GABA受体拮抗剂荷包牡丹碱和CGP52432对厚壳贻贝幼虫附着变态的影响

为研究GABA及其受体拮抗剂荷包牡丹碱（GABA_A受体）和CGP52432 （GABA_B受体）在厚壳贻贝幼虫附着变态中的调控作用,实验通过分析厚壳贻贝不同发育阶段幼虫转录组数据获得GABA受体相关蛋白基因（GABARAP）和GABA_B2受体基因(GABA_B2R),发现GABA_B2R在变态前期的眼点幼虫阶段相较于其他阶段幼体显著高表达。通过实时荧光定量PCR验证也得到相似的表达模式,显示GABA_B2R可能参与调控厚壳贻贝幼虫变态发育。药理学实验结果显示,10^-4 mol/L GABA对厚壳贻贝幼虫的变态具有诱导活性（27.2%±3.0%）。在拮抗剂实验中,10^-6～10^-4 mol/L荷包牡丹碱和CGP52432均显著抑制了厚壳贻贝幼虫变态,且抑制作用随浓度升高而增强。实验还发现GABA及其受体拮抗剂都抑制了厚壳贻贝幼虫的游泳行为。研究表明,GABA_A或GABA_...     

脂多糖对细菌生物被膜形成及厚壳贻贝幼虫变态的影响

为探究脂多糖(LPS)对海洋细菌生物被膜形成、海洋贝类幼虫变态所产生的作用,本研究使用不同浓度的LPS直接刺激厚壳贻贝幼虫,观察其对幼虫附着变态的直接作用;同时在海假交替单胞菌形成生物被膜的过程中添加不同浓度的LPS,通过分析生物被膜生物学特性的变化,及变化后的生物被膜对厚壳贻贝幼虫附着变态的影响。研究结果显示,3种浓度的LPS均可直接诱导厚壳贻贝幼虫的变态;10 mg/L浓度LPS处理后的生物被膜其细菌密度、膜厚明显降低(p<0.05),胞外产物中多糖(如可乐酸)、脂类显著增加(p<0.05),同时其对幼虫变态的诱导作用也显著提高(p<0.05),因此推测,在厚壳贻贝幼虫的附着变态过程中,LPS具有直接诱导作用;除此之外,脂多糖还可以通过调控生物被膜胞外物质,特别是可乐酸的生成,从而间接影响厚壳贻贝幼虫的附着变态。     

纤维素对海假交替单胞菌生物被膜生物学特性及厚壳贻贝幼虫附着变态的影响

为探究纤维素对海假交替单胞菌生物被膜的生物学特性及其对厚壳贻贝幼虫附着变态的影响,实验设置海假交替单胞菌(初始细菌密度5×10⁸个/m L)分别与浓度为0.02、0.20、2.00、20.00 mg/L的纤维素共孵育形成生物被膜,检测形成的生物被膜对厚壳贻贝幼虫变态的诱导作用变化,比较生物被膜成膜能力、胞外产物等生物学特性变化,并分析其与厚壳贻贝幼虫附着变态的关系。纤维素浓度为2.00或20.00 mg/L时,所形成的生物被膜对幼虫附着变态的诱导活性显著降低。通过分析加入纤维素后海假交替单胞菌形成的生物被膜生物学特性发现,随着纤维素浓度升高,生物被膜中细菌的分布更为分散,细菌密度和膜厚呈逐渐降低趋势,生物被膜胞外产物中α-多糖、β-多糖和脂质的生物量显著降低,而蛋白无明显变化。在海假交替单胞菌生物被膜形成过程中,纤维素主要通过降低胞外α-多糖、β-多糖和脂质的产生,进而间接调控厚壳贻贝幼虫附着变态。研究结果为探究海假交替单胞菌纤维素对生物被膜形成及对厚壳贻贝附着变态调控分子机制提供理论依据,为整治生物污损等实际问题提供新思路。     

维生素B7和B12对细菌生物被膜形成及厚壳贻贝幼虫变态的影响

为探究B族维生素对海洋细菌生物被膜形成、海洋贝类幼虫变态所产生的作用,本研究首先使用维生素B7 (VB7)和B12 (VB12)直接刺激厚壳贻贝(Mytilus coruscus)幼虫,观察其对变态的直接诱导活性;然后通过添加VB7和VB12,与海假交替单胞菌(Pseudoalteromonas marina)共同形成生物被膜,分析B族维生素对生物被膜形成及其生物学特性的影响;同时检测生物被膜变化对厚壳贻贝幼虫变态发育的影响。研究结果显示,0.02 mmol/L浓度VB7和VB12可以直接诱导厚壳贻贝幼虫的变态,且效果最为显著(P<0.05);0.02 mmol/L浓度VB7和VB12处理后的海假交替单胞菌生物被膜对幼虫附着变态的诱导作用均显著提高(P<0.05);进一步通过细菌密度计数、膜厚度分析、可拉酸染色和定量等方法,揭示VB7和VB12处理后生物被膜细菌密度、膜厚度以及胞外多糖、蛋白和脂质均显著增加(P<0.05)。研究结果证实,VB7和VB12可能通过改变海洋细菌生物被膜的生物学特性,进而调控厚壳贻贝幼虫变态发育。本研究为探究厚壳贻贝幼虫附着变态的分子机制提供了新的理论依据和创新思路,同时为B族维生素在提高厚壳贻贝人工育苗技术、改善厚壳贻贝养殖产业问题和促进海洋牧场生态修复建设等方面的应用提供了理论基础。     

化学物质诱导泥蚶幼虫附着变态的研究↑（*）

1996年8～9月间在室内用8种化学诱导物对泥蚶变态前期幼虫进行附着变态诱导实验。结果表明,8种药物对泥蚶幼虫的变态均有一定的诱导作用。其中,3-HT、EPI和NE对幼虫变态的诱导作用极显著,有效浓度范围较广;KCl和Ala的诱导作用较显著,有效浓度范围较窄;而Try、CABA和Ach只有某些浓度组的诱导效果明显,诱导效率对浓度的信赖性较强。从诱导物浓度来看,10-4mol/L的Ala、GABA和10-5mol/L的EPI诱导效果最好,与对照组相比,变态率均提高12%以上。不同处理时间试验表明,处理时间的长短与幼虫的变态率无一致的相关性关系,因药而异;总的来看,处理时间不超过8h的诱导效果较好,时间过长诱导率降低(GABA除外)。其中,用10-5mol/L的Ala处理1～4h或10-5mol/L的EPI处理4h效果最好,与对照组相比,变态率均提高25%以上。8种药物中,只有Try表现出对幼虫明显的毒性影响,Try的浓度越高,处理时间越长,幼虫的死亡率越高,用10-4mol/L的Try处理幼虫3h,死亡率达89%。     

不同温度下形成的深海菌膜对厚壳贻贝幼虫变态的影响

为探讨深海细菌生物被膜对温度的适应性及对厚壳贻贝幼虫变态的影响,分别在4、18、25、37°C条件下培养生物被膜,调查温度对细菌密度、膜厚和胞外产物等生物学特性的影响,以及被膜对幼虫变态发育的影响。结果显示,4个温度条件下产生的生物被膜均可有效促进幼虫变态发育。其中,深海菌株Virgibacillus sp. 1在18°C时生物被膜的变态诱导活性最高(35%),且α胞外多糖含量较高,诱导活性与温度和细菌密度均显著相关;同时温度与其细菌密度和膜厚均显著相关。2株深海假交替单胞菌诱导活性与细菌密度均显著相关,温度仅影响Pseudoalteromonas sp. 33细菌密度。研究表明,3株深海细菌均有很好的温度适应性,且都能形成生物被膜;温度变化导致生物被膜的生物学特性改变,最终影响其对幼虫的诱导效果。     

化学物质对不同发育天数海湾扇贝幼虫变态的诱导

自海湾扇贝(Argopectenirradians)受精后第10天起,定期取一定量海湾扇贝幼虫,分别置于不同浓度梯度的KCl、肾上腺素、去甲肾上腺素、L-DOPA、5-羟色胺、GABA、茶碱和咖啡因等8种诱导物中,处理时间为8h。实验后第14天取出幼虫观察显示,这8种诱导物对不同发育天数海湾扇贝幼虫的变态有着不同的诱导作用。13.42×10-3和20.13×10-3mol/L的KCl对第12天幼虫的变态有抑制作用,变态提高率为负值;当幼虫发育至第13和14天时,上述两浓度的KCl能够明显诱导幼虫变态,变态率均提高20%以上,而对于第16天的幼虫诱导作用有所减弱,变态提高率有所降低;26.85×10-3mol/L的KCl对第12和13天幼虫的变态均有抑制作用,变态提高率为负值,对第14和16天幼虫的变态却有明显持续的诱导作用,变态率分别提高22.98%和37.5%。神经递质肾上腺素、去甲肾上腺素、L-DOPA、5-羟色胺和GABA的诱导作用规律基本相似,即对第13天海湾扇贝幼虫的变态有明显的抑制作用,变态提高率均为负值,而对第14天幼虫的诱导作用较显著。茶碱和咖啡因作为影响细胞内cAMP的物质,其诱导作用规律与神经递质有所不同,对第13天海湾扇贝幼虫变态的诱导效果最好。     

G-蛋白偶联受体与信号通路对紫贻贝幼体附着变态的调控作用

为了阐明紫贻贝Mytilus galloprovincialis幼体附着变态的内在机理,在天然诱导物-微生物膜的诱导参照下,通过使用G蛋白偶联受体（GPCRs）与信号通路的活化剂与抑制剂初步研究了其对该种幼体附着变态过程中调控作用。结果显示,GPCRs活化剂Gpp[NH]p与抑制剂GDP-β-S没有诱导和抑制活性,表明可能非GPCRs的其他细胞反应/过程参与了M. galloprovincialis幼体的附着变态。腺苷酸环化酶/环腺苷酸（AC/cAMP）信号通路相关、理论上具有诱导活性的6种神经性试剂,除了磷酸二脂酶抑制剂（IBMX）外,其它的在10-8-10-3 M浓度下48 h、72 h后都没有活性,并且高浓度下表现出较高的神经性药物毒性。IBMX仅在10-4 M 浓度72 h后呈现出一定的诱导活性,但是与微生物膜48 h就具有非常高的活性相比显著性较低且时间滞后。磷脂酰肌醇/二酰基甘油/蛋白激酶（PI/DAG/PKC）信号通路相关的2种活化剂48 h、72 h均无诱导活性,但是其抑制剂H-7在10-6 M的低浓度下48 h后表现出显著性的抑制效果,72 h后效果丧失,抑制浓度上升到10-5 M。以上活性浓度与作用时间两个方面的结果明确表明了PI/DAG/PKC信号通路可能参与调控M. galloprovincialis幼体的附着变态,而AC/cAMP信号通路可能作为次要通路,或者由于所用神经性试剂的毒性造成的一种实验假象。本研究有助于推进该品种的发育生物学研究以及通过切断信号通路实现生物污损防治技术的开发。     

邻苯二甲酸二丁酯对厚壳贻贝抗氧化防疫系统的影响

邻苯二甲酸二丁酯（dibutyl phthalate，DBP）作为增塑剂，已被中国列入优先控制的污染物名单，其对海洋生物的影响已引起人们的广泛关注。为研究邻苯二甲酸酯类化合物（phthalic acid esters，PAEs）对海洋贝类抗氧化防疫系统的影响，本文以厚壳贻贝（）为受试生物，研究了不同质量浓度DBP（0 mg/L、100 mg/L、200 mg/L、280 mg/L、360 mg/L）胁迫以及清水释放对其鳃和内脏团中抗氧化系统相关指标超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）活性以及丙二醛（malondialdehyde，MDA）含量变化的情况。结果表明：厚壳贻贝鳃和内脏团SOD、CAT活性以及MDA含量呈现不完全相同的变化规律。DBP胁迫阶段，鳃和内脏团中SOD活性基本呈先诱导后抑制再诱导的趋势，CAT活性则先抑制后诱导，MDA含量基本呈不断升高趋势；清水释放阶段，鳃和内脏团中SOD活性基本呈先抑制后诱导的趋势，CAT活性基本处于诱导状态，之后内脏团CAT活性被抑制，MDA含量处于先升高，之后内脏团MDA含量出现降低的趋势，浓度-效应和时间-效应较为明显。研究表明，在DBP胁迫下，厚壳贻贝鳃和内脏团产生了氧化损伤，可诱导机体产生脂质过氧化现象，且15 d清水释放期间不足以让厚壳贻贝恢复到正常水平。本研究旨在为海洋贝类的健康养殖和风险评价提供科学依据。     

硅烷化表面海洋细菌对厚壳贻贝稚贝附着的影响

为探讨厚壳贻贝稚贝对自然微生物膜中海洋细菌的附着行为反应,本论文研究了厚壳贻贝（Mytilus coruscus）稚贝附着与硅烷基化附着基表面、微生物膜密度以及细菌种属系统发育之间的相互关系。研究表明所测海洋细菌均能显著促进厚壳贻贝稚贝的附着;其中Staphylococcus sp. 1和Cobetia sp. 1表现出较低诱导活性,且这两株细菌形成的微生物膜密度与稚贝附着率之间无显著相关性;其他7株海洋细菌均表现出中等程度诱导活性,且所形成的微生物膜密度与稚贝附着率之间呈显著相关性。系统发育分析表明所测海洋细菌对厚壳贻贝稚贝附着的诱导活性与细菌种属无关。因而,硅烷基化表面的海洋附着细菌对厚壳贻贝附着有着显著性促进作用,本研究将为后续开展厚壳贻贝稚贝附着机制奠定了基础。     

Effect of different ions on larval metamorphosis of the mussel Mytilus galloprovincialis

Metamorphic responses of pediveliger larvae of Mytilus galloprovincialis to different ions were investigated through a series of bioassays. Effects of tetraethylammonium chloride (TEA) on inductive effects of these above ions were also investigated. Excess ions including Li⁺, Cs⁺, Rb⁺ and Ca²⁺ induced larval metamorphosis at 10^?3 M to 5 × 10^?2 M in 24‐h exposure assays. In continuous exposure assays, only excess Ca²⁺ showed inductive activity and induced 25% metamorphosis at 5 × 10^?2 M. Larval responses to Li⁺ and Rb⁺were inhibited by TEA, while induction of metamorphosis by Cs⁺and Ca²⁺ was independent of the presence of TEA. Thus, these ions used can be useful inducers of larval metamorphosis for application in the aquaculture industry. The finding provides new insights on the biochemical mechanism controlling larval metamorphosis in this species.     

不同来源海洋弧菌微生物被膜对厚壳贻贝稚贝附着的影响

为探讨广泛存在于近海环境中海洋弧菌和贝类附着的相互作用关系,实验从自然微生物被膜和厚壳贻贝成体肠道内分离了海洋弧菌,测定其种属及亲缘性,调查了这些不同来源弧菌形成的微生物被膜与厚壳贻贝稚贝附着的调控作用。结果显示,测试弧菌形成的微生物被膜密度随着初始细菌密度的增加呈现不同程度的增加。源于自然微生物被膜和贻贝肠道内的10株弧菌均能显著促进厚壳贻贝稚贝的附着,不同菌株形成微生物被膜的诱导活性存在显著差异,附着率变化范围为17%~67%,其中稚贝在Vibrio sp.17微生物被膜上的附着率为67%±2%。微生物被膜对稚贝附着诱导活性与被膜密度呈显著相关性,其中弧菌V.crassostreae ECSMB14106的相关性系数最高,为0.8992。此外,微生物被膜的诱导活性与弧菌的来源无关。本实验初步探明了海洋弧菌对厚壳贻贝稚贝附着的影响,有助于进一步理解微生物被膜调控厚壳贻贝的附着分子机理。     

厚壳贻贝Wnt4基因时空表达

为探究Wnt4基因在厚壳贻贝幼虫发育阶段和组织生长过程中的作用,通过RACE技术克隆了厚壳贻贝Wnt4基因c DNA全长序列,该序列全长3342 bp,开放阅读框为1074 bp,编码357个氨基酸。该序列与人、小鼠、海胆、栉孔扇贝和长牡蛎等物种的同源性分别为61%、61%、60%、71%和76%。通过实时荧光定量PCR(q RT-PCR)分析Wnt4基因在厚壳贻贝成体多个组织中(外套膜、闭壳肌、鳃、雌雄性腺、足和消化腺)均有表达,其中在外套膜中表达量最高,推测可能与贝壳形成有关;Wnt4基因在厚壳贻贝幼虫发育阶段高表达主要集中在壳顶期,并推测Wnt4基因可能参与了贝壳形态结构发生转变的过程以及某些器官的形成与发育。本研究为进一步开展双壳贝类Wnt基因家族的功能研究提供了理论依据。     

培养基对微生物被膜形成和厚壳贻贝附着的影响

为探讨微生物被膜生物学特性、培养基以及大型生物附着三者之间的相互关系,本实验通过微生物分子生态学和海洋贝类生态学等方法调查了不同培养基对海洋细菌所形成微生物被膜的影响及其对厚壳贻贝幼虫附着的影响。结果显示,厚壳贻贝稚贝率与培养基类型和细菌初始密度显著相关,进一步分析表明,Zo Bell 2216E和Seawater Luriabertani(SWLB)条件下分别有6株和9株细菌形成的微生物被膜密度与稚贝率显著相关。扫描电镜结果显示,Staphylococcus sp.3在Zo Bell 2216E培养基条件下形成微生物被膜的细菌分布较为紧密,Pseudoalteromonas sp.8则在SWLB培养基条件下微生物被膜细菌分布较为紧密,且形态变为短杆状。SDS-PAGE结果显示,相比Zo Bell 2216E培养基,Staphylococcus sp.3在SWLB培养基条件下9条条带蛋白显著下降,其中2个条带完全消失;Pseudoalteromonas sp.8则在SWLB培养基条件下明显增加5条蛋白条带。研究表明,微生物被膜的形成受到培养基的影响,培养基的不同导致微生物被膜的形态结构、分布和蛋白有所差异,最终导致微生物被膜诱导厚壳贻贝幼虫附着变态的活性差异,本研究为后续开展厚壳贻贝附着的分子机制奠定良好基础。     

Formalin as an alternative to trifluralin as prophylaxis against fungal infection in mud crab Scylla serrata (Forsskål) larvae

The toxicity of formalin and trifluralin to the larval stages of the mud crab Scylla serrata was compared in a static bioassay. Prophylactic doses of 5, 10, 15, 20 and 25 μg L⁻¹ formalin and 0.05, 0.1, 0.2, 0.4 and 0.8 μg L⁻¹ trifluralin were used. Toxicity was assessed on the basis of survival of larvae after 24, 48, 72 and 96 h exposure to the test chemicals and metamorphosis to the next larval stage. Result shows that larval survival in all stages was significantly reduced at concentrations of 20 and 25 μg L⁻¹ formalin whereas larvae were able to tolerate all trifluralin treatments. However, larvae became more tolerant to high formalin concentrations as the larval stage progressed. Survival was better at 5, 10 and 15 μg L⁻¹ formalin and in all trifluralin treatments than the control in almost all the larval stages. Faster metamorphosis was observed at 5 and 10 μg L⁻¹ formalin and 0.05, 0.1 and 0.2 μg L⁻¹ trifluralin concentrations. Doses of formalin and trifluralin obtained from the toxicity experiments were applied as prophylaxis to newly hatched larvae in white plastic basins. Prophylactic doses of 5 and 10 μg L⁻¹ formalin and 0.05 and 0.1 μg L⁻¹ trifluralin applied every other day were found to be effective in enhancing survival and larval development to megalopa compared with control. However, no megalopae survived to crab instar in all formalin treatments. Although the use of fungicides in rearing systems resulted in higher survival compared with controls, other strategies (i.e. maintenance of good water quality and hygienic practices in the hatchery) should be further investigated as an alternative to the use of chemicals in hatcheries.     

肾上腺素对岩牡蛎幼虫变态的诱导

为了得到壳型规则、大小均一的单体牡蛎,本研究采用肾上腺素对岩牡蛎单体苗种生产的诱导条件进行研究,阐明了岩牡蛎单体苗种的最适诱导浓度、诱导时间和诱导密度。结果显示,肾上腺素能显著诱导岩牡蛎幼虫的不固着变态,最适诱导浓度为5×10–5 mol/L,最适诱导时间为1 h,提高诱导浓度和延长诱导时间导致岩牡蛎幼虫的不固着变态率、稚贝壳高和存活率显著降低;肾上腺素对低于8个/mL幼虫密度的诱导效果差异不显著,但稚贝壳高和存活率在8个/mL的培育密度下显著低于0.5～4个/mL,研究表明利用肾上腺素诱导岩牡蛎单体时,可大批量处理眼点幼虫,但稚贝充气培养的最适培养密度应不高于4个/mL。     

Effect of temperature on survival,growth and development of Mytilus galloprovincialis larvae

Mussel aquaculture is widely prevalent worldwide, but generally relies on natural seed collection, which does not always meet the needs of the producers. Thus, development of mussel hatcheries is of economic interest in some parts of the world, such as Europe; it provides opportunities not only on annual reliability of seed but also on genetic improvements. To broaden knowledge on mussel larval physiology, we carried out temperature treatments (17, 20 and 24 °C) on Mytilus galloprovincialis larvae under laboratory conditions. The trials ended when 30% of the larval population was in the post‐larval stage. The temperature coefficient Q₁₀ indicated a strong relationship between temperature and increase in growth from 17 to 20 °C, but not between 20 and 24 °C. Exposure of M. galloprovincialis larvae to 17 °C resulted in poor growth, low survival and a delayed development and was considered to be inadequate for M. galloprovincialis larval culture. Rearing the larvae at 20 or 24 °C produced better growth, higher survival rates and faster metamorphosis as compared with 17 °C. The temperature region within 20 and 24 °C was suggested as adequate for the mussel M. galloprovincialis larval culture, and implications of these results on the development of commercial hatcheries were discussed.     

Effects of stocking density and algal concentration on the survival,growth and metamorphosis of Bobu Ivory shell,Babylonia formosae habei (Neogastropoda: Buccinidae) larvae

Independent and combined effects of stocking density and algal concentration on the survival, growth and metamorphosis of the Bobu Ivory shell Babylonia formosae habei larvae were assessed using a 5 × 5 factorial design with densities of 0.25, 0.5, 0.75, 1.00 and 1.50 larvae mL⁻¹ and algal concentrations of 5, 10, 15, 20 and 25 × 10⁴ cells mL⁻¹ in the laboratory. Larval growth, survival and metamorphosis were significantly affected by both the independent effects of stocking density and algal concentration and by their interaction. The highest per cent survival (72.5%) and metamorphosis (49.5%), fastest growth (41.57 μm day⁻¹) and shortest time to initial metamorphosis (10 days) all occurred at the lowest stocking density and the highest algal concentration. Both crowding and food limitation had independently negative impacts on the survival, growth and metamorphosis of larvae, and these negative impacts were further strengthened by the interaction of a higher stocking density and a lower algal concentration. Moreover, the results suggest that stocking density and algal concentration obviously played different roles in determining larval survival and growth. To maximize survival and growth, B. formosae habei larvae should be reared at a lower stoking density of 0.25 larvae mL⁻¹ and fed a higher algal concentration of 25 × 10⁴ cells mL⁻¹ in large-scale hatchery seed culture.     

Effect of larval stocking density on growth,survival and whole body cortisol of the Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819) larvae reared under laboratory conditions

Interest of mussel hatchery development has increased during the last years as natural seed collection does not always satisfy the demand of producers. Success on larval bivalve culture depends on several factors such as diet, temperature or stocking density. In this study, the effect of larval density in mussel culture success has been assessed testing three larval densities: high (50 larvae mL^?1), medium (25 larvae mL^?1) and low (5 larvae mL^?1). Results showed higher growth and survival at low density but the amount of postlarvae was low. The highest number of settled larvae was observed at high density but mortality was also high and a delay on the larval development was detected producing a prolongation on the culture length. However, medium density resulted in a good survival and growth and also in a considerable amount of postlarvae, as this density could be adequate for massive mussel seed production. Cortisol content on the larvae and postlarvae was also analysed and significant differences were obtained among groups probably due to the stress associated to settlement and metamorphosis.