Japonicus Samples Research Articles

Diversity and Isolation of Endophytic Fungi in Panax japonicus and Biotransformation Activity on Saponins.

This study reports the diversity and community structure differences of the endophytic fungi of Panax japonicus of different ages to obtain novel endophytic fungi with glycoside hydrolytic activity for rare saponins production. This study used the high-throughput sequencing method to analyze the diversity and community structure of endophytic fungi of P. japonicus. The endophytic fungi were processed by traditional isolation, culture, conservation, and ITS rDNA sequence analyses. Then the total saponins of P. japonicus were used as the substrate to evaluate the glycoside hydrolytic activity. The composition analysis of the community structure showed that the abundance, evenness, and diversity of endophytic fungi of nine-year-old P. japonicus were the best among all samples. A total of 210 endophytic fungi were isolated from P. japonicus samples and further annotated by sequencing the internal transcribed spacer. Then the biotransformation activity of obtained strains was further examined on total saponins of P. japonicus (TSPJ), with a strain identified as Fusarium equiseti (No.30) from 7-year-old P. japonicus showing significant glycoside hydrolytic activity on TSPJ, including ginsenoside Ro→zinglbroside R1, pseudoginsenoside RT1→pseudoginsenoside RP1, chikusetsusaponin IV→tarasaponin VI and chikusetsusaponin IVa →calenduloside E. These results reveal the diversity and community structure differences of the endophytic fungi of P. japonicus with different ages and establish a resource library of endophytic fungi of P. japonicus. More importantly, we identified a valuable endophytic fungus with glycoside hydrolytic activity and provided a promising convenient microbial transformation approach to produce minor deglycosylated ginsenosides.

Protected Geographical Indication Discrimination of Zhejiang and Non-Zhejiang Ophiopogonis japonicus by Near-Infrared (NIR) Spectroscopy Combined with Chemometrics: The Influence of Different Stoichiometric and Spectrogram Pretreatment Methods.

This paper presents a method for the protected geographical indication discrimination of Ophiopogon japonicus from Zhejiang and elsewhere using near-infrared (NIR) spectroscopy combined with chemometrics. A total of 3657 Ophiopogon japonicus samples from five major production areas in China were analyzed by NIR spectroscopy, and divided into 2127 from Zhejiang and 1530 from other areas ('non-Zhejiang'). Principal component analysis (PCA) was selected to screen outliers and eliminate them. Monte Carlo cross validation (MCCV) was introduced to divide the training set and test set according to a ratio of 3:7. The raw spectra were preprocessed by nine single and partial combination methods such as the standard normal variable (SNV) and derivative, and then modeled by partial least squares regression (PLSR), a support vector machine (SVM), and soft independent modeling of class analogies (SIMCA). The effects of different pretreatment and chemometrics methods on the model are discussed. The results showed that the three pattern recognition methods were effective in geographical origin tracing, and selecting the appropriate preprocessing method could improve the traceability accuracy. The accuracy of PLSR after the standard normal variable was better, with R2 reaching 0.9979, while that of the second derivative was the lowest with an R2 of 0.9656. After the SNV pretreatment, the accuracy of the training set and test set of SVM reached the highest values, which were 99.73% and 98.40%, respectively. The accuracy of SIMCA pretreated with SNV and MSC was the highest for the origin traceability of Ophiopogon japonicus, which could reach 100%. The distance between the two classification models of SIMCA-SNV and SIMCA-MSC is greater than 3, indicating that the SIMCA model has good performance.

Susceptibility of kuruma shrimp to the infection with Decapod iridescent virus 1

Infection with Decapod iridescent virus 1 (iDIV1), an important emerging disease of shrimps and crabs, has been included in the Quarterly Aquatic Animal Disease Report (QAAD) by the Network of Aquaculture Centres in Asia-Pacific (NACA) and listed by the World Organization for Animal Health (WOAH). China has classified iDIV1 as a Class II animal pandemic disease. In the present study, to determine the susceptibility of Penaeus japonicus to Decapod iridescent virus 1 (DIV1), healthy kuruma shrimp were artificially infected with DIV1 (isolate SHIV 20141215) by per os (the pathway that mimics natural transmission) and intramuscular injection (invasive pathway). The infected P. japonicus showed clinical signs such as anorexia, retardation, evident reddish body, swollen and whitish lymphoid organs, and mortalities of almost 100%. Real-time PCR showed that all the challenged individuals by per os or intramuscular routes were DIV1-positive with an average virus load between 10(9.09 ± 0.58) and 10(8.94 ± 0.45) copies/μg-DNA, respectively. Histological examination revealed karyopyknosis, and eosinophilic inclusions and minute basophilic stains were combined in lymphoid organs, hematopoietic tissue and gills of diseased individuals. In addition, lymphoid organs showed disorganization of the tubule matrix. In situ DIG-labeling loop-mediated isothermal amplification (ISDL) also demonstrated the presence of DIV1 signals existed in lymphoid organs, hemopoietic tissue, gills, epithelial tissue, hepatopancreas and muscle. Ultrathin sections examined using transmission electron microscopy (TEM) revealed the presence of DIV1 virions, the virogenic stroma, and the nucleocapsid production process in infected cells. In addition, pathogen surveillance of cultured samples showed that the DIV1 detection rate of farmed P. japonicus samples from five coastal provinces in China was 5.3% (9/157) in 2022. The results mentioned above support that P. japonicus is a newly confirmed susceptible host for DIV1, enhancing the pathogen ecological understanding of pathogens and giving more support for developing DIV1 preventive and control strategies.

Selection and validation of reference genes for RT-qPCR-based analyses of Anastatus japonicus Ashmead (Hymenoptera: Helicopteridae).

RT-qPCR remains a vital approach for molecular biology studies aimed at quantifying gene expression in a range of physiological or pathological settings. However, the use of appropriate reference genes is essential to attain meaningful RT-qPCR results. Anastatus japonicus Ashmead (Hymenoptera: Helicopteridae) is an important egg parasitoid wasp and natural enemy of fruit bugs and forest caterpillars. While recent transcriptomic studies have analyzed gene expression profiles in A. japonicus specimens, offering a robust foundation for functional research focused on this parasitoid, no validated A. japonicus reference genes have yet been established, hampering further research efforts. Accordingly, this study sought to address this issue by screening for the most stable internal reference genes in A. japonicus samples to permit reliable RT-qPCR analyses. The utility of eight candidate reference genes (ACTIN, TATA, GAPDH, TUB, RPL13, RPS6, EF1α, RPS3a) was assessed under four different conditions by comparing developmental stages (larvae, pupae, adults), tissues (abdomen, chest, head), sex (male or female adults), or diapause states (diapause induction for 25, 35, 45, or 55days, or diapause termination). RefFinder was used to calculate gene stability based on the integration of four algorithms (BestKeeper, Normfinder, geNorm, and ΔCt method) to determine the optimal RT-qPCR reference gene. Based on this approach, RPS6 and RPL13 were found to be the most reliable reference genes when assessing different stages of development, while ACTIN and EF1α were optimal when comparing adults of different sexes, RPL13 and EF1α were optimal when analyzing different tissues, and TATA and ACTIN were optimal for different diapause states. These results provide a valuable foundation for future RT-qPCR analyses of A. japonicus gene expression and function under a range of experimental conditions.

The shelf life extension of refrigerated Nemipterus japonicus fillets by chitosan coating incorporated with propolis extract

In this study, chitosan coatings incorporated with propolis extract (PE) were utilized to extend the shelf-life of refrigerated Nemipterus japonicus fillets. Microbiological (total mesophilic bacteria (TMB) and total psychrotrophic count (TPC)), physicochemical (pH, total volatile bases nitrogen (TVB-N), 2-thiobarbituric acid reactive substances (TBARS), free fatty acid (FFA)) and sensory (color, odor and preference) characteristics of coated fillets were evaluated during 12days. The results showed that incorporating PE into chitosan solution could significantly (P<0.05) improve the quality of fish fillets. The results showed that the chitosan+PE samples reduced the extent of lipid oxidation, as judged by TBARS and FFA, suggesting that there is a synergistic effect between chitosan coating and propolis. Moreover, the application of chitosan coatings with PE improved the TVB-N and pH values of the Nemipterus japonicus samples significantly compared to untreated sample, thus extending the shelf life of Nemipterus japonicus fillet approximately >10days. All wrapped fillets in treatment samples incorporated with PE had lower bacterial counts compared to control (P<0.05). However, the bacterial counts of chitosan coating combined with PE were lower than those treated by chitosan or PE alone during the storage. The fillets treated with chitosan and PE had the best sensory properties. The increase (>10days) in the shelf-life was attributed to the antioxidant and antimicrobial characteristics of chitosan and PE. These results confirmed that chitosan coating enriched with PE can be an improving method to reducing deterioration of refrigerated Nemipterus japonicus fillets.

Discrimination of dried sea cucumber (Apostichopus japonicus) products from different geographical origins by sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS)-based proteomic analysis and chemometrics

The article reports on discrimination of dried sea cucumber Apostichopus japonicus (A. japonicus) products from different geographical regions in China (Xiapu, Jiaonan, Weihai, Yantai, Dalian). For this purpose, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS)-based proteomic analysis combined with chemometrics was evaluated. Proteome variations of A. japonicus samples determined by SWATH-MS using ultra-high performance liquid chromatography coupled to quadrupole time of flight mass spectrometer were mined by chemometric tools to clarify the correlation between protein quantitation data and geographical origin. A differentiating OPLS-DA model was constructed with 55 SWATH-MS quantified proteins. A. japonicus samples from five locations were clearly classified by the developed OPLS-DA model and 100% prediction accuracy was achieved in validation testing as well as real-life sample analysis. Seventeen proteins were identified as discriminatory biomarkers allowing geographical provenance. The proposed method might be applied in origin authentication of commercially dried A. japonicus products.

A simple multi-residue method for determination of plant growth retardants in Ophiopogon japonicus and soil using ultra-performance liquid chromatography–tandem mass spectrometry

Plant growth retardants may play an important role in regulation of yield and quality of crops, fruits, and vegetables. Such compounds have begun to be used in the cultivation of traditional Chinese medicines (TCMs), especially for root medicines. Although the potential risks to human health of these compounds has attracted increasing attention, analytical methods for detection of plant growth retardants in TCMs remain poorly investigated. In this study, an effective and reliable method for simultaneous determination of 11 plant growth retardants in Ophiopogon japonicus and soil samples was developed by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS). Extraction was conducted in acetonitrile containing 1.0% (v/v) acetic acid with ultrasonication. The octadecylsilyl (C18) and MgSO4 were used as the dispersive-solid phase extraction (d-SPE) sorbent and provided satisfactory recoveries for the analytes. The conditions of extraction and LC-MS/MS were optimized to achieve the highest recovery and sensitivity. Good linearity was achieved within a wide range with all correlation coefficients exceeding 0.9950. The recoveries of all analytes in O. japonicus and soil samples ranged from 57.37% (choline chloride) to 99.93% (trinexapac-ethyl) and from 54.37% (daminozide) to 94.82% (triadimenol), respectively. The limits of quantifications ranged from 0.03 to 3.54 μg/L. The proposed method was successfully applied to detect and quantify 11 plant growth retardants in empirical O. japonicus and soil samples. High frequency of paclobutrazol and choline chloride was found in O. japonicus samples. In addition, paclobutrazol showed a high residual concentration (>1100 μg/kg) in the soil of O. japonicus indigenous production.

Speciation Analysis of Trace Mercury in Sea Cucumber Species of Apostichopus japonicus Using High-Performance Liquid Chromatography Conjunction With Inductively Coupled Plasma Mass Spectrometry.

In this paper, a simple and cost-effective method using high-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry with a rapid ultrasound-assisted extraction was used for analysis speciation of trace mercury in sea cucumber species of Apostichopus japonicus. The effective separation of inorganic mercury, methylmercury, and ethylmercury was achieved within 10min using Agilent ZORBAX SB-C18 analytical and guard columns with an isocratic mobile phase consisting of 8% methanol and 92% H2O containing 0.12% L-cysteine (m/v) and 0.01mol/L ammonium acetate. Mercury species were extracted from A. japonicus samples using a solution containing 2-mercaptoethanol, L-cysteine, and hydrochloric acid and sonicating for 0.5h. The limits of detection of inorganic mercury, methylmercury, and ethylmercury were 0.12, 0.08, and 0.20μg/L, and the minimum detectable concentrations (measured at 0.500g sample volume in 10.00mL) were 2.4, 1.6, and 4.0μg/kg, respectively. Analysis of a scallop certified reference material (GBW 10024) revealed accordance between the experimental and certified values. This study provides a reference for the evaluation of mercury speciation in sea cucumber and other seafood.

Studies on Quality Control of Domestic Leonurus japonicus Houttuyn

Background: Leonurine is a the aerial part of Leonurus japonicus Houttuyn, which has been used as a traditional medicines and is registered in the Korean Pharmacopoeia. Methods and Results: In the present study we performed a heavy metals and thin layer chromatography and measured loss on drying, as well as the contents of total ash, acid-insoluble ash, ethanol soluble compounds, and leonurine, using 15 domestically collected L. japonicus samples. The methods were performed according to the ‘crude drugs test of the general test, processes and apparatus’, published by MFDS, Korea (2014). The purity test (heavy metals) indicated that levels of Pb and Hg were 0.35 - 3.64, and 0.001 ppm, respectively, whereas the levels of As and Cd were undetectable, and stachydrine was identified by thin layer chromatography (Rf : 0.15). We found that 5.93 - 10.62% (average: 8.58 ± 1.8%) of the sample mass was lost during drying, and the contents of total ash, acid-insoluble ash, ethanol soluble compounds, and leonurine were 7.87 - 10.84% (average: 9.62 ± 0.82%), 0.99 - 1.76% (average: 1.38 ± 0.24%), 16.70 - 23.11% (average: 19.49 ± 2.14%) and 0.04 - 0.17% (average: 0.11 ± 0.04%) respectively. In addition, HPLC profiling detected leonurine (5.94 min), rutin (16.43 min) and myricetin (26.78 min). Conclusions: We hope that this the rusult of the present study will contribute to the standardization and quality control of Korean herbal medicines.

A complete mitochondrial genome from Echinochasmus japonicus supports the elevation of Echinochasminae Odhner, 1910 to family rank (Trematoda: Platyhelminthes)

The complete mitochondrial (mt) genome of the trematode Echinochasmus japonicus Tanabe, 1926 was fully determined and annotated. The circular mt molecule of this species is 15,865bp in length, containing 12 protein-coding genes (arranged in the following order: cox3-cob-nad4L-nad4-atp6-nad2-nad1-nad3-cox1-cox2-nad6-nad5), two ribosomal RNA genes (rrnL and rrnS) and 22 transfer RNA genes (trnH; trnQ; trnF; trnM; trnV; trnA; trnD; trnN; trnP; trnI; trnK; trnS1(AGN); trnW; trnT; trnC; trnL1(CUN); trnS2(UCN); trnL2(UUN); trnG; and trnE). The atp8 gene is absent. The 3′ end of nad4L overlaps the 5′ end of nad4 by 40bp. An array of eight identical tandem repeats (240bp each) was found between trnG and trnE in the long non-coding region of the individual worm sequenced. Numbers of these repeats varied among E. japonicus samples. Phylogenetic analysis of concatenated mt protein sequences of 40 trematode species/strains supports the elevation of Echinochasminae Odhner, 1910 to family rank, close to the families Echinostomatidae and Fasciolidae. As echinochasmid and echinostomatid species can parasitize humans, the future characterization of additional mt genomes is needed for development of mt markers for identification and phylogenetic, population, epidemiological and hybridization studies.

Newly discovered population of Aedes japonicus japonicus (Diptera: Culicidae) in Upper Bavaria, Germany, and Salzburg, Austria, is closely related to the Austrian/Slovenian bush mosquito population.

BackgroundThe German mosquito surveillance instrument ‘Mueckenatlas’ requests the general public to collect and submit mosquito specimens. Among these, increasing numbers of individuals of invasive species have been registered. Specimens of the Asian bush mosquito Aedes japonicus japonicus submitted from German Upper Bavaria, where this species had not previously been recorded, triggered regional monitoring in mid-2015.MethodsThe search for Ae. j. japonicus breeding sites and developmental stages concentrated on cemeteries in the municipality of origin of the submitted specimens and, subsequently, in the whole region. A virtual grid consisting of 10 × 10 km2 cells in which up to three cemeteries were checked, was laid over the region. A cell was considered positive as soon as Ae. j. japonicus larvae were detected, and regarded negative when no larvae could be found in any of the cemeteries inspected. All cells surrounding a positive cell were screened accordingly. A subset of collected Aedes j. japonicus specimens was subjected to microsatellite and nad4 sequence analyses, and obtained data were compared to individuals from previously discovered European populations.ResultsBased on the grid cells, an area of approximately 900 km2 was populated by Ae. j. japonicus in Upper Bavaria and neighbouring Austria. Genetic analyses of microsatellites and nad4 gene sequences generated one genotype out of two previously described for Europe and three haplotypes, one of which had previously been found in Europe only in Ae. j. japonicus samples from a population in East Austria and Slovenia. The genetic analysis suggests the new population is closely related to the Austrian/Slovenian population.ConclusionAs Ae. j. japonicus is well adapted to temperate climates, it has a strong tendency to expand and to colonise new territories in Central Europe, which is facilitated by human-mediated, passive transportation. The new population in Upper Bavaria/Austria is the seventh separate population described in Europe. According to our data, it originated from a previously detected population in eastern Austria/Slovenia and not from an introduction event from abroad. The dispersal and population dynamics of Ae. j. japonicus should be thoroughly surveyed, as this species is a potential vector of disease agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1447-z) contains supplementary material, which is available to authorized users.

Divergent Metabolic Responses of Apostichopus japonicus Suffered from Skin Ulceration Syndrome and Pathogen Challenge

Skin ulceration syndrome (SUS) is the main limitation in the development of Apostichopus japonicus culture industries, in which Vibrio splendidus has been well documented as one of the major pathogens. However, the intrinsic mechanisms toward pathogen challenge and disease outbreak remain largely unknown at the metabolic level. In this work, the metabolic responses were investigated in muscles of sea cucumber among natural SUS-diseased and V. splendidus-challenged samples. The pathogen did not induce obvious biological effects in A. japonicus samples after infection for the first 24 h. An enhanced energy storage (or reduced energy demand) and immune responses were observed in V. splendidus-challenged A. japonicus samples at 48 h, as marked by increased glucose and branched chain amino acids, respectively. Afterward, infection of V. splendidus induced significant increases in energy demand in A. japonicus samples at both 72 and 96 h, confirmed by decreased glucose and glycogen, and increased ATP. Surprisingly, high levels of glycogen and glucose and low levels of threonine, alanine, arginine, glutamate, glutamine, taurine and ATP were founded in natural SUS-diseased sea cucumber. Our present results provided essential metabolic information about host-pathogen interaction for sea cucumber, and informed that the metabolic biomarkers induced by V. splendidus were not usable for the prediction of SUS disease in practice.

Identification of natural hybrids in Korean Phragmites using haplotype and genotype analyses

To elucidate natural hybridization of Korean Phragmites, we collected Phragmites plants from 29 regions in South Korea. Haplotypes of the samples, which were determined using two known chloroplast intergenic sequences in this study, were combined with previously known haplotypes. Phylogenetic analysis identified that 30 Korean Phragmites were grouped with two different haplotypes, ‘P’ or ‘W’, respectively, indicating that introduced Phragmites samples from other continents were not present in Korea. The vast majority (26) of the 27 test samples were grouped with the P haplotype, while the E4 sample and the three control Phragmites japonicus samples were grouped with haplotype W. Interestingly, parsimony network analysis revealed that Phragmites australis in Korea might have originated from various regions including Busan (S1), Icheon (M2), and Ansan (W2). Genotype analysis using the PhaHKT1 nuclear gene identified the M3 sample as Phragmites japonicus. For the first time, we found two hybrids (E4 and M3) in the wild by haplotype and genotype analyses, implying that the phenotype of Phragmites australis might be dominant in the hybrids. In summary, we suggest that hybrid speciation might be an important factor in the genetic diversity of Korean Phragmites.

Genetic variation in two sea cucumber (Apostichopus japonicus) stocks revealed by ISSR markers

Sea cucumber Apostichopus japonicus samples were collected in Changdao, Penglai (PL), 27 individuals, and Lingshandao, Qingdao (QD), 30 individuals, in the Shandong Peninsula, China. Ten SSR primers were used to assess the genetic variation and relationship between and within the two stocks. Respectively, for each stock, the percentage of polymorphic bands was 85.2% and 86.9%; the gene diver- sity was 0.360 5 and 0.342 8; and the Shannon's information index was 0.515 0 and 0.499 0. At species level, the percentage of polymorphic bands was 92.2%, the total gene diversity was 0.378 9 and the Shannon's information index was 0.550 4. The coefficient of overall genetic differentiation and the ge- netic distances between the stocks were also calculated to be 0.073 0 and 0.079 6 using the POPGENE program. Results show that the genetic diversity of the two stocks is still large but the genetic distance between the two stocks is close. A dendrogram was constructed for the 57 individuals from the two stocks, showing that the genetic structure was unitary for PL stock but complex for QD stock.

Distribution and dietary relationships of the Japanese temperate bass Lateolabrax japonicus juveniles with two contrasting copepod assemblages in estuarine nursery grounds in the Ariake Sea, Japan

Early juvenile Japanese temperate bass Lateolabrax japonicus samples were collected during four cruises to study the spatial and temporal patterns of distribution and feeding habits of the fish in Chikugo estuary, Ariake Sea, Japan. Gut contents were studied by separating, identifying and counting the prey organisms. Plankton samples were collected during each cruise to study the numerical abundance of copepods in the water. Copepod dry biomass and gut content dry mass were estimated. Juveniles were distributed over wide estuarine areas in salinities ranging from as low as 0·37 to as high as 28·81. Considerable spatial and temporal variations were observed in the copepod distribution in the environment and in the food habits of the fish. Two distinctly different copepod assemblages were identified along the estuary: one in the upper river, dominated by a single species Sinocalanus sinensis, and the other a multi‐specific assemblage in the lower estuary dominated by common coastal copepods such as Acartia omorii, Oithona davisae, Paracalanus parvus and Calanus sinicus. The gut content composition corresponded strongly with the copepod composition in the environment, i.e. a single species (S. sinensis) dietary habit at the upper river and a multi‐specific dietary habit dominated by the common coastal copepods in the lower estuary. Ivlev's electivity index showed that the fish strongly preferred larger copepods and avoided smaller ones. Higher dry biomass of copepods in the water as well as higher dry masses of the gut contents were recorded in the low‐to‐medium saline upper river areas, indicating that these areas are of particular importance as nursery grounds for the juvenile Japanese temperate bass. It was speculated that ascending to the upstream nursery areas to utilize S. sinensis, which is the single dominant copepod in these areas, is one of the most important survival strategies of the Japanese temperate bass juveniles in the Chikugo estuary.