三种水产药物对近江牡蛎(Crassostrea ariakensis)的影响

用3种水产药物硫酸铜、孔雀石绿、甲醛分别设置浓度梯度,对正常和天然患病近江牡蛎进行处理,连续观察7d,发现3种药物对正常近江牡蛎的影响强度为孔雀石绿〉甲醛〉硫酸铜。对患病近江牡蛎的致死率显著高于对正常近江牡蛎的致死率,即使用同一药物的更低浓度处理患病近江牡蛎,其死亡率也显著高于更高浓度处理的正常近江牡蛎的死亡率。在患病近江牡蛎药物处理实验中,对照组有明显死亡,但死亡率低于药物处理组。实验结果表明水产药物残留对近江牡蛎,特别是对患病近江牡蛎有较大影响,会导致其死亡率明显上升,这也可能是引起近江牡蛎大规模死亡的原因之一。     

近江牡蛎吊养养成技术标准

近江牡蛎是我国重要的养殖经济贝类,其养殖历史约两个世纪,但是,一直缺乏该牡蛎的养殖技术规范(标准)。为了近江牡蛎优质高产,特制定了该牡蛎吊养技术标准。该标准由以下几个方面组成:养殖条件,包括养殖环境、海水温度范围(6-32℃)、海水深度(低潮线以下2-8m)、盐度(8‰-30‰),吊养设施结构,吊养方法,饲养过程中的管理和起捕条件与方法等等。     

热休克诱导近江牡蛎对高温的耐受性

设计6个温度组成的梯度,即38℃、39℃、40℃、41℃、42℃、43℃,对近江牡蛎加热处理1.5h后放回室温海水中连续观察3d,发现致死温度为42℃,半致死温度是40℃,亚致死温度为36℃。经亚致死温度36℃热休克1.5h后,在正常环境温度下恢复6h,再经受致死温度42℃处理1.5h后,放回室温海水中连续观察3d,发现三次重复的平均存活率为96.7%,显著高于对照组的存活率(0),结果表明预先的热休克极显著地增强了近江牡蛎对致死温度(42℃)的耐受力。     

盐度对长牡蛎和近江牡蛎及其杂交稚贝生长和存活的影响

2011年8月以长牡蛎自繁组GG(Crassostrea gigas♀×C.gigas♂),近江牡蛎自繁组AA(C.ariakensis♀×C.ariakensis♂)、正交组GA(Crassostrea gigas♀×C.ariakensis♂)、反交组AG(C.ariakensis♀×C.gigas♂)为实验材料,开展了稚贝对盐度的适应性研究。结果发现长牡蛎的最适生存盐度为15—35,最适生长盐度为25—35;近江牡蛎的最适生存盐度为10—25,最适生长盐度为20—25;GA的最适生存盐度为15—30,最适生长盐度为15—30,AG的最适生存盐度为20—30,最适生长盐度为20—25。GG对低盐度敏感,AA对高盐度敏感,AG具有高盐度存活的杂种优势,在盐度30时,中亲杂种优势HG×A为13.32,单亲杂种优势HGA和HAG分别为1.89和27.88,在盐度40时,HAG上升到400,GA和AG都不具有生长优势。杂种稚贝对盐度适应介于双亲之间,且表现出一定程度的父系遗传特点。     

近江牡蛎肠道细菌及其产酶能力的研究

从近江牡蛎(Jinjiang Ostrea vivularisGould)肠道中分离出21株菌,其中9株革兰氏阳性菌,12株革兰氏阴性菌。并检测其蛋白酶、脂肪酶、淀粉酶、纤维素酶的产酶能力。结果表明,13株菌(61.9%)能分泌蛋白酶,13株(61.9%)分泌淀粉酶;11株(52.4%)产脂肪酶;7株(33.3%)产纤维素酶。产4种酶的有5株,产3种酶的3株,产2种酶的5株,产1种酶的3株,不产酶的仅有5株。产酶菌株的比例高达76.2%(16/21),可见近江牡蛎肠道细菌对食饵消化有重要作用。     

近江牡蛎HSP70基因对溶藻弧菌感染的反应

采用实时荧光定量RT-PCR方法,检测了注射溶藻弧菌(Vibiro alginolyticus)后近江牡蛎鳃,闭壳肌,消化腺,外套膜,心脏以及血细胞中HSP70基因的表达变化。结果显示近江牡蛎这五种器官组织中的HSP70基因表达量均出现显著性高表达,且在鳃、外套膜和血细胞中的HSP70基因表达变化规律表现为典型的时间依赖性。血细胞中,显著高表达的峰值出现在24h,至72h恢复到对照水平,高表达持续时间最长:鳃中表达峰值出现时间较早,在第3h,随后在第12h便恢复到对照水平;外套膜,消化腺以及心脏中的峰值分别出现在6h,6h和3h,而在闭壳肌组织中,没出现显著性高表达。由此可见,近江牡蛎HSP70s可能在机体抗菌免疫过程中起了重要作用。     

受孔雀石绿胁迫的近江牡蛎HSP90基因的表达变化

HSP90是重要抗逆分子,为了探讨近江牡蛎Crassostrea hongkongensis HSP90分子在抗逆中的作用,本研究建立实时荧光定量PCR方法,研究了在孔雀石绿胁迫下近江牡蛎HSP90基因在外套膜、消化腺、鳃、闭壳肌4种器官组织中的表达变化规律.结果显示,在浓度1μg/L的孔雀石绿处理下,4种器官组织中的...     

菌胁迫对近江牡蛎子代母源C型凝集素mRNA表达及抗菌影响

为了揭示近江牡蛎（Crassostrea rivularis）C型凝集素（C-type lectin,CTL）母源免疫在子代胚胎发育早期过程中的免疫防御影响,文章运用荧光定量PCR、人工促排、免疫相关酶活测定等实验,开展了菌胁迫近江牡蛎子代母源CTL m RNA表达及抗菌影响。结果表明：近江牡蛎子代胚胎发育早期可检测到母源CTL转录本,母贝分别在不同菌刺激后,其子代在受精卵、32细胞期、桑葚期、囊胚期CTL m RNA相对表达均出现不同程度的升高,与相应空白组比较差异显著（P<0.05）;其子代在受精卵期、32细胞期的Cu/Zn-SOD活力和抑菌活性显著高于相应空白组（P<0.05）;其子代在原肠胚期、担轮幼虫期累计死亡率均显著低于空白组（P<0.05）。结论：该近江牡蛎CTL具有母源免疫传代和子代免疫时效,当母源受到适当的免疫刺激可以增加其子代胚胎发育早期对相同病原感染的免疫防御能力。     

近江牡蛎共附生耐热真菌C051的鉴定、生理学特性及抗菌活性研究

为进一步开发利用耐热海洋真菌,以1株近江牡蛎共附生耐热真菌C051为出发菌株,对其进行系统鉴定、生理学特性及抗菌活性研究。采用形态学鉴定并结合18S rRNA序列分析,确定菌株C051为烟曲霉(Aspergillus fumigatus)。3种培养基比较,适合基础培养基为PDA;生长温度范围为15～50℃,最适生长温度45℃,具有明显的耐热真菌特性;生长pH范围3～9,最适生长pH 6;生长盐浓度0%～16%,最适生长盐浓度3.5%,具兼性海洋真菌的特点。其发酵产物对铜绿假单胞菌(Pseudomons aeruginosa)、金黄色葡萄球菌(Staphylococcus aureus)、交替假单胞菌属SL-1(Pseudoalteromonas sp.)、金黄杆菌属SL-4(Chryseobacterium sp.)均有良好的抑菌效果,最大抑菌圈直径达22.15 mm,为耐热海洋真菌的开发与研究提供参考。     

黄河口西南侧小岛河河口天然牡蛎礁的牡蛎种群结构

牡蛎礁是生态系统服务价值高、但退化最严重及受关注度最高的海洋生境之一,牡蛎礁修复亦成为国际海洋生态修复的热点。掌握牡蛎自然种群状况及动态变化是评估牡蛎礁修复效果的基础。目前,我国天然牡蛎礁的牡蛎种群状况相关的背景资料较为缺乏。在黄河口西南侧的小岛河河口新发现天然活体牡蛎礁,但该牡蛎礁曾被大规模的商业采捕,亟需推进针对性的保护和修复研究工作。基于2021年11月对该牡蛎礁开展的牡蛎种群生态调查,分析其种类组成、年龄结构及生长特征。结果显示:该牡蛎礁分布有近江牡蛎(Crassostrea ariakensis)和长牡蛎(Crassostrea gigas)。牡蛎礁上以活体牡蛎为主,死亡牡蛎壳体数仅占6.1%-6.7%。活体牡蛎的密度和生物量分别为(2811±778)个/m²和(21.97±30.43)kg/m²,近江牡蛎较多,其密度和生物量分别占比55.7%和76.4%。近江牡蛎和长牡蛎的年龄分别介于0+-4+ 龄和0+-2+ 龄,它们都以壳高介于30-40 mm及壳质量<5 g的0+ 龄个体数量居多(>80%)。近江牡蛎的壳体形态参数均值都高于同龄组长牡蛎的相应值。两种牡蛎壳体均呈负异速增长,不同龄级的壳体延展方向不同。拟合von Bertalanffy生长方程得到,近江牡蛎和长牡蛎的渐近壳高分别为286 mm和173 mm,估算的拐点年龄分别为5.47 龄和2.56 龄,两种牡蛎的生长曲线分布存在极显著差异(P<0.001)。以上结果表明,小岛河河口的天然牡蛎礁的牡蛎自然种群资源较丰富,具有高密度、低龄和低死亡率等特点,有较好的活力和扩张潜力,有利于被采捕后的礁体的恢复。两种牡蛎中,近江牡蛎因其具有较高的生物量和较长的生长年龄,对礁体形成和扩繁可能更为重要。建议对该天然牡蛎礁及牡蛎种群开展周期>3年的原位保护、修复和连续监测计划。     

Eleven novel microsatellite markers for the Asian oyster Crassostrea ariakensis

Clones from four partial genomic libraries of the Asian oyster Crassostrea ariakensis were screened to reveal 64 microsatellite-containing sequences. Polymerase chain reaction primers were designed for 34 of these loci. Reactions were optimized for 11 primer pairs and markers were evaluated in eight family crosses and one wild population from northern China. Zero to three null alleles per locus were identified in family crosses, and two of the 11 loci showed allele frequency deviations from Hardy-Weinberg equilibrium in the wild population. These markers have proven useful for genetic studies of C. ariakensis.     

Purification and antigenic characteristics of a rickettsia-like organism from the oyster Crassostrea ariakensis

A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants.     

Interacting effects of temperature and salinity on Bonamia sp. parasitism in the Asian oyster Crassostrea ariakensis

The proposition to introduce the Asian oyster Crassostrea ariakensis to the mid-Atlantic region of the USA is being considered with caution, particularly after the discovery of a novel microcell haplosporidian parasite, Bonamia sp., in North Carolina. Although this parasite was found to be pathogenic in C. ariakensis under warm euhaline conditions, its persistence in C. ariakensis exposed to various temperature and salinity combinations remained unresolved. In this laboratory experiment, we tested the influence of temperature in combination with a wide range of salinities (10, 20 and 30 psu) on Bonamia sp. Temperature was either changed from warm (>20 °C) to cold (6 °C for 6 weeks) and back to warm or maintained constant and warm. Warm temperature was associated with higher host mortality than cold temperature, suggesting that temperature influenced Bonamia sp. pathogenicity. The effect of salinity was revealed under warm temperature with highest mortality levels observed in infected C. ariakensis exposed to 30 psu. When temperature was increased following low-temperature exposure, Bonamia sp. was not detected; however sub-optimal experimental conditions may have contributed to this result, making it difficult to draw conclusions regarding the reemergence of the parasite after low-temperature exposure. Although the overwintering of Bonamia sp. in C. ariakensis will need to be further investigated, the results presented here suggest that Bonamia sp. may be able to persist in C. ariakensis under a combination of low temperature and meso- to euhaline salinities.     

Effect of simultaneous variation in temperature and ammonia concentration on percent fertilization and hatching in Crassostrea ariakensis

The combined effects of temperature and ammonia concentration on the percent fertilization and percent hatching in Crassostrea ariakensis were examined under laboratory conditions using the central composite design and response surface methodology. The results indicated: (1) The linear effects of temperature and ammonia concentration on the percent fertilization were significant (P<0.05), and the quadratic effects were highly significant (P<0.01). The interactive effect between temperature and ammonia concentration on the percent fertilization was not significant (P>0.05). (2) The linear effect of temperature on the percent hatching was highly significant (P<0.01), and that of ammonia concentration was nonsignificant (P>0.05). The quadratic effects of temperature and ammonia concentration on the percent hatching were highly significant (P<0.01). The interaction on the percent hatching was not significant (P>0.05). Temperature was more important than ammonia in influencing the fertilization and hatching in C. ariakensis. (3) The model equations of the percent fertilization and hatching towards temperature and ammonia concentration were established, with the coefficients of determination R²=99.4% and 99.76%, respectively. Through the lack-of-fit test, these models were of great adequacy. The predictive coefficients of determination for the two model equations were as high as 94.6% and 98.03%, respectively, showing that they could be used for practical projection. (4) Via the statistical simultaneous optimization technique, the optimal factor level combination, i.e., 25 °C/0.038 mg mL⁻¹, was derived, at which the greatest percent fertilization 95.25% and hatching 83.26% was achieved, with the desirability being 97.81%. Our results may provide advantageous guidelines for the successful reproduction of C. ariakensis.     

Co‐localized Crassostrea virginica and Crassostrea ariakensis Oysters differ in bioaccumulation,retention and depuration of microbial indicators and human enteropathogens

Aims: To evaluate the bioaccumulation, retention and depuration rates of nine pathogens and surrogates when two oyster species were co‐localized in tanks of seawater. Methods and Results: Crassostrea ariakensis (n = 52) and Crassostrea virginica (n = 52) were exposed to five virus types, two protozoan and two microsporidian species for 24 h. Oysters were then placed in depuration tanks, and subsets were removed and analysed for micro‐organisms at weekly intervals. The odds of C. ariakensis oysters harbouring mouse norovirus‐1 (MNV‐1), human norovirus (NoV) or haepatitis A virus (HAV) were significantly greater than the odds of C. virginica oysters harbouring the same viruses (MNV‐1 OR = 5·05, P = 0·03; NoV OR = 6·97, P = 0·01; HAV OR = 7·40, P < 0·001). Additionally, compared to C. virginica, C. ariakensis retained significantly higher numbers of transmissive stages of all protozoan and microsporidian species (P < 0·01). Crassostrea ariakensis oysters are also capable of retaining multiple human pathogens for at least 1 month. Conclusions: Crassostrea ariakensis oysters were statistically more likely to harbour enteropathogens and microbial indicators, compared to C. virginica. Individual C. ariakensis were also statistically more likely to retain multiple viruses, protozoa and microsporidia than C. virginica, highlighting the role the species may play in the transmission of multiple diseases. Significance and Impact of the Study: Nonnative Crassostrea ariakensis oysters are under review for their introduction into the Chesapeake Bay. The results of this study suggest that nonnative C. ariakensis oysters may present a serious public health threat to people consuming the oysters raw from contaminated sites.     

The repellent effect of two pyrethroid insecticides on the honey bee*

ABSTRACT. . An improved model for the repellent effect of pyrethroid insecticides on insects was developed using small colonies of honey bees, Apis mellifera L., in flight cages. Conditioning to scented feeders allowed the separation of foraging bees from a single colony into paired treatment and control groups. The repellent response was characterized as a sublethal toxic effect resulting in transitory inhibition of activity. Permethrin and cypermethrin were shown to be contact repellents to honey bees; exposure was primarily to the tarsi and abdominal venter. Repellency was fully reversible within 24 h. No permanent effects on either memory function or foraging efficiency were observed following acute exposure.