Hybrid de novo whole-genome assembly and annotation of the model tapeworm Hymenolepis diminuta

Despite the use of Hymenolepis diminuta as a model organism in experimental parasitology, a full genome description has not yet been published. Here we present a hybrid de novo genome assembly based on complementary sequencing technologies and methods. The combination of Illumina pairedend, Illumina mate-pair and Oxford Nanopore Technology reads greatly improved the assembly of the H. diminuta genome. Our results indicate that the hybrid sequencing approach is the method of choice for obtaining high-quality data. The fnal genome assembly is 177Mbp with contig N50 size of 75kbp and a scafold N50 size of 2.3Mbp. We obtained one of the most complete cestode genome assemblies and annotated 15,169 potential protein-coding genes. The obtained data may help explain cestode gene function and better clarify the evolution of its gene families, and thus the adaptive features evolved during millennia of co-evolution with their hosts

Fine-Tuning of DADA2 Parameters for Multiregional Metabarcoding Analysis of 16S rRNA Genes from Activated Sludge and Comparison of Taxonomy Classification Power and Taxonomy Databases

Taxonomic classification using metabarcoding is a commonly used method in microbiological studies of environmental samples and during monitoring of biotechnological processes. However, it is difficult to compare results from different laboratories, due to the variety of bioinformatics tools that have been developed and used for data analysis. This problem is compounded by different choices regarding which variable region of the 16S rRNA gene and which database is used for taxonomic identification. Therefore, this study employed the DADA2 algorithm to optimize the preprocessing of raw data obtained from the sequencing of activated sludge samples, using simultaneous analysis of three frequently used regions of 16S rRNA (V1–V3, V3–V4, V4–V5). Additionally, the study evaluated which variable region and which of the frequently used microbial databases for taxonomic classification (Greengenes2, Silva, RefSeq) more accurately classify OTUs into taxa. Adjusting the values of selected parameters of the DADA2 algorithm, we obtained the highest possible numbers of OTUs for each region. Regarding biodiversity within regions, the V3–V4 region had the highest Simpson and Shannon indexes, and the Chao1 index was similar to that of the V1–V3 region. Beta-biodiversity analysis revealed statistically significant differences between regions. When comparing databases for each of the regions studied, the highest numbers of taxonomic groups were obtained using the SILVA database. These results suggest that standardization of metabarcoding of short amplicons may be possible

Chemerin Impact on Alternative mRNA Transcription in the Porcine Luteal Cells

Chemerin participates in the regulation of processes related to physiological and disorder mechanisms in mammals, including metabolism, obesity, inflammation, and reproduction. In this study, we have investigated chemerin influence on alternative mRNA transcription within the porcine luteal cell transcriptome, such as differential expression of long non-coding RNAs (DELs) and their interactions with differentially expressed genes (DEGs), differences in alternative splicing of transcripts (DASs), and allele-specific expression (ASEs) related to the single nucleotide variants (SNVs) frequency. Luteal cells were collected from gilts during the mid-luteal phase of the oestrous cycle. After in vitro culture of cells un-/treated with chemerin, the total RNA was isolated and sequenced using the high-throughput method. The in silico analyses revealed 24 DELs cis interacting with 6 DEGs and trans-correlated with 300 DEGs, 137 DASs events, and 18 ASEs. The results enabled us to analyse metabolic and signalling pathways in detail, providing new insights into the effects of chemerin on the corpus luteum functions related to inflammatory response, leukocyte infiltration, the occurrence of luteotropic and luteolytic signals (leading to apoptosis and/or necroptosis). Validation of the results using qPCR confirmed the predicted expression changes. Chemerin at physiological concentrations significantly modifies the transcription processes in the porcine luteal cells

Transcriptome, Spliceosome and Editome Expression Patterns of the Porcine Endometrium in Response to a Single Subclinical Dose of Salmonella Enteritidis Lipopolysaccharide

Endometrial infections at a young age can lead to fertility issues in adulthood. Bacterial endotoxins, such as lipopolysaccharide (LPS), can participate in long-term molecular changes even at low concentrations. Lipopolysaccharide plays a crucial role in the progression of septic shock, inflammation and auto-immune diseases. The aim of this study was to describe transcriptomic modulations in the porcine endometrium, induced in vivo by a single subclinical dose of LPS from Salmonella Enteritidis. which did not produce clinical symptoms of toxicity. The RNA-seq methodology was applied to reveal 456 differentially expressed regions, including 375 genes, four long noncoding RNAs, and 77 other unclassified transcripts. Two independent methods confirmed 118 alternatively spliced genes that participate i.a., in the formation of the MHC-I complex and the adaptive immune response. Single nucleotide variant-calling algorithms supported the identification of 3730 allele-specific expression variants and 57 canonical A-to-I RNA editing sites. The results demonstrated that the differential expression of genes involved in inflammation, immune response, angiogenesis and endometrial development may be maintained for up to 7 days after exposure to LPS. RNA editing sites and long noncoding RNAs (lncRNAs) play an important role in transcriptional regulatory machinery in the porcine endometrium in response to LPS administration

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the transcriptome of aryl hydrocarbon receptor (AhR) knock-down porcine granulosa cells

Background 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical, adversely affecting reproductive processes. The well-characterized canonical mechanism of TCDD action involves the activation of aryl hydrocarbon receptor (AhR) pathway, but AhR-independent mechanisms were also suggested. By applying RNA interference technology and Next Generation Sequencing (NGS) we aimed to identify genes involved in the mechanism of TCDD action in AhR knock-down porcine granulosa cells. Methods Porcine granulosa cells were transfected with small interfering RNAs targeting mRNA of AhR. After transfection, medium was exchanged and the AhR knock-down cells were treated with TCDD (100 nM) for 3, 12 or 24 h, total cellular RNA was isolated and designated for NGS. Following sequencing, differentially expressed genes (DEGs) were identified. To analyze functions and establish possible interactions of DEGs, the Gene Ontology (GO) database and the Search Tool for the Retrieval of Interacting Genes (STRING) database were used, respectively. Results The AhR gene expression level and protein abundance were significantly decreased after AhR-targeted siRNAs transfection of the cells. In TCDD-treated AhR knock-down cells we identified 360 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change [log2FC] ≥ 1.0). The functional enrichment analysis of DEGs revealed that TCDD influenced the expression of genes involved, among other, in the metabolism of vitamin A, follicular development and oocyte maturation, proliferation and differentiation as well as inflammation, stress response, apoptosis and oncogenesis. The three-time point study demonstrated that TCDD-induced changes in the transcriptome of AhR knock-down porcine granulosa cells were especially pronounced during the early stages of the treatment (3 h). Conclusions TCDD affected the transcriptome of AhR knock-down porcine granulosa cells. The molecules involved in the AhR-independent action of TCDD were indicated in the study. The obtained data contribute to better understanding of molecular processes induced by xenobiotics in the ovary

Flutamide-induced alterations in transcriptional profiling of neonatal porcine ovaries

Abstract Background Androgens are involved in the regulation of ovarian development during fetal/neonatal life. Environmental chemicals displaying anti-androgenic activities may affect multiple signal transduction pathways by blocking endogenous androgen action. The aim of the current study was to examine effects of the anti-androgen flutamide on the expression of coding transcripts and long non-coding RNAs (lncRNAs) in neonatal porcine ovaries. By employing RNA-Seq technology we aimed to extend our understanding of the role of androgens in neonatal folliculogenesis and examine the impact of the anti-androgen flutamide on ovarian function. Method Piglets were subcutaneously injected with flutamide (50 mg/kg BW) or corn oil (controls) between postnatal days 1 and 10 (n = 3/group). Ovaries were excised from the 11-day-old piglets and total cellular RNAs were isolated and sequenced. Results Flutamide-treated piglet ovaries showed 280 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change ≥1.0) and 98 differentially expressed lncRNAs (DELs; P-adjusted < 0.05 and log2FC ≥ 1.0). The DEGs were assigned to GO term, covering biological processes, molecular functions and cellular components, which linked the DEGs to functions associated with cellular transport, cell divisions and cytoskeleton. In addition, STRING software demonstrated strongest interactions between genes related to cell proliferation. Correlations between DEGs and DELs were also found, revealing that a majority of the genes targeted by the flutamide-affected lncRNAs were associated with intracellular transport and cell division. Conclusions Our results suggest that neonatal exposure of pigs to flutamide alters the expression of genes involved in ovarian cell proliferation, ovarian steroidogenesis and oocyte fertilization, which in turn may affect female reproduction in adult life

Transcription Analysis of the Chemerin Impact on Gene Expression Profile in the Luteal Cells of Gilts

Chemerin is a recently discovered adipokine that participates in the regulation of many physiological and disorder-related processes in mammals, including metabolism, inflammatory reactions, obesity, and reproduction. We investigated how chemerin affects the transcriptome profile of porcine luteal cells. The luteal cells were acquired from mature gilts. After the in vitro culturing with and without chemerin, the total RNAs were isolated and high-throughput sequencing was performed. Obtained datasets were processed using bioinformatic tools. The study revealed 509 differentially expressed genes under the chemerin influence. Their products take part in many processes, important for the functions of the corpus luteum, such as steroids and prostaglandins synthesis, NF-&kappa;B and JAK/STAT signal transducing pathways, and apoptosis. The expression of the CASP3, HSD3B7, IL1B, and PTGS2 genes, due to their important role in the physiology of the corpus luteum, was validated using the quantitative real-time polymerase chain reaction (qPCR) method. The qPCR confirmed the changes of gene expression. Chemerin in physiological concentrations significantly affects the expression of many genes in luteal cells of pigs, which is likely to result in modification of physiological processes related to reproduction

Playing Peekaboo with a Master Manipulator: Metagenetic Detection and Phylogenetic Analysis of Wolbachia Supergroups in Freshwater Invertebrates

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.Funding Agencies|COST Action [CA18239]; COST (European Cooperation in Science and Technology); National Science Centre, Poland [2017/01/X/NZ8/01873, 2021/43/D/NZ8/00344]; Young Scientists competition of University of Gdansk, Poland [538/L260/B149/18]; University of Gdansk, Poland [1220/146/2021]; European Molecular Biology Organization (EMBO) [7862]; Vetenskapsradets [2017-04951]; ELLIIT Call C grant; Spanish Ministry of Science and Innovation [FJC2021-046991-I]</p