Quantitative sandwich ELISA for the determination of tropomyosin from crustaceans in foods

The ubiquitous muscle protein tropomyosin has been identified as the major shrimp allergen and is suggested to be a cross-reacting allergen. Previously, only a few methods for the detection of tropomyosin in food have been published. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of tropomyosin from crustaceans in foods has been developed and validated. A polyclonal rabbit antitropomyosin capture antibody and the biotinylated conjugate of the same antibody for detection were the basis for the ELISA, which was specific for crustaceans. The ELISA was able to quantitate tropomyosin in various food matrixes, had a detection limit of 1 microg/g, and cross-reacted to some extent with cockroach. Recoveries ranged from 63 to 120%, and the intra and interassay coefficients of variation were <6 and <14%, respectively.     

Implications of intraguild predation for sea turtle nest protection

In the United States, raccoons Procyon lotor are often removed from sea turtle nesting beaches to decrease egg mortality. However, raccoons also consume ghost crabs Ocypode quadrata, another common egg predator. Reducing predator populations can benefit secondary predators, inflating total predation pressure and leading to a decline in prey species. We used track and burrow counts to compare raccoon and ghost crab abundance at four beaches in Florida, USA, that differ in management activity and determined predation rates on loggerhead Caretta caretta nests by each predator. Mean raccoon abundance (range 0.12-0.46 tracks plot⁻¹ night⁻¹) and ghost crab density (0.09-0.19 burrows m⁻²) were inversely correlated. Ghost crabs were largest at the site with the fewest raccoons. The stable nitrogen isotope ratios of ghost crabs (mean 9.8‰) were positively correlated with body mass, indicating larger ghost crabs feed at a higher trophic level and suggesting large ghost crabs may consume more loggerhead eggs. The highest rates of egg predation by both predators (31%) occurred where raccoon abundance was lowest and ghost crab abundance was highest, suggesting ghost crab burrows may facilitate predation by raccoons. Our data suggest that predation by raccoons limits ghost crabs and that removing raccoons can increase ghost crab abundance and sea turtle egg mortality. Although predator removal can be effective when nest predation rates are quite high, maintaining moderate raccoon densities may be important for controlling ghost crabs. These results highlight the importance of understanding food web connectivity in developing management strategies to achieve conservation goals, especially when the species of concern are threatened or facing extinction.     

A reliable and sensitive immunoassay for the determination of crustacean protein in processed foods

Among food allergens, crustacea such as shrimps, crabs, and lobsters are a frequent cause of adverse food reactions in allergic patients. The major allergen has been identified as a muscular protein, tropomyosin. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of crustacean protein in processed foods was developed using the sample dilution buffer that is added to porcine tropomyosin. The sandwich ELISA method was highly specific for the Decapoda group, apart from minor cross-reactivities to other crustacea and mollusks. The recovery ranged from 85 to 141%, while the intra- and interassay coefficients of variation were less than 2.8 and 8.4%, respectively.     

铁蟹过敏原的分离、鉴定和快速纯化

应用免疫印迹(Western blot) 的方法鉴定铁蟹过敏原组分,然后利用电泳洗脱的方法快速纯化主要过敏原。取磷酸盐缓冲液制备的铁蟹肉浸出液,经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,测定各组分的相对分子量,然后利用18 例蟹过敏患者的血清进行免疫印迹,鉴定出主要和次要过敏原,利用普通垂直电泳槽快速洗脱主要过敏原蛋白,并鉴定活性。结果显示,SDS-PAGE显示铁蟹肉可辨条带有16条,相对分子质量在16.5～168 kD 之间。Western blotting结果表明,铁蟹肉浸出液共有10条过敏条带,其中相对分子质量为76kD的蛋白条带,阳性反应率为100%,纯化后获取了76 kD的主要过敏原,经过免疫印迹鉴定其具有免疫活性。表明相对分子质量76 kD的组分为铁蟹主要过敏组分,快速电洗脱可以纯化相对分子量为76kD的过敏原组分。     

Freshwater crabs and the biodiversity crisis: Importance, threats, status, and conservation challenges

Freshwater ecosystems in the tropics host a diverse endemic fauna including freshwater crabs, but the rapid loss and deterioration of habitat means that many species are now under imminent threat. Studies on freshwater fish and amphibians suggest a third to half of the species in some tropical freshwaters is either extinct or endangered, but the status of the freshwater crabs is not known. Freshwater crabs, with 1280 species, represent one-fifth of all the World’s brachyurans. We therefore undertook a comprehensive IUCN Red List assessment of the freshwater crabs, which was the first time that such a study had been attempted on a global scale for any group of freshwater invertebrates. The conservation status of all known species from the Americas, Africa, Europe, Asia, and Australasia revealed unexpectedly high threat levels. Here we show that about one-sixth of all freshwater crab species have an elevated risk of extinction, only one-third are not at-risk, and although none are actually extinct, almost half are too poorly known to assess. Out of 122 countries that have populations of freshwater crabs, 43 have species in need of protection. The majority of threatened species are restricted-range semi-terrestrial endemics living in habitats subjected to deforestation, alteration of drainage patterns, and pollution. This is illustrated with a case study of one such species found in Singapore. This underlines the need to prioritize and develop conservation measures before species decline to levels from which they cannot recover. The proportion of freshwater crabs threatened with extinction is equal to that of reef-building corals, and exceeds that of all other groups that have been assessed except for amphibians. These results represent a baseline that can be used to design strategies to save the World’s threatened freshwater crab species.     

Differential Sex, Morphotype and Tissue Accumulation of Mercury in the Crab Carcinus maenas

Carcinus maenas is an invasive species of recognised economical and ecological importance in which mercury accumulation could be a pathway for bioamplification through food webs. Little information is available about differential accumulation between crab sexes and morphotypes. Taking this in mind, a set of different industrial discharge scenarios were investigated in 96-h laboratory experiments for assessing the accumulation of inorganic mercury from contaminated seawater into the tissues of C. maenas. Three groups of crabs (green males, green and red females) where exposed to 5, 50 and 250 ??g Hg L^?1. Differences among sexes, morphotypes and tissues were detected, depending on the mercury concentration. The muscle did not show differential accumulation between sexes or morphotypes. For mercury-exposed crabs, the contaminant was accumulated preferably in the gills (more than 75%) while, in control experiments, it was in the internal organs, muscle and hepatopancreas, and gills corresponded to less than 31% of the total mercury quantified. The different tissue contamination seems dependent on the major pathway of exposure, diet or water. Mercury accumulation by the crab was a rapid process and could represent a risk for the environment only after 96 h. In a scenario of a discharge point of 250 ??g L^?1, all tissues of crabs exposed would attain a very close, or even exceed the threshold concentration value for human consumption (0.5 mg kg^?1).     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

Habitat fragmentation in marine landscapes: relative effects of habitat cover and configuration on juvenile crab survival in California and North Carolina seagrass beds

Processes that dictate faunal abundance (e.g. predation) may be linked primarily to the configuration of habitat (e.g. patch size, patch isolation, proximity to edge) or simply to the amount of habitat (i.e. habitat cover) in marine and terrestrial systems. I asked whether juvenile crab survival in marine seagrass habitats is linked to habitat cover or to habitat configuration, and whether relationships between crab survival and habitat features were similar in two widely separated seagrass landscapes (Bodega Harbor, California and Back Sound, North Carolina, USA). I also evaluated the independent effects of seagrass structural complexity (shoot density and shoot biomass per unit area) and habitat fragmentation on crab survival. Juvenile red rock crabs (Cancer productus: 1+year class) were tethered in Bodega Harbor and juvenile blue crabs (Callinectes sapidus: 0+year class) were tethered in Back Sound. Seagrass cover in both landscapes ranged from <10 to 100% over 10×10 m areas. Crab survival was correlated with elements of landscape structure and structural complexity in both landscapes, but relationships between crab survival and specific habitat features differed between the two landscapes. In California, juvenile red rock crab survival was negatively correlated with seagrass cover and was positively correlated with seagrass shoot density, and was marginally (P=0.06) negatively correlated with seagrass shoot biomass. In North Carolina, juvenile blue crab survival was positively correlated with log₁₀ patch area and was negatively correlated with seagrass shoot biomass. The results indicate that (1) both seagrass cover and configuration may influence crab survival; (2) seagrass structural complexity influences crab survival independently of landscape structure; (3) the relative influence of cover, configuration and structural complexity on survival likely differ among seagrass landscapes; and (4) increasing structural complexity (e.g. shoot biomass) may not always lead to increased faunal survival. The results correspond to those of recent studies in marine and terrestrial landscapes showing widely variable faunal responses to landscape structure among species, geographic location and through time, and imply that seagrass restoration and conservation strategies should incorporate species-specific responses to habitat structure at multiple scales.     

Associations Between Dioxins/Furans and Dioxin-Like PCBs in Estuarine Sediment and Blue Crab

The objective of the present study was to evaluate the relationships between the quantity, toxicity, and compositional profile of dioxin/furan compounds (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in estuarine sediment and in the blue crab (Callinectes sapidus). Sediment and blue crab samples were collected in three small urban estuaries that are in relatively close proximity to each other. Results show that differences between PCDD/F and DL-PCB mass concentrations and total toxic equivalents (TEQ) toxicity in sediments of the three estuaries are reflected in those of the blue crab. TEQs are higher in the hepatopancreas of the crabs than in the sediment, but the concentration factor is inversely proportional to the TEQ in the sediments. Congener profiles in the crabs are systematically different from those in the sediments, and the difference is more pronounced for PCDD/Fs than for DL-PCBs, possibly due to differences in metabolization rates. Compared with sediment profiles, more lesser-chlorinated PCDD/Fs that have higher TEFs accumulate in crab hepatopancreas. This selective bioaccumulation of PCDD/Fs results in a TEQ augmentation in crab hepatopancreas compared with sediments. The bioaccumulation in the blue crab is also selective for PCDD/Fs over DL-PCBs.     

Effect of oils and oil-products on crustaceans

The effect of oil and oil products on the 5 species of crustaceans in the Caspian sea was investigated. Two species of shrimp (Palaemon elegans, P. adspersus), crab (Thithropanopeus harrisii tridentatus), amphipoda (Pontogammarus maeoticus), and cirripeda crayfish (Balanus improvisus) are classified among them. Various oil products (oil, phenol, fuel oil, xerosene, gasoline, solar oil) were used in the experiments. The influence of oil on the respiration and mass increase, quality of generation, and larval stages of the crustaceans was studied. Minimal critical concentrations for the crustaceans are 0.001 mg L^?1 of fenol, 0.4 mg L^?1 of oil, 0.1 mg L^?1 of solar oil, 0.01 mg L^?1 of gasoline and fuel oil.     

拟穴青蟹软壳蟹与硬壳蟹营养品质和风味差异综合比较分析

为明确在最佳收集时间段内不同时间点的软壳蟹和硬壳蟹营养与风味物质的差异,本试验采用顶空固相微萃取(SPME)、气质联用(GS-MS)方法比较了拟穴青蟹硬壳蟹与蜕壳后0~48 h软壳蟹肌肉和肝胰腺2种组织中水分、灰分、氨基酸、矿物质元素、脂肪酸及挥发性物质含量。结果表明,软壳蟹2种组织在蜕壳0 h水分含量最高,硬壳蟹灰分含量显著(P<0.05)高于各时间点软壳蟹。除肝胰腺中胱氨酸和丙氨酸外,2种组织硬壳蟹其他氨基酸含量均高于软壳蟹,其中亮氨酸含量差异显著(P<0.05);在肌肉和肝胰腺组织中,分别有12和14种氨基酸含量均在蜕壳后6 h最高。软壳蟹2种组织Ca、Mg和Fe含量高于硬壳蟹,但差异不显著;蜕壳0 h 软壳蟹肝胰腺组织中Ca含量显著高于其他时间点(P<0.05)。蜕壳后24 h软壳蟹肌肉和0 h肝胰腺组织中∑PUFA均高于其他时间点。2种组织中挥发性物质包括烷烃类、醛类、酮类、醇类、芳香烃类、酯类、酚类和醚类化合物,总体相对含量有差异,但差异不显著。综上比较,拟穴青蟹软壳蟹蜕壳后0~6 h肝胰腺和肌肉比其他时间点更具营养、风味更加,是制取软壳蟹最佳时间。本研究结果为软壳蟹生产、营养和风味评价、市场价值评估提供了一定的参考。     

一次性施肥对稻田蟹及水稻产量的影响

在稻蟹种养模式下,采用田间微区法系统地研究了一次性施肥技术条件下,不同有机无机氮肥配比对耕层土壤和田面水中的NH4+和NO3-含量变化以及对河蟹和水稻产量、效益的影响。结果表明:施肥能明显增加水稻土壤、水面中的NH4+和NO3-含量,施肥后3~7 d左右达到最高值,随后迅速下降并趋于稳定。水田氮素形态以NH4+为主、NO3-次之;与常规施肥(FP)和测土推荐施肥(OPT)相比,A1配方肥一次性施用对河蟹的生长及产量均有较好的效果,同时水稻产量也较高,达到10620.5 kg hm-2,接近于OPT处理;河蟹成活率达66.7%,明显好于其他处理;亩效益分别比FP和OPT处理增加了165.4元和120.7元。而OPT处理只在水稻产量反应较好,对河蟹生长有一定的影响。说明,合理的有机无机肥配比能够实现稻蟹双丰收的目标。     

Fast real-time PCR for the detection of crustacean allergen in foods

Crustaceans are one of the most common allergens causing severe food reaction. These food allergens are a health problem, and they have become very important; there are various regulations that establish that labeling must be present regarding these allergens to warn consumers. In the present work a fast real-time PCR, by a LNA probe, was developed. This allows the detection of crustaceans in all kinds of products, including processed products in which very aggressive treatments of temperature and pressure during the manufacturing process are used. This methodology provides greater sensitivity and specificity and reduces the analysis time of real-time PCR to 40 min. This methodology was further validated by means of simulating products likely to contain this allergen. For this, products present on the market were spiked with crustacean cooking water. The assay is a potential tool in issues related to the labeling of products and food security to protect the allergic consumer.     

蟹脚壳肉分离装置的设计与试验

针对河蟹蟹脚人工壳肉分离劳动强度大、效率低下且蟹肉易污染等问题,设计了一种辊筒挤压式蟹脚壳肉分离装置。该装置通过倾斜的输送滑道将蟹脚输送到一对直径相同、转动方向相反,转速相同的圆柱辊筒之间,利用其挤压作用完成蟹脚的壳肉分离。为确定蟹脚壳肉分离装置的最佳工作参数,以分离效率、得肉率及蟹肉品质为性能指标,以送料角度、辊筒间隙和辊筒转速为影响因素对样机开展了正交试验。结果表明:影响壳肉分离效率的极显著因素为辊筒转速,送料角度和辊筒间隙影响不显著;影响得肉率的极显著因素为辊筒间隙,显著因素为送料角度,辊筒转速影响不显著;影响蟹肉品质的极显著因素为辊筒间隙,送料角度和辊筒转速影响不显著。试验结果表明:最佳工作参数为:送料角度40?,辊筒转速35 r/min、辊筒间隙1.2 mm,在此最优组合参数下,分离率23.1 kg/h,得肉率98.5%,蟹肉品质8.9分,该研究可为后续的设计优化与性能提高提供参考。     

中华绒螯蟹平面运动三维观测和动力学分析

为给适应复杂路面的多足仿生机器人或步行移动平台的研究提供理论依据,该文以中华绒螯蟹为研究对象,使用三维运动图像采集系统采集其运动图像,并使用三维运动图像分析系统得到中华绒螯蟹质心的运动学参数,分析结果表明：中华绒螯蟹在平面运动时主要使用跳跃步态;质心动能、重力势能以及总机械能呈正弦波动,动能与重力势能同相;使用恢复系数表示动能和重力势能相互转换的效率,其平均值为31.73%±17.29%,恢复系数和平均速率没有相关关系;质心机械能比质量功率随平均速率的增长呈线性增加;质心水平动能比质量功率和重力势能比质量功率对中华绒螯蟹身体的加速和抬升起主要作用。该研究可为研究适应复杂路面的仿生机器人或移动平台提供理论依据。     

液氮冻结软壳青蟹在冻藏期间的营养物质变化

为探讨液氮(LN)冻结拟穴青蟹软壳蟹在冻藏期间的营养物质变化,本研究采用基于核磁共振(NMR)的代谢组学技术分析了LN冻结软壳蟹可食组织代谢谱在短期(7 d)和长期(8个月)冻藏期间的变化。结果表明,-20℃冻藏和LN冻结均对短期冻藏的软壳蟹代谢谱无显著影响(P>0.05),但 -20℃ 冻藏导致软壳蟹肌肉的二甲胺和肝胰腺的肌苷水平倍增,而LN冻结能抑制这两种物质的增加。长期冻藏可导致LN冻结软壳蟹肌肉的肌苷、麦芽糖和葡萄糖以及肝胰腺的肌苷、谷氨酰胺和尿苷水平显著升高(P<0.05)。表明LN冻结可有效缓解长期冻藏软壳蟹的质量下降。本研究结果为揭示软壳蟹冻藏过程中的品质变化提供了理论参考。     

贮藏温度对炝蟹品质的影响

为阐明不同贮藏温度下炝蟹品质的变化和食用安全性,本试验以三疣梭子蟹为材料制作炝蟹,探讨冻藏(-18℃)和冷藏(4℃)条件下炝蟹感官品质、菌落总数(TVC)、挥发性盐基氮(TVB-N)和生物胺的变化规律,并对其生物胺与其他品质指标进行相关性分析。结果表明,贮藏温度对炝蟹感官品质TVC、TVB-N等指标的劣变具有显著的影响。-18℃冻藏和4℃冷藏条件下,炝蟹感官品质分别于第10天和第6个月超出可接受限值;随着贮藏时间的延长,4℃冷藏条件下炝蟹TVC值显著增加,但至贮藏16 d时仍小于5 lg(CFU·g^-1),TVB-N值于贮藏12 d时超出一级鲜度临界值;-18℃冻藏过程中炝蟹TVC值略有增加,但变化不显著,TVB-N值于第6个月超出一级鲜度临界值。4℃冷藏过程中炝蟹中共检测出8种生物胺,而-18℃冻藏过程中共检测出5种生物胺。此外,色胺、腐胺和酪胺是炝蟹贮藏过程中产生的主要生物胺,且与其他品质均具有显著相关性。本研究结果为炝蟹在贮藏过程中的安全性和货架期预测提供了理论依据。     

Effect of maillard reaction on allergenicity of scallop tropomyosin

Scallop tropomyosin (TM), the major allergen of shellfish, was prepared from adductor muscles and reacted with four reducing sugars to investigate the effect of the Maillard reaction on the allergenicity of TM. The IgE-binding ability of TM increased significantly with the progress of the reaction with glucose, ribose, and maltose, but not with maltotriose. The allergenicity was enhanced at the early stage of the Maillard reaction, and the trend of the effect depended on the type of reducing sugar used. 2,4,6-Trinitrobenzenesulfonic acid treatment of the lysine residues in TM showed that the protein surface charge resulting from the Maillard reaction had no effect on the enhancement of the allergenicity. Thus, the change in the allergenicity would be closely related to the structural change caused by the Maillard reaction.     

基于塘内循环自净的河蟹生态养殖系统设计与试验

为了提高河蟹养殖品质和养殖池塘水质净化能力,该研究提出了一种通过养殖池塘内部结构改造实现池塘水体内部循环自净的技术,并设计了一套针对河蟹的生态养殖系统。应用软围隔将养殖塘划分为两个相对独立的功能区(养殖区和自净区),设计了浮式气提推水装置和射流装置作为塘内水循环动力系统,在推水装置的作用下,养殖区的水体流入自净区,经过滤、吸附、杀菌及降温等环节重新回到养殖区,形成"九分养蟹一分养水"的河蟹循环自净生态养殖模式。在崇明宝岛蟹业面积约为9 600 m~2的河蟹养殖池塘进行实施和试验,试验表明:合理配置气提推水装置可实现河蟹养殖池塘水体日循环2次以上;试验塘循环自净状态相较静水状态,水温均衡度提高10.17%,下层溶解氧水平提高18.57%,氨氮平均质量浓度下降19.2%;同时,养殖效果抽样对比显示试验塘200 g以上公蟹和150 g以上母蟹较对照塘分别增加了45%和35%。该研究设计的塘内循环自净的河蟹生态养殖系统能较好地净化养殖水体,有利于河蟹的生长,可为河蟹池塘生态高效养殖模式的构建和推广提供参考。     

河蟹养殖中气象条件的影响及趋利避害对策研究

通过对洪泽湖周边河蟹生长各阶段在自然条件与人工调节气象条件下生长差异的实地对比观测研究，得出：在育幼期合理调节气象条件，蟹苗成活率可提高到50％～60％，全育期缩短15～20d，节约成本30％以上；在生长期合理利用气象条件，河蟹死亡率、发病率明显降低，取得了明显的经济效益。进一步提出了河蟹生长各阶段气象服务要点和防御对策。