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miRNAs represent ubiquitous regulators of gene expression and play an important and pivotal regulatory role in viral disease pathogenesis and virus–host interactions. Although previous studies have provided basic data for understanding the role of miRNAs in the molecular mechanisms of viral infection in birds, the role of miRNAs in the regulation of host responses to chicken astrovirus (CAstV) infection in chickens is not yet understood. In our study, we applied next-generation sequencing to profile miRNA expression in CAstV-infected chickens and to decipher miRNA-targeted specific signaling pathways engaged in potentially vital virus-infection biological processes. Among the 1354 detected miRNAs, we identified 58 mature miRNAs that were significantly differentially expressed in infected birds. Target prediction resulted in 4741 target genes. GO and KEGG pathway enrichment analyses showed that the target genes were mainly involved in the regulation of cellular processes and immune responses. [ABSTRACT FROM AUTHOR]

The infamous "master manipulators"—intracellular bacteria of the genus Wolbachia—infect a broad range of phylogenetically diverse invertebrate hosts in terrestrial ecosystems. Wolbachia has an important impact on the ecology and evolution of their host with documented effects including induced parthenogenesis, male killing, feminization, and cytoplasmic incompatibility. Nonetheless, data on Wolbachia infections in non-terrestrial invertebrates are scarce. Sampling bias and methodological limitations are some of the reasons limiting the detection of these bacteria in aquatic organisms. In this study, we present a new metagenetic method for detecting the co-occurrence of different Wolbachia strains in freshwater invertebrates host species, i.e., freshwater Arthropoda (Crustacea), Mollusca (Bivalvia), and water bears (Tardigrada) by applying NGS primers designed by us and a Python script that allows the identification of Wolbachia target sequences from the microbiome communities. We also compare the results obtained using the commonly applied NGS primers and the Sanger sequencing approach. Finally, we describe three supergroups of Wolbachia: (i) a new supergroup V identified in Crustacea and Bivalvia hosts; (ii) supergroup A identified in Crustacea, Bivalvia, and Eutardigrada hosts, and (iii) supergroup E infection in the Crustacea host microbiome community. [ABSTRACT FROM AUTHOR]

In the high-throughput sequencing (HTS) era, a metabarcoding technique based on the bacterial V3–V4 hypervariable region of 16S rRNA analysis requires sophisticated bioinformatics pipelines and validated methods that allow researchers to compare their data with confidence. Many commercial laboratories conduct extensive HTS analyses; however, there is no available information on whether the results generated by these vendors are consistent. In our study, we compared the sequencing data obtained for the same marine microbiome community sample generated by three commercial laboratories. Additionally, as a sequencing control to determine differences between commercial laboratories and two 16S rRNA databases, we also performed a "mock community" analysis of a defined number of microbial species. We also assessed the impact of the choice of two commonly used 16S rRNA databases, i.e., Greengenes and SILVA, on downstream data analysis, including taxonomic classification assignment. We demonstrated that the final results depend on the choice of the laboratory conducting the HTS and the reference database of ribosomal sequences. Our findings showed that the number of produced ASVs (amplicon sequence variants) ranged from 137 to 564. Different putative bacterial endosymbionts could be identified, and these differences correspond to the applied 16S rRNA database. The results presented might be of particular interest to researchers who plan to perform microbiome community analysis using the 16S rRNA marker gene, including the identification of putative bacterial endosymbionts, and serve as a guide for choosing the optimum pipeline to obtain the most accurate and reproducible data. [ABSTRACT FROM AUTHOR]

The European beaver (Castor fiber L.) is an important free-living rodent that inhabits Eurasian temperate forests. Beavers are often referred to as ecosystem engineers because they create or change existing habitats, enhance biodiversity and prepare the environment for diverse plant and animal species. Beavers are protected in most European Union countries, but their genomic background remains unknown. In this study, gene expression patterns in beaver testes and the variations in genetic expression in breeding and non-breeding seasons were determined by high-throughput transcriptome sequencing. Paired-end sequencing in the Illumina HiSeq 2000 sequencer produced a total of 373.06 million of high-quality reads. De novo assembly of contigs yielded 130,741 unigenes with an average length of 1,369.3 nt, N50 value of 1,734, and average GC content of 46.51%. A comprehensive analysis of the testicular transcriptome revealed more than 26,000 highly expressed unigenes which exhibited the highest homology with Rattus norvegicus and Ictidomys tridecemlineatus genomes. More than 8,000 highly expressed genes were found to be involved in fundamental biological processes, cellular components or molecular pathways. The study also revealed 42 genes whose regulation differed between breeding and non-breeding seasons. During the non-breeding period, the expression of 37 genes was up-regulated, and the expression of 5 genes was down-regulated relative to the breeding season. The identified genes encode molecules which are involved in signaling transduction, DNA repair, stress responses, inflammatory processes, metabolism and steroidogenesis. Our results pave the way for further research into season-dependent variations in beaver testes. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the influence of simultaneous, long-term exposure to increasing concentrations of three classes of antimicrobials (β-lactams, fluoroquinolones and nitroimidazoles) on: (1) the efficiency of anaerobic digestion of sewage sludge, (2) qualitative and quantitative changes in microbial consortia that participate in methane fermentation, and (3) fate of antibiotic resistance genes (ARGs). Long-term supplementation of sewage sludge with a combination of metronidazole, amoxicillin and ciprofloxacin applied at different doses did not induce significant changes in process parameters, including the concentrations of volatile fatty acids (VFAs), or the total abundance of ARGs. Exposure to antibiotics significantly decreased methane production and modified microbial composition. The sequencing analysis revealed that the abundance of OTUs characteristic of Archaea was not correlated with the biogas production efficiency. The study also demonstrated that the hydrogen-dependent pathway of methylotrophic methanogenesis could significantly contribute to the stability of anaerobic digestion in the presence of antimicrobials. The greatest changes in microbial biodiversity were noted in substrate samples exposed to the highest dose of the tested antibiotics, relative to control. The widespread use of antimicrobials increases antibiotic concentrations in sewage sludge, which may decrease the efficiency of anaerobic digestion, and contribute to the spread of antibiotic resistance (AR). [ABSTRACT FROM AUTHOR]

In birds, the zona pellucida (ZP) matrix that surrounds the ovulated oocyte—called the inner perivitelline layer—is involved in sperm–zona interaction and successful fertilization. To identify the important genes and proteins connected with the final step of egg development, next-generation sequencing and two-dimensional electrophoresis, combined with mass spectrometry, were used for the analysis of mature oocytes at the F1 developmental stage. A total of 8161 genes and 228 proteins were annotated. Six subfamilies of genes, with codes ZP, ZP1–4, ZPD, and ZPAX, were identified, with the dominant expression of ZPD. The main expression site for ZP1 was the liver; however, granulosa cells may also participate in local ZP1 secretion. A ubiquitination system was identified in mature oocytes, where ZP1 was found to be the main ubiquitinated protein. Analysis of transcripts classified in estrogen receptor (ESR) signaling indicated the presence of ESR1 and ESR2, as well as a set of estrogen-dependent genes involved in both genomic and nongenomic mechanisms for the regulation of gene expression by estrogen. Oxidative phosphorylation was found to be a possible source of adenosine triphosphate, and the nuclear factor erythroid 2-related factor 2 signaling pathway could be involved in the response against oxidative stress. Oocyte–granulosa cell communication by tight, adherens, and gap junctions seems to be essential for the final step of oocyte maturation. [ABSTRACT FROM AUTHOR]

Anisakis simplex (s.l.) is a complex of three sibling (biological) species of parasitic nematodes of marine mammals, including A. berlandi, A. pegreffii and A. simplex (s.s.). It is characterized by a complex life cycle in which humans can become accidental hosts by consuming dishes made of raw or undercooked fish containing L3 larvae, which in many regions of the world is related to the national or regional culinary tradition. This has spurred scientific efforts to develop new methods for treating the disease, called anisakiasis, and to neutralize invasive L3. Thiabendazole (TBZ) is a wide-spectrum anthelminthic with a higher efficacy than albendazole, a drug whose long-term use induces resistance in many parasitic species. Cytochromes P450 participate in TBZ metabolism, and the expression of their genes is controlled by nuclear hormone receptors (NHR). This study aimed to examine the effects of TBZ on the above-described pathway in invasive larvae of A. simplex (s.l.). The efficacy of TBZ against A. simplex (s.l.) larvae was observed for the first time. Larvae were cultured in vitro for 72 h in a medium containing TBZ at five concentrations from 0.5 to 1.5 mM. However, the survival curves did not significantly differ from each other. This means that all of the concentrations of TBZ had a similar effect on the A. simplex (s.l.) L3 larvae during in vitro culture. Nevertheless, TBZ modified the expression of nhr-48, cyp13a3 and cyp1a1 genes in the L3 of A. simplex (s.l.). [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are suggested to play an important role in the sperm biological processes. We performed de novo transcriptome assembly to characterize lncRNAs in spermatozoa, and to investigate the role of the potential target genes of the differentially expressed lncRNAs (DElncRNAs) in sperm freezability. We detected approximately 4007 DElncRNAs, which were differentially expressed in spermatozoa from boars classified as having good and poor semen freezability (GSF and PSF, respectively). Most of the DElncRNAs were upregulated in boars of the PSF group and appeared to significantly affect the sperm's response to the cryopreservation conditions. Furthermore, we predicted that the potential target genes were regulated by DElncRNAs in cis or trans. It was found that DElncRNAs of both freezability groups had potential cis- and trans-regulatory effects on different protein-coding genes, such as COX7A2L, TXNDC8 and SOX-7. Gene Ontology (GO) enrichment revealed that the DElncRNA target genes are associated with numerous biological processes, including signal transduction, response to stress, cell death (apoptosis), motility and embryo development. Significant differences in the de novo assembled transcriptome expression profiles of the DElncRNAs between the freezability groups were confirmed by quantitative real-time PCR analysis. This study reveals the potential effects of protein-coding genes of DElncRNAs on sperm functions, which could contribute to further research on their relevance in semen freezability. [ABSTRACT FROM AUTHOR]

Genetic markers have been used to assess the freezability of semen. With the advancement in molecular genetic techniques, it is possible to assess the relationships between sperm functions and gene polymorphisms. In this study, variant calling analysis of RNA-Seq datasets was used to identify single nucleotide polymorphisms (SNPs) in boar spermatozoa and to explore the associations between SNPs and post-thaw semen quality. Assessment of post-thaw sperm quality characteristics showed that 21 boars were considered as having good semen freezability (GSF), while 19 boars were classified as having poor semen freezability (PSF). Variant calling demonstrated that most of the polymorphisms (67%) detected in boar spermatozoa were at the 3'-untranslated regions (3'-UTRs). Analysis of SNP abundance in various functional gene categories showed that gene ontology (GO) terms were related to response to stress, motility, metabolism, reproduction, and embryo development. Genomic DNA was isolated from sperm samples of 40 boars. Forty SNPs were selected and genotyped, and several SNPs were significantly associated with motility and membrane integrity of frozen-thawed (FT) spermatozoa. Polymorphism in SCLT1 gene was associated with significantly higher motility and plasma membrane integrity of FT spermatozoa from boars of the GSF group compared with those of the PSF group. Likewise, polymorphisms in MAP3K20, MS4A2, and ROBO1 genes were significantly associated with reduced cryo-induced lipid peroxidation and DNA damage of FT spermatozoa from boars of the GSF group. Candidate genes with significant SNP associations, including APPL1, PLBD1, FBXO16, EML5, RAB3C, OXSR1,PRICKLE1, and MAP3K20 genes, represent potential markers for post-thaw semen quality, and they might be relevant for future improvement in the selection procedure of boars for cryopreservation. The findings of this study provide evidence indicating that polymorphisms in genes expressed in spermatozoa could be considered as factors associated with post-thaw semen quality. [ABSTRACT FROM AUTHOR]