The efficacy of DNA barcoding in the classification,genetic differentiation,and biodiversity assessment of benthic macroinvertebrates

Macroinvertebrates have been recognized as key ecological indicators of aquatic environment and are the most commonly used approaches for water quality assessment. However, species identification of macroinvertebrates (especially of aquatic insects) proves to be very difficult due to the lack of taxonomic expertise in some regions and can become time‐consuming. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in seven orders (Insecta: Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, and Odonata) from four large transboundary rivers of northwest China and further explored its potential application to biodiversity assessment. A total of 1,144 COI sequences, belonging to 176 species, 112 genera, and 53 families were obtained and analyzed. The barcoding gap analysis showed that COI gene fragment yielded significant intra‐ and interspecific divergences and obvious barcoding gaps. NJ phylogenetic trees showed that all species group into monophyletic species clusters whether from the same population or not, except two species (Polypedilum. laetum and Polypedilum. bullum). The distance‐based (ABGD) and tree‐based (PTP and MPTP) methods were utilized for grouping specimens into Operational Taxonomic Units (OTUs) and delimiting species. The ABGD, PTP, and MPTP analysis were divided into 177 (p = .0599), 197, and 195 OTUs, respectively. The BIN analysis generated 186 different BINs. Overall, our study showed that DNA barcoding offers an effective framework for macroinvertebrate species identification and sheds new light on the biodiversity assessment of local macroinvertebrates. Also, the construction of DNA barcode reference library of benthic macroinvertebrates in Eurasian transboundary rivers provides a solid backup for bioassessment studies of freshwater habitats using modern high‐throughput technologies in the near future.     

Interacting effects of water temperature and dietary protein level on hematological parameters in Nile tilapia juveniles,Oreochromis niloticus (L.) and mortality under Streptococcus iniae infection

Based on central composite rotatable experimental design and response surface method, the interacting effects of temperature (20 °C–34 °C) and dietary protein level (25%–50%) on hematological parameters including red blood cell (RBC), white blood cell (WBC) and hemoglobin (Hb) of juvenile Oreochromis niloticus were studied under laboratory conditions. The experiment lasted for 7 weeks. After the feeding trial, fish were challenged with Streptococcus iniae and mortality was recorded for within 8 days. Results showed that the linear and quadratic effects of temperature on RBC, WBC and Hb were highly significant (P < 0.01). When the dietary protein level was 25%–50%, the RBC, WBC and Hb were increased firstly and then decreased, but the linear and quadratic effects of protein level were insignificant (P > 0.05). The interacting effects of temperature and protein level on RBC and Hb were significant (P < 0.05). The regression equations of RBC, WBC and Hb toward the two factors of interest were established, with the coefficients of determination being 0.870, 0.836 and 0.881, respectively (P < 0.01). These equations could be used for prediction in practice. After the challenge, the mortalities for the combinations of 22.1 °C/28.7% and 20.0 °C/37.5% were significantly higher than 27.0 °C/37.5% (P < 0.05). The optimal temperature/dietary protein level combination was obtained at 27.9 °C/38.1% at which the lowest mortality (13.76%) was attained. This value was close to the optimal temperature/dietary protein level combination (29.4 °C/41.9%) for the greatest levels of RBC (2.560 × 10⁶ μL^?1), WBC (270.648 × 10³ μL^?1) and Hb (92.851 g L^?1). The results of this study indicated that preferred temperature/dietary protein level combination might strengthen the non-specific immunity and reduce susceptibility to S. iniae.     

Effects of dietary live yeast Hanseniaspora opuntiae C21 on the immune and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus

A feeding experiment was conducted to determine effects of Hanseniaspora opuntiae C21 on immune response and disease resistance against Vibrio splendidus infection in juvenile sea cucumbers Apostichopus japonicus. Sea cucumbers were fed with either diets containing C21 at 10⁴, 10⁵ and 10⁶ CFU g^?1 feed or a control diet for 30–50 days, respectively. After feeding for 30 days and 45 days, five sea cucumbers from each tank were sampled for immunological analyses. Results indicated that C21 significantly improved the phagocytic activity in coelomocytes of sea cucumbers (P < 0.05). Moreover, C21 administration significantly enhanced lysozyme (LSZ), phenoloxidase activity (PO), total nitric oxide synthase (T-NOS), superoxide dismutase (SOD), alkaline phosphatase (AKP) and acid phosphatase (ACP) activities in coelomic fluid, and LSZ, T-NOS, AKP and ACP activities in coelomocytes lysate supernatant (CLS) of sea cucumbers (P < 0.05). After feeding for 45 days, 10 sea cucumbers from each dose group were challenged with V. splendidus NB13. Cumulative incidence and mortality of sea cucumbers fed with C21 were found to be lower than those of control group. After feeding for 50 days, sea cucumbers in 10⁴ CFU g^?1 C21 treatment and control tanks were subjected to acute salinity changes (from 30 to 20) for 24 h in the laboratory, and the immunological parameters were measured to evaluate the immune capacities of the A. japonicus. Phagocytic, LAZ and T-NOS activities of C21-treated group were higher than those of control group, indicating that salinity stress tolerance of sea cucumber was enhanced by C21. The present results showed that a diet supplemented with C21 could stimulate the immune system of juvenile A. japonicus thus enhancing their resistance against V. splendidus.     

Heterogeneous interactome between Litopenaeus vannamei plasma proteins and Vibrio parahaemolyticus outer membrane proteins

A great loss has been suffered by microbial infectious diseases under intensive shrimp farming in recent years. In this background, the understanding of shrimp innate immunity becomes an importantly scientific issue, but little is known about the heterogeneous protein–protein interaction between pathogenic cells and hosts, which is a key step for the invading microbes to infect internet organs through bloodstream. In the present study, bacterial outer membrane (OM) protein array and pull-down approaches are used to isolate both Vibrio parahaemolyticus OM proteins that bind to shrimp serum proteins and the shrimp serum proteins that interact with bacterial cells, respectively. Three interacting shrimp serum proteins, hemocyanin, β-1,3-glucan binding protein and LV_HP_RA36F08r and thirty interacting OM proteins were determined. They form 63 heterogeneous protein–protein interactions. Nine out of the 30 OM proteins were randomly demonstrated to be up-regulated or down-regulated when bacterial cells were cultured with shrimp sera, indicating the biological significance of the network. The interesting findings uncover the complexity of struggle between host immunity and bacterial infection. Compared with our previous report on heterogeneous interactome between fish grill and bacterial OM proteins, the present study further extends the investigation from lower vertebrates to invertebrates and develops a bacterial OM protein array to identify the OM proteins bound with shrimp serum proteins, which elevates the frequencies of the bound OM proteins. Our results highlight the way to determine and understand the heterogeneous interaction between hosts and microbes.     

遗传性肾炎的遗传学研究进展

遗传性肾炎(hereditary nephritis,HN)是一组与遗传有关,主要累及肾小球的肾脏疾病,常伴有其它器官的损伤.HN呈家族聚集性,可表现为常染色体显性遗传、常染色体隐性遗传和X连锁遗传,有些家系还表现为非孟德尔遗传和线粒体遗传.对HN主要疾病的临床表型、遗传学和动物模型的总结和对HN疾病的深入研究有可能找到疾病的致病突变,以及更好地了解疾病的分子机制.     

实时定量PCR构建法夫酵母内参基因β-actin、 gpd、18S rRNA标准品质粒和标准曲线

为建立检测法夫酵母JMU-MVP14中虾青素合成相关基因在不同生长时期表达水平的实时定量PCR方法,构建法夫酵母JMU-MVP14的管家基因β-actin、gpd、18S rRNA的标准质粒,进行实时定量PCR,制作标准曲线及回归方程.β-actin基因标准曲线相关系数（R2）=0.9956,扩增效率（E） =96.93％;gpd基因标准曲线相关系数（R2） =0.9901,扩增效率（E） =93.78％;18S rRNA基因标准曲线相关系数（R2） =0.9981,扩增效率（E）=98.76％.3个基因片段的熔解曲线均呈单峰;扩增曲线呈典型的S型动力学曲线,指数期和平台期明显,为理想的熔解曲线和扩增曲线.用geNorm软件对三个管家基因的稳定性进行分析,三个基因的稳定性排序为β-actin＞ 18S rRNA＞ gpd,故β-actin和18S rRNA较适合作为研究法夫酵母JMU-MVP14定量实验的内参基因.