Two new species of the genus Sectonema Thorne, 1930 (Nematoda, Dorylaimida, Aporcelaimidae) from Iran, with new insights into its evolutionary relationships.

Two new species of the genus Sectonema found in northern Iran are characterized, including morphological descriptions and molecular (18S-, 28S-rDNA) analyses. Sectonema tehranense sp. nov. is distinguished by its 7.22 - 8.53 mm long body, lip region offset by constriction and 24 - 31 µm wide with perioral lobes and abundant setae- or cilia-like projections covering the oral field, mural tooth 15.5 - 17 µm long at its ventral side, neck 1091 - 1478 µm long, pharyngeal expansion occupying 61 - 71% of the total neck length, female genital system diovarian, uterus simple and 3.9 - 4.2 times the corresponding body diameter long, transverse vulva (V = 49 - 59), tail short and rounded (44 - 65 µm, c = 99 - 162, c' = 0.6 - 0.8), spicules 111 - 127 µm long, and 7 - 10 spaced ventromedian supplements with hiatus. Sectonema noshahrense sp. nov. displays a 4.07 - 4.73 mm long body, lip region offset by constriction and 23 - 25 µm wide with perioral lobes and abundant setae- or cilia-like projections covering the oral field, odontostyle 14 - 14.5 µm long, neck 722 - 822 µm long, pharyngeal expansion occupying 66 - 68% of the total neck length, female genital system diovarian, uterus simple and 2.4 - 2.7 times the corresponding body diameter long, transverse vulva (V = 54 - 55), tail convex conoid (39 - 47 µm, c = 91 - 111, c' = 0.8 - 0.9), spicules 82 µm long, and seven spaced ventromedian supplements with hiatus. Molecular analyses confirm a maximally supported (Epacrolaimus + Metaporcelaimus + Sectonema) clade and a tentative biogeographical pattern, with sequences of Indolamayan taxa forming a clade separated from those of Palearctic ones. Parallel or convergent evolution processes might be involved in the phylogeny of the species currently classified under Sectonema. This genus is certainly more heterogeneous than previously assumed.

Insights into the Adolescent Cystic Fibrosis Airway Microbiome Using Shotgun Metagenomics.

Cystic fibrosis (CF) is an inherited genetic disorder which manifests primarily in airway disease. Recent advances in molecular technologies have unearthed the diverse polymicrobial nature of the CF airway. Numerous studies have characterised the genus-level composition of this airway community using targeted 16S rDNA sequencing. Here, we employed whole-genome shotgun metagenomics to provide a more comprehensive understanding of the early CF airway microbiome. We collected 48 sputum samples from 11 adolescents and children with CF over a 12-month period and performed shotgun metagenomics on the Illumina NextSeq platform. We carried out functional and taxonomic analysis of the lung microbiome at the species and strain levels. Correlations between microbial diversity measures and independent demographic and clinical variables were performed. Shotgun metagenomics detected a greater diversity of bacteria than culture-based methods. A large proportion of the top 25 most-dominant species were anaerobes. Samples dominated by Staphylococcus aureus and Prevotella melaninogenica had significantly higher microbiome diversity, while no CF pathogen was associated with reduced microbial diversity. There was a diverse resistome present in all samples in this study, with 57.8% agreement between shotgun metagenomics and culture-based methods for detection of resistance. Pathogenic sequence types (STs) of S. aureus, Pseudomonas aeruginosa, Haemophilus influenzae and Stenotrophomonas maltophilia were observed to persist in young CF patients, while STs of S. aureus were both persistent and shared between patients. This study provides new insight into the temporal changes in strain level composition of the microbiome and the landscape of the resistome in young people with CF. Shotgun metagenomics could provide a very useful one-stop assay for detecting pathogens, emergence of resistance and conversion to persistent colonisation in early CF disease.

Non-traumatic osteonecrosis of the femoral head induced by steroid and alcohol exposure is associated with intestinal flora alterations and metabolomic profiles.

OBJECTIVE: Osteonecrosis of the femoral head (ONFH) is a severe disease that primarily affects the middle-aged population, imposing a significant economic and social burden. Recent research has linked the progression of non-traumatic osteonecrosis of the femoral head (NONFH) to the composition of the gut microbiota. Steroids and alcohol are considered major contributing factors. However, the relationship between NONFH caused by two etiologies and the microbiota remains unclear. In this study, we examined the gut microbiota and fecal metabolic phenotypes of two groups of patients, and analyzed potential differences in the pathogenic mechanisms from both the microbial and metabolic perspectives. METHODS: Utilizing fecal samples from 68 NONFH patients (32 steroid-induced, 36 alcohol-induced), high-throughput 16 S rDNA sequencing and liquid chromatography with tandem mass spectrometry (LC-MS/MS) metabolomics analyses were conducted. Univariate and multivariate analyses were applied to the omics data, employing linear discriminant analysis effect size to identify potential biomarkers. Additionally, functional annotation of differential metabolites and associated pathways was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Subsequently, Spearman correlation analysis was employed to assess the potential correlations between differential gut microbiota and metabolites. RESULTS: High-throughput 16 S rDNA sequencing revealed significant gut microbial differences. At the genus level, the alcohol group had higher Lactobacillus and Roseburia, while the steroid group had more Megasphaera and Akkermansia. LC-MS/MS metabolomic analysis indicates significant differences in fecal metabolites between steroid- and alcohol-induced ONFH patients. Alcohol-induced ONFH (AONFH) showed elevated levels of L-Lysine and Oxoglutaric acid, while steroid-induced ONFH(SONFH) had increased Gluconic acid and Phosphoric acid. KEGG annotation revealed 10 pathways with metabolite differences between AONFH and SONFH patients. Correlation analysis revealed the association between differential gut flora and differential metabolites. CONCLUSIONS: Our results suggest that hormones and alcohol can induce changes in the gut microbiota, leading to alterations in fecal metabolites. These changes, driven by different pathways, contribute to the progression of the disease. The study opens new research directions for understanding the pathogenic mechanisms of hormone- or alcohol-induced NONFH, suggesting that differentiated preventive and therapeutic approaches may be needed for NONFH caused by different triggers.

In silico assessment of 18S rDNA metabarcoding markers for the characterization of nematode communities.

Nematodes are keystone actors of soil, freshwater and marine ecosystems, but the complexity of morphological identification has limited broad-scale monitoring of nematode biodiversity. DNA metabarcoding is increasingly used to assess nematode diversity but requires universal primers with high taxonomic coverage and high taxonomic resolution. Several primers have been proposed for the metabarcoding of nematode diversity, many of which target the 18S rRNA gene. In silico analyses have a great potential to assess key parameters of primers, including taxonomic coverage, resolution and specificity. Based on a recently-available reference database, we tested in silico the performance of fourteen commonly used and one newly optimized primer for nematode metabarcoding. Most primers showed very good coverage, amplifying most of the sequences in the reference database, while four markers showed limited coverage. All primers showed good taxonomic resolution. Resolution was particularly good if the aim was the identification of higher-level taxa, such as genera or families. Overall, species-level resolution was higher for primers amplifying long fragments. None of the primers was highly specific for nematodes as, despite some variation, they all amplified a large number of other eukaryotes. Differences in performance across primers highlight the complexity of the choice of markers appropriate for the metabarcoding of nematodes, which depends on a trade-off between taxonomic resolution and the length of amplified fragments. Our in silico analyses provide new insights for the identification of the most appropriate primers, depending on the study goals and the origin of DNA samples. This represents an essential step to design and optimize metabarcoding studies assessing nematode diversity.

Employing a triple metabarcoding approach to differentiate active, dormant and dead microeukaryotes in sediments.

Microbial communities are commonly characterised through the metabarcoding of environmental DNA. This DNA originates from both viable (including dormant and active) and dead organisms, leading to recent efforts to distinguish between these states. In this study, we further these approaches by distinguishing not only between viable and dead cells but also between dormant and actively growing cells. This is achieved by sequencing both rRNA and rDNA, in conjunction with propidium monoazide cross-linked rDNA, to partition the active, dormant and relic fractions in environmental samples. We apply this method to characterise the diversity and assemblage structure of these fractions of microeukaryotes in intertidal sediments during a wet-dry-rewet incubation cycle. Our findings indicate that a significant proportion of microeukaryotic phylotypes detected in the total rDNA pools originate from dormant and relic microeukaryotes in the sediments, both in terms of richness (dormant, 13 ± 2%; relic, 47 ± 5%) and read abundance (dormant, 20 ± 7%; relic, 14 ± 5%). The richness and sequence proportion of dormant microeukaryotes notably increase during the transition from wet to dry conditions. Statistical analyses suggest that the dynamics of diversity and assemblage structure across different activity fractions are influenced by various environmental drivers. Our strategy offers a versatile approach that can be adapted to characterise other microbes in a wide range of environments.

Distinct compositions and functions of circulating microbial DNA in the peripheral blood compared to fecal microbial DNA in healthy individuals.

The crucial function of circulating microbial DNA (cmDNA) in peripheral blood is gaining recognition because of its importance in normal physiology and immunity in healthy individuals. Evidence suggests that cmDNA in peripheral blood is derived from highly abundant, translocating gut microbes. However, the associations with and differences between cmDNA in peripheral blood and the gut microbiome remain unclear. We collected blood, urine, and fecal samples from volunteers to compare their microbial information via 16S rDNA sequencing. The results revealed that, compared with gut microbial DNA, cmDNA in peripheral blood was associated with reduced diversity and a distinct microbiota composition. The cmDNA in the blood reflects the biochemical processes of microorganisms, including synthesis, energy conversion, degradation, and adaptability, surpassing that of fecal samples. Interestingly, cmDNA in blood showed a limited presence of DNA from anaerobes and gram-positive bacteria, which contrast with the trend observed in fecal samples. Furthermore, analysis of cmDNA revealed traits associated with mobile elements and potential pathologies, among others, which were minimal in stool samples. Notably, cmDNA analysis indicated similarities between the microbial functions and phenotypes in blood and urine samples, although greater diversity was observed in urine samples. Source Tracker analysis suggests that gut microbes might not be the main source of blood cmDNA, or a selective mechanism allows only certain microbial DNA into the bloodstream. In conclusion, our study highlights the composition and potential functions associated with cmDNA in peripheral blood, emphasizing its selective presence; however, further research is required to elucidate the mechanisms involved.IMPORTANCEOur research provides novel insights into the unique characteristics and potential functional implications of circulating microbial DNA (cmDNA) in peripheral blood. Unlike other studies that analyzed sequencing data from fecal or blood microbiota in different study cohorts, our comparative analysis of cmDNA from blood, urine, and fecal samples from the same group of volunteers revealed a distinct blood-specific cmDNA composition. We discovered a decreased diversity of microbial DNA in blood samples compared to fecal samples as well as an increased presence of biochemical processes microbial DNA in blood. Notably, we add to the existing knowledge by documenting a reduced abundance of anaerobes and gram-positive bacteria in blood compared to fecal samples according to the analysis of cmDNA and gut microbial DNA, respectively. This observation suggested that a potential selective barrier or screening mechanism might filter microbial DNA molecules, indicating potential selectivity in the translocation process which contrasts with the traditional view that cmDNA primarily originates from random translocation from the gut and other regions. By highlighting these differences, our findings prompt a reconsideration of the origin and role of cmDNA in blood circulation and suggest that selective processes involving more complex biological mechanisms may be involved.

Effects of chronic unpredictable mild stress on gut microbiota and fecal amino acid and short-chain fatty acid pathways in mice.

Depression is a serious disease that has a significant impact on social functioning. However, the exact causes of depression are still not fully understood. Therefore, it is necessary to explore new pathways leading to depression. In this study, we used 16 S rDNA to examine changes in gut microbiota and predict related pathways in depression-like mice. Additionally, we employed liquid chromatography-mass spectrometry (LC-MS) to identify changes in amino acids and gas chromatography-mass spectrometry (GC-MS) to identify changes in short-chain fatty acids (SCFAs) in fecal samples. We conducted Pearson/Spearman correlation analysis to investigate the associations between changes in amino acids/SCFAs and behavioral outcomes. The 16 S rDNA sequencing revealed significant alterations in gut microbiota at the phylum and genus levels in mice subjected to chronic unpredictable mild stress (CUMS). The relative abundances of Bacteroidetes, Proteobacteria, Bacteroides, and Alloprevotella were increased, while Firmicutes, Verrucomicrobia, Actinobacteria, Lactobacillus, Akkermansia, Lachnospirillum, and Enterobacter were decreased in the CUMS mice. We used PICRUSt software to annotate the kyoto encyclopedia of genes and genomes (KEGG) pathway function related to depression-like behavior in mice. Our analysis identified sixty functional pathways associated with the gut microbiota of mice exhibiting depression-like behavior. In the amino acid concentration analysis, we observed decreased levels of hydroxyproline and tryptophan, and increased levels of alanine in CUMS mice. In the SCFAs concentration assay, we found decreased levels of butyric acid and valeric acid, and increased levels of acetic acid in CUMS mice. Some of these changes were significantly correlated with depressive-like behaviors. Our study contributes to the understanding of the mechanism of the gut-brain axis in the occurrence and development of depression.

First molecular identification and phylogenetic illustration of Sarcocystis species infection in Red Sea shortfin mako shark (Isurus oxyrinchus Rafinesque, 1810).

BACKGROUND: members of the genus Sarcocystis are intracellular obligate protozoan parasites classified within the phylum Apicomplexa and have an obligate heteroxenous life cycle involving two hosts. A more comprehensive understanding of the prevalence and geographic range of different Sarcocystis species in marine ecosystems is needed globally and nationally. Hence, the objective of this study was to document the incidence of Sarcocystis infection in sharks within the aquarium ecosystem of Egypt and to identify the species through the characterization of the SSU rDNA gene. METHODS: All organs of the mako shark specimen underwent macroscopic screening to detect the existence of a Sarcocystis cyst. Ten cysts were collected from the intestine and processed separately to extract the genomic DNA. The polymerase chain reaction (PCR) was accomplished by amplifying a specific 18S ribosomal RNA (rRNA) gene fragment. Subsequently, the resulting amplicons were subjected to purification and sequencing processes. RESULTS: Macroscopic examination of the mako shark intestinal wall sample revealed the presence of Sarcocystis cysts of various sizes and shapes, and sequencing of the amplicons from Sarcocystis DNA revealed a 100% nucleotide identity with the sequence of Sarcocystis tenella recorded from sheep in Iran; The mako shark sequence has been deposited in the GeneBank with the accession number OQ721979. This study presents the first scientific evidence demonstrating the presence of the Sarcocystis parasite in sharks, thereby documenting this specific marine species as a novel intermediate host in the Sarcocystis life cycle. CONCLUSIONS: This is the first identification of Sarcocystis infection in sharks, and we anticipate it will be an essential study for future screenings and establishing effective management measures for this disease in aquatic ecosystems.

Isolation of endophytes from Dioscorea nipponica Makino for stimulating diosgenin production and plant growth.

KEY MESSAGE: Both bacterial and fungal endophytes exhibited one or more plant growth-promoting (PGP) traits. Among these strains, the Paenibacillus peoriae SYbr421 strain demonstrated the greatest activity in the direct biotransformation of tuber powder from D. nipponica into diosgenin. Endophytes play crucial roles in shaping active metabolites within plants, significantly influencing both the quality and yield of host plants. Dioscorea nipponica Makino accumulates abundant steroidal saponins, which can be hydrolyzed to produce diosgenin. However, our understanding of the associated endophytes and their contributions to plant growth and diosgenin production is limited. The present study aimed to assess the PGP ability and potential of diosgenin biotransformation by endophytes isolates associated with D. nipponica for the efficient improvement of plant growth and development of a clean and effective approach for producing the valuable drug diosgenin. Eighteen bacterial endophytes were classified into six genera through sequencing and phylogenetic analysis of the 16S rDNA gene. Similarly, 12 fungal endophytes were categorized into 5 genera based on sequencing and phylogenetic analysis of the ITS rDNA gene. Pure culture experiments revealed that 30 isolated endophytic strains exhibited one or more PGP traits, such as nitrogen fixation, phosphate solubilization, siderophore synthesis, and IAA production. One strain of endophytic bacteria, P. peoriae SYbr421, effectively directly biotransformed the saponin components in D. nipponica. Moreover, a high yield of diosgenin (3.50%) was obtained at an inoculum size of 4% after 6 days of fermentation. Thus, SYbr421 could be used for a cleaner and more eco-friendly diosgenin production process. In addition, based on the assessment of growth-promoting isolates and seed germination results, the strains SYbr421, SYfr1321, and SYfl221 were selected for greenhouse experiments. The results revealed that the inoculation of these promising isolates significantly increased the plant height and fresh weight of the leaves and roots compared to the control plants. These findings underscore the importance of preparing PGP bioinoculants from selected isolates as an additional option for sustainable diosgenin production.

Transferability of Human and Environmental Microbiome on Clothes as a Tool for Forensic Investigations.

Considering the growing importance of microbiome analyses in forensics for identifying individuals, this study explores the transfer of the skin microbiome onto clothing, its persistence on fabrics over time, and its transferability from the environment and between different garments. Furthermore, this project compares three specific QIAGEN microbiome extraction kits to test their extraction efficiency on fabric samples. Additionally, this study aims to check if these extracts contain human DNA, providing a chance to obtain more information from the same evidence for personal identification. The results obtained show: (1) variations in the skin microbiome between the volunteers, potentially due to their different sex; (2) differences in microbial composition between worn and unworn clothing; (3) the influence of the environment on the microbial signature of unworn clothing; (4) the potential use of certain phyla as biomarkers to differentiate between worn and unworn garments, even over extended periods; (5) a tendency towards extraction biases in the QIAampMP® DNA microbiome kit among the three tested ones; and (6) none of the extraction kits allow for the typing of human genetic profiles suitable for comparison. In conclusion, our study offers supplementary insights into the potential utility of time-transferred microbiome analysis on garments for forensic applications.

Comparing massa medicata fermentata before and after charred in terms of digestive promoting effect via metabolomics and microbiome analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Massa Medicata Fermentata, a fermented Chinese medicine, is produced by the fermentation of six traditional Chinese medicines. Liu Shenqu (LSQ) and charred Liu Shenqu (CLSQ) have been used for strengthening the spleen and enhancing digestion for over a thousand years, and CLSQ is commonly used in clinical practice. However, it is unclear whether there is a difference in the spleen strengthening and digestion effects between LSQ and CLSQ, as well as their mechanisms of action. AIM OF STUDY: This study aims to compare the effects of LSQ and CLSQ on the digestive function of functional dyspepsia (FD) rats and reveal their mechanisms of action. MATERIALS AND METHODS: SPF grade SD rats were randomly divided into 6 groups: control group, model group, Liu Shenqu decoction low-dosage (LSQ LD) group, Liu Shenqu decoction high-dosage (LSQ HD) group, charred Liu Shenqu decoction low-dosage (CLSQ LD) group, and charred Liu Shenqu decoction high-dosage (CLSQ HD) group. Rats were injected intraperitoneally with reserpine to create an FD model and then treated by intragastric administration. During this period, record the weight and food intake of the animals. After 18 days of treatment, specimens of the gastric antrum, spleen, and duodenum of rats were taken for pathological staining and immunohistochemical detection of Ghrelin protein expression. Enzyme linked immunosorbent assay (ELISA) was used to determine the concentration of relevant gastrointestinal hormones in serum. The 16 S rDNA sequencing method was used to evaluate the effect of cecal contents on the structure of the gut microbiota in experimental rats. Plasma metabolomics analysis was performed using ultra high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) to further reveal their mechanism of action. RESULTS: LSQ and CLSQ improved the pathological tissue histological structure of FD rats and increased the levels of MTL and GAS hormones in serum and the levels of ghrelin in the gastric antrum, spleen, and duodenum, while reducing VIP, CCK, and SP hormone levels. The above results showed that the therapeutic efficacy of CLSQ is better than that of LSQ. Futhermore, the mechanism of action of LSQ and CLSQ were revealed. The 16 S rDNA sequencing results showed that both LSQ and CLSQ can improve the composition and diversity of the gut microbiota. And metabolomic analysis demonstrated that 20 metabolites changed after LSQ treatment, and 16 metabolites underwent continuous changes after CLSQ treatment. Further analysis revealed that LSQ mainly intervened in the metabolic pathways of glycerol phospholipid metabolism and arginine and proline metabolism, but CLSQ mainly intervened in the metabolic pathways of ether lipid metabolism, sphingolipid metabolism, and glycerophospholipid metabolism. CONCLUSIONS: Both LSQ and CLSQ can improve functional dyspepsia in FD rats, but CLSQ has a stronger improvement effect on FD. Although their mechanisms of action are all related to regulating gastrointestinal hormone secretion, significantly improving intestinal microbiota disorders, and improving multiple metabolic pathways, but the specific gut microbiota and metabolic pathways they regulate are different.

Mammal dung-dung beetle trophic networks: an improved method based on gut-content DNA.

Background: Dung beetles provide many important ecosystem services, including dung decomposition, pathogen control, soil aeration, and secondary seed dispersal. Yet, the biology of most dung beetles remains unknown. Natural diets are poorly studied, partly because previous research has focused on choice or attraction experiments using few, easily accessible dung types from zoo animals, farm animals, or humans. This way, many links within natural food webs have certainly been missed. In this work, we aimed to establish a protocol to analyze the natural diets of dung beetles using DNA gut barcoding. Methods: First, the feasibility of gut-content DNA extraction and amplification of 12s rDNA from six different mammal dung types was tested in the laboratory. We then applied the method to beetles caught in pitfall traps in Ecuador and Germany by using 12s rDNA primers. For a subset of the dung beetles caught in the Ecuador sampling, we also used 16s rDNA primers to see if these would improve the number of species we could identify. We predicted the likelihood of amplifying DNA using gut fullness, DNA concentration, PCR primer, collection method, and beetle species as predictor variables in a dominance analysis. Based on the gut barcodes, we generated a dung beetle-mammal network for both field sites (Ecuador and Germany) and analyzed the levels of network specificity. Results: We successfully amplified mammal DNA from dung beetle gut contents for 128 specimens, which included such prominent species as Panthera onca (jaguar) and Puma concolor (puma). The overall success rate of DNA amplification was 53%. The best predictors for amplification success were gut fullness and DNA concentration, suggesting the success rate can be increased by focusing on beetles with a full gut. The mammal dung-dung beetle networks differed from purely random network models and showed a moderate degree of network specialization (H2': Ecuador = 0.49; Germany = 0.41). Conclusion: We here present a reliable method of extracting and amplifying gut-content DNA from dung beetles. Identifying mammal dung via DNA reference libraries, we created mammal dung-dung beetle trophic networks. This has benefits over previous methods because we inventoried the natural mammal dung resources of dung beetles instead of using artificial mammal baits. Our results revealed higher levels of specialization than expected and more rodent DNA than expected in Germany, suggesting that the presented method provides more detailed insights into mammal dung-dung beetle networks. In addition, the method could have applications for mammal monitoring in many ecosystems.

Integrative description of a new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Shennongjia, Central China.

A new species of the genus Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Xiangxi River, Shennongjia Forestry District, Hubei Province, China, is described on the basis of an integrative approach, involving morphology, and molecular systematics. The new species Dugesia saccaria A-T. Wang & Sluys, sp. nov. is characterized by the following features: a dumb-bell-shaped, muscularized hump located just anterior to the knee-shaped bend in the bursal canal; a ventrally displaced ejaculatory duct, which, however, opens terminally through the dorsal portion of the blunt tip of the penis papilla; a ventrally located seminal vesicle, giving rise to a vertically running duct that eventually curves downwards to communicate with the ejaculatory duct via a small diaphragm; oviducts opening asymmetrically into the dorsal portion of the common atrium and at the knee-shaped part of the bursal canal. The phylogenetic position of the new species was determined using four molecular markers (18S rDNA; ITS-1; 28S rDNA; COI), which suggested that it groups with other species of Dugesia from the Australasian and Oriental biogeographical regions.

Description of Saccocoelioides miguelmontesi n. sp. (Digenea: Haploporidae) from characid fishes in the Iguazu River Basin based on morphological and molecular evidence.

Here we describe a new species of the genus Saccocoelioides found parasitizing Astyanax dissimilis Garavello & Sampaio, Psalidodon bifasciatus (Garavello and Sampaio) and Bryconamericus ikaa Casciotta, Almirón & Azpelicueta from the Iguazu National Park, Misiones province, Argentina. Saccocoelioides miguelmontesi n. sp. was studied based on morphological and molecular (28S rDNA and COI mtDNA sequences) data. The COI mtDNA tree indicated that the specimens collected from the three fish hosts are conspecific, with an intragroup p-distance of 0%. The new species shows an intermediate morphological configuration between the diminutive and robust forms described for Saccocoelioides by Curran (2018). Although, in the 28S rDNA tree, it is placed in a well-supported clade with the two robust species analysed (S. elongatus and S. magnus; p-distance of 1 and 2%, respectively), it differs from the robust group by the range of body size, mature egg size, oral and ventral sucker size, sucker ratio, oral sucker to pharynx ratio, and post-cecal or post-testis/body length percentage. Our results led us to redefine the robust group as having eggs shorter or equal in length to the pharynx. Saccocoelioides miguelmontesi n. sp. the 10th species reported from Argentina and the 7th species within the robust group.

Are there any completely sterile organs or tissues in the human body? Is there any sacred place?

The human microbiome comprises an ample set of organisms that inhabit and interact within the human body, contributing both positively and negatively to our health. In recent years, several research groups have described the presence of microorganisms in organs or tissues traditionally considered as 'sterile' under healthy and pathological conditions. In this sense, microorganisms have been detected in several types of cancer, including those in 'sterile' organs. But how can the presence of microorganisms be detected? In most studies, 16S and internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing has led to the identification of prokaryotes and fungi. However, a major limitation of this technique is that it cannot distinguish between living and dead organisms. RNA-based methods have been proposed to overcome this limitation, as the shorter half-life of the RNA would identify only the transcriptionally active microorganisms, although perhaps not all the viable ones. In this sense, metaproteomic techniques or the search for molecular metabolic signatures could be interesting alternatives for the identification of living microorganisms. In summary, new technological advances are challenging the notion of 'sterile' organs in our body. However, to date, evidence for a structured living microbiome in most of these organs is scarce or non-existent. The implementation of new technological approaches will be necessary to fully understand the importance of the microbiome in these organs, which could pave the way for the development of a wide range of new therapeutic strategies.

Ribotin: automated assembly and phasing of rDNA morphs.

MOTIVATION: The ribosomal DNA (rDNA) arrays are highly repetitive and homogenous regions which exist in all life. Due to their repetitiveness, current assembly methods do not fully assemble the rDNA arrays in humans and many other eukaryotes, and so variation within the rDNA arrays cannot be effectively studied. RESULTS: Here, we present the tool ribotin to assemble full length rDNA copies, or morphs. Ribotin uses a combination of highly accurate long reads and extremely long nanopore reads to resolve the variation between rDNA morphs. We show that ribotin successfully recovers the most abundant morphs in human and nonhuman genomes. We also find that genome wide consensus sequences of the rDNA arrays frequently produce a mosaic sequence that does not exist in the genome. AVAILABILITY AND IMPLEMENTATION: Ribotin is available on https://github.com/maickrau/ribotin and as a package on bioconda.

A comparison of two gene regions for assessing community composition of eukaryotic marine microalgae from coastal ecosystems.

Two gene regions commonly used to characterise the diversity of eukaryotic communities using metabarcoding are the 18S ribosomal DNA V4 and V9 gene regions. We assessed the effectiveness of these two regions for characterising diverisity of coastal eukaryotic microalgae communities (EMCs) from tropical and temperate sites. We binned amplicon sequence variants (ASVs) into the high level taxonomic groups: dinoflagellates, pennate diatoms, radial centric diatoms, polar centric diatoms, chlorophytes, haptophytes and 'other microalgae'. When V4 and V9 generated ASV abundances were compared, the V9 region generated a higher number of raw reads, captured more diversity from all high level taxonomic groups and was more closely aligned with the community composition determined using light microscopy. The V4 region did resolve more ASVs to a deeper taxonomic resolution within the dinoflagellates, but did not effectively resolve other major taxonomic divisions. When characterising these communities via metabarcoding, the use of multiple gene regions is recommended, but the V9 gene region can be used in isolation to provide high-level community biodiversity to reflect relative abundances within groups. This approach reduces the cost of sequencing multiple gene regions whilst still providing important baseline ecosystem function information.

Description of Saprolegnia velencensis sp. n. (Oomycota), a novel water mold species from Lake Velence, Hungary.

Here, we describe a novel water mold species, Saprolegnia velencensis sp. n. from Lake Velence, in Hungary. Two strains (SAP239 and SAP241) were isolated from lake water, and characterized using morphological and molecular markers. In addition, phylogenetic analyses based on ITS-rDNA regions and on the RNA polymerase II B subunit (RPB2) gene complemented the study. The ITS-rDNA of the two strains was 100% identical, showed the highest similarity to that of S. ferax (with 94.4% identity), and they formed a separate cluster in both the ITS-rDNA and RPB2-based maximum likelihood phylogenetic trees with high bootstrap support. Although mature oogonia and antheridia were not seen under in vitro conditions, the S. velencensis sp. n. could be clearly distinguished from its closest relative, S. ferax, by the length and width of sporangia, as the new species had shorter and narrower sporangia (163.33±70.07 and 36.69±8.27 µm, respectively) than those of S. ferax. The two species also differed in the size of the secondary cysts (11.63±1.77 µm), which were slightly smaller in S. ferax. Our results showed that S. velencensis sp. n. could not be identified with any of the previously described water mold species, justifying its description as a new species.

High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA.

The Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA) is known for detecting various protein targets; however, its ability to detect nucleic acid sequences is not well established. Here, the capabilities of the AlphaLISA technology were expanded to include direct detection of DNA (aka: oligo-Alpha) and was applied to the detection of Listeria monocytogenes. Parameters were defined that allowed the newly developed oligo-Alpha to differentiate L. monocytogenes from other Listeria species through the use of only a single nucleotide polymorphism within the 16S rDNA region. Investigations into the applicability of this assay with different matrices demonstrated its utility in both milk and juice. One remarkable feature of the oligo-Alpha is that greater sensitivity could be achieved through the use of multiple acceptor oligos compared to only a single acceptor oligo, even when only a single donor oligo was employed. Additional acceptor oligos were easily incorporated into the assay and a tenfold change in the detection limit was readily achieved, with detection limits of 250 attomole of target being recorded. In summary, replacement of antibodies with oligonucleotides allows us to take advantage of genotypic difference(s), which both expands its repertoire of biological markers and furthers its use as a diagnostic tool.

Genotyping of ticks: first molecular report of Hyalomma asiaticum and molecular detection of tick-borne bacteria in ticks and blood from Khyber Pakhtunkhwa, Pakistan.

Multiple ticks (Acari: Ixodoidea) carrying Rickettsiales bacteria have significant importance for both human and animal health. Thus, the purpose of this work was to genetically analyze tick species and their associated Rickettsiales bacteria in animal hosts. In order to achieve these objectives, various animals (including camels, cattle, goats, sheep, dogs, and mice) were inspected in four districts (Mardan, Peshawar, Kohat, and Karak) of Khyber Pakhtunkhwa to collect ticks, while blood samples were collected from all the symptomatic and asymptomatic cattle in all four districts. A total of 234 ticks were obtained from 86 out of 143 (60.14%) host animals, which were morphologically identified as Rhipicephalus turanicus, Rhipicephalus microplus, Haemaphysalis cornupunctata, and Hyalomma asiaticum. Among these, their representative ticks (126/234, 53.85%) were processed for molecular confirmation using cytochrome c oxidase (cox1) gene. Obtained cox1 sequences of four different tick species showed 99.72%-100% maximum identity with their corresponding species reported from Pakistan, China, India, and Kazakhstan and clustered phylogenetically. This study presented the first genetic report of Hy. asiaticum ticks in Pakistan. Moreover, genetically confirmed tick species were molecularly analyzed by PCR for detection of Rickettsiales DNA using partial fragments of 16S rDNA, 190-kDa outer membrane protein A (ompA), and 120-kDa outer membrane protein B (ompB) genes. In addition, blood samples were analyzed to identify Rickettsiales bacteria using the aforementioned genes. Rickettsiales bacteria were found in 24/126 (19.05%) ticks and 4/16 (25.00%) in symptomatic cattle's blood. The obtained ompA and ompB sequences from Hy. asiaticum ticks showed 99.73%-99.87% with Candidatus Rickettsia shennongii and unidentified Rickettsia sp., whereas the obtained 16S rDNA sequences from cattle's blood and ticks (Hae. cornupunctata) showed 99.67% highest identity with Anaplasma phagocytophilum. The 16S rDNA sequence of Rickettsiales DNA from Rh. turanicus ticks showed 100% identity with Ehrlichia canis and unidentified Ehrlichia sp. Obtained sequences of Rickettsiales bacteria were grouped along with their respective species in phylogenetic trees, which were previously reported in Greece, Cuba, Iraq, Turkey, Pakistan, South Korea, and China (mainland and Taiwan). This extensive study explores the wide range of damaging ticks and their corresponding tick-borne bacteria in the area, suggesting a possible danger to both livestock and human communities.