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We examined gelatinous zooplankton from off eastern Australia for lepocreadiid trematode metacercariae. From 221 specimens of 17 species of cnidarian medusae and 218 specimens of four species of ctenophores, infections were found in seven cnidarian and two ctenophore species. Metacercariae were distinguished using cox1 mtDNA, ITS2 rDNA and morphology. We identified three species of Prodistomum Linton, 1910 [P. keyam Bray & Cribb, 1996, P. orientale (Layman, 1930), and Prodistomum Type 3], two species of Opechona Looss, 1907 [O. kahawai Bray & Cribb, 2003 and O. cf. olssoni], and Cephalolepidapedon saba Yamaguti, 1970. Two species were found in cnidarians and ctenophores, three only in cnidarians, and one only in a ctenophore. Three Australian fishes were identified as definitive hosts; four species were collected from Scomber australasicus and one each from Arripis trutta and Monodactylus argenteus. Transmission of trematodes to these fishes by ingestion of gelatinous zooplankton is plausible given their mid-water feeding habits, although such predation is rarely reported. Combined morphological and molecular analyses of adult trematodes identified two cox1 types for C. saba, three cox1 types and species of Opechona, and six cox1 types and five species of Prodistomum of which only two are identified to species. All three genera are widely distributed geographically and have unresolved taxonomic issues. Levels of distinction between the recognised species varied dramatically for morphology, the three molecular markers, and host distribution. Phylogenetic analysis of 28S rDNA data extends previous findings that species of Opechona and Prodistomum do not form monophyletic clades., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

The genus Geobacillus is composed of thermophilic bacteria that exhibit diverse biotechnological potentialities. Specifically, Geobacillus stearothermophilus is included as a test bacterium in commercial microbiological inhibition methods, although it exhibits limited sensitivity to aminoglycosides, macrolides, and quinolones. Therefore, this article evaluates the antibiotic susceptibility profiles of five test bacteria (G. stearothermophilus subsp. calidolactis C953, Geobacillus thermocatenulatus LMG 19007, Geobacillus thermoleovorans LMG 9823, Geobacillus kaustophilus DSM 7263 and Geobacillus vulcani 13174). For that purpose, the minimum inhibitory concentrations (MICs) of 21 antibiotics were determined in milk samples for five test bacteria using the radial diffusion microbiological inhibition method. Subsequently, the similarities between bacteria and antibiotics were analyzed using cluster analysis. The dendrogram of this multivariate analysis shows an association between a group formed by G. thermocatenulatus and G. stearothermophilus and another by G. thermoleovorans, G. kaustophilus and G. vulcani. Finally, future microbiological methods could be developed in microtiter plates using G. thermocatenulatus as test bacterium, as it exhibits similar sensitivities to G. stearothermophilus. Conversely, G. vulcani, G. thermoleovorans and G. kaustophilus show higher MICs than G. thermocatenulatus., (Copyright © 2023 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Hongqu rice wine, a famous traditional fermented alcoholic beverage, is brewed with traditional Hongqu (mainly including Gutian Qu and Wuyi Qu). This study aimed to compare the microbial communities and metabolic profiles in the traditional brewing of Hongqu rice wines fermented with Gutian Qu and Wuyi Qu. Compared with Hongqu rice wine fermented with Wuyi Qu (WY), Hongqu rice wine fermented with Gutian Qu (GT) exhibited higher levels of biogenic amines. The composition of volatile flavor components of Hongqu rice wine brewed by different fermentation starters (Gutian Qu and Wuyi Qu) was obviously different. Among them, ethyl acetate, isobutanol, 3-methylbutan-1-ol, ethyl decanoate, ethyl palmitate, ethyl oleate, nonanoic acid, 4-ethylguaiacol, 5-pentyldihydro-2(3H)-furanone, ethyl acetate, n-decanoic acid etc. were identified as the characteristic aroma-active compounds between GT and WY. Microbiome analysis based on high-throughput sequencing of full-length 16S rDNA/ITS-5.8S rDNA amplicons revealed that Lactococcus, Leuconostoc, Pseudomonas, Serratia, Enterobacter, Weissella, Saccharomyces, Monascus and Candida were the predominant microbial genera during the traditional production of GT, while Lactococcus, Lactobacillus, Leuconostoc, Enterobacter, Kozakia, Weissella, Klebsiella, Cronobacter, Saccharomyces, Millerozyma, Monascus, Talaromyces and Meyerozyma were the predominant microbial genera in the traditional fermentation of WY. Correlation analysis revealed that Lactobacillus showed significant positive correlations with most of the characteristic volatile flavor components and biogenic amines. Furthermore, bioinformatical analysis based on PICRUSt revealed that microbial enzymes related to biogenic amines synthesis were more abundant in GT than those in WY, and the enzymes responsible for the degradation of biogenic amines were less abundant in GT than those in WY. Collectively, this study provides important scientific data for enhancing the flavor quality of Hongqu rice wine, and lays a solid foundation for the healthy and sustainable development of Hongqu rice wine industry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The etiology of allergic rhinitis (AR) is complicated. Traditional therapy of AR still has challenges, such as low long-term treatment compliance, unsatisfactory therapeutic outcomes, and a high financial burden. It is urgent to investigate the pathophysiology of allergic rhinitis from different perspectives and explore brand-new possible preventative or treatment initiatives., Objective: The aim is to apply a multi-group technique and correlation analysis to explore more about the pathogenesis of AR from the perspectives of gut microbiota, fecal metabolites, and serum metabolism., Methods: Thirty BALB/c mice were randomly divided into the AR and Con(control) groups. A standardized Ovalbumin (OVA)-induced AR mouse model was established by intraperitoneal OVA injection followed by nasal excitation. We detected the serum IL-4, IL-5, and IgE by enzyme-linked immunosorbent assay (ELISA), evaluated the histological characteristics of the nasal tissues by the hematoxylin and eosin (H&E) staining, and observed the nasal symptoms (rubs and sneezes) to evaluate the reliability of the AR mouse model. The colonic NF-κB protein was detected by Western Blot, and the colonic histological characteristics were observed by the H&E staining to evaluate inflammation of colon tissue. We analyzed the V3 and V4 regions of the 16S ribosomal DNA (rDNA) gene from the feces (colon contents) through 16S rDNA sequencing technology. Untargeted metabolomics was used to examine fecal and serum samples to find differential metabolites. Finally, through comparison and correlation analysis of differential gut microbiota, fecal metabolites, and serum metabolites, we further explore the overall impact of AR on gut microbiota, fecal metabolites, and host serum metabolism and its correlation., Results: In the AR group, the IL-4, IL-5, IgE, eosinophil infiltration, and the times of rubs and sneezes were significantly higher than those in the Con group, indicating the successful establishment of the AR model. No differences in diversity were detected between the AR and Con groups. However, there were modifications in the microbiota's structure. At the phylum level, the proportion of Firmicutes and Proteobacteria in the AR group increased significantly, while the proportion of Bacteroides decreased significantly, and the ratio of Firmicutes/Bacteroides was higher. The key differential genera, such as Ruminococcus , were increased significantly in the AR group, while the other key differential genera, such as Lactobacillus , Bacteroides , and Prevotella , were significantly decreased in the Con group. Untargeted metabolomics analysis identified 28 upregulated and 4 downregulated differential metabolites in feces and 11 upregulated and 16 downregulated differential metabolites in serum under AR conditions. Interestingly, one of the significant difference metabolites, α- Linoleic acid (ALA), decreased consistently in feces and serum of AR. KEGG functional enrichment analysis and correlation analysis showed a close relationship between differential serum metabolites and fecal metabolites, and changes in fecal and serum metabolic patterns are associated with altered gut microbiota in AR. The NF-κB protein and inflammatory infiltration of the colon increased considerably in the AR group., Conclusion: Our study reveals that AR alters fecal and serum metabolomic signatures and gut microbiota characteristics, and there is a striking correlation between the three. The correlation analysis of the microbiome and metabolome provides a deeper understanding of AR's pathogenesis, which may provide a theoretical basis for AR's potential prevention and treatment strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, He, Wei, Liu, Xu, Lin, Chen, Lin, Wang, Li and Xu.)

Although polymerase chain reaction (PCR) amplification and sequencing of the bacterial 16S rDNA region has numerous scientific applications, it does not provide DNA methylation information. Herein, we propose a simple extension for bisulfite sequencing to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical isolates or flora. Multiple displacement amplification without DNA denaturation was used to preferentially pre-amplify single-stranded bacterial DNA after bisulfite conversion. Following the pre-amplification, the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing, enabling the simultaneous identification of DNA methylation status and sequence data. We used this approach (termed sm16S rDNA PCR/sequencing) to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii and different methylation motifs among Enterococcus faecalis strains from small volumes of clinical specimens. Further, our analysis suggested that M. MmnI may be correlated to erythromycin resistance. Thus, sm16S rDNA PCR/sequencing is a useful extension method for analyzing the DNA methylation of 16S rDNA regions in a microflora, providing additional information not provided by conventional PCR. Given the relationship between DNA methylation status and drug resistance in bacteria, we believe this technique can be effectively applied in clinical sample testing., (© 2023. The Author(s).)

The biological agents are carried from deserts and dried lands to long distances by high dust volumes. Their adverse effects can be reduced by specifying and controlling dust sources and their related biological agents. Thus, the current work examined the relationship between the bacteria in air and soil samples by taking samples from the soil surface of two dust sources, as well as from air samples during spring from Khorramshahr and Abadan cities. The dust event is the most influential factor on airborne bacteria. There is an insignificant negative (-0.06), insignificant positive (0.14), and weak positive (0.24) correlation between airborne bacteria and UV radiation, relative humidity, and temperature, respectively. After preparing a 16S ribosomal DNA (rDNA) clone library from the soil and air samples, operational taxonomic unit picking and taxonomic assignment were conducted using QIIME Virtual Box. In the present work, Bacillus was the dominant species. The relationship between dust sources and air samples was determined by principal component analysis. Bacteria in the Hoor-Al-Azim dust source and airborne bacteria on dusty and non-dusty days showed a more significant correlation compared to bacteria in the Shadegan dust source. Source Tracker software was used to estimate the contribution of dust sources. The primary source of dust was associated with the dried areas of Hoor-Al-Azim on the non-dusty and dusty days. Finally, the long transport of airborne bacteria was assessed by moderate resolution imaging spectroradiometer (MODIS) and the back trajectory model of Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) data. The research findings can help decision-makers prioritize dust sources to control the adverse effects of dust., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Heat stress is an important issue in dairy cattle feeding management affecting summer health and economic efficiency. This experiment combined 16S rDNA sequencing(3,864,982 tags, 30 sequencing data), metagenomic sequencing(1,269,441,128 reads, 18 sequencing data), metabolomics analysis(72 sequencing data) and blood index analysis. Ten cows in each animal type (growing heifers, heifers, and lactating cows) were selected for sample collection in April and August. Here, we characterized both the changes in metabolites, rumen microbial communities and their functional potential and the effects of heat stress on serum biochemical, immune, oxidative stress, and hormonal indices derived from rumen fluid and serum samples from cows during different growth stages and in different climates. The generated data expand the resources for the rumen microbiome related to heat stress and age and provide useful datasets for research on developing therapeutic strategies to achieve high summer milk production in cows. These datasets will help researchers study the effects of heat stress on the physiological metabolism of Holstein cows and the time-dependent changes associated with growth stages., (© 2022. The Author(s).)

In this study, we characterize the changes in nucleolar morphology and its dynamics induced by the recently introduced compound CX-5461, an inhibitor of ribosome synthesis. Time-lapse imaging, immunofluorescence and ultrastructural analysis revealed that exposure of cells to CX-5461 has a profound impact on their nucleolar morphology and function: nucleoli acquired a compact, spherical shape and display enlarged, ring-like masses of perinucleolar condensed chromatin. Tunnels consisting of chromatin developed as transient structures running through nucleoli. Nucleolar components involved in rRNA transcription, fibrillar centres and dense fibrillar component with their major constituents ribosomal DNA, RNA polymerase I and fibrillarin maintain their topological arrangement but become reduced in number and move towards the nucleolar periphery. Nucleolar changes are paralleled by an increased amount of the DNA damage response indicator γH2AX and DNA unwinding enzyme topoisomerase I in nucleoli and the perinucleolar area suggesting that CX-5461 induces torsional stress and DNA damage in rDNA. This is corroborated by the irreversibility of the observed altered nucleolar phenotypes. We demonstrate that incubation with CX-5461, apart from leading to specific morphological alterations, increases senescence and decreases cell replication. We discuss that these alterations differ from those observed with other drugs interfering with nucleolar functions., (© 2022. The Author(s).)

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions., (© 2021. The Author(s).)

The present study describes three new species of monogenean parasites of characid fishes from the Upper Paraná River basin, Brazil: Characithecium paranapanemense n. sp. on Psalidodon paranae and Psalidodon bockmanni, Diaphorocleidus magnus n. sp. on Astyanax lacustris and Psalidodon fasciatus, and Diaphorocleidus neotropicalis n. sp. on Astyanax lacustris and P. bockmanni. An amendment for Diaphorocleidus is proposed, since additional characters observed in the new species required to extend the generic diagnostic features mainly to include: articulation process connecting the base of the MCO with accessory piece present or absent, and accessory piece with variable shapes (plate-like, pincer-shaped, wrench-shaped, sheath-shaped), divided or not into subunits. Characithecium paranapanemense n. sp. can be distinguished from other congeners by the morphology of its MCO and accessory piece. Diaphorocleidus magnus n. sp. differs from most of its congeners by the morphology of its accessory piece, the presence of articulation process connecting the base of the MCO with accessory piece, and the morphology of the sclerotized structures of the haptor. Diaphorocleidus neotropicalis n. sp. can be easily distinguished from its congeners by the morphology of the accessory piece, the sclerotized structures of the haptor and the morphology of the vagina. Molecular data of the new species (partial 28S rDNA and mitochondrial cytochrome oxidase I) were obtained and the first phylogenetic analysis based on 28S rDNA gene sequences for species of Characithecium and Diaphorocleidus are provided. Although Diaphorocleidus and Characithecium share some morphological similarities, phylogenetic analysis indicates that species of these two genera are not closely related., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fasciola gigantica and Fasciola hepatica are digenetic trematodes causing fasciolosis in ruminants. The host and geographical distribution of both Fasciola species are influenced by environmental and climatic conditions favouring survival and development of free-living stages and intermediate hosts, and livestock management practices. The aim of the present study was to describe the host distribution of the two Fasciola species in buffalo, cattle, goats, and sheep in the Balochistan and Punjab provinces of Pakistan. 359 flukes were collected from a total of 32 livers from the four livestock species. Deep amplicon sequencing of the internal transcribed spacer region 2 of ribosomal DNA (rDNA ITS-2) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND-1) loci confirmed co-infection of F. hepatica and F. gigantica in Balochistan and single species F. gigantica infection in Punjab. In Balochistan, co-infections and hybrids of both Fasciola species were identified in cattle, with more F. hepatica detected than F. gigantica. However, F. hepatica was the only species identified in goats, and F. gigantica was the only species identified in buffalo. In Punjab, all flukes were confirmed as F. gigantica in each of the four livestock species. Overall, the results indicate differences in the host and geographical distribution of F. gigantica and F. hepatica, and provide useful knowledge for the development of control strategies for livestock and humans., (Copyright © 2021. Published by Elsevier B.V.)

Mud volcanoes (MVs) are visible signs of oil and gas reserves present deep beneath land and sea. The Marac MV in Trinidad is the only MV associated with natural hydrocarbon seeps. Petrogenic polyaromatic hydrocarbons (PAHs) in its sediments must undergo biogeochemical cycles of detoxification as they can enter the water table and aquifers threatening ecosystems and biota. Recurrent hydrocarbon seep activity of MVs consolidates the growth of hydrocarbonoclastic fungal communities. Fungi possess advantageous metabolic and ecophysiological features for remediation but are underexplored compared to bacteria. Additionally, indigenous fungi are more efficient at PAH detoxification than commercial/foreign counterparts and remediation strategies remain site-specific. Few studies have focused on hydrocarbonoclastic fungal incidence and potential in MVs, an aspect that has not been explored in Trinidad. This study determined the unique biodiversity of culturable fungi from the Marac MV capable of metabolizing PAHs in vitro and investigated their extracellular peroxidase activity to utilize different substrates ergo their extracellular oxidoreductase activity (> 50% of the strains decolourized of methylene blue dye). Dothideomycetes and Eurotiomycetes (89% combined incidence) were predominantly isolated. ITS rDNA sequence cluster analysis confirmed strain identities. 18 indigenous hydrocarbonoclastic strains not previously reported in the literature and some of which were biosurfactant-producing, were identified. Intra-strain variability was apparent for PAH utilization, oil-tolerance and hydroxylase substrate specificity. Comparatively high levels of extracellular protein were detected for strains that demonstrated low substrate specificity. Halotolerant strains were also recovered which indicated marine-mixed substrata of the MV as a result of deep sea conduits. This work highlighted novel MV fungal strains as potential bioremediators and biocatalysts with a broad industrial applications., (© 2021. The Author(s).)

Myrmicinosporidium durumHölldobler (1933) is a widely distributed fungal endoparasite of ants. However, little is known about its biology, ecology, or evolutionary history. Our study investigated the phylogenetics of this entomopathogenic fungus using a molecular approach. Samples of M. durum were obtained from infected Solenopsis fugax workers collected in Warsaw (Poland). Analyses of rDNA markers revealed that M. durum belongs to a phylum of primarily aquatic fungi, Blastocladiomycota. It is currently the only species from this group known to parasitise hymenopterans. Our findings have clarified this fungus' taxonomy and suggest future directions for research into its biology, ecology, and infection dynamics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Previously, it was suggested that haemadipsid leeches represent an important vector of trypanosomes amongst native animals in Australia. Consequently, Chtonobdella bilineata leeches were investigated for the presence of trypanosome species by polymerase chain reaction (PCR), DNA sequencing and in vitro isolation. Phylogenetic analysis ensued to further define the populations present. PCR targeting the 28S rDNA demonstrated that over 95% of C. bilineata contained trypanosomes; diversity profiling by deep amplicon sequencing of 18S rDNA indicated the presence of four different clusters related to the Trypanosoma (Megatrypanum) theileri. Novy–MacNeal–Nicolle slopes with liquid overlay were used to isolate trypanosomes into culture that proved similar in morphology to Trypanosoma cyclops in that they contained a large numbers of acidocalcisomes. Phylogeny of 18S rDNA/GAPDH/ND5 DNA sequences from primary cultures and subclones showed the trypanosomes were monophyletic, with T. cyclops as a sister group. Blood-meal analysis of leeches showed that leeches primarily contained blood from swamp wallaby (Wallabia bicolour), human (Homo sapiens) or horse (Equus sp.). The leech C. bilineata is a host for at least five lineages of Trypanosoma sp. and these are monophyletic with T. cyclops; we propose Trypanosoma cyclops australiensis as a subspecies of T. cyclops based on genetic similarity and biogeography considerations.

In the present study, we combined morphological and phylogenetic methods to characterize Myxobolus pseudonobilis n. sp. infecting Hypophthalmichthys molitrix Valenciennes, 1844 from Chongqing, China. The morphology and molecular characteristics of M. pseudonobilis n. sp. were distinct from those of other previously described Myxobolus species. Mature myxospores were ovoid in frontal view with spore dimensions of 10.0 ± 0.4 (9.3-10.9) μm in length and 8.5 ± 0.2 (7.9-9.0) μm in width. Two polar capsules occupying approximately half of the myxospore length were unequal in size. The larger polar capsule containing 6 to 7 filament coils measured 5.2 ± 0.3 (4.5-5.8) μm in length and 3.6 ± 0.2 (3.2-3.9) μm in width, while the smaller capsule with 4 to 5 filament coils measured 3.9 ± 0.3 (3.0-4.4) μm in length and 2.5 ± 0.3 (2.1-3.6) μm in width. The comparison of molecular characteristics demonstrated similarities and genetic distances of 18S rDNA sequences of 95.19% - 98.20% and 1.82% - 5.46%, respectively, between M. pseudonobilis n. sp. and its morphologically similar species, and secondary structures were also distinctly different. Moreover, phylogenetic analysis showed that M. pseudonobilis n. sp. was clustered with other myxobolids possessing spores with a blunt anterior end and branched independently. In addition, the morphology of myxosporeans as an important indicator was discussed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The genus Anisakis is a well-known group of nematodes that parasitize cetaceans as the final host and cause mucosal damage to their stomach. However, little has been done to precisely identify the nematodes recovered from the final hosts, especially in the Western Pacific, because of taxonomic confusion about the discrimination of sibling species and the difficulties of obtaining specimens from cetaceans. We describe the results of genetic identification and histopathological observations of specimens recovered from an ulcerated lesion and stomach contents in the forestomach of a female harbor porpoise accidentally caught by a set net fishery in Usujiri, southern Hokkaido, Japan. All the specimens arbitrarily collected from the lesion and stomach contents were identified as Anisakis simplex sensu stricto according to their ITS rDNA sequences. The size of the ulcer was approximately 6.3 mm in diameter and it was infected with 119 individual nematodes, mostly consisting of L3 and L4 stage larvae (95.0%). Histological sections were characterized by a locally extensive ulcer with the parasites penetrating into the muscularis externa that caused a thickening of the surrounding mucosa., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Children with Type 1 diabetes mellitus (T1DM) have an elevated risk of abnormal blood pressure (BP) measurements and patterns. Both hypertension and T1DM are well-known risk factors for cardiovascular disease and kidney failure. The human microbiome has been linked to both diabetes and hypertension, but the relationship between the gut microbiome and BP in children with T1DM is not well-understood. In this cross-sectional study, we examined the relationship between resting office BP and gut microbiota composition, diversity, and richness in children with T1DM and healthy controls. We recruited 29 pediatric subjects and divided them into three groups: healthy controls (HC, n = 5), T1DM with normal BP (T1DM-Normo, n = 17), and T1DM with elevated BP (T1DM-HBP, n = 7). We measured the BP, dietary and clinical parameters for each subject. We collected fecal samples to perform the 16s rDNA sequencing and to measure the short-chain fatty acids (SCFAs) level. The microbiome downstream analysis included the relative abundance of microbiota, alpha and beta diversity, microbial markers using Linear Discriminant effect size analysis (LEfSe), potential gut microbial metabolic pathways using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) and metabolic pathways validation using Statistical Inference of Associations between Microbial Communities And host phenotype (SIAMCAT) machine learning toolbox. Our study results showed that T1DM-HBP group had distinct gut microbial composition (at multiple taxonomic levels) and reduced diversity (richness and abundance) compared with T1DM-Normo and HC groups. Children with T1DM-HBP showed a significant reduction of Bifidobacterium levels (especially B. adolescentis, B. bifidum, and B. longum) compared to the T1DM-Normo group. We also observed unique gut-microbial metabolic pathways, such as elevated lipopolysaccharide synthesis and glutathione metabolism in children with T1DM-HBP compared to T1DM-Normo children. We can conclude that the reduction in the abundance of genus Bifidobacterium could play a significant role in elevating the BP in pediatric T1DM subjects. More studies are needed to corroborate our findings and further explore the potential contributing mechanisms we describe., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Trematodes of the genus Rhytidodoides are parasitic in marine turtles. Of the already known species, Rhytidodoides similis Price, 1939, occurs especially in the gall bladder. In this study, we surveyed 73 green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan, and detected Rhytidodoides sp. from the gall bladders of 18 turtles. A detailed morphological analysis revealed that the forebody of Rhytidodoides sp. differed slightly in shape from that of R. similis. There has been no information on DNA sequences of the family Rhytidodidae. A molecular phylogeny based on 28S rDNA sequences of Rhytidodoides sp. and related taxa suggested that the Rhytidodidae is sister to the other families of Echinostomatoidea. The intraspecific diversity of Rhytidodoides sp. was examined by using DNA sequences of mitochondrial cytochrome c oxidase subunit 1 gene (COI). The population genetic features of the COI haplotypes demonstrated that Rhytidodoides sp. is highly diverse in the Ogasawara Islands. The DNA sequences determined in this study will contribute to the species identification of congeners and the taxonomic reconsideration of the Echinostomatoidea., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The advancement in metagenomic techniques has provided novel tools for profiling human parasites in environmental matrices, such as water and wastewater. However, application of metagenomic techniques for the profiling of protozoan parasites in environmental matrices is not commonly reported in the literature. The key factors leading to the less common use of metagenomics are the complexity and large eukaryotic genome, the prevalence of small parasite populations in environmental samples compared to bacteria, difficulties in extracting DNA from (oo)cysts, and limited reference databases for parasites. This calls for further research to develop optimized methods specifically looking at protozoan parasites in the environment. This study reviews the current workflow, methods and provide recommendations for the standardization of techniques. The article identifies and summarizes the key methods, advantages, and limitations associated with metagenomic analysis, like sample pre-processing, DNA extraction, sequencing approaches, and analysis methods. The study enhances the understanding and application of standardized protocols for profiling of protozoan parasite community from highly complexe samples and further creates a resourceful comparison among datasets without any biases., (Copyright © 2021. Published by Elsevier B.V.)

Coelozoic parasites of the family Myxidiidae were observed in fish of the order Characiformes captured in the middle Tocantins River, Maranhão, Brazil, within the transition between the Cerrado savanna biome and the eastern extreme of the Brazilian Amazon Forest. The analysis of the morphological characteristics of the parasites, complemented with a Bayesian phylogenetic analysis, supported the description of new specie Myxidium imperatrizensis n. sp., found parasitising the gallbladder of specimens of the fish Triportheus angulatus. The study is also only the second report of the occurrence of Myxidium parasites in fish of the family Triportheidae in Brazilian rivers. The results of the present study expand the known distribution of the genera Myxidium the basin of the Tocantins River, the largest hydrographic basin located entirely within Brazil, which encompasses parts of both the Cerrado and Amazon biomes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Porrocaecum angusticolle is a nematode species mainly parasitic in the birds of Accipitriformes and Strigiformes. However, some aspects of the morphology of P. angusticolle remain insufficiently known. In the present study, the detailed morphology of P. angusticolle was studied using light and, for the first time, scanning electron microscopy, based on newly collected specimens from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic. Some previously unreported morphological features of taxonomic significance were observed. The nuclear and mitochondrial DNA markers, including partial large ribosomal DNA (28S), complete internal transcribed spacer (ITS-1 + 5.8S + ITS-2), cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2) of P. angusticolle were sequenced for molecular identification of this species. There was no intraspecific genetic variation detected in the 28S and ITS regions among different individuals of P. angusticolle, but low level of intraspecific nucleotide divergence was found in the cox1 (0.26-0.78%) and cox2 regions (1.0%). The 28S and cox2 of P. angusticolle were sequenced for the first time. Our molecular evidence supported the validity of both P. angusticolle and P. depressum. The newly obtained genetic data are helpful for further studies of DNA-based taxonomy, population genetics and phylogeny of the genus of Porrocaecum., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The present study describes a new coelozoic, eukaryotic microparasite of the genus Ellipsomyxa Køie, 2003 (Ceratomyxidae: Myxozoa) found parasitizing the gallbladder of Satanoperca jurupari Heckel, 1840 collected in the Curiaú River Environmental Protection Area in Macapá, Amapá state, Brazil. The fish were collected using mesh cast net. The gallbladders were examined, preserved in 80% alcohol for molecular analysis (SSU rDNA gene), and fixed in Davidson for histological slide preparation. The new parasite had a prevalence of 81% in the gallbladder, asymmetric plasmodia, irregular free spores in the bladder fluid, with no cyst formation. The spores are elliptical, with characteristics of the genus Ellipsomyxa, and they had a mean length of 10.11 (8.56-10.5) μm, mean width of 7.81 (5.96-9.56) μm, and thick walls. The polar capsules are sub-spherical in shape, slightly asymmetrical, with a mean length of 3.12 (2.31-3.99) μm and mean width of 2.5 (2.22-2.95) μm, containing polar filament with five or six coils perpendicular to the longitudinal axis of the capsule. The Bayesian Inference assigned the new species to a subclade formed by a lineage of Ellipsomyxa species from the Amazon region. Ellipsomyxa tucujuensis n. sp. is the sixth species of this genus described in fish from the Amazon region, and the first for the state of Amapá., (Copyright © 2021. Published by Elsevier B.V.)

Amphistome species belonging to the genus Catadiscus are poorly studied intestinal trematodes found primarily in Neotropical anurans. Herein, developmental stages of an amphistome species found during herpetological and malacological surveys in a temporary marsh pond from Brazil were subjected to morphological (light and scanning electron microscopy) and molecular analyses. Adult parasites recovered from anurans were identified as Catadiscus marinholutzi. Amphistome cercariae found in the planorbid snails Drepanotrema depressissimum and Drepanotrema lucidum from the same waterbody were used for experimental and molecular studies. Immature parasites, morphologically compatible with members of Catadiscus, were experimentally obtained in laboratory-reared tadpoles. Sequencing of a partial region of 28S rDNA gene of both adult and cercariae revealed 100% similarity between these developmental stages, confirming their conspecificity. Phylogenetic analyses were attempted for the first time to reveal the position of a species of Catadiscus in the superfamily Paramphistomoidea. Catadiscus marinholutzi falls in a virtual polytomy together with other paramphistomoids, which leaves its phylogenetic relationships within the group unclear. Moreover, the high genetic divergence to Diplodiscus spp. (10.06–10.84%) cast doubts on the placement of Catadiscus within Diplodiscidae. Hence the species composition of the Diplodiscidae should be re-evaluated in further studies using a broader spectrum of related taxa.

Objective: Airborne microbial communities include a significant number of uncultured and poorly characterized bacteria. No effective method currently exists to evaluate the health risks of such complex bacterial populations, particularly for pneumonia., Methods: We developed a method to evaluate risks from airborne microorganisms, guided by the principle that closer evolutionary relationships reflect similar biological characteristics, and thus used 16S rDNA sequences of 10 common pneumonia-related bacterial pathogens. We calculated a risk of breath-related ( Rbr ) index of airborne bacterial communities and verified effectiveness with artificial flora and a clinical project., Results: We suggested applying Rbr80 to evaluate the health risks of airborne bacterial communities that comprise 80% of dominant operational taxonomic units (OTUs). The feasibility of Rbr80 was confirmed by artificial flora and by pneumonia data from a hospital. A high Rbr80 value indicated a high risk of pneumonia from airborne bacterial communities., Conclusion: Rbr80 is an effective index to evaluate the pneumonia-associated risk from airborne bacteria. Values of Rbr80 greater than 15.40 are considered high risk., (Copyright © 2021 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Acanthocephalans of the order Echinorhynchida are one of the most diverse groups in their phylum, with approximately 470 species classified into 11 families that largely consist of parasites of freshwater, brackish and marine fishes and, sporadically, reptiles and amphibians distributed worldwide. Previous phylogenies inferred with molecular data have supported the paraphyly or polyphyly of some families, suggesting that most of them have been diagnosed based on unique combinations of characters, rather than shared derivative features. We expand the taxonomic sampling of several genera such as Acanthocephalus, Echinorhynchus and Pseudoacanthocephalus of Echinorhynchidae from diverse biogeographical zones in the Americas, Europe and Asia with the aim of testing the monophyly of the family by using two molecular markers. Sequences from small (SSU) and large (LSU) subunits of ribosomal DNA were obtained for six species representing the genera Acanthocephalus and Echinorhynchus from the Neotropical, Nearctic, Palearctic and Oriental regions. These sequences were aligned with other sequences available in the GenBank dataset from Echinorhynchidae. Phylogenetic trees inferred with the combined (SSU + LSU) and the individual data sets consistently placed the genera Acanthocephalus, Pseudoacanthocephalus and Echinorhynchus into three independent lineages. Two families, Paracanthocephalidae Golvan, 1960, and Pseudoacanthocephalidae Petrochenko, 1956, were resurrected to accommodate the genera Acanthocephalus and Pseudoacanthocephalus, respectively. The species of the genus Acanthocephalus from the Nearctic, Palearctic and Oriental biogeographic regions formed a clade that was well supported. However, Acanthocephalus amini from the Neotropical region was nested inside Arhythmacanthidae. Therefore, the genus Calakmulrhynchus was created to accommodate A. amini and resolve the paraphyly of Acanthocephalus. Finally, the diagnoses of the families Echinorhynchidae and Arhythmacanthidae were amended. The molecular phylogenies should be used as a taxonomic framework to find shared derived characters (synapomorphies) and build a more robust classification scheme that reflects the evolutionary history of the acanthocephalans., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Saccocoelioides is a genus of trematodes associated with fishes from the Americas. In the current research, morphologically distinct specimens of Saccocoelioides spp. were collected from six countries in Middle America. Specimens were sequenced using three molecular markers, the domains D1-D3 of the large subunit (LSU) from the nuclear rDNA, the cytochrome c oxidase subunit 1 (cox1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) from mitochondrial DNA. A total of 74 new sequences were compared and aligned with other sequences available in GenBank. Maximum likelihood and Bayesian inference analyses were inferred from the LSU and cox1 datasets, revealing unequivocally that all the specimens correspond to S. lamothei. A haplotype network was built with 119 sequences of the nad1 gene. The network detected 57 distinct haplotypes divided into three haplogroups. To explore morphological differences among samples of S. lamothei, 17 morphological features were measured from 53 specimens from three fish families: Eleotridae, Mugilidae and Gobiidae. Principal component analysis yielded three main polygons that corresponded with each family analysed, suggesting host-induced phenotypic plasticity. The current evidence suggests that S. lamothei infects at least five fish families along the Pacific coasts of Mexico, Guatemala, El Salvador, Honduras, Nicaragua and Costa Rica.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to confirm the species identity of Dicrocoeliid flukes collected from the Chitral valley in the Himalayan ranges of Pakistan. The morphology of 48 flukes belonging to eight host populations was examined; but overlapping traits prevented accurate species designation. Phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess within and between species variation and re-affirm the use of species-specific single nucleotide polymorphism markers. PCR and sequencing of 34 corresponding fragments of ribosomal DNA and 14 corresponding fragments of mitochondrial DNA from the Chitral valley flukes, revealed 10 and 4 unique haplotypes, respectively. These confirmed for the first time the molecular species identity of Pakistani lancet liver flukes as D. dendriticum. This work provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The two black rhinoceros subspecies (Diceros bicornis bicornis and D. b. minor) in South African conservation areas are managed as separate metapopulations. Since infection with Babesia bicornis can be fatal in black rhinoceroses, occurrence of this and other piroplasms in the two metapopulations was determined to assess possible risk. Blood specimens were collected from 156 black rhinoceroses: 80 from D. b. bicornis and 76 from D. b. minor. DNA was extracted; the V4 hypervariable region of the parasite 18S rRNA gene was amplified and subjected to the Reverse Line Blot (RLB) hybridization assay. There was a significant difference in occurrence of piroplasms: 18/80 (23%) in D. b. bicornis and 39/76 (51%) in D. b. minor. Theileria bicornis occurred in significantly more of the D. b. minor population (36/76; 47%) than the D. b. bicornis population (1/80; 1%); with B. bicornis the difference was not significant: D. b. bicornis 5/80 (6%) and D. b. minor 9/76 (11%). Three individuals were infected with Theileria equi. Results were confirmed using molecular characterization of the near full-length parasite 18S rRNA gene of 13 selected specimens. We identified four (Tb1, Tb2, Tb3 and Tb4) 18S rDNA sequence types for T. bicornis, two for B. bicornis (Bb1 and Bb2) and one for T. equi (Teq1). We furthermore identified T. bicornis haplotypes H1, H3 and H4 in 10 rhinoceroses; H3 was the most common haplotype identified. Rhinoceroses inhabiting more arid areas are apparently free of T. bicornis and B. bicornis, probably due to the absence or scarcity of vectors. When individuals are relocated for metapopulation management purposes, appropriate prophylactic action should be taken to minimise the risk of babesiosis, which could be fatal., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Plasmodia containing myxospores belonging to the genus Henneguya Thélohan, 1892 were found in the gills of Eugerres brasilianus (Cuvier, 1830). Despite the economic importance, few parasitological studies have been done with this species. We describe Henneguya lagunensis n. sp. using morphological and molecular data. The mature myxospores were rounded, measuring 29.1 ± 2.2 μm in total length, 8.2 ± 1.0 μm in body length, 7.9 ± 0.2 μm in body width, 20.7 ± 2.4 μm in tail length and 4.8 ± 1.0 μm in thickness. The polar capsules measured 3.3 ± 0.4 in length and 1.7 ± 0.3 μm in width. Polar filaments had 4-5 turns, helical. Phylogenetic analysis showed Henneguya lagunensis n. sp. as a sister species of Henneguya cynoscioni Dyková, Buron, Roumillat and Fiala, 2011, within a clade that contained mostly Henneguya species that parasitize marine fish of the order Perciformes. This is the first report of a species of Henneguya parasitizing Eugerres brasilianus., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Trematodes are one of the largest taxa of mollusk parasites. The clam Leukoma thaca is an economically exploited bivalve found along the south-eastern Pacific coast of Peru and Chile. This bivalve is parasitized by various unidentified larval stages of digeneans in the mantle, gonads and digestive gland. The aims of this study were to determine and describe the different larval stages of the digeneans based on morphological characteristics, to identify them at the species level by performing molecular analyses, and to evaluate pathologies associated with the parasites of this clam. Individuals of L. thaca were collected in San Jorge Bay (23°S), Chile, between November 2018 and February 2019. Morphological description was carried out using in vivo and fixed specimens, and analyses including histological and scanning electron microscopy were performed. Individuals were also isolated for molecular analysis using nuclear ribosomal internal transcribed spacer 1 (ITS1), including partial subunit 18S rDNA (18S) and small subunit 5.8S gene (5.8S). Morphological characteristics indicated that the metacercaria larval stage belongs to the family Gymnophallidae, genus Parvatrema, which was supported by molecular analysis. Molecular results revealed that metacercaria, sporocysts and cercaria stages found in this clam belong to the same species of Parvatrema (genetic distance 0%), evidencing that this species uses L. thaca as the first and second intermediate host. Pathologies examined in the host were similar in nature to those reported in other gymnophallids in bivalves, but high prevalence of cercariae (20%) in gonads suggested an important castrator effect on the host., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we describe two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the nearly complete rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota-a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates that their host specificity is not as strict as generally assumed.

The morphology, morphogenesis and molecular phylogeny of Parabistichella dieckmanni (Foissner, 1998) Foissner, 2016, isolated from north China, were investigated. The Chinese population was characterized as having five to seven frontal cirri in corona, four to seven buccal cirri arranged in a row, two to four short frontal rows and two long frontoventral rows, three to seven transverse cirri, four macronuclear nodules, three dorsal kineties following a Gonostomum-pattern, and caudal cirri absent. Morphogenetic research reveals that the main characteristics during binary fission are as follows: (1) the long left frontoventral row is formed by two or three anlagen; (2) the posterior part of the parental adoral zone of membranelles is renewed, and the oral primordium of the opisthe is formed intrakinetally; (3) FVT-anlagen I to VI (or V, VII) produce each a frontal cirrus to form the frontal corona; (4) development of dorsal kineties follows the Gonostomum-pattern. Phylogenetic analyses showed that P. dieckmanni does not group with other Parabistichella species. Therefore, the genus Parabistichella is polyphyletic. Additionally, Parabistichella variabilis n. comb. (basionym: Bistichella variabilis He & Xu, 2011) and Parabistichella cheni n. nom. (basionym: Parabistichella variabilis Jiang et al., 2013) were suggested., (© 2020 International Society of Protistologists.)

The morphology and phylogeny of two metopid ciliates, collected from anaerobic habitats in China, were investigated using live observation, protargol staining method, and SSU rDNA sequencing. The new species Metopus paravestitus nov. spec. can be distinguished by a combination of the following features: oblong cell with densely arranged ectobiotic prokaryotes perpendicular to cell surface, filiform intracytoplasmic structures packed in the anterior portion of the cell. Our work also demonstrates the wide geographical distribution of Metopus es (Müller, 1776) Lauterborn, 1916. The order Metopida is consistently depicted as a paraphylum in SSU rDNA phylogeny. Metopus paravestitus nov. spec. is closely related to its marine congeners than to freshwater forms. The present study confirms once again the non-monophyly of the genus Metopus and genus Metopidae., (© 2020 International Society of Protistologists.)

Balantioides coli is a ciliated parasite that can cause diarrhea when inhabiting the colon and cecum of pigs and humans. However, information regarding the changes in structure and composition of the gut microbiome in piglets infected with B. coli remains scarce. In this study, 16S rDNA amplicon sequencing was conducted on fecal samples from both B. coli-positive piglets with diarrhea and B. coli-negative piglets without diarrhea. The results showed that Firmicutes and Bacteroidetes were the major phyla in the two groups, and the relative abundance of the phylum Firmicutes, including Ruminococcaceae and Clostridiaceae, was significantly lower in the B. coli-positive samples than in the B. coli-negative ones (p < 0.05). Compared with B. coli-negative samples, Alloprevotella and Faecalibacterium showed distinctly higher relative abundances (p < 0.05) in the B. coli-positive samples, and the abundances of some potential pathogenic bacteria, including Escherichia-Shigella and Campylobacter, were positively correlated with B. coli infection and diarrhea of weaned piglets. In addition, there were statistically significant differences in fecal microbiota diversity and abundances of predicted functional genes between B. coli-colonized and B. coli-negative samples (p <  0.05). Taken together, these findings suggest that there were significant differences in the bacterial community composition, diversity, and functions between the B. coli-positive and B. coli-negative piglets, and the colonization by B. coli may be associated with the dysbiosis of gut microbiota structure in weaned piglets., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lasiotocus Looss, 1907 is the largest genus within the Monorchiidae Odhner, 1911, with 52 species currently considered valid. Species belonging to this genus exhibit wide morphological variation and it is likely that many of them belong in other genera; however, testing the validity of the group has been hampered by a lack of molecular sequence data, especially for the type-species. Here, we report the first DNA sequences for Lasiotocus mulli (Stossich, 1883) Odhner, 1911, the type-species of the genus, and only the sixth Lasiotocus species to be sequenced. Sequences were generated for three ribosomal DNA markers, the ITS2, 18S and 28S regions, and for one mitochondrial DNA marker, the cox1 region. Phylogenetic analyses show that the six sequenced species form four clearly distinct clades, each of which we argue require separate genera. On the basis of these relationships and review of the morphology of all species in the genus, we propose a system of six genera. Four genera, Lasiotocus, the resurrected concept of Ancylocoelium Nicoll, 1912, Infundiburictus n. gen., and Sinistroporomonorchis n. gen., represent the four molecular clades. Two genera, Paralasiotocus n. gen. and Alloinfundiburictus n. gen., are proposed on the basis of morphology only. Ancylocoelium is resurrected for Ancylocoelium typicum Nicoll, 1912, Infundiburictus is erected for Lasiotocus arrhichostoma Searle, Cutmore & Cribb, 2014, Sinistroporomonorchis is erected for Lasiotocus glebulentus Overstreet, 1971and Lasiotocus lizae Liu, 2002, Paralasiotocus is erected for Lasiotocus okinawaensis Machida, 2011, and Alloinfundiburictus is erected for Lasiotocus cacuminatus Nicoll, 1915. Of the 52 species presently recognised in Lasiotocus, 43 are distributed among these genera; nine are considered species inquirendae or are transferred to other genera. We think it likely that more genera will be required for the species reviewed here but more sequence data are essential to further refine the classification., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Several species of trypanosomes can infect bats (Chiroptera), but current information about bat trypanosomes in Colombia is scarce. The objectives of this study were to estimate the infection rate and to characterize the trypanosome species infecting bats from three rural regions near the municipality of Cumaribo in Vichada, Colombia. Blood samples were collected from 39 bats. DNA was extracted from the blood samples and analyzed using nuclear genetic markers (SSU rDNA, ITS rDNA, and cathepsin genes) to discriminate among trypanosome species. Trypanosomes were detected in 66.7 % (26/39) of blood samples using PCR; 61.5 % (24/39) of infections were identified as Trypanosoma theileri and 5.1 % (2/39) as T. wauwau-like parasites. The phylogeographic analysis revealed that our T. theileri sequences were associated with the TthIIB genotype from cattle in Brazil and Venezuela. The T. wauwau-like parasites represent a new genotype of the species and were found in Molossus molossus and Platyrrhinus helleri bats. These data represent the first evidence of this trypanosome in both Colombia, and in these species of bats. Bat infections with T. theileri suggest an important role of these hosts in maintaining this genotype, probably acquired by ingesting insect vectors. The T. wauwau-like genotype in new mammalian host species supports the 'bat seeding' hypothesis of the T. cruzi clade. The epidemiological and evolutionary implications of these findings are discussed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania., Competing Interests: The authors have declared that no competing interests exist.

The armored Harttia catfishes present great species diversity and remarkable cytogenetic variation, including different sex chromosome systems. Here we analyzed three new species, H. duriventris , H. villasboas and H. rondoni , using both conventional and molecular cytogenetic techniques (Giemsa-staining and C-banding), including the mapping of repetitive DNAs using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) experiments. Both H. duriventris and H. villasboas have 2n = ♀56/♂55 chromosomes, and an X 1 X 1 X 2 X 2 /X 1 X 2 Y sex chromosome system, while a proto or neo-XY system is proposed for H. rondoni (2n = 54♀♂). Single motifs of 5S and 18S rDNA occur in all three species, with the latter being also mapped in the sex chromosomes. The results confirm the general evolutionary trend that has been noticed for the genus: an extensive variation on their chromosome number, single sites of rDNA sequences and the occurrence of multiple sex chromosomes. Comparative genomic analyses with another congeneric species, H. punctata , reveal that the X 1 X 2 Y sex chromosomes of these species share the genomic contents, indicating a probable common origin. The remarkable karyotypic variation, including sex chromosomes systems, makes Harttia a suitable model for evolutionary studies focusing on karyotype differentiation and sex chromosome evolution among lower vertebrates.

Morphological and molecular evaluation of tapeworms of the genus Bothriocephalus Rudolphi, 1808 (Cestoda: Bothriocephalidea), based on newly collected and uniformly fixed worms from freshwater fishes in Canada and the United States has revealed unexpected diversity. With a combination of selected morphological features and 4 molecular markers (18S rDNA V8 region, ITS1, ITS2, and COI gene sequences), the following morphotypes and lineages of the Bothriocephalus cuspidatus Cooper, 1917 complex were identified, several of which are specific to their respective fish definitive hosts and may represent separate species: B. cuspidatus sensu stricto from walleye, Sander vitreus (type host), which likely includes a miniature morphotype from Johnny darter, Etheostoma nigrum (both Percidae); Bothriocephalus morphotype from pumpkinseed, Lepomis gibbosus (Centrarchidae); and Bothriocephalus morphotype from rock bass, Ambloplites rupestris (Centrarchidae). The Bothriocephalus morphotype from goldeye, Hiodon alosoides (Hiodontidae), may also represent a separate lineage (possibly Bothriocephalus texomensisSelf, 1954) but requires additional studies. A morphotype from smallmouth bass, Micropterus dolomieu, based on a single specimen, is morphologically and genetically very similar to the morphotype from rock bass. Morphological study of the scolex and strobila of heat-killed and fixed specimens has revealed consistent differences, often subtle, that allowed us to differentiate between these morphotypes., (© American Society of Parasitologists 2020.)

The Indonesian island of Sulawesi is a globally significant biodiversity hotspot with substantial undescribed biota, particularly blood-borne parasites of endemic wildlife. Documenting the blood parasites of Sulawesi's murine rodents is the first fundamental step towards the discovery of pathogens likely to be of concern for the health and conservation of Sulawesi's endemic murines. We screened liver samples from 441 specimens belonging to 20 different species of murine rodents from 2 mountain ranges on Sulawesi, using polymerase chin reaction (PCR) primers targeting the conserved 18S rDNA region across the protozoan class Kinetoplastea. We detected infections in 156 specimens (10 host species) with a mean prevalence of 35.4% (95% confidence interval [CI] = 30.9-39.8%). Sequences from these samples identified 4 infections to the genus Parabodo, 1 to Blechomonas, and the remaining 151 to the genus Trypanosoma. Within Trypanosoma, we recovered 17 haplotypes nested within the Trypanosoma theileri clade infecting 117 specimens (8 host species) and 4 haplotypes nested within the Trypanosoma lewisi clade infecting 34 specimens (6 host species). Haplotypes within the T. theileri clade were related to regional Indo-Australian endemic trypanosomes, displayed geographic structuring but with evidence of long-term connectivity between mountains, and had substantial phylogenetic diversity. These results suggest T. theileri clade parasites are native to Sulawesi. Conversely, T. lewisi clade haplotypes were recovered from both endemic and introduced rodents, demonstrated complete geographic separation between clades, and had low genetic diversity. These results suggest that the T. lewisi clade parasites invaded Sulawesi recently and likely in 2 separate invasion events. Our results provide the first records of metakinetoplastids in Sulawesi's rodents and highlight the need for more extensive sampling for pathogens in this biodiversity hotspot., (© American Society of Parasitologists 2020.)

Sculpin fishes belonging to the family Cottidae represent a large and complex group, inhabiting a wide range of freshwater, brackish-water, and marine environments. Numerous studies based on analysis of their morphology and genetic makeup frequently provided controversial results. In the present work, we sequenced complete mitochondrial (mt) genomes and fragments of nuclear ribosomal DNA (rDNA) of the fourhorn sculpin Myoxocephalus quadricornis and some related cottids to increase the power of phylogenetic and taxonomic analyses of this complex fish group. A comparison of the My. quadricornis mt genomes obtained by us with other complete mt genomes available in GenBank has revealed a surprisingly low divergence (3.06 ± 0.12%) with Megalocottus platycephalus and, at the same time, a significantly higher divergence (7.89 ± 0.16%) with the species of the genus Myoxocephalus . Correspondingly, phylogenetic analyses have shown that My. quadricornis is clustered with Me. platycephalus but not with the Myoxocephalus species. Completely consistent patterns of divergence and tree topologies have been obtained based on nuclear rDNA. Thus, the multi-gene data in the present work indicates obvious contradictions in the relationships between the Myoxocephalus and Megalocottus species studied. An extensive phylogenetic analysis has provided evidence for a closer affinity of My. quadricornis with the species of the genus Megalocottus than with the species of the genus Myoxocephalus . A recombination analysis, along with the additional GenBank data, excludes introgression and/or incorrect taxonomic identification as the possible causative factors responsible for the observed closer affinity between the two species from different genera. The above facts necessitate realignment of the genera Myoxocephalus and Megalocottus . The genetic data supports the two recognized genera, Myoxocephalus and Megalocottus , but suggests changing their compositions through transferring My. quadricornis to the genus Megalocottus . The results of the present study resolve the relationships within a complex group of sculpin fishes and show a promising approach to phylogenetic systematics (as a key organizing principle in biodiversity research) for a better understanding of the taxonomy and evolution of fishes and for supplying relevant information to address various fish biodiversity conservation and management issues.

Hypotrichs are a highly differentiated and very diverse group of ciliated protists. Their systematics and taxonomy are challenging and call for detailed investigations on their general morphology, ultrastructure, ontogenesis, and molecular phylogeny. Here, a comprehensive study is conducted on a brackish water population of Parabistichella variabilis using light and electron microscopy and phylogenetic analyses based on small subunit ribosomal DNA sequence data. Its morphology, including the infraciliature, pellicle, nuclei, buccal seal, and extrusomes, is documented. The present findings indicate that in P. variabilis: (i) the cortical granules are extrusomes, which differ from those of other hypotrichs; (ii) the buccal seal is bounded by the plasma membrane and contains a single layer of longitudinal microtubules; (iii) two contractile vacuoles might be present rather than one; and (iv) the pharyngeal disks are bounded by a single membrane. Early-to-middle stages of ontogenesis are described for the first time, enabling the complete characterization of this process. Phylogenetic analyses indicate that Parabistichella variabilis is closely related to several species from different genera, such as Orthoamphisiella breviseries, Uroleptoides magnigranulosus, and Tachysoma pellionellum. However, ultrastructural and gene sequence data for more taxa are needed in order to resolve the systematics of Parabistichella., (© 2020 International Society of Protistologists.)

Ornithodoros cerradoensis n. sp. is described from field-collected and laboratory reared nymphs, males, females, and larvae parasitizing the rodents Cavia aperea and Thrichomys sp. in the Brazilian Savannah. This new species is morphologically and genetically related with the Ornithodoros talaje group and can be separated from other Neotropical species using the following combination of characters: larva with 18 pairs of setae on dorsum (seven anterolateral, four central and seven posterolateral), hypostome with median dentition 2/2; adults provided with large mammillae; dorsal disks surrounded by bulked marginal ridges delimiting barely pebbled areas; three disks in the anterolateral file, and median disk not merging with the posteromedian file. Feeding assays in the laboratory demonstrated that (1) larvae of O. cerradoensis are slow-feeders (∼6 days), (2) first nymphal instar (N1) molts to second instar (N2) without feeding, and (3) N2 and third nymphal instar (N3) engorge rapidly (minutes). With the exception of Ornithodoros hasei nymphs that depict flattened bodies, O. cerradoensis N1, N2, and N3 highly resemble homologous instars of other species in O. talaje sensu lato, therefore are not suitable for morphological comparisons within the group. In addition to morphological signature of larvae and adults that separate this new species; results of cross-mating attempts between O. cerradoensis and Ornithodoros guaporensis a morphologically and phylogenetically closely related species that also parasitizes rodents in the Brazilian Savannah; a Principal Component Analysis using larval characters; and a phylogenetic analysis using mitochondrial markers, support O. cerradoensis as an independent lineage within the Ornithodorinae., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Larvae of Ornithodoros knoxjonesi collected at five localities in three countries were studied using morphological and molecular methods to confirm this species' taxonomic validity. The larva of O. knoxjonesi is characterized as having 14 pairs of dorsal setae, eight pairs of ventral setae, plus a posteromedian seta; an elongate dorsal plate, tapered anteriorly; and a hypostome that is narrower near its midlength, with posteriorly projecting denticles. Although the larvae of O. knoxjonesi and Ornithodoros peropteryx are morphologically quite similar, the larva of O. knoxjonesi is characterized as having dorsal setae that are wider at the tip than at the base, while in O. peropteryx these setae are narrower at the tip than at the base; moreover, the dorsal setae are shorter in O. knoxjonesi (Al 0.037-0.065; Pl 0.035-0.059) than in O. peropteryx (Al 0.120-0.132; Pl 0.080-0.096). These species also differ in that O. knoxjonesi possesses only the Al seta on tarsus I, whereas O. peropteryx has both Al and Pl setae. And while both species have two setae on coxae I-III, in O. knoxjonesi the anterior seta is tapering and smooth and the posterior is fringed, while both setae are fringed in O. peropteryx. At the molecular level, based on a maximum likelihood analysis using approximately 400 bp of the mitochondrial 16S rDNA gene, O. knoxjonesi appears as an independent lineage, separated from O. peropteryx by a genetic distance of 16.28 %. Balantiopteryx plicata is a common host of O. knoxjonesi; however, in this work we report Pteronotus personatus and Pteronotus gymnonotus as new hosts of this tick species., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Grape pomace (GP) is an abundant by-product from wine production and is rich in phenolic compounds, unsaturated fatty acids, dietary fibre and beneficial bacteria. In this study, weaned piglets were fed a basic diet supplemented with 5% GP for 4 weeks. Compared with those in the control (CON) group, it was found that the proportion of Lactobacillus delbrueckii, Olsenella umbonata and Selenomonas bovis in the caecum and the villus height and villus height/crypt depth ratio (VCR) of the jejunum were both significantly increased in the GP group ( p < 0.05). Meanwhile, at the mRNA expression level, several proinflammatory cytokines ( IL-1β, IL-8, IL-6 and TNF-α ) were significantly downregulated ( p < 0.05) in piglet caecal tissue, and the short-chain fatty acid receptors ( GPR41 and GPR43 ) were not significantly upregulated. In contrast, the levels of IgG was significantly increased ( p < 0.05) in the sera of weaned piglets in the GP group. However, no difference in growth performance between the two groups of piglets was detected. These results show that GP had no adverse effects on the growth performance of piglets, but GP can promote the content of some beneficial bacteria in the caecum; this effect is conducive to improving the disease resistance potential of piglets.

Postpasteurization contamination (PPC) with gram-negative bacteria adversely affects the quality and shelf-life of milk through the development of flavor, odor, texture, and visual defects. Through evaluation of milk quality at 4 large fluid milk processing facilities in the northeast United States, we examined the efficacy of 3 strategies designed to reduce the occurrence of PPC in fluid milk: (1) employee training (focusing on good manufacturing practices) alone and (2) with concurrent implementation of modified clean-in-place chemistry and (3) preventive maintenance (PM) focused on replacement of wearable rubber components. Despite increases in employee knowledge and self-reported behavior change, microbiological evaluation of fluid milk before and after interventions indicated that neither training alone nor training combined with modified clean-in-place interventions significantly decreased PPC. Furthermore, characterization of gram-negative bacterial isolates from milk suggested that specific bacterial taxonomic groups (notably, Pseudomonas sequence types) continued to contribute to PPC even after interventions and that no major changes in the composition of the spoilage-associated microbial populations occurred as a consequence of the interventions. More specifically, in 3 of 4 facilities, gram-negative bacteria with identical 16S rDNA sequence types were isolated on multiple occasions. Evaluation of a PM intervention showed that used rubber goods harbored PPC-associated bacteria and that PPC may have been less frequent following a PM intervention in which wearable rubber goods were replaced (reduction from 3/3 samples with PPC before to 1/3 samples after). Overall, our findings suggest that commonly used "broad stroke interventions" may have a limited effect on reducing PPC. Our case study also demonstrates the inherent complexities of identifying and successfully addressing sanitation problems in large and complex fluid milk processing facilities. For example, broad changes to sanitation practices without improvements in PM and sanitary equipment design may not always lead to reduced PPC. Our data also indicate that although short-term evaluations, such as pre- and post-tests for employee training, may suggest improvements after corrective and preventive actions, extensive microbial testing, ideally in combination with isolate characterization, may be necessary to evaluate return on investment of different interventions., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Disseminated phaeohyphomycosis is a rare and unusual infection that can be caused by dematiaceous fungi. Cyphellophora is an uncommon aetiological agent of cutaneous and systemic fungal infections. This study describes a case of disseminated phaeohyphomycosis caused by C. ludoviensis in Iran. The molecular identification of the isolates was established by DNA sequencing of the internal transcribed spacer (ITS) region of rDNA. The patient was successfully treated with an 8-month course of systemic voriconazole., (© 2020 The Australasian College of Dermatologists.)

Myxobolus pseudodispar Gorbunova, 1936 (Myxozoa) was originally described as a parasite of common roach, Rutilus rutilus (Linnaeus), with developing stages in muscles and spores disseminated in macrophage centres of different organs and tissues. Later, this parasite was described from several other cyprinids, but with relatively large intraspecific differences based on SSU rDNA gene sequences. Within our long-term study on myxozoan biodiversity, we performed a broad microscopic and molecular screening of various freshwater fish species (over 450 specimens, 36 species) from different localities. We investigated the cryptic species status of M. pseudodispar. Our analysis revealed four new unique SSU rDNA sequences of M. pseudodispar as well as an infection in new fish host species. Myxobolus pseudodispar sequence analysis showed clear phylogenetic grouping according to fish host criterion forming 13 well-recognised clades. Using 1% SSU rDNA-based genetic distance criterion, at least ten new species of Myxobolus Bütschli, 1882 may be recognised in the group of M. pseudodispar sequences. Our analysis showed the paraphyletic character of M. pseudodispar sequences and the statistical tests rejected hypothetical tree topology with the monophyletic status of the M. pseudodispar group. Myxobolus pseudodispar represents a species complex and it is a typical example of myxozoan hidden diversity phenomenon confirming myxozoans as an evolutionary very successful group of parasites with a great ability to adapt to a new hosts with subsequent speciation events.