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European foulbrood (EFB) is a prevalent disease in the European honey bee (Apis mellifera) in the United States, which can lead to colony decline and collapse. The bacterial components of EFB are well-studied, but the diversity of viral infections within infected colonies has not been explored. In this study, we use meta-transcriptomics sequencing of 12 honey bee hives, symptomatic (+, n = 6) and asymptomatic (-, n = 6) for EFB, to investigate viral infection associated with the disease. We assembled 41 viral genomes, belonging to three families (Iflaviridae, Dicistroviridae, and Sinhaliviridae), all previously reported in honey bees, including Lake Sinai virus, deformed wing virus, sacbrood virus, Black queen cell virus, and Israeli acute paralysis virus. In colonies with severe EFB, we observed a higher occurrence of viral genomes (34 genomes) in contrast to fewer recovered from healthy colonies (seven genomes) and a complete absence of Dicistroviridae genomes.We observed specific Lake Sinai virus clades associated exclusively with EFB + or EFB - colonies, in addition to EFB-afflicted colonies that exhibited an increase in relative abundance of sacbrood viruses. Multivariate analyses highlighted that a combination of site and EFB disease status influenced RNA virome composition, while EFB status alone did not significantly impact it, presenting a challenge for comparisons between colonies kept in different yards. These findings contribute to the understanding of viral dynamics in honey bee colonies compromised by EFB and underscore the need for future investigations to consider viral composition when investigating EFB.IMPORTANCEThis study on the viromes of honey bee colonies affected by European foulbrood (EFB) sheds light on the dynamics of viral populations in bee colonies in the context of a prevalent bacterial brood disease. The identification of distinct Lake Sinai virus and sacbrood virus clades associated with colonies affected by severe EFB suggests a potential connection between viral composition and disease status, emphasizing the need for further investigation into the role of viruses during EFB infection. The observed increase in sacbrood viruses during EFB infection suggests a potential viral dysbiosis, with potential implications for honey bee brood health. These findings contribute valuable insights related to beekeeping practices, offering a foundation for future research aimed at understanding and mitigating the impact of bacterial and viral infection in commercial honey bee operations and the management of EFB.

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BACKGROUND: Most researches on sponge holobionts focus primarily on symbiotic microbes, yet data at the level of the sponge hologenome are still relatively scarce. Understanding of the sponge host and its microbial gene expression profiles and the host-microbes interplay in different niches represents a key aspect of sponge hologenome. Using the Hawaiian demosponge Mycale grandis in different niches as a model, i.e. on rocks, on the surface of coral Porites compressa, under alga Gracilaria salicornia, we compared the bacterial and fungal community structure, functional gene diversity, expression pattern and the host transcriptome by integrating open-format (deep sequencing) and closed-format (GeoChip microarray) high-throughput techniques. RESULTS: Little inter-niche variation in bacterial and fungal phylogenetic diversity was detected for M. grandis in different niches, but a clear niche-dependent variability in the functional gene diversity and expression pattern of M. grandis host and its symbiotic microbiota was uncovered by GeoChip microarray and transcriptome analyses. Particularly, sponge host genes related to innate immunity and microbial recognition showed a strong correlation with the microbial symbionts' functional gene diversity and transcriptional richness in different niches. The cross-niche variability with respect to the symbiont functional gene diversity and the transcriptional richness of M. grandis holobiont putatively reflects the interplay of niche-specific selective pressure and the symbiont functional diversity. CONCLUSIONS: Niche-dependent gene expression profiles of M. grandis hologenome and the host-microbes interplay were suggested though little inter-niche variation in bacterial and fungal diversity was detected, particularly the sponge innate immunity was found to be closely related to the symbiotic microbes. Altogether, these findings provide novel insights into the black box of one sponge holobiont in different niches at the hologenome level.

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Flowers are colonized by a diverse community of microorganisms that can alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, members of the genera Pseudomonas and Pantoea, isolated from apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between the two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower commensal strains did not competitively exclude E. amylovora from the stigma habitat, as both bacteria and the pathogen co-existed on the stigma of apple flowers and in vitro. This suggests that plant protection might be mediated by other mechanisms than competitive niche exclusion. Using a synthetic stigma exudation medium, ternary co-culture of the bacterial strains led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Importantly, the gene expression profiles for the ternary co-culture were not just additive from binary co-cultures, suggesting that some functions only emerged in multipartite co-culture. Additionally, the ternary co-culture of the strains resulted in a stronger acidification of the growth milieu than mono- or binary co-cultures, pointing to another emergent property of co-inoculation. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their potential impact on plant health and pathogen behavior. IMPORTANCE: Fire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen's behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.

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China is the leading country for pumpkin production in the world. As other cucurbits, diseases caused by viruses are among the serious threats to pumpkin production, but our knowledge on the virus species infecting pumpkin plants is fragmentary. To understand the occurrence of viral diseases on pumpkin, we determined the geographical distribution characteristics, relative abundance and evolutionary relationship of pumpkin infected viruses by meta-transcriptome sequencing (RNA-seq) and viromic analysis of 159 samples exhibited typical viral symptoms collected across China in this study. Totally, 11 known and 3 new viruses were identified. Interestingly, 3 new viruses identified in this study should be positive-sense single-stranded RNA virus whose hosts are prokaryotes. The viruses identified in different sampling locations exhibit significant variations in term of virus species and relative abundance. Here, the results provide valuable information for understanding the virus species and their diversity in cultivated pumpkin across major growing regions of China.

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Tick-borne viruses (TBVs) have increasingly caused a global public health concern. This study collected Rhipicephalus ticks in Guangdong, southern China to identify RNA viruses. Meta-transcriptome analysis revealed the virome in Rhipicephalus ticks, resulting in the discovery of 10 viruses, including Lihan tick virus, Brown dog tick phlebovirus 1 and 2 in the family Phenuiviridae, Mivirus and Wuhan tick virus 2 in the family Chuviridae, Wuhan tick virus 1 in the family Rhabdoviridae, bovine hepacivirus in the family Flaviviridae, Guangdong tick quaranjavirus (GTQV) in the family Orthomyxoviridae, Guangdong tick orbivirus (GTOV) in the family Reoviridae, and Guangdong tick Manly virus (GTMV) of an unclassified family. Phylogenetic analysis showed that most of these TBVs were genetically related to the strains in countries outside China, and GTQV, GTOV, and GTMV may represent novel viral species. These findings provided evidence of the long-distance spread of these TBVs in Guangdong, southern China, suggesting the necessity and importance of TBV surveillance.

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Xylanases are hemicellulases that break down xylan to soluble pentoses. They are used for industrial purposes, such as paper whitening, beverage clarification, and biofuel production. The second-generation bioethanol production is hindered by the enzymatic hydrolysis step of the lignocellulosic biomass, due to the complex arrangement established among its constituents. Xylanases can potentially increase the production yield by improving the action of the cellulolytic enzyme complex. We prospected endo-ß-1,4-xylanases from meta-transcriptomes of the termite Heterotermes tenuis. In silico structural characterization and functional analysis of an endo-ß-1,4-xylanase from a symbiotic protist of H. tenuis indicate two active sites and a substrate-binding groove needed for the catalytic activity. No N-glycosylation sites were found. This endo-ß-1,4-xylanase was recombinantly expressed in Pichia pastoris and Escherichia coli cells, presenting a molecular mass of approximately 20 kDa. Enzymatic activity assay using recombinant endo-ß-1,4-xylanase was also performed on 1% xylan agar stained with Congo red at 30 °C and 40 °C. The enzyme expressed in both systems was able to hydrolyze the substrate xylan, becoming a promising candidate for further analysis aiming to determine its potential for application in industrial xylan degradation processes.

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Autophagy is a critical mechanism deployed by eukaryotic cells in response to stress, including viral infection, to boost the innate antimicrobial responses. However, an increasing number of pathogens hijack the autophagic machinery to facilitate their own replication. Influenza A virus (IAV), responsible for several global pandemics, has an intricate dependence on autophagy for successful replication in mammalian cells. To elucidate key chokepoints in the host stress responses facilitating IAV replication, we constructed a meta-transcriptome of IAV and host gene expression dynamics during early (1-3 hpi), mid (4-6 hpi), and late (8-12 hpi) stages of the viral replication cycle at two multiplicities of infection (MOI): 1 and 5. We supplemented the global transcriptome study with phosphoproteomic analysis of stress-activated protein kinase (SAPK/JNK) signaling in lung carcinoma (predominantly used as an in vitro model of IAV replication) and normal human bronchial epithelial cells. We report significant differences in the activation profiles of autophagy regulating genes upon IAV infection at the two MOI as well as divergent dependence on ULK1 signaling within the normal and cancer cells. Regardless of the cell model, JNK-Thr187 signaling was crucial for the production of infectious viral particles.

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Diet-induced acidosis imposes a health risk to young calves. In this study, we aimed to investigate the host jejunum transcriptome changes, along with its microbial community variations, using our established model of feed-induced ruminal acidosis in young calves. Eight bull calves were randomly assigned to two diet treatments beginning at birth (a starch-rich diet, Aci; a control diet, Con). Whole-transcriptome RNA sequencing was performed on the jejunum tissues collected at 17 weeks of age. Ribosomal RNA reads were used for studying microbial community structure variations in the jejunum. A total of 853 differentially expressed genes were identified (402 upregulated and 451 downregulated) between the two groups. The cell cycle and the digestion and absorption of protein in jejunal tissue were affected by acidosis. Compared to the control, genera of Campylobacter, Burkholderia, Acidaminococcus, Corynebacterium, and Olsenella significantly increased in abundance in the Aci group, while Lachnoclostridium and Ruminococcus were significantly lower in the Aci group. Expression changes in the AXL gene were associated with the abundance variations of a high number of genera in jejunum. Our study provided a snapshot of the transcriptome changes in the jejunum and its associated meta-transcriptome changes in microbial communities in young calves with feed-induced acidosis.

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Sertraline hydrochloride (Ser-HCl) is an effective and commonly used antidepressant drug, which is also frequently detected in aquatic environments. Our previous research showed that Ser-HCl changes the community composition of aquatic microbiome, but the understanding of the expression of functional pathways in microbial communities is still incomplete; to address this knowledge gap, we used meta-transcriptomics analysis to evaluate the toxicity of Ser-HCl to natural aquatic microbial communities cultured in laboratory microcosms. Meta-transcriptomic results show that a 15-day exposure to 50 µg/L Ser-HCl significantly changed the functional expression activity of aquatic microbial communities. Pathways related to lipid metabolism, energy metabolism, membrane transport function, and genetic information processing in the aquatic microbial community were severely inhibited under Ser-HCl treatment, but metabolism of cofactors and vitamins to alleviate biological toxicity after Ser-HCl exposure was enhanced. Our study thus reveals details of the effects of sertraline on the functioning of aquatic microbiome. Due to the extensive use of Ser-HCl and its strong biological activity, it should not continue to be an overlooked pollutant. Therefore, more attention should be paid to the negative effects of such biologically active drugs on the expression of aquatic microbiome.

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The gut microbiota produces metabolites that regulate host immunity, thereby impacting disease resistance and susceptibility. The extent to which commensal bacteria reciprocally respond to immune activation, however, remains largely unexplored. Herein, we colonized mice with four anaerobic symbionts and show that acute immune responses result in dramatic transcriptional reprogramming of these commensals with minimal changes in their relative abundance. Transcriptomic changes include induction of stress-response mediators and downregulation of carbohydrate-degrading factors such as polysaccharide utilization loci (PULs). Flagellin and anti-CD3 antibody, two distinct immune stimuli, induced similar transcriptional profiles, suggesting that commensal bacteria detect common effectors or activate shared pathways when facing different host responses. Immune activation altered the intestinal metabolome within 6 hours, decreasing luminal short-chain fatty acid and increasing aromatic metabolite concentrations. Thus, intestinal bacteria, prior to detectable shifts in community composition, respond to acute host immune activation by rapidly changing gene transcription and immunomodulatory metabolite production.

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The growing tips of plants grow sterile; therefore, disease-free plants can be generated from them. How plants safeguard growing apices from pathogen infection is still a mystery. The shoot apical meristem (SAM) is one of the three stem cells niches that give rise to the above ground plant organs. This is very well explored; however, how signaling networks orchestrate immune responses against pathogen infections in the SAM remains unclear. To reconstruct a transcriptional framework of the differentially expressed genes (DEGs) pertaining to various SAM cellular populations, we acquired large-scale transcriptome datasets from the public repository Gene Expression Omnibus (GEO). We identify here distinct sets of genes for various SAM cellular populations that are enriched in immune functions, such as immune defense, pathogen infection, biotic stress, and response to salicylic acid and jasmonic acid and their biosynthetic pathways in the SAM. We further linked those immune genes to their respective proteins and identify interactions among them by mapping a transcriptome-guided SAM-interactome. Furthermore, we compared stem-cells regulated transcriptome with innate immune responses in plants showing transcriptional separation among their DEGs in Arabidopsis. Besides unleashing a repertoire of immune-related genes in the SAM, our analysis provides a SAM-interactome that will help the community in designing functional experiments to study the specific defense dynamics of the SAM-cellular populations. Moreover, our study promotes the essence of large-scale omics data re-analysis, allowing a fresh look at the SAM-cellular transcriptome repurposing data-sets for new questions.

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Development of a properly functioning gastrointestinal tract (GIT) at an early age is critical for the wellbeing and lifetime productivity of dairy cattle. The role of early microbial colonization on GIT development in neonatal cattle and the associated molecular changes remain largely unknown, particularly for the small intestine. In this study, we performed artificial dosing of exogenous rumen fluid during the early life of the calf, starting at birth through the weaning transition at 8 wk. Six calves were included in this study. At 8 wk of age, tissue from the ileum was collected and subjected to host transcriptome and microbial metatranscriptome analysis using RNA sequencing. A total of 333 genes showed significant differential expression (DE) (fold-change ≥2; adjusted P < 0.1, mean read-count ≥10) between the treated and control calves. Gene ontology analysis indicated that these DE genes are predominantly associated with processes related to the host immune response (P < 0.0001). Association analysis between the host gene expression and the microbial genus abundance identified 57 genes as having significant correlation with the ileum microbial genera (P < 0.0001). Of these, three genes showed significant association with six microbial genera: lysozyme 2 (LYZ2), fatty acid binding protein 5 (FABP5), and fucosyltransferase (FUT1). Specifically, the profound increase in expression of LYZ2 in treated calves suggests the initiation of antibacterial activity and innate response from the host. Despite the limitation of a relatively small sample size, this study sheds light on the potential impact of early introduction of microbes on the small intestine of calves.

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Silver nanoparticles (AgNPs) are widely used because of their excellent antibacterial properties. They are, however, easily discharged into the water environment, causing potential adverse environmental effects. Meta-transcriptomic analyses are helpful to study the transcriptional response of prokaryotic and eukaryotic aquatic microorganisms to AgNPs. In the present study, microcosms were used to investigate the toxicity of AgNPs to a natural aquatic microbial community. It was found that a 7-day exposure to 10 µg L-1 silver nanoparticles (AgNPs) dramatically affected the structure of the microbial community. Aquatic micro eukaryota (including eukaryotic algae, fungi, and zooplankton) and bacteria (i.e., heterotrophic bacteria and cyanobacteria) responded differently to the AgNPs stress. Meta-transcriptomic analyses demonstrated that eukaryota could use multiple cellular strategies to cope with AgNPs stress, such as enhancing nitrogen and sulfur metabolism, over-expressing genes related to translation, amino acids biosynthesis, and promoting bacterial-eukaryotic algae interactions. By contrast, bacteria were negatively affected by AgNPs with less signs of detoxification than in case of eukaryota; various pathways related to energy metabolism, DNA replication and genetic repair were seriously inhibited by AgNPs. As a result, eukaryotic algae (mainly Chlorophyta) dominated over cyanobacteria in the AgNPs treated microcosms over the 7-d exposure. The present study helps to understand the effects of AgNPs on aquatic microorganisms and provides insights into the contrasting AgNPs toxicity in eukaryota and bacteria.

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Next-generation approaches have enabled researchers to deeply study the plant microbiota and to reveal how microbiota associated with plant roots has key effects on plant nutrition, disease resistance, and plant development. Although early "omics" experiments focused mainly on the species composition of microbial communities, new "meta-omics" approaches such as meta-transcriptomics provide hints about the functions of the microbes when interacting with their plant host. Here, we used an RNA-seq dataset previously generated for tomato (Solanum lycopersicum) plants growing on different native soils to test the hypothesis that host-targeted transcriptomics can detect the taxonomic and functional diversity of root microbiota. Even though the sequencing throughput for the microbial populations was limited, we were able to reconstruct the microbial communities and obtain an overview of their functional diversity. Comparisons of the host transcriptome and the meta-transcriptome suggested that the composition and the metabolic activities of the microbiota shape plant responses at the molecular level. Despite the limitations, mining available next-generation sequencing datasets can provide unexpected results and potential benefits for microbiota research.

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We present the genome organization and molecular characterization of the three Formica exsecta viruses, along with ORF predictions, and functional annotation of genes. The Formica exsecta virus-4 (FeV4; GenBank ID: MF287670) is a newly discovered negative-sense single-stranded RNA virus representing the first identified member of order Mononegavirales in ants, whereas the Formica exsecta virus-1 (FeV1; GenBank ID: KF500001), and the Formica exsecta virus-2 (FeV2; GenBank ID: KF500002) are positive single-stranded RNA viruses initially identified (but not characterized) in our earlier study. The new virus FeV4 was found by re-analyzing data from a study published earlier. The Formica exsecta virus-4 genome is 9,866 bp in size, with an overall G + C content of 44.92%, and containing five predicted open reading frames (ORFs). Our bioinformatics analysis indicates that gaps are absent and the ORFs are complete, which based on our comparative genomics analysis suggests that the genomes are complete. Following the characterization, we validate virus infection for FeV1, FeV2 and FeV4 for the first time in field-collected worker ants. Some colonies were infected by multiple viruses, and the viruses were observed to infect all castes, and multiple life stages of workers and queens. Finally, highly similar viruses were expressed in adult workers and queens of six other Formica species: F. fusca, F. pressilabris, F. pratensis, F. aquilonia, F. truncorum and F. cinerea. This research indicates that viruses can be shared between ant species, but further studies on viral transmission are needed to understand viral infection pathways.

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Next-generation DNA sequencing technologies, such as RNA-Seq, currently dominate genome-wide gene expression studies. A standard approach to analyse this data requires mapping sequence reads to a reference and counting the number of reads which map to each gene. However, for many transcriptome studies, a suitable reference genome is unavailable, especially for meta-transcriptome studies which assay gene expression from mixed populations of organisms. Where a reference is unavailable, it is possible to generate a reference by the de novo assembly of the sequence reads. However, the high cost of generating high-coverage data for de novo assembly hinders this approach and more importantly the accurate assembly of such data is challenging, especially for meta-transcriptome data, and resulting assemblies frequently suffer from collapsed regions or chimeric sequences. As an alternative to the standard reference mapping approach, we have developed a k-mer-based analysis pipeline (DiffKAP) to identify differentially expressed reads between RNA-Seq datasets without the requirement for a reference. We compared the DiffKAP approach with the traditional Tophat/Cuffdiff method using RNA-Seq data from soybean, which has a suitable reference genome. We subsequently examined differential gene expression for a coral meta-transcriptome where no reference is available, and validated the results using qRT-PCR. We conclude that DiffKAP is an accurate method to study differential gene expression in complex meta-transcriptomes without the requirement of a reference genome.

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The proper analysis of high-throughput sequencing datasets of mixed microbial communities (meta-transcriptomics) is substantially more complex than for datasets composed of single organisms. Adapting commonly used RNA-seq methods to the analysis of meta-transcriptome datasets can be misleading and not use all the available information in a consistent manner. However, meta-transcriptomic experiments can be investigated in a principled manner using Bayesian probabilistic modeling of the data at a functional level coupled with analysis under a compositional data analysis paradigm. We present a worked example for the differential functional evaluation of mixed-species microbial communities obtained from human clinical samples that were sequenced on an Illumina platform. We demonstrate methods to functionally map reads directly, conduct a compositionally appropriate exploratory data analysis, evaluate differential relative abundance, and finally identify compositionally associated (constant ratio) functions. Using these approaches we have found that meta-transcriptomic functional analyses are highly reproducible and convey significant information regarding the ecosystem.

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