Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra

Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquetra is one of the most common bloom-forming species in estuarine and coastal waters worldwide. Although this species does not produce toxins, unlike some other Heterocapsa species, the high density of its blooms can cause significant ecological damage. We developed a H. triquetra species-specific nuclease protection assay sandwich hybridization (NPA-SH) probe that targets the large subunit of ribosomal RNA (LSU rRNA). We tested probe specificity and sensitivity with five other dinoflagellates that also cause red tides. Our assay detected H. triquetra at a concentration of 1.5×10⁴ cells/mL, more sensitive than required for a red-tide guidance warning by the Korea Ministry of Oceans and Fisheries in 2015 (3.0×10⁴ cells/mL). We also used the NPA-SH assay to monitor H. triquetra in the Tongyeong region of the southern sea area of Korea during 2014. This method could detect H. triquetra cells within 3 h. Our assay is useful for monitoring H. triquetra under field conditions.     

Detection of Prorocentrum donghaiense using sandwich hybridization integrated with nuclease protection assay

Prorocentrum donghaiense is an important harmful algae bloom (HAB) causing creature in China’s seas, and the conventional visual detection can not cope with long-term monitoring and high-throughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay (NPA-SH) was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, ...     

An alkaline unwinding assay for the detection of DNA damage in aquatic organisms

This paper reports a rapid and facile procedure for obtaining of DNA from small, whole aquatic organisms (fathead minnow, Pimephales promelas) as well as from the liver of another species (bluegill sunfish, Lepomis macrochirus) and the subsequent estimation of its double strandedness. DNA was isolated from various tissues by homogenization in a basic pH solution containing detergent followed by differential extraction using organic solvents. Additional purification was accomplished by molecular sieve chromatography. Under defined alkaline unwinding conditions, the amount of double-stranded and single-stranded DNA was determined using the bisbenzimidazole dye Hoechst 33258. The technique was used to detect the occurrence of strand breaks in DNA of fathead minnows chemically exposed to benzo[a]pyrene.     

环介导恒温扩增技术快速检测米氏凯伦藻方法的建立

米氏凯伦藻(Karenia mikimotoi,Hansen)属于裸甲藻目(Gymnodiniales),裸甲藻科(Gymnodi-niaceae),凯伦藻属(Karenia)。它能产生溶血性(hemolytic)毒素和鱼毒(ichthyotoxins),溶解鱼类细胞,破坏鱼鳃组织结构,使鱼类无法正常呼吸而窒息死亡[1]。近年来,米氏凯伦藻引起的赤潮在世界各海域屡次发生,日本海域,墨西哥湾,新西兰、韩国、苏格兰和澳大利亚海域都有米氏凯伦藻引发赤潮的报道,对渔业造成很大损失[2]。     

Movements in the harlequin fish (Othos dentex), a large predatory serranid from southern Australia: implications for detection and protection

The harlequin fish (Othos dentex) is the largest serranid found in the temperate waters of southern Australia. Acoustic telemetry was used to continuously track the movements and activity patterns of 10 harlequin fish (330–620?mm total length; 0.5–3?kg weight) for a 16-month period at a coastal reef site. Data showed that the harlequin fish is a site-attached, diurnal predator, with a relatively small home range in comparison with other temperate reef fishes from Australia and New Zealand. These characteristics indicate that the harlequin fish is susceptible to localised depletions from fishing, but that it can be protected within no-take marine protected areas and can be detected with appropriate daytime monitoring techniques. Individuals also displayed discrete depth preferences on the reef slope, evidence of a nocturnal home base, and homing ability following disturbance from an extreme storm event.     

Particulate peptidoglycan in seawater determined by the silkworm larvae plasma (SLP) assay

Peptidoglycan (PG) is a biopolymer found exclusively in the cell wall of bacteria. Recent chemical analysis of particulate organic matter suggests that a major amount of the muramic acid, an amino sugar present only in PG, could not be accounted for in terms of bacterial cells (Benner and Kaiser, 2003); however, data on particulate PG is quite sparse. In the present study, conducted in 1996, the PG was examined at 5 sampling sites in the northwestern Pacific Ocean, and in natural seawater cultures. Particulate PG, which was concentrated using a 96-well filtration plate equipped with Durapore filters (pore size, 0.22 μm), was measured by the silkworm larvae plasma (SLP) assay. The PG concentration generally decreased with depth and correlated significantly with bacterial abundance throughout the entire water column. However, the ratio of particulate PG to bacterial abundance varied with depth. The average ratio was 0.61 ± 0.53 (average ± SD, n = 40) between 50 and 2000 m, which agreed with the bacterial cellular PG content from 0.63 to 1.1 fg cell⁻¹ obtained in seawater cultures. On the other hand, the ratios of PG to bacteria from the surface to 50 m (3.7 ± 2.6, n = 29) and below 2,000 m (2.1 ± 1.7, n = 7) were significantly higher than that between 50 and 2,000 m. These results may suggest that, in the surface and deep layers, a significant fraction of particulate PG was present in bacterial detritus, whereas this fraction was reduced in the middle layer.     

Combined effects of ethanol and cinnamaldehyde in the Japanese medaka embryo-larval assay (MELA)

The Japanese medaka (Oryzias latipes) was used in the medaka embryo-larval assay (MELA) to determine possible adverse developmental effects of ethanol and the spice component, cinnamaldehyde (CAD). Fish may be exposed to waterborne ethanol and a variety of natural products from non-point sources or leaks during ethanol use as a fuel and from point source processing plant effluents. Consumption of ethanol during human pregnancy is known to cause fetal alcohol syndrome (FAS), a collection of birth defects including craniofacial abnormalities thought to be caused by the generation of free radicals during ethanol metabolism by both alcohol and aldehyde dehydrogenase(s). Fish are also susceptible to FAS (Dasmahapatra et al., meeting abstract). The activity of aldehyde dehydrogenase is inhibited by CAD [Biochem. J. 282 (1992) 353-360; Biochem. Pharmacol. 45 (1993) 1621-1630], and CAD is known to cause developmental abnormalities in the rat [Food Chem. Toxicol. 27 (1989) 781-786]. Therefore, the combined effects of treatment with both ethanol and CAD would be expected to produce additive or greater than additive effects in the MELA assay. Medaka were exposed to ethanol at 100 mM, CAD at 10, 1.0, 0.67 or 0.50 mM, to ethanol and CAD combined, or were non-treated controls. Ethanol at 100 mM was without effect. CAD alone at 10 mM and 1.0 mM was lethal by 1 dpf. Embryos exposed to 100 mM ethanol and 0.67 mM CAD exhibited cardiovascular and pigmentation defects and delayed hatching. Embryos exposed to 0.50 mM CAD alone had less severe cardiovascular problems as compared to the combined ethanol and CAD treatment. Taken together the results indicate that the combined effects of ethanol and CAD are greater than the individual effects and indicate the need to monitor effluents in fish nursery areas to protect natural fish populations. Supported by PHS/NIH ES07929.     

流行性造血器官坏死病毒（EHNV）PCR快速检测试剂盒的研制

以流行性造血器官坏死病毒（EHNV）中的主衣壳蛋白基因保守区序列作为扩增靶序列,设计合成了一对特异性引物,应用快速的模板制备方法和优化PCR扩增条件,建立了EHNV病毒PCR快速检测技术,并开发成简便、快速、实用的检测试剂盒.该试剂盒的检测灵敏度相当于31个病毒粒子,模板制备时间约30 min,约4 h即可得到准确的结果;且无非特异性扩增带,适用于由EHNV病毒感染的鱼类苗种和水产品的检疫及水质环境的监测.     

On the external pressure capacity of deepwater sandwich pipes with inter-layer adhesion conditions

Sandwich pipes (SP), a composite structure consisting of two concentric steel tubes and a polymeric or cement-based core has been proposed to be well-insulated and withstand high installation and operational loads in deepwater oil and gas field. In the paper, the post-buckling responses and pressure capacity of sandwich pipes filled with the solid polypropylene annular were investigated. The degree of the inter-layer adhesion condition between the core layer and the surrounding pipes was modeled by the contact surfaces adopting different maximum shear strength values to allow the relative displacement between the layers. The effects such as inter-layer adhesion interactions, thickness-to-radius ratios, the core thickness, the material parameters, the relative initial ovality directions and the inelastic anisotropy on the collapse pressure of SPs were discussed. More than 2000 finite element (FE) models of the sandwich pipes with practical configurations were constructed and analyzed using the programming language Python within the FE software package ABAQUS. Based on the FE results, a simplified equation was developed and recommended for the collapse pressure calculation of sandwich pipes with the polypropylene annular.     

东海原甲藻抗体的制备及酶联免疫检测方法的建立

通过制备针对东海原甲藻细胞破碎物的多克隆和单克隆抗体,建立了基于双抗体酶联免疫分析定量检测东海原甲藻(Prorocentrum donghaiense)的检测方法。利用该方法对单一藻种、混合藻种和现场样品中的东海原甲藻进行检测的结果与镜检结果相一致,最低检测限度为1×103 cells/m L。该方法的建立对中国近海赤潮暴发的实时监控具有重要意义。     

一种增强型YOLOv3的合成孔径雷达(SAR)舰船检测方法

近年来,海战场成为现代战争的主要作战区域之一,舰船目标逐渐成为海上重点监测对象,能否快速准确地识别海战场舰船目标的战术意图,给指挥员的决策提供必要的支持,这关系到一场海上战役的成败.随着合成孔径雷达(synthetic aperture radar,SAR)成像技术的不断发展,大量SAR图像可用于舰船目标检测与识别.利...     

二温式RT-PCR检测对虾Taura综合征病毒的研究

设计了一对能扩增大小为231bp对虾Taura综合征病毒(TSV)某段基因的特异性引物,优化建立了能快速检测TSV的二温式RT-PCR。特异性和敏感性试验结果表明。二温式RT-PCR能对3个试验用TSV毒株的RNA进行扩增,并得到与预期大小一致的231bp的扩增产物,而其它3种对虾病原则无相同大小的特异性扩增产物出现;RT-PCR最低能检测到1pg的TSV RNA。应用RT-PCR对320份分别来自广西沿海不同对虾养殖场的对虾样品进行检测,结果共有85份检出TSV。说明TSV在广西沿海地区养殖对虾中已呈现出区域性流行趋势。同时也显示了RT-PCR在TSV临床检测中具有较高的实用性。     

长江口北支上段岸滩侵蚀及保护实施效果分析

受上下游工程、来水来沙影响,长江口北支上段近期岸滩侵蚀后退严重。为揭示北支上段近期演变机理,控制河势变化和保护生态基底,收集2001年以来长江口北支全河道水下地形资料,并开展了2017—2020年工程区段水下地形精细实测,基于水动力数学模型研究了岸滩保护措施合理性,以分形理论评价了保护措施实施后效果。结果表明：①长江口北支进口江心洲发育,导致上段“S”形主轴弯曲度加大,加剧了暗沙、江心洲和岸滩动荡;②在冲刷最严重的岸滩采用丁坝群加护坎可有效阻止岸滩侵蚀后退;③多年水下地形实测和分形理论计算评价,岸滩趋于稳定,生态基底得以恢复。鉴于新江心沙洲发育和岸滩、深槽变化加剧了北支上段演变的复杂性,建议尽快成立综合协调部门,统筹提出满足水利、防洪、供水、航运、生态等多目标需求的综合整治方案。     

Development and validation of a TaqManTM fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda

Edwardsiella tarda is one of the most important emerging pathogens in the global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ-PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （log₁0 n_c as x; n_c is copy number） of pGEM-16S rDNA was generated. The results of intra-and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.     

Quantitative determination of lysozyme in the hemolymph of Mytilus edulis by the enzyme-linked immunosorbent assay (ELISA)

The defensive responses of the bay mussel, Mytilus edulis, to invading organisms and toxicants is primarily cellular. Hemolymph cells as well as the other organs and tissues of the mussel through their normal functioning and interactions with toxic substances influence the chemical composition of the animal's hemolymph. It has been found that the levels of specific molecules in the hemolymph fluctuate as a consequence of trace metal insult. It was proposed that one way in which trace metal toxicity may be expressed is through an influx of free lysosomal hydrolases in the hemolymph.Reported here is the development of an enzyme-linked immunosorbent assay (ELISA) with which micro-samples of mussel hemolymph can be analyzed quantitatively for the lysosomal hydrolytic enzyme, lysozyme. During development comparisons were made of the results acquired by spectrophotometric determinations of lysozyme activity and ELISA-determined lysozyme concentrations in the hemolymph of mussels. The hemolymph from unstressed and copper-stressed mussels was examined. It was found that the overall trends in lysozyme fluctuations were indicated by both techniques. However, on occasion in copper-stressed populations a divergence between the two sets of data was observed. This suggested an effect of copper on lysozyme molecular configuration.     

Determining the sensitive developmental stages of intersex induction in medaka (Oryzias latipes) exposed to 17 beta-estradiol or testosterone.

Certain environmentally persistent compounds can adversely affect reproduction by acting as steroid hormone agonists or antagonists. The goal of the present study was to determine the developmental stage most susceptible to exogenous hormone (estradiol and testosterone) exposure using a small teleost model. In the first (pilot study) of two experiments, medaka (Oryzias latipes), at varying developmental stages, were bath-exposed to 5 micrograms/l 17 beta-estradiol for 24 h. At 5 months of age, fecundity, fertility and embryo and larval viability (reproductive success) were investigated in control and exposed groups. Fish at 1, 1.5, 2 and 5.5 months of age were also sampled, processed and examined histologically for gonadal alteration. No significant differences in mortality, gonadal morphology, body weight, sex-ratio or time to maturity were seen between control and exposed fish. At 5 months, however, when exposure groups were compared to controls, significant differences were seen in reproductive success and viability of offspring. A second experiment exposed embryo stage 10, and 1-, 7- and 21-day-old larvae for 6 days to 15 micrograms/l 17 beta-estradiol or 100 micrograms/l testosterone. No significant differences were seen at 5 months in mortality, body weight, or time to sexual maturity. However, sex-ratios were significantly biased toward female in the stage 10, 1- and 7-day post-hatch estradiol exposure groups. No significant changes in sex-ratio were associated with testosterone exposure at any developmental stage. Further, intersex gonads were observed in fish from all groups exposed to 15 micrograms/l estradiol. Only those fish exposed as newly hatched fry or at 1 week post-hatch displayed intersex gonads following 100 micrograms/l testosterone exposure. Data from these experiments show that newly hatched fry are that life stage most sensitive to hormone exposure and the most appropriate to use in determining effects of known endocrine-disrupting compounds.     

近岸补沙养护海滩研究综述

在海平面上升、风暴频率增加等自然因素和人工挖沙、河流水利及海岸工程建设等人为因素的影响下。海滩侵蚀的现象越来越普遍。我国厦门岛、北戴河、青岛汇泉湾等黄金海滩滩面变陡,颗粒粗化,岸线萎缩,可用沙滩面积大幅减少,海滩资源的损失已成为滨海城市旅游业发展的瓶颈。     

Performances of multi-element multi-user detection strategies in ashallow-water acoustic network (SWAN)

A multi-element receiver strategy is proposed in this paper for a multi-user shallow-water acoustic network (SWAN). The base station receiver, equipped with prior knowledge of the synchronization and training sequences of all intended users, has the task of demodulating the received signals of each user independent of the presence of other users. The adopted receiver strategy enables robust communications through the challenging underwater environment which is limited by both environmental and system factors. The channel is characterized by inter-symbol interference due to multipath propagation and multiple access interference. In this paper, we propose a number of multi-user detection receiver structures employing adaptive decision feedback equalization and spatial diversity to mitigate the effect of these two types of interference. Computer simulations and experimental sea trials conducted in the North Sea in 1999 were used to test the receiver strategies' performance for a two user near far scenario. Amongst a number of strategies tested, the structure based on recursive successive interference cancellation demonstrated improved performance overall     

Evaluating the effects of protection on two benthic habitats at Tavolara-Punta Coda Cavallo MPA (North-East Sardinia, Italy)

In this paper, two benthic habitats have been investigated in a fully protected site and two control sites at the Tavolara-Punta Coda Cavallo marine protected area (MPA) (NE Sardinia). Overall, a protection effect on the benthic assemblages was highlighted in the shallow subtidal (5 m deep) rather than at intertidal algal turf habitat. Structure of assemblages at the shallow subtidal habitat is different in the fully protected site vs. controls, while this was not true for the intertidal habitat. At the subtidal, this finding is probably linked to indirect effects due to an increase of consumers in the protected site (e.g., sea urchins), while the lack of direct impacts in the intertidal at control sites is indicative of very similar assemblages. Cover of encrusting algae was significantly higher at the subtidal protected site suggesting a possible higher grazing pressure. Possible causes underlying the inconsistency of results obtained between habitats include the possibility that trophic cascade effects have a different influence at different heights on the shore. The need to estimate the interconnection among benthic habitats through trophic links is also highlighted to provide an estimate of the vulnerability to protection of various habitats.     

粉沙海床管道在联锁排防护下的冲刷试验研究

采用当地海床的天然粉沙进行试验床面的设计,对海底管道在实际海床受到的极限波浪和最大水流作用进行试验,在管道上方铺设混凝土联锁排,对联锁排块体稳定性和其对管道防护的有效性进行研究。通过物理模型试验,研究了在极限波浪和最大水流作用下,管道在粉沙床面最大冲刷深度。通过不同水深中波流条件下研究了混凝土联锁排防护效果及其周围床面的冲刷变化,观测了联锁排的稳定性。试验结果表明:按半经验半理论简化公式得出的联锁排厚度能满足稳定性的要求,比水利部规范计算的联锁排厚度偏大的公式更加适合应用,试验期间混凝土联锁排的排面稳定,能有效保护管道,在该防护下管道没有冲刷,只在联锁排上下游与床面接触的来流段和尾端有冲刷,但该冲刷深度不影响联锁排整体的稳定性。