complete mitogenome of Tylototriton shanjing is 16,661 bp in length with GenBank accession number KR154461, which contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), 2 ribosomal RNA genes (rRNA), and 1 control region (CR). The overall base composition of this mitogenome is biased toward AT content at 59.45%. Most of the PCGs and tRNA genes are located on the H-strand, except for ND6 subunit gene and eight tRNA genes, which were distributed on the L-strand. The PCGs used "ATG" and "GTG" as the start codons, while "TAA", "TAG", "AGA", and "T-" are used as stop codons. Almost all tRNA genes were folded into typical cloverleaf secondary structures. The T. shanjing genome had two tandem repeat sequences in the cob-noncoding region. The mitogenomic phylogenetic analyses shows that the genera Echinotriton and Tylototriton were clustered into a strong supported monophyletic clade, which is a sister clade to the genus Pleurodeles, this confirms the previous phylogenetic results.

Thermoregulation in mixed-species colonies of honeybees (Apis cerana and Apis mellifera).

Apis cerana and Apis mellifera normally display different strategies in cooling hive temperature, raising the question whether they would coordinate their efforts in to achieve stable thermoregulation in mixed colonies. The results show that the normal temperatures in the brood area in mixed colonies are more similar to those of pure A. cerana colonies than pure A. mellifera colonies. Under heat stress, A. cerana workers are more sensitive, and initiate fanning earlier than A. mellifera workers. In mixed colonies, the former become the main force for thermoregulation. When worker bees of both species were fanning together at the entrance, their own species-specific postures were adopted, but due to a significantly smaller number of A. mellifera workers engaged in fanning, the cooling efficiency of mixed colonies were closest to that of pure A. cerana colonies.