Ribosomal DNA Sequences Research Articles

Characteristics of Microbial Distribution in Different Oral Niches of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC), one of the most common malignant tumors of the head and neck, is closely associated with the presence of oral microbes. However, the microbiomes of different oral niches in OSCC patients and their association with OSCC have not been adequately characterized. In this study, 305 samples were collected from 65 OSCC patients, including tumor tissue, adjacent normal tissue (paracancerous tissue), cancer surface tissue, anatomically matched contralateral normal mucosa, saliva, and tongue coat. 16S ribosomal DNA (16S rDNA) sequencing was used to compare the microbial composition, distribution, and co-occurrence network of different oral niches. The association between the microbiome and the clinical features of OSCC was also characterized. The oral microbiome of OSCC patients showed a regular ecological distribution. Tumor and paracancerous tissues were more microbially diverse than other oral niches. Cancer surface, contralateral normal mucosa, saliva, and tongue coat showed similar microbial compositions, especially the contralateral normal mucosa and saliva. Periodontitis-associated bacteria of the genera Fusobacterium, Prevotella, Porphyromonas, Campylobacter, and Aggregatibacter, and anaerobic bacteria were enriched in tumor samples. The microbiome was highly correlated with tumor clinicopathological features, with several genera (Lautropia, Asteroleplasma, Parvimonas, Peptostreptococcus, Pyramidobacter, Roseburia, and Propionibacterium) demonstrating a relatively high diagnostic power for OSCC metastasis, potentially providing an indicator for the development of OSCC.

Restoration of the Genus Paraunisaccoides Martin, 1973 (Digenea: Haploporidae) and Description of P. elegans n. sp. and Unisaccus halongi n. sp. from Mugilid Fish in Vietnam

We restore the genus Paraunisaccoides (Haploporidae), synonymised earlier with the genus Skrjabinolecithum. Adult worms, detected in Vietnamese mullet fish, were highly similar to trematodes described as P. lobolecithum via digestive and genital system structures and relative organ arrangement. Differences are expressed as absence and presence of pads on the hermaphrodite duct, respectively, and the disjunction of some metric parameter values, namely body, ovary and eggs. Ribosomal DNA sequences, based on the phylogenetic analysis of Haploporidae, indicates that new worms represent a sister clade to Unisaccus tonkini. Genetic divergence between new worms and Skrjabinolecithum species can be interpreted as intergeneric. Based on morphological and molecular data, we recognise Paraunisaccoides as a valid genus within Waretrematinae and worms from Vietnam as a new species of this genus, P. elegans n. sp. Other worms detected in Vietnamese mugilids are morphologically similar to representatives of Paraunisaccoides и Skrjabinolecithum. However, molecular-based phylogenetic analysis showed that these trematodes are closely related to Unisaccus tonkini; the genetic divergence between them is at the interspecific level, despite considerable differences in vitellarium structure as intergeneric character. Accepting the priority of molecular results, we include these new worms into the genus Unisaccus as new species, Unisaccus halongi n. sp.

A novel Ceratomyxa species (Myxozoa: Cnidaria) infecting an Amazonian catfish.

Parasites are important organisms for the health of ecosystems. While the Amazon Basin is home to a great diversity of ichthyofauna, our knowledge of myxozoan diversity in the biome remains relatively limited. The present study describes a new myxozoan species, Ceratomyxa mandii n. sp., parasitizing the gallbladder of the Amazonian catfish Pimelodina flavipinnis (Pimelodidae) from the Solimões River, in the region of Manaus, Brazil. Light and electron microscopy, small subunit ribosomal DNA (SSU rDNA) sequencing and phylogenetic analysis were performed. The new species exhibited worm-like plasmodia with undulatory motility. The SSU rDNA based phylogenetic analysis revealed it to be a sister taxon of C. gracillima, which also parasitizes an Amazonian pimelodid fish, possibly reflecting a host-parasite co-speciation process. This study contributes to our understanding of this little sampled group of organisms.

Morphological and molecular characterization of some popular cultivated mushrooms of India

Morphological and molecular characterization of some popular cultivated mushrooms of IndiaSanjeev Kumar1*, Anand Sagar2, Shwet Kamal3 and Jagriti Rana41. Associate professor, Govt. College Bilaspur.2. Dean of Life Sciences, H.P. Univ.. Shimla. 3. Principal Scientist, DMR Solan.4. PhD research Scholar, Department of Biosciences, H.P. Univ.Shimla.* Corresponding author, E-mail: sanjeev5112@gmail.com +919418045021 AbstractThe mushroom cultivation is a highly lucrative agribusiness to produce nutritious food and earn money in short space and time. The present studies were conducted to analyze morphological and molecular characteristics of five cultivated strains of mushrooms Agaricus bisporus (NBS-5), Calocybe indica (DMRO-302), Flammulina velutipes (DMRO-253), Pleurotus florida (DMRP-136) and Volvariella volvacea (DMRO-484). The fruiting bodies were procured from ICAR-Directorate of Mushroom Research, Solan, HP (India) and screened for morphological and molecular details. Macroscopic features like shape, size and colour of stipe and pileus were recorded. Microscopic details were worked out by cutting free hand sections of material and observed under research microscope and scanning electron microscope. Molecular identification was done through internal transcribed spacer (ITS) regions of ribosomal DNA sequencing. The nucleotide sequences of each sample were analyzed for Basic local Alignment Search Tool (BLAST) search program in US National Centre for Biotechnology Information (NCBI) for identification. The DNA sequences obtained in this study were submitted in the Gen Bank database at NCBI with accession numbers MW718994.1 (A. bisporus), MW714627.1 (C. indica), MW719474.1 (F. velutipes), MW719254.1 (P. florida) and MW720603.1 (V. volvacea).Keywords: Mushroom, Molecular identification, ITS, rRNA, Nucleotide sequencing, Gen Bank database

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis.

In recent years, the “milky disease” caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/μL) and internal transcribed spacer (ITS; 6.74 × 105 copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

Correlations between dominant vegetation type and composition and diversity of soil bacterial communities in a subtropical forest

AbstractSingle and mixed vegetation types have significant effects on soil parameters and the composition and diversity of soil bacterial community. To understand the influence of different vegetation types on the structure of soil bacterial community across soil depth in a subtropical forest, we assessed the relative abundance of edaphic bacterial community and soil parameters, including pH, cation exchange capacity (CEC), and dissolved organic C in Daiyun Mountain Nature Reserve in Fujian Province, southeastern China. The study area constitutes pine coniferous forest (CF) of Pinus taiwanensis Hayata, broad‐leaved forest (BF) of Castanopsis fabri Hance, and a mixed forest comprising CF and BF (MF). Quantitative polymerase chain reaction (PCR) and Illumina sequencing of 16S ribosomal DNA were used to analyze the abundance, diversity, and composition of soil bacteria. The results showed that the pH, CEC, and diversity of tree species are all associated with the composition of bacterial community in the soil. It was found that CEC, soluble organic N, and pH largely affected the structure of soil bacterial community in A horizon, whereas CEC, moisture content, and organic P affected the structure of soil bacterial community in B horizon. We found that the dominant taxa in the CF and BF were Proteobacteria and Acidobacteria, respectively. The results of both mental and random forest analyses displayed groups according to vegetation types, indicating that the bacterial communities in the research site were significantly influenced by vegetation types in subtropical forests. The study highlights the ecological effects of forest management and elucidates the differences in the functional structure of soil bacterial communities under different vegetation types and soil depths.

Lactobacillus Ameliorates SD-Induced Stress Responses and Gut Dysbiosis by Increasing the Absorption of Gut-Derived GABA in Rhesus Monkeys

Sleep deprivation (SD) has become a health problem in the modern society. Although probiotics supplementation has been proven to improve SD-induced gut dysbiosis, the potential neuroendocrine mechanisms remain elusive. In this study, thirty rhesus monkeys (RMs) were recruited. Paradoxical sleep, bright light, and noise were used to build an RM SD model. We examined the plasma γ-aminobutyric acid (GABA), stress hormones, and inflammatory cytokines using ELISAs. 16S ribosomal DNA sequencing and untargeted metabolomics sequencing were employed to detect gut microbial community and metabolites, respectively. The results of our study showed that RMs subjected to SD had elevated plasma stress hormones (such as cortisol and norepinephrine) and proinflammatory cytokines (such as TNF-α, IL-6, and IL-8), and a decreased anti‐inflammatory cytokine IL-10 level. Additionally, SD could give rise to a significant change in gut microbiota and metabolites. The differential gut microbiota and metabolites caused by SD were enriched in the signaling pathways related to GABA metabolism. Pearson correlation analysis revealed that there is a significant correlation between plasma GABA and SD-induced stress responses and gut dysbiosis. The supplementation of GABA-producing probiotics could significantly increase the relative abundance of Lactobacillus and plasma GABA levels, and reverse SD‐induced stress responses and gut dysbiosis. Therefore, we speculated that SD-induced stress response and gut dysbiosis might be an outcome of reduced gut-derived GABA absorption. The supplementation of GABA-producing Lactobacillus might be beneficial for the treatment of SD-induced intestinal dysfunction.

Clostridium species diversity in gut microbiota of patients with renal failure

The Pathology of digestive tract has long been known to be correlated with chronic kidney disease (CKD). The member of the major Firmicutes phylum especially Clostridium subcluster XIVa altered quantitatively and qualitatively in the gut microbiota of patients with End Stage Renal Disease (ESRD) and CKD. Therefore, in this study, the abundance of the species of Clostridium genus of Firmicutes phylum compared between intestine microbiota of patients with kidney failure and healthy individual. Fresh fecal specimens of 20 patients at different stages of CKD and 20 healthy individuals were collected. Bacterial DNA of samples were extracted to use for 16S ribosomal DNA sequencing targeting the V3–V4 region. Next generation sequencing (NGS) method at MiSeq system was used to find the diversity of gut microbiota composition. Totally, 11 (1.68%) of 651 bacterial strains which were isolated from forty fecal samples of both healthy volunteers and CKD/ESRD patients, were identified as Clostridium species. Eight genera of 11 Clostridium genera were related to Clostridium sensu stricto, and 3 other genera were as follows Vallitalea, Acidaminobacter and Caloramator. Among both group, the highest abundance was dedicated to Clostridium celatum genera. Sarcina maxima were not identified. The composition of Clostridium spp. showed the same frequency among CKD/ESRD and healthy groups (p < 0.05). The abundance of Clostridium spp. is virtually the same and not differs among healthy individuals and CKD/ESRD patients. Results of the present indicate despite of critical role of gut microbiota, some pathogens and their metabolites have no role on hemostasis and pathogenesis of kidney disorders.

Intraspecific Genetic Variation of Anisakis typica in Indian Mackerel Caught from the Gulf of Thailand, Samut Songkhram Province.

Anisakis nematodes infecting Indian mackerel (Rastrelliger kanagurta) were initially discovered in Thailand in our preliminary investigation. Nevertheless, the species of Anisakis collected has not been determined nor has its genetic variation been researched. Thus, this study aimed to molecularly identify the species of Anisakis specimens using the internal transcribed spacer (ITS) region of ribosomal DNA sequences. In addition, the intraspecific genetic variation was also determined using mitochondrial cytochrome oxidase subunit II (COII) gene sequences. The phylogenetic relationships of the ITS region classified all samples into Anisakis typica; however, the genetic variation between them could not be distinguished. By contrast, the phylogenetic tree analysis of the COII region identified all samples as A. typica, with 17 different haplotypes by 66 polymorphic sites and five of the substitutions resulted in amino acid change. Additionally, the distribution pattern of the COII region can be separated into two groups between South America and Asian countries. All our haplotypes belong to Asian countries. Compared with the two genetic markers used in this investigation, COII appears to be a better candidate for studying genetic variation sensitive to environmental changes and intermediate or definitive host behavioral changes.

Short-term adaptability to non-hyperthermal stress: Antioxidant, immune and gut microbial responses in the red swamp crayfish, Procambarus clarkii

Thermal stress is a common experience suffered in rice-crayfish integrated systems (RCIS) by the red swamp crayfish, Procambarus clarkii. Based on our previous studies, the adaptability of this crayfish to non-hyperthermal stress over a specified period was explored within a short term by investigating the antioxidant capacity, immune function, HSP70 expression and intestinal microbial changes. Under the non-hyperthermal stress temperature (30 °C) determined by cumulative survivals, the malondialdehyde (MDA) content, total antioxidant capacity (T-AOC), total super oxide dismutase (T-SOD) and catalase (CAT) activities in hepatopancreas significantly increased to the peak values during at 12 h or 24 h (P < 0.05), respectively, and then gradually recovered to the levels of the controls (24 °C) (P > 0.05); the immune-related enzymatic activities (alkaline phosphatase, AKP; acid phosphatase, ACP; lysozyme, LZM) and gene expressions (TNFAIP, TNF and Toll) rapidly increased and reach the peaks at 12 h and 48 h, respectively, and maintained subsequently the higher levels compared with the controls (P < 0.05) except for TNFAIP. The relative expressions of HSP70 in the hepatopancreas, intestine and gill all increased sharply and reached the peak at 6 h (P < 0.05), then decreased fast and roughly returned to the levels of the controls at 96 h (P > 0.05), while, there was still a relatively high expression during the later stages in the gill. High-throughput sequencing of 16S ribosomal DNA revealed the thermal stress altered the diversity and composition of intestinal microbiota. The increased abundance of Proteobacteria and decreased abundance of Bacteroidetes (mainly Bacteroides) and Firmicutes might be indicative of the elevated microbial instability and the unnecessity for high level of nutrient metabolism in the host. The abundance increase of relevant metabolic pathways (e.g., the biosynthesis and degradation of amino acids and lipids) predicted by MetaCyc might be caused by the abundance increase of Proteobacteria. The decreased abundance of two phyla (Actinobacteria and Fusobacteria) and genus Anaerorhabdus could adversely affect the crayfish's intestinal health status. While, the obvious abundance decrease of putatively opportunistic genera (Shewanella and Acinetobacter), combined with the abundance increase of beneficial phylum Tenericutes and genus Rhodobacter, suggested that there concurrently existed some adaptability to the non-thermal stress, which did not resulted in apparent risks of intestinal disorder. This study provides insights into the underling adaptive mechanisms associated with the anti-oxidation, immunity, intestinal microflora and nutrient metabolism of P. clarkii in response to the short-term non-hyperthermal stress.

Exploration of plant growth-promoting endophytic bacteria from Pisum sativum and Cicer arietinum from South-West Haryana.

In the present study, nonrhizobial endophytes were isolated from Pisum sativum and Cicer arietinum from Haryana, India. A total of 355 bacterial endophytes were screened for plant growth promoting traits. Out of all, 96 bacterial endophytes were selected based on morphological characters and multi-PGP traits, and their diversity analyzed by amplified ribosomal DNA restriction analysis. Based on their ARDRA profile, the 25 representative isolates (12 from P. sativum and 13 from C. arietinum), were selected and identified by 16S ribosomal DNA sequencing. Genetic relatedness based on BLAST analysis revealed the similarity of these isolates with members of three prominent phyla, that is, Proteobacteria, Firmicutes, and Actinobacteria. The dominant cluster, Firmicutes, constituted 60% of the isolates, assigned to four different genera, Bacillus, Staphylococcus, Ornithinibacillus, and Lysinibacillus. Phylum α-proteobacteria included two genera, namely Paenochrobactrum and Ochrobactrum and three genera in phylum γ-proteobacteria, namely Pseudomonas, Pantoea and Proteus. The phylum Actinobacteria was constituted of two genera, Microbacterium and Arthrobacter. Bacillus zhangzhouensis, Bacillus safensis, Arthrobacter enclensis from P. sativum and Bacillus haynesii, Paenochrobactrum sp. from C. arietinum are documented as plant growth promoting endophytic bacteria for the first time in the present study. The in vitro and in vivo assessment based on bonitur score revealed that the endophytic isolates Bacillus mojavensis PRN2, Pseudomonas chlororaphis PHN9, B. safensis PRER2, Pseudomonas sp. RCP1, Pseudomonas lini PRN1 and B. haynensii RCP3 from P. sativum and C. arietinum significantly enhanced the plant growth parameters. Therefore, these potential isolates can be further harnessed for preparation of bioformulations to enhance sustainable agriculture.

Spirulina platensis aqueous extracts ameliorate colonic mucosal damage and modulate gut microbiota disorder in mice with ulcerative colitis by inhibiting inflammation and oxidative stress.

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease (IBD) that has become a major gastroenterologic problem during recent decades. Numerous complicating factors are involved in UC development such as oxidative stress, inflammation, and microbiota disorder. These factors exacerbate damage to the intestinal mucosal barrier. Spirulina platensis is a commercial alga with various biological activity that is widely used as a functional ingredient in food and beverage products. However, there have been few studies on the treatment of UC using S. platensis aqueous extracts (SP), and the underlying mechanism of action of SP against UC has not yet been elucidated. Herein, we aimed to investigate the modulatory effect of SP on microbiota disorders in UC mice and clarify the underlying mechanisms by which SP alleviates damage to the intestinal mucosal barrier. Dextran sulfate sodium (DSS) was used to establish a normal human colonic epithelial cell (NCM460) injury model and UC animal model. The mitochondrial membrane potential assay 3-‍‍(4,5-dimethylthiazol-2-yl)-2,‍5-diphenyltetrazolium bromide (MTT) and staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and Hoechst 33258 were carried out to determine the effects of SP on the NCM460 cell injury model. Moreover, hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), western blot, and 16S ribosomal DNA (rDNA) sequencing were used to explore the effects and underlying mechanisms of action of SP on UC in C57BL/6 mice. In vitro studies showed that SP alleviated DSS-induced NCM460 cell injury. SP also significantly reduced the excessive generation of intracellular reactive oxygen species (ROS) and prevented mitochondrial membrane potential reduction after DSS challenge. In vivo studies indicated that SP administration could alleviate the severity of DSS-induced colonic mucosal damage compared with the control group. Inhibition of inflammation and oxidative stress was associated with increases in the activity of antioxidant enzymes and the expression of tight junction proteins (TJs) post-SP treatment. SP improved gut microbiota disorder mainly by increasing antioxidant enzyme activity and the expression of TJs in the colon. Our findings demonstrate that the protective effect of SP against UC is based on its inhibition of pro-inflammatory cytokine overproduction, inhibition of DSS-induced ROS production, and enhanced expression of antioxidant enzymes and TJs in the colonic mucosal barrier.

Resilience or susceptibility to traumatic stress: Potential influence of the microbiome

Exposure to traumatic stress is a major risk factor for development of neuropsychiatric disorders in a sub-population of individuals, while others remain resilient. The mechanisms and contributing factors differentiating between these phenotypes are still unclear. We hypothesize that inter-individual differences in the microbial composition and function contribute to host resilience or susceptibility to stress-induced psychopathologies. The current study aimed to characterize gut microbial community before and after exposure to traumatic stress in an animal model of PTSD. Sprague-Dawley male rats were randomly divided into unstressed controls and experimental group subjected to Single Prolonged Stress (SPS). After 14 days, behavioral analyses were performed using Open Field, Social Interaction and Elevated Plus Maze tests. Based on the anxiety measures, the SPS group was further subdivided into resilient (SPS-R) and susceptible (SPS–S) cohorts. The animals were sacrificed after the last behavioral test and cecum, colon, hippocampus, and medial prefrontal cortex were dissected. Prior to SPS and immediately after Open Field test, fecal samples were collected from each rat for 16S V3–V4 ribosomal DNA sequencing, whereas urine samples were collected before SPS, 90 min into immobilization and on the day of sacrifice to measure epinephrine and norepinephrine levels. Analyses of the fecal microbiota revealed significant differences in microbial communities and in their predictive functionality among the groups before and after SPS stressors. Before SPS, the SPS-S subgroup harbored microbiota with an overall pro-inflammatory phenotype, whereas SPS-R subgroup had microbiota with an overall anti-inflammatory phenotype, with predictive functional pathways enriched in carbohydrate and lipid metabolism and decreased in amino acid metabolism and neurodegenerative diseases. After SPS, the gut microbial communities and their predictive functionality shifted especially in SPS cohorts, with volatility at the genus level correlating inversely with Anxiety Index. In line with the alterations seen in the gut microbiota, the levels of cecal short chain fatty acids were also altered, with SPS-S subgroup having significantly lower levels of acetate, valerate and caproate. The levels of acetate inversely correlated with Anxiety Index. Interestingly, urinary epinephrine and norepinephrine levels were also higher in the SPS-S subgroup at baseline and during stress, indicative of an altered sympathoadrenal stress axis. Finally, shorter colon (marker of intestinal inflammation) and a lower claudin-5 protein expression (marker for increased blood brain barrier permeability) were observed in the SPS-S subgroup. Taken together, our results suggest microbiota is a potential factor in predisposing subjects either to stress susceptibility or resilience. Moreover, SPS triggered significant shifts in the gut microbiota, their metabolites and brain permeability. These findings could lead to new therapeutic directions for PTSD possibly through the controlled manipulation of gut microbiota. It may enable early identification of individuals more likely to develop prolonged anxiogenic symptoms following traumatic stress.

In Vitro Characterization and Identification of Potential Probiotic Yeasts Isolated from Fermented Dairy and Non-Dairy Food Products.

This study is about the isolation of yeast from fermented dairy and non-dairy products as well as the characterization of their survival in in vitro digestion conditions and tolerance to bile salts. Promising strains were selected to further investigate their probiotic properties, including cell surface properties (autoaggregation, hydrophobicity and coaggregation), physiological properties (adhesion to the HT-29 cell line and cholesterol lowering), antimicrobial activities, bile salt hydrolysis, exopolysaccharide (EPS) producing capability, heat resistance and resistance to six antibiotics. The selected yeast isolates demonstrated remarkable survivability in an acidic environment. The reduction caused by in vitro digestion conditions ranged from 0.7 to 2.1 Log10. Bile salt tolerance increased with the extension in the incubation period, which ranged from 69.2% to 91.1% after 24 h. The ability of the 12 selected isolates to remove cholesterol varied from 41.6% to 96.5%, and all yeast strains exhibited a capability to hydrolyse screened bile salts. All the selected isolates exhibited heat resistance, hydrophobicity, strong coaggregation, autoaggregation after 24 h, robust antimicrobial activity and EPS production. The ability to adhere to the HT-29 cell line was within an average of 6.3 Log10 CFU/mL after 2 h. Based on ITS/5.8S ribosomal DNA sequencing, 12 yeast isolates were identified as 1 strain for each Candida albicans and Saccharomyces cerevisiae and 10 strains for Pichia kudriavzevii.

Taxonomy and Systematics of Two New Species of Myxobolus (Cnidaria: Myxobolidae) Parasitizing the Gills of Mugil curema (Mugilidae) from the Brazilian Coast

We describe two new species of Myxobolus (Myxobolidae) found parasitizing Mugil curema from two locations in Brazil: Myxobolus curemae n. sp. from gill arch and rays and Myxobolus maceioensis n. sp. from gill lamellae. We based the descriptions on myxospore morphology, histology, and small-subunit ribosomal DNA sequences. Myxospores of the two new species had similar morphology and morphometry but differed in spore body width and length, and ssrDNA sequences differed by 10.5%. These data supported the diagnosis of the parasites as distinct and novel species. The phylogenetic analysis showed a subclade formed by species that parasitize Mugiliformes, with M. maceioensis n. sp. as a sister species of Myxobolus episquamalis and Myxobolus bizerti, while there is a group of six species that are sister related to M. curemae n. sp. Our analysis was consistent with previous studies suggesting that orders of the hosts are strongly correlated with phylogenetic signals in the Myxobolidae. Myxobolus curemae n. sp. and M. maceioensis n. sp. are new species identified parasitizing M. curema.

Exploring evolutionary trends within the Pennellidae (Copepoda: Siphonostomatoida) using molecular data.

The family Pennellidae comprises ecto- and mesoparasitic copepods on marine fishes. Although a preliminary scheme of phylogenetic relationships of pennellids based on morphological characters exists, it is difficult to objectively define character states because of their highly modified bodies and reduced appendages. This molecule-based study analysed phylogenetic relationships among seven genera and 12 species of pennellids, using 18S and 28S ribosomal DNA sequences in order to infer evolutionary trends within the family. Our molecular analysis recovered three clades (Clade-I, Peniculus; Clade-II, Haemobaphes-Lernaeocera-Phrixocephalus-Exopenna-Lernaeenicus radiatus; and Clade-III, Pennella-Lernaeenicus spp.). This result was congruent with some of the morphology-based phylogenetic relationships previously proposed but did not support a sister group comprising Exopenna, Phrixocephalus and Pennella. The second and third offshoots after the divergence of Clade-I species are characterized by reduced body tagmosis and changes in lifestyle from ectoparasites to mesoparasites. In some gill parasites of Clade-II, their sigmoid-shaped bodies and coiled egg strings have likely evolved in adaptation to the limited available space within the gill cavities of the hosts. Phrixocephalus is an eye parasite in Clade-II, which also has coiled egg strings, may have descended from an ancestral gill parasite. All species of Clade-III are characterized by the possession of a head region with processes deeply embedded into the host tissues and functioning as an anchor.

Efficacy and Mechanism of Thymol/KGM/LG Edible Coating Solution on Inhibition of Mucor circinelloides Isolated From Okra.

With the increasing demand and quality requirement for the natural nutritious food in modern society, okra has attracted much attention because of its high nutritional value and remarkable functionality. However, the occurrence of postharvest diseases of fresh okra severely limited the application and the value of okra. Therefore, in this study, the dominant pathogens causing postharvest diseases such as soft rot were isolated from naturally decaying okra. It was identified as Mucor circinelloides by its morphological characteristics and standard internal transcribed spacer ribosomal DNA sequence. Furthermore, the biological characteristics of M. circinelloides were studied, and the inhibitory effect of thymol/KGM/LG (TKL) edible coating solution on M. circinelloides and its possible mechanism was discussed. In addition, TKL edible coating solution had a dose-dependent inhibitory effect on M. circinelloides, with a 50% inhibitory concentration (EC50) of 113.55 mg/L. The TKL edible coating solution at 960 mg/L of thymol completely inhibited mycelial growth and spore germination of M. circinelloides. The results showed that the best carbon source of M. circinelloides was maltose, the best nitrogen source was beef extract and potassium nitrate, the best pH was 6, the best temperature was 28°C, the best NaCl concentration was 0.5%, and the light was conducive to the growth of M. circinelloides. It was also observed by scanning electron microscope (SEM) that TKL was more likely to destroy the cell wall integrity of M. circinelloides, inhibit spore morphology and change mycelium structure. Meanwhile, the activity of chitinase (CHI), an enzyme related to cell wall synthesis of M. circinelloides, was significantly decreased after being treated by TKL with thymol at 100 mg/L (TKL100). The content of Malondialdehyde (MDA) in M. circinelloides decreased significantly from 12 h to 48 h, which may cause oxidative damage to the cell membrane. The activity polygalacturonase (PG), pectin methylgalacturonase (PMG), and cellulase (Cx) of M. circinelloides decreased significantly. Therefore, the results showed that TKL had a good bacteriostatic effect on okra soft rot pathogen, and the main bacteriostatic mechanism might be the damage of cell membrane, degradation of the cell wall, inhibition of metabolic activities, and reduction of metabolites, which is helpful to further understand the inhibitory effect of TKL on okra soft rot pathogen and its mechanism.

Nucleospora hippocampi n. sp., an Intranuclear Microsporidian Infecting the Seahorse Hippocampus erectus From China

The life cycle, ultrastructure, and molecular phylogeny of a new intranuclear microsporidian, Nucleospora hippocampi n. sp., infecting the intestine of the Hippocampus erectus, were described. The histopathology revealed an extensive infection, mainly in the columnar epithelium of the intestinal mucosa layer. The enterocytes were the important target cell for Nucleospora hippocampi n. sp. infection. Transmission electron microscopy results showed that this microsporidian developed directly within the host cell nucleoplasm. In the intranuclear life cycle, the transformation from meront to sporogonial plasmodium was recognized by forming electron-dense disc structures, which were considered the polar tube precursors. The microsporidian showed the typical morphological characteristics of the family Enterocytozoonidae in the formation and development of spore organelles prior to the division of the sporogonial plasmodium. According to wet smear observation, eight spores were generally formed in a single host nucleus. Mature spores were elongated ovoids that were slightly bent and measured 1.93 × 0.97 μm. The isofilar polar tube was arranged in 7~8 coils in one row. Phylogenetic analysis of its small subunit ribosomal DNA sequences demonstrated that the parasite belonged to the Nucleospora group clade. The histological, ultrastructural, and molecular data support the emergence of a new species in the genus Nucleospora. This is the first report of Nucleospora species in Asia and threatened syngnathid fishes.

Threat of cercarial dermatitis in Hungary: A first report of Trichobilharzia franki from the mallard (Anas platyrhynchos) and European ear snail (Radix auricularia) using molecular methods.

Cercarial dermatitis in humans is a re-emerging zoonotic disease caused by infectious larvae of avian blood flukes within the Schistosomatidae family. Upon water contact, these avian schistosome larvae directly penetrate human skin and cause irritation. Between September 2018 and September 2020, carcasses of 94 mallards (Anas platyrhynchos), two green-winged teals (Anas crecca) along with one ferruginous duck (Aythya nyroca) were examined. Birds were collected within 12 regions of Hungary, representative of 9 different counties. Inspecting both morphological characters and molecular data, the cytochrome oxidase subunit I (COI) and large subunit ribosomal DNA (LSU rDNA) sequences, Bilharziella polonica and Dendritobilharzia pulverulenta were each identified. Most importantly, Trichobilharzia franki was found for the first time in Hungary and in 5 dispersed counties in conjunction with of 3 counties where other avian schistosomes were found. Of note, these avian blood flukes were predominately encountered in wild mallards (∼50%) and not in birds reared for hunting (∼1%). In total, 245 European ear snails (Radix auricularia), a known intermediate host of Trichobilharzia spp., were collected from an urban pond in Eger, Hungary. Five snails (∼2%) consistently shed numerous furcocercariae of T. franki that were confirmed by molecular methods. Our findings help to pinpoint a contemporary life cycle of this avian schistosome within an urban environment, a location regularly visited by various wild waterfowl and the possible species of avian schistosomes responsible for cercarial dermatitis in Hungary. Taken as a whole, we demonstrate the actual and potential risk zone for cercarial dermatitis, particularly with reference to R. auricularia distributions, within Hungary and across Central Europe.

Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia

Fargesia, the largest genus within the temperate bamboo tribe Arundinarieae, has more than 90 species mainly distributed in the mountains of Southwest China. The Fargesia bamboos are important components of the subalpine forest ecosystems that provide food and habitat for many endangered animals, including the giant panda. However, species-level identification of Fargesia is difficult. Moreover, the rapid radiation and slow molecular evolutionary rate of Fargesia pose a significant challenge to using DNA barcoding with standard plant barcodes (rbcL, matK, and ITS) in bamboos. With progress in the sequencing technologies, complete plastid genomes (plastomes) and nuclear ribosomal DNA (nrDNA) sequences have been proposed as organelle barcodes for species identification; however, these have not been tested in bamboos. We collected 196 individuals representing 62 species of Fargesia to comprehensively evaluate the discriminatory power of plastomes and nrDNA sequences compared to standard barcodes. Our analysis indicates that complete plastomes have substantially higher discriminatory power (28.6%) than standard barcodes (5.7%), whereas nrDNA sequences show a moderate improvement (65.4%) compared to ITS (47.2%). We also found that nuclear markers performed better than plastid markers, and ITS alone had higher discriminatory power than complete plastomes. The study also demonstrated that plastomes and nrDNA sequences can contribute to intrageneric phylogenetic resolution in Fargesia. However, neither of these sequences were able to discriminate all the sampled species, and therefore, more nuclear markers need to be identified.