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Zooplankton monitoring is important for understanding their population dynamics and life history, ecosystem health, and environmental changes. Compared with traditional morphological identification, environmental DNA (eDNA) analysis allows for more sensitive and efficient monitoring of zooplankton diversity. Previous eDNA studies have primarily used metabarcoding approaches to reveal their richness and composition, whereas its performance in predicting zooplankton abundance remains understudied. We conducted water and bulk sampling in Lake Biwa, Japan, showing that the number of sequence reads by metabarcoding moderately correlated with eDNA concentrations estimated by quantitative real-time PCR (qPCR). In addition, the eDNA read number was significantly related to cladoceran and copepod abundance estimated by microscopy sorting, although there remained too much uncertainty in the read-abundance relationship. Moreover, there was a significant difference in species composition between eDNA metabarcoding and sorting. Although our results indicated the potential applicability of eDNA metabarcoding for quantifying multiple zooplankton abundance, several methodological validations in eDNA metabarcoding would also be required to optimize its performance in the future. [ABSTRACT FROM AUTHOR]

Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood. [ABSTRACT FROM AUTHOR]

The effectiveness of selenium nanoparticle (SeNP)-enriched Lactiplantibacillus plantarum and Lactobacillus acidophilus was studied against Shiga toxin-producing Escherichia coli O157:H7 infection on the intestinal fragments and kidney tissue of BALB/c mice. Gut microbiota-targeted bacteria and E. coli O157:H7 counts were obtained by qPCR and PCR. Histology of ileum, colon, and kidney tissues and Stx secretions were analyzed until one-week post-infection. Mice fed with SeNP Lpb. plantarum in the preinfection feeding groups have lower E. coli O157:H7 counts and lower intestinal damage than those in the infected group. The lowest mean fecal probiotic counts were in the L. acidophilus group (7.61 log 10). In pretreatment groups of SeNP L. acidophilus and L. acidophilus, the mean counts of bacteria decreased to 104 CFU/g by day 7. The lowest Stx copy number was demonstrated in SeNP Lpb. plantarum feeding groups' day 7 (P < 0.05). Feeding groups with SeNP Lpb. plantarum had significantly higher members of Lactobacilli in their fecal microbiota than the control group on day 7. It was clarified that Se-enriched Lpb. plantarum and L. acidophilus can be useful as a method of preventing STEC infections. The viability of STEC infection exposure to selenium-enriched Lactobacillus spp. was decreased more than for non-Se-enriched Lactobacillus spp. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) analysis can facilitate surveillance of invasive aquatic plant's distribution. However, it is still questionable how accurately eDNA concentration reflects and predicts aquatic plant abundance. Although previous studies investigated relationships between aquatic plant eDNA concentration and their abundance in freshwater environments, the conclusions were not consistent between the studies. Here, a systematic literature search was conducted to summarize previous findings and meta-analyze the relationships between eDNA concentration and aquatic plant abundance. Hierarchical random-effect meta-analyses showed significant positive, but weak, relationships between them (r = 0.45 in average), meaning that eDNA concentration accounted for approximately 20% of the observed variation in plant abundance. Additionally, the relationships tended to be rather stronger in the field than in the laboratory environments, which may be attributed to limited quantifiability and species specificity of plant coverage as abundance metrics, heterogeneous dispersion of plant eDNA particles, and phenology-dependent eDNA production from plant bodies. The finding indicates that aquatic plant eDNA particles could reflect more regional (but not local) abundance, and thus, eDNA-based abundance estimation might be more appropriate at a broader scale (i.e., lower spatial resolution). Further studies focusing on various environmental parameters and plant ecotypes are needed to clarify the relationship. [ABSTRACT FROM AUTHOR]

Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (β) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future. [ABSTRACT FROM AUTHOR]

Wheat blast is an important disease that affects cross-border food security. The causal pathogen, Pyricularia oryzae (syn. Magnaporthe oryzae) Triticum pathotype (MoT), forms a phylogenetic group distinct from the other P. oryzae pathotypes pathogenic on various gramineous plants. Here we developed MoT-specific primer sets for qPCR and LAMP detection methods, and their amplification specificity and efficiency were tested in comparison with previous reported MoT-specific primers. The amplification efficiency and specificity were affected by the polymerase premix. To evaluate the detection specificity, purified DNA samples of 15 MoT isolates, 89 non-MoT isolates of Pyricularia spp., and crude DNA from wheat plants inoculated with MoT were used. We conclude that the Sp3 primer sets for qPCR and the MoT3L primer sets for LAMP were the best in this research. [ABSTRACT FROM AUTHOR]

Aims: Arbuscular mycorrhizal (AM) fungi establish root symbioses that can improve acquisition of nutrients such as nitrogen (N) from soils, leading to improvements in host plant growth. Previously, we showed that applying AM fungal necromass to the rhizosphere could stimulate plant growth. Here we investigated the interactions between AM fungi (living or dead) and ammonia oxidizers (AO), a key group of soil prokaryotes involved in soil N cycling, to understand the effects of these interactions on plant N levels. We were interested in whether the necromass contained any significant biological nitrification inhibitory (BNI) activity that could account for the previously suggested antagonism between AM fungi and AO. Methods: We set up a compartmented pot experiment with Andropogon gerardii as a host plant, which included rhizosphere (amended with living and/or dead AM fungal biomass) and a root-free zone. The latter contained a mesh bag with 15N-labelled plant litter. Results: Inoculation with living AM fungus enhanced plant biomass and nutrient acquisition, including increased transfer of 15N from the mesh bags to the plants. Additionally, AO bacteria but not archaea were significantly suppressed in the mesh bags. In contrast, AM fungal necromass had minimal effect on plant biomass and nutrient acquisition. Surprisingly, it stimulated the growth of both AO bacteria and archaea in the rhizosphere of non-mycorrhizal plants. Conclusions: Based on the above, we found no support for AM fungi suppressing the AO through an elusive BNI production. Rather, it seems that the main mechanism of AM fungal-AO interaction is substrate competition. [ABSTRACT FROM AUTHOR]

The pond smelt Hypomesus nipponensis and Japanese icefish Salangichthys microdon are economically important fishery resources in Japan. The catching of these fish species has decreased owing to environmental pollution and habitat loss caused by human impacts. We developed species-specific primer–probe sets to detect and quantify environmental DNA (eDNA) in these fish species. The primer–probe sets were specific to each target species with the detection limits of two (H. nipponensis) and one (S. microdon) copies per reaction, sufficient for eDNA analysis in the field. Using these assays, the eDNA was detected with a high probability (100% and 86% for H. nipponensis and S. microdon, respectively) at the sites where each fish species was captured by a cast net survey in the Hirata–Funagawa River (one of the inflowing rivers of Lake Shinji, Shimane Prefecture, Japan), where it was frequently detected without cast net capture. The eDNA was detected in S. microdon a little earlier than in H. nipponensis, and the eDNA concentrations of both species gradually decreased after peaking in July and reached negligible levels by October. The utility of the assays for estimating the seasonal appearances/disappearances of both species in this river was demonstrated. The species-specific primer–probe sets of the eDNA-based method represent a potentially useful monitoring tool that requires minimal labor and time and could be useful for managing H. nipponensis and S. microdon fisheries in the future. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) analysis is a promising tool for the sensitive and effective monitoring of species distribution and abundance. Traditional eDNA analysis has targeted mitochondrial DNA (mtDNA) fragments due to their abundance in cells; however, the quantification may vary depending on cell type and physiology. Conversely, some recent eDNA studies have targeted multi-copy nuclear DNA (nuDNA) fragments, such as ribosomal RNA genes, in water, and reported a higher detectability and more rapid degradation than mitochondrial eDNA (mt-eDNA). These properties suggest that nuclear eDNA (nu-eDNA) may be useful for the accurate estimation of species abundance relative to mt-eDNA, but which remains unclear. In this study, we compiled previous studies and re-analyzed the relationships between mt- and nu-eDNA concentration and species abundance by comparing the R2 values of the linear regression. We then performed an aquarium experiment using zebrafish (Danio rerio) to compare the relationships across genetic regions, including single-copy nuDNA. We found more accurate relationships between multi-copy nu-eDNA and species abundance than mt-eDNA in these datasets, although the difference was not significant upon weighted-averaging the R2 values. Moreover, we compared the decay rate constants of zebrafish eDNA across genetic regions and found that multi-copy nu-eDNA degraded faster than mt-eDNA under pH 7, implying a quick turnover of multi-copy nu-eDNA in the field. Although further empirical studies of nu-eDNA applications are necessary to support our findings, this study provides the groundwork for improving the estimation accuracy of species abundance via eDNA analysis. [ABSTRACT FROM AUTHOR]

To explore the relationship between NF2 promoter gene mutation and the risk of medulloblastomas (MBs). We collected tissues from 16 MB patients and 7 age-matched non-MB controls. Gene sequencing, qPCR (real-time quantitative polymerase chain reaction), IHC (immunohistochemistry), and WB (Western blot) were used to analyze the changes in the NF2 gene sequence and expression between patients and controls. We found that NF2 promoter gene mutations occurred in MB patients. The NF2 mRNA expression was higher in the controls than in patients (p = 0.03 < 0.05); however, the results of IHC and WB demonstrated that the NF2 protein expression was significantly higher in patients than in the controls (IHC: p = 0.0001; WB: p = 0.01). There was no significant difference in the CRL4 mRNA and protein levels. In addition, NF2 protein was mainly expressed in the nucleus in MB patients, while the NF2 protein was mainly expressed in the cytoplasm in the controls. NF2 promoter mutations exist in MB patients. NF2 mRNA expression was higher in controls than patients; whereas NF2 protein level was higher in patients than in controls. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal (AM) fungi can significantly contribute to plant nitrogen (N) uptake from complex organic sources, most likely in concert with activity of soil saprotrophs and other microbes releasing and transforming the N bound in organic forms. Here, we tested whether AM fungus (Rhizophagus irregularis) extraradical hyphal networks showed any preferences towards certain forms of organic N (chitin of fungal or crustacean origin, DNA, clover biomass, or albumin) administered in spatially discrete patches, and how the presence of AM fungal hyphae affected other microbes. By direct 15N labeling, we also quantified the flux of N to the plants (Andropogon gerardii) through the AM fungal hyphae from fungal chitin and from clover biomass. The AM fungal hyphae colonized patches supplemented with organic N sources significantly more than those receiving only mineral nutrients, organic carbon in form of cellulose, or nothing. Mycorrhizal plants grew 6.4-fold larger and accumulated, on average, 20.3-fold more 15N originating from the labeled organic sources than their nonmycorrhizal counterparts. Whereas the abundance of microbes (bacteria, fungi, or Acanthamoeba sp.) in the different patches was primarily driven by patch quality, we noted a consistent suppression of the microbial abundances by the presence of AM fungal hyphae. This suppression was particularly strong for ammonia oxidizing bacteria. Our results indicate that AM fungi successfully competed with the other microbes for free ammonium ions and suggest an important role for the notoriously understudied soil protists to play in recycling organic N from soil to plants via AM fungal hyphae. [ABSTRACT FROM AUTHOR]

The value of chromatin diminution (CD) in different species of freshwater cyclopoid copepods can differ significantly. The biological and evolutionary roles of these differences remain unclear. To expand the knowledge on CD distribution and magnitude in this group of copepods, a quick method for its evaluation was required. This study proposes a simple approach for CD assessment in copepods using quantitative realtime PCR (qPCR). The magnitude of changes in the genome size was assessed by comparing fluorescence curves of qPCR fragments of target genes for pre- and post-diminution materials. The method was tested on four cyclopoid copepods species. In Cyclops kolensis, CD was assessed as 95.3 ± 1.2; in Acanthocyclops vernalis it was assessed at 94.6 ± 0.8%; at C. insignis, it was 82.3 ± 5.2%; and for the first time, CD was found in Megacyclops viridis at 91.1 ± 2.6%. The advantages of our approach are its rapidity, simplicity and minimal requirements of materials studied. [ABSTRACT FROM AUTHOR]

Eucommia ulmoides is a focus of considerable attention because it is used to produce gutta-percha latex and raw materials for traditional Chinese medicine. The species has also been widely planted for vegetable oil production due to the high content of alpha-linolenic acid (ALA) in the seeds. However, little was known about fatty acid biosynthesis in the seeds. Here, we present transcriptome analyses that identified genes related to the fatty acid biosynthesis pathways in the seeds collected 70 and 160 days after flowering (DAF) from two varieties: 'Huazhong No. 6' (ALA content of 38.7 %) and 'Huazhong No. 10' (ALA content of 67.6 %). De novo assembly generated 96,469 unigenes with an average length of 690 bp. Among them, 11,260 unigenes identified by functional annotation against the Kyoto Encyclopedia of Genes and Genomes database were mapped to 117 metabolic pathways. 65 unigenes involved in fatty acid biosynthesis include 5 coding for 3-oxoacyl-ACP reductase, 2 coding for β-hydroxyacyl-ACP dehydratase, 10 coding for β-ketoacyl-ACP synthase II, and 2 coding for enoyl-ACP reductase I. Expression of the nine differentially expressed unigenes coding for major enzymes in fatty acid biosynthesis was validated by real-time fluorescent quantitative PCR (qPCR) using RNA from six seed stages (70, 88, 106, 124, 142, and 160 DAF) in 'Huazhong No. 10'. The qPCR analysis showed that expression of these 9 unigenes all changed significantly at 88 DAF. This study provides a large-scale transcriptome annotation of E. ulmoides, which is fundamental information for further characterizing gene functionals involved in the process of seed development. [ABSTRACT FROM AUTHOR]

Monitoring populations of arbuscular mycorrhizal fungi (AMF) in roots is a pre-requisite for improving our understanding of AMF ecology and functioning of the symbiosis in natural conditions. Among other approaches, quantification of fungal DNA in plant tissues by quantitative real-time PCR is one of the advanced techniques with a great potential to process large numbers of samples and to deliver truly quantitative information. Its application potential would greatly increase if the samples could be preserved by drying, but little is currently known about the feasibility and reliability of fungal DNA quantification from dry plant material. We addressed this question by comparing quantification results based on dry root material to those obtained from deep-frozen roots of Medicago truncatula colonized with Rhizophagus sp. The fungal DNA was well conserved in the dry root samples with overall fungal DNA levels in the extracts comparable with those determined in extracts of frozen roots. There was, however, no correlation between the quantitative data sets obtained from the two types of material, and data from dry roots were more variable. Based on these results, we recommend dry material for qualitative screenings but advocate using frozen root materials if precise quantification of fungal DNA is required. [ABSTRACT FROM AUTHOR]

Quantitative real-time PCR (qPCR), as an important quantitative technique for nucleic acids, has been widely used in many fields including clinical diagnosis, molecular biology, and cancer research. However, non-specific amplification products are still a frequent problem in qPCR. In this study, we investigated the effects of QDs on real-time amplification based on either SYBR Green I or EvaGreen. It was found that QDs could raise the amplification sensitivity and thus enhance the efficiency using SYBR Green I detection system. In the case of EvaGreen detection systems, addition of QDs also led to a better correlation coefficient than without QDs. EvaGreen-based system gave sharper peaks for melting curves than SYBR Green I. The experiments indicated that the polymerase activity could be partially blocked by QDs at the pre-PCR temperatures, resulting in the improvement of PCR specificity. These results indicated that CdTe QDs could be used as a descent qPCR enhancer. Good amplification fidelity in QDs-facilitated qPCR was also a plus that has not been reported elsewhere. [ABSTRACT FROM AUTHOR]

Hantaan viruses cause two severe diseases lacking efficient treatment, yet no effective prophylactic vaccines are available. Continued exploration of alternative antiviral agents to treat hantavirus-related syndromes remains compulsory. The fluorescence-based quantitative real-time PCR (qPCR) has become the touchstone for target gene quantification. In the present study, standard curves for Hantaan virus (HTNV), mouse, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated by serial 10-fold dilutions of the constructed recombinant plasmid pGEM-T/HTNV, pGEM-T/mouse-GAPDH, and pGEM-T/human-GAPDH, respectively. Comparisons between the indirect immunofluorescence assay and qPCR assay in the detection of HTNV-infected Vero E6 cells showed improved detection limit and sensitivity of latter method. To characterize the inhibitory effect of several conventional antivirals (arbidol and ribavirin) and unconventional antivirals (indomethacin and curcumin) on HTNV, the levels of viral RNAs were measured for 4 days post-treatment of HTNV-infected Vero E6 cells and 18 days post-inoculation of HTNV-infected suckling mice. Our results validated that HTNV was sensitive to ribavirin and arbidol treatment, while indomethacin and curcumin may also be therapeutically effective in treating HTNV infection. As a result, the establishment and application of qPCR may be a useful tool for the evaluation of potential antivirals for Hantaan virus infection in vitro and in vivo. [ABSTRACT FROM AUTHOR]