母体暴露环境浓度盐酸四环素对F1代斑马鱼胚胎骨骼发育的影响

抗生素被广泛应用于兽药和饲料添加剂中,尤其是四环素等广谱抗生素,长期滥用于养殖业中.四环素是目前用量最大、应用范围最广泛的一种抗生素.抗生素在体内蓄积,或者以原型随粪便排入环境中,造成环境污染.实验用斑马鱼为模式动物来评价低浓度四环素对斑马鱼下一代(F1)发育的毒性影响.选取4月龄亲代斑马鱼(F0),分别给予0.1、1和100 μg·L-1盐酸四环素(tetracycline hydrochloride,TCH)处理30 d后,实施交配后获得F1代斑马鱼胚胎.结果表明,F1代胚胎随母体TCH暴露浓度的升高而孵化率降低、畸形率和死亡率增加.同时斑马鱼幼鱼下颌骨长度、下颌弓长度变长、下颌骨宽度和舌骨角长度缩短.进一步检测与幼鱼骨骼发育相关基因表达,发现TCH抑制了runx1、sox9a、sox10和col2α1a mRNA的表达.以上研究结果表明,TCH的残留和污染可能会影响斑马鱼胚胎的发育,尤其是对胚胎软骨发育有影响.     

BDE-47对斑马鱼胚胎-幼鱼的急性毒性及氧化应激作用

为研究2,2',4,4'-四溴联苯醚(BDE-47)对斑马鱼胚胎-幼鱼急性毒性、氧化应激及细胞凋亡的影响,以受精后3 h的斑马鱼胚胎为染毒对象,用概率单位法计算BDE-47对斑马鱼胚胎-幼鱼的96 h-LC₅₀;再参照96 h-LC₅₀按一定比例级差设置0.25、0.5、1.0、2.0 mg·L^-1 4个浓度组和1个对照组(0 mg·L^-1)进行96 h半静水式毒性试验,检测斑马鱼超氧化物歧化酶(SOD)及过氧化氢酶(CAT)活性、丙二醛(MDA)含量和细胞凋亡情况。结果表明,BDE-47对斑马鱼96 h的急性毒性LC₅₀为3.77 mg·L^-1(95%可信区间1.93～10.27 mg·L^-1);2.0 mg·L^-1剂量组与对照组相比,SOD活性和MDA含量显著增加,且CAT活性与BDE-47染毒浓度之间存在明显的剂量-效应关系;0.5 mg·L^-1 BDE-47染毒96 h后,即观察到斑马鱼幼鱼出现明显细胞凋亡,主要集中于神经管和脑部。研究表明,BDE-47可以影响斑马鱼体内抗氧化防御系统,并能诱导细胞凋亡;BDE-47导致神经组织的氧化损伤可能在动物神经毒性中起重要作用。     

丙硫菌唑对斑马鱼的安全性评价及其生物富集行为研究

丙硫菌唑是一种市场前景非常好的新型广谱杀菌剂。本文研究了丙硫菌唑对水生生物斑马鱼的急性毒性和生物累积风险。通过斑马鱼的急性毒性试验获得丙硫菌唑对斑马鱼的96 h-LC₅₀为2.06 mg a.i. L^-1。随后,采用0.02 mg L^-1 (1/100LC₅₀)和0.2 mg L^-1 (1/10LC₅₀)2个浓度的丙硫菌唑,通过8 d实验,获得其在斑马鱼体内的生物累积效应。在0.02 mg L^-1组中,第8天时,斑马鱼体内的浓度达到0.733 mg kg^-1,生物富集系数(BCF_{8 d})缓慢增长到34.36。而在0.2 mg L^-1组,第8天时,斑马鱼组织内丙硫菌唑浓度为4.198 mg kg^-1, BCF_{8 d}值为19.72。结果表明,丙硫菌唑对斑马鱼的毒性等级为中毒,同时其在斑马鱼体内具有中等生物累积效应。因此,在使用过程中,需要考虑其对水生生物的毒性和水生食物链的放大效应。     

甲状腺激素脱碘酶在孕哺期BDE 209暴露致子代小鼠神经发育毒性中的潜在作用

研究孕哺期BDE 209暴露对母鼠胎盘和子代脑组织甲状腺激素脱碘酶(deiodinase, DI)基因表达的影响,及其在子鼠神经发育毒性效应中的作用。将75只雌性昆明小鼠随机分为对照组、低剂量组和高剂量组,暴露BDE 209 10 d后,与雄鼠合笼,每组选取怀孕时间相近(相差不过2 d)的8只母鼠孕期持续染毒至子鼠断乳。采用实时荧光定量PCR检测孕17～18 d胎盘、出生后60 d子鼠脑组织三种类型脱碘酶基因相对表达;利用Morris水迷宫评价出生后60 d子鼠学习记忆能力;测量第2、16、30和60 d子鼠体重,观察孕哺期BDE 209暴露对子代生长发育的作用。结果显示,BDE 209对子鼠出生时体重未见明显影响;出生后30 d,高剂量BDE 209暴露组雌性子鼠体重显著低于对照组子鼠体重(p<0.05),而低、高剂量暴露组雄性子鼠体重均显著低于对照组子鼠体重(p<0.05,p<0.01);出生后60 d,BDE 209对体重影响不明显。BDE 209暴露能够显著延长出生后60 d雌、雄性子鼠逃避潜伏期(p<0.05或p<0.01)。BDE 209暴露显著降低母鼠胎盘中三种类型脱碘酶(主要是DI-3)基因表达(p<0.05或p<0.01);同时,BDE 209暴露可诱导出生60 d后雄性子鼠脑组织中DI-1基因表达(p<0.05或p<0.01),抑制雄性子鼠脑组织中DI-3基因表达(p<0.05);BDE209暴露对出生后60 d雌鼠脑组织脱碘酶未见明显影响(p>0.05)。研究结果表明,孕哺期BDE 209暴露可能通过影响母鼠胎盘组织脱碘酶(特别是DI-3)基因表达,导致子鼠神经发育毒性效应-学习记忆能力障碍。     

全氟辛烷基磺酸钾(PFOS)和纳米氧化锌(Nano-ZnO)复合暴露对斑马鱼下丘脑-垂体-甲状腺轴功能的影响

全氟辛烷基磺酸钾(PFOS)和纳米氧化锌(Nano-Zn O)广泛存在于环境中,但是它们复合暴露对水生生物的潜在毒性机制尚未明确。本文探讨PFOS和Nano-Zn O复合暴露对斑马鱼下丘脑-垂体-甲状腺轴(HPT轴)毒性的影响。将斑马鱼胚胎从孵化开始暴露于PFOS(0.2、0.4、0.8 mg·L~(-1))、Nano-Zn O(6.75、12.5、25 mg·L~(-1))、PFOS+Nano-Zn O(0.2+6.75、0.4+12.5、0.8+25 mg·L~(-1))溶液中15 d后,分析幼鱼的发育毒性,体内的甲状腺激素(甲状腺素(T4)和三碘甲状腺氨酸(T3)含量和与甲状腺有关基因(CRF、TSH、NIS、TG和TPO)的表达情况。结果发现复合暴露组与单独暴露组相比,前者显著诱导了幼鱼的畸形率,降低了幼鱼的存活率,并抑制了幼鱼的体长。复合暴露组显著增加了幼鱼体内T3含量,同时抑制体内T4的含量。与单独暴露组相比,复合暴露组显著诱导了CRF和NIS基因的表达,同时抑制了TSHβ和TG基因的表达。而TPO基因的表达水平在单独和复合暴露组中没有显著差异。本研究首次证明了PFOS和Nano-Zn O复合暴露对斑马鱼幼鱼甲状腺轴的干扰效果并对其进行了机制探讨。     

中华圆田螺对沉积物中十溴联苯醚工业品DE-83的生物积累与转化

为研究沉积物中十溴联苯醚(BDE-209)的生物有效性,将淡水生态系统中常见的底栖动物中华圆田螺(Cipangopaludina cahayensis)暴露于商品DE-83(主要包含92%的BDE-209、6%的BDE-206、1.5%的BDE-207和0.5%的BDE-208)染毒的沉积物,进行60 d养殖实验。根据暴露期间中华圆田螺体内PBDEs含量与同系物组成的变化,探讨了田螺对DE-83的富集动力学、积累常数以及可能发生的生物转化。实验结果显示,DE-83主要同系物均能够被中华圆田螺积累,但是生物有效性非常低,BDE-209、BDE-207和BDE-206的吸收速率常数(k_s)为0.029～0.042 d^-1,BDE-207 > BDE-206 > BDE-209。依据动力学参数推算的生物-沉积物积累因子(BSAF)非常低,分别为0.05(BDE-209)和0.02(BDE-206和BDE-207)。暴露20 d后,中华圆田螺体内有低溴代同系物检出,其含量随暴露时间延长而增加,说明BDE-209在中华圆田螺体内发生了生物转化。     

评估斑马鱼胚胎毒性试验在农药危害筛选中的应用

考虑到人类社会中大量使用化学物质，想要准确有效地评估这些化学物质对人类和生态受体的潜在风险，研发有效的手段和方法是至关重要的。鱼胚胎急性毒性试验是其中一种工具，已表现出与幼鱼急性毒性标准试验的预测结果高度吻合，而幼鱼试验对资源消耗更多。然而，也有证据表明，对于某些类型的化学物质，包括神经毒素，鱼类胚胎的敏感性低于幼鱼。本文利用已发表的斑马鱼胚胎毒性数据，与3种常用检测用鱼幼鱼(虹鳟鱼、蓝鳃太阳鱼、羊鲷)的半致死浓度50%(LC₅₀)数据进行比较，研究了鱼类胚胎对农药危害评估的效用。在将农药视为单因素的情况下，斑马鱼胚胎和幼鱼毒性数据相关性较差，差异显著(r²=0.28；p2=0.64；p 精选自Glaberman, S., Padilla, S. and Barron, M. G. (2017), Evaluating the zebrafish embryo toxicity test for pesticide hazard screening. Environmental Toxicology and Chemistry, 36: 1221–1226. doi: 10.1002/etc.3641
详情请见http://onlinelibrary.wiley.com/doi/10.1002/etc.3641/full
     

环丙沙星(CIP)对鲤鱼早期生长阶段的影响

我们依据经济合作与发展组织的210指导性文件,利用鲤鱼受精卵,进行环丙沙星(CIP)的毒性试验。测试浓度设置为1、100、500、1 000和3 000 μg?L^L-1。我们发现在所有测试组中,暴露于CIP会加速孵化,但在暴露于最高浓度CIP(3000 μg?L^L-1)的测试组,出现了显著的生长下降的现象。在实验的第6天(孵化之后),可以观察到大量增加的肉眼可见的形态异常现象。最高数量肉眼可见的形态异常现象是在自由胚胎和幼虫CIP暴露浓度为100、500、1 000和3 000 μg?L^L-1的测试组中产生的(测试样本的20%~23%)。在所有CIP暴露试验组中,均检测出GST活性的呈逐步降低趋势,但是在CIP浓度为500和3 000 μg?L^L-1的测试组中出现了显著差异(p < 0.01)。其中在大多数测试浓度下,谷胱甘肽过氧化物酶和过氧化氢酶的活性增加(p < 0.01,p < 0.05),而在CIP暴露浓度为500和3 000 μg?L^L-1的测试组,谷胱甘肽还原酶出现活性降低的现象(p < 0.05)。在所有的CIP暴露组中,硫代巴比妥酸活性物质的浓度明显降低(p < 0.01)。可测的CIP最低有效浓度(LOEC)是1 μg?L^L-1。\这些结果表明,孵化过程,早期的个体发育,形态异常的产生,以及鱼类体内的抗氧化和生物转化酶活性和脂质过氧化反应,都会受到CIP影响。
精选自Dana Zivna, Lucie Plhalova, Lucie Chromcova, Jana Blahova, Miroslav Prokes, Misa Skoric, Petr Marsalek, Eva Praskova, Stanislava Stepanova, Zdenka Svobodova. The effects of ciprofloxacin on early life stages of common carp (Cyprinus carpio). Environmental Toxicology and Chemistry: Volume 35, Issue 7, pages 1733–1740, July 2016. DOI: 10.1002/etc.3317
详情请见http://onlinelibrary.wiley.com/doi/10.1002/etc.3317/full
     

Di (2-ethylhesyl)(DEHP)和Monoethylphthalate(MEHP)长期暴露对海洋青鳉(Oryzias melastigma)内分泌干扰效应的评价

为探讨邻苯二甲酸二(2-乙基己)酯(DEHP)和邻苯二甲酸单乙基己基酯(MEHP)长期暴露对海水生物的内分泌干扰效应及机制,将孵化后1周的海洋青鳉(Oryzias melastigma)分别暴露于DEHP(0.1 mg·L^-1和0.5 mg·L^-1)和MEHP(0.1 mg·L^-1和0.5 mg·L^-1)6个月。结果显示：DEHP显著增加了雌性和雄性海洋青鳉的肝指数,而MEHP只在高剂量时显著增加雄性青鳉的肝指数。对于雌性青鳉,DEHP暴露后肝脏雌激素相关基因ERα、ERβ、ERγ、VTG1、VTG2、ChgH和ChgL的表达水平显著上调,而对于雄性青鳉,DEHP暴露后,只有肝脏ERβ的表达水平显著上调。相比之下,MEHP暴露对雌性和雄性青鳉肝VTG和Chg基因表达无显著影响。DEHP激活了雌性和雄性青鳉的肝过氧化物增殖激活受体PPARα和PPARγ,而MEHP只在低剂量时上调了雄性青鳉PPARγ的表达。在雌性和雄性青鳉体内,VTG和Chg的表达与ERα和ERγ的表达显著相关,并且ER与PPAR也显著相关。研究表明,DEHP长期暴露可通过激活肝性激素受体调控肝雌激素响应基因(VTG和Chg)和过氧化物增殖激活受体(PPARα和PPARγ)的表达而对海洋青鳉产生内分泌干扰效应,并且显示出性别特异性。MEHP对海洋青鳉的内分泌干扰效应弱于DEHP。     

磷酸三(2,3-二氯丙基)酯阻燃剂对稀有鮈鲫的毒性效应

为研究新型阻燃剂磷酸三(2,3-二氯丙基)酯(TDCPP)的毒性效应,以稀有鮈鲫(Gobiocypris rarus)为实验生物,采用半静态实验方法,分别进行96 h和28 d的染毒,研究TDCPP对稀有鮈鲫的急性毒性和慢性毒性效应,并通过对稀有鮈鲫脑组织中与神经纤维的生长、发育、轴突再生等相关基因mRNA表达量的检测,初步探讨了TDCPP的神经毒性作用。结果表明：TDCPP对稀有鮈鲫的96 h-LC₅₀为2.99(2.20～3.38) mg·L^-1,根据化学物质对鱼类毒性分级标准TDCPP属于剧毒性。经0.9、1.5、2.1和2.7 mg·L^-1 TDCPP染毒28 d,与对照组相比,暴露组稀有鮈鲫肝脏及脑组织中SOD和GSH-Px的活性均受到抑制,且随着TDCPP的暴露浓度增加,其抑制作用显著增强;暴露组稀有鮈鲫脑组织中与神经纤维的生长、发育相关的微管蛋白α/β、神经丝NF-M以及关键蛋白GAP-43等基因的mRNA表达量,与对照相比均发生显著下调。可知,TDCPP暴露可以诱发稀有鮈鲫神经毒性作用。     

三氯化铝在酸性环境中对斑马鱼幼鱼运动及学习记忆能力的影响

以斑马鱼胚胎和幼鱼为实验对象,探讨三氯化铝(Al Cl3)在酸性环境中对斑马鱼幼鱼运动能力及学习记忆的影响。将受精后6 h(hours post-fertilization)的斑马鱼胚胎分成2个大组,分别为对照组和三氯化铝组,以观察三氯化铝的毒性作用。每一大组又分为中性(pH7.4)、微酸(pH6.4)和酸性(pH5.4)组,以观察酸性环境分别及与三氯化铝的共同毒性作用。分别采用72 hpf斑马鱼幼鱼机械逃避反射实验、144 hpf斑马鱼幼鱼自发运动、168 hpf单一多次光刺激斑马鱼幼鱼实验等探讨在中性、微酸和酸性环境下暴露铝离子对斑马鱼幼鱼运动能力以及学习记忆的影响。结果显示:与中性环境下的三氯化铝组相比,微酸和酸性环境下的三氯化铝组可以使斑马鱼幼鱼机械逃避次数减少(P0.05),平均速度和移动距离下降(P0.001),斑马鱼幼鱼运动轨迹杂乱无章,自发触壁活动减少;单一多次光刺激实验结果发现,微酸和酸性环境下的三氯化铝组使斑马鱼幼鱼到达平台速度的光照次数显著增加且差异具有统计学意义(P0.001)。以上结果说明,三氯化铝在微酸和酸性环境下,能导致斑马鱼幼鱼运动能力及学习记忆能力下降,并且酸度值越小,对斑马鱼幼鱼运动和学习记忆的影响更显著。酸度值与三氯化铝之间存在协同作用,酸性环境可以使三氯化铝毒性增加。     

Characteristics of feeding on dinoflagellates by newly hatched larval crabs

The zoeal larvae of brachyuran crabs must feed soon after hatching on a diet that includes large micro- and mesozooplankton in order to satisfy nutritional requirements. However, newly hatched larvae have been shown to ingest a variety of dinoflagellates, perhaps using microbial carbon sources to sustain them until they encounter more favored prey. Ingestion of dinoflagellates by larval crabs has been documented previously under conditions in which the larvae were exposed to algae provided in monoculture or in defined mixtures of cells. We report here on experiments conducted on the hatching stage of five crab species to determine if ingestion of dinoflagellates occurred when they were provided in combination with Artemia sp. nauplii or after a period of feeding on mesozooplankton. Quantitative measurements of chl a in the larval guts provided evidence of ingestion of algal cells. Active ingestion of the dinoflagellate Prorocentrum micans at specified intervals during an extended feeding period was determined on larvae of two crab species using fluorescently labeled cells provided for brief periods at prescribed time intervals. Stage 1 larvae of four of the five crab species ingested dinoflagellates when they were provided in combination with nauplii and larvae of all five species ingested cells after feeding solely on nauplii for 24 h. Ingestion of algal cells was first evident in the larval guts after 6 h of feeding at both low (200 cell ml⁻¹) and high (1,000 cells ml⁻¹) prey densities. Higher prey densities resulted in higher gut chl a. Larvae continuously exposed to dinoflagellates actively ingested cells at every 3 h interval tested over a 36 h period. Results confirm previous studies that larvae will ingest dinoflagellates even when they are encountered in a mixed prey field or when having previously fed. Ingestion of cells may occur on a continual basis over time.     

Biochemical adaptations of the mudskipper Boleophthalmus boddaerti to a lack of oxygen

Specimens of the mudskipper Boleophthalmus boddaerti, collected along the estuarine canal at Pasir Ris, Singapore between April 1987 and December 1989, were examined in the laboratory. After being exposed to environmental hypoxia for 6 h, no accumulation of lactate, alanine or succinate was observed in the muscle tissue of B. boddaerti. In addition, the blood lactate content and muscle creatine phosphate (CrP), adenosine triphosphate (ATP) and glycogen contents were not significantly different from those of the control. During normoxic recovery, oxygen debt repayment was only a small fraction of the oxygen deficit incurred during the 6 h of hypoxic exposure. Therefore, it would appear that B. boddaerti coped with environmental hypoxia by undergoing metabolic rate reduction. After anoxic exposure, the CrP, ATP and adenosine diphosphate (ADP) contents in the muscle tissue of B. boddaerti were significantly lower than the corresponding control values. Lactate accumulated in the blood and muscle tissue of the anoxic fish though the muscle energy charge and glycogen content remained constant. Hence, B. boddaerti was able to cope with such a period of anoxia without increasing its glycolytic rate. Succinate, which was not detected in the muscle tissue of the control and hypoxia-exposed fish, accumulated in significant quantities in the muscle tissue of B. boddaerti exposed to environmental anoxia and functional hypoxia. In the case of functional hypoxia, there was a significant decrease in the muscle glycogen content, and the muscle lactate content increased 17.3-fold, indicating that glycolysis was activated to provide the source of energy during muscular exercise.     

六溴环十二烷(HBCD)对H4细胞和SK-N-AS细胞脱碘酶2和3表达的影响

六溴环十二烷(hexabromocyclododecane,HBCD)与多溴联苯醚(polybrominated diethyl ethers,PBDEs)复合污染体系,对人类健康尤其神经系统所造成的潜在危害及其机制一直是笔者课题组的研究方向。HBCD是广泛使用的溴化阻燃剂,与PBDEs一样,会通过干扰内分泌系统、影响甲状腺激素分泌及损伤神经系统,对生物体产生发育神经毒性。作为系列研究之一,本研究以H4人脑神经胶质瘤细胞和SK-N-AS人神经母细胞瘤细胞为体外生物模型,通过观察HBCD对H4细胞Ⅱ型脱碘酶(Dio2)和SK-N-AS细胞Ⅲ型脱碘酶(Dio3)表达的调控,初步探讨了HBCD对神经系统局部甲状腺激素水平的潜在影响。H4细胞和SK-N-AS细胞分别暴露于0、1、3和9μmol·L-1HBCD 24 h后,采用MTT法检测细胞活力,Western Blot和RT-PCR法分别分析Dio2和Dio3蛋白和基因的表达,酶联免疫吸附测定(ELISA)法检测H4细胞脑源性神经细胞营养因子(BDNF)的分泌。结果表明,HBCD以剂量依赖方式降低H4细胞和SK-N-AS细胞生存率,引起H4细胞Dio2蛋白和基因表达下调,而致SK-N-AS细胞Dio3蛋白和基因表达上调。此外,HBCD还降低H4细胞BDNF的分泌。这表明,HBCD很可能通过影响神经和胶质细胞脱碘酶的表达,影响脑局部甲状腺激素水平,从而引起神经系统损伤及发育神经毒性。     

芴对斑马鱼不同发育阶段毒性效应的比较

为考察多环芳烃芴对斑马鱼的毒性效应,将斑马鱼成鱼以及0 hpf(孵化后0 h)和10 hpf(孵化后10 h)的斑马鱼胚胎分别暴露于不同浓度的芴溶液中,观察各时间段半数致死浓度(LC50)及各毒理学终点的半数效应浓度(EC50)。结果表明,成鱼、0hpf胚胎、10 hpf胚胎的48 h-LC50分别为4.013、6.074、28.980 mg·L-1,三者对芴的致死敏感性为成鱼0 hpf胚胎10 hpf胚胎。芴的毒性作用主要发生在胚胎的分节期(10 h)之前,0 hpf胚胎各项指标敏感性依次为:36 h心率异常48 h心包囊肿24 h无主动运动48 h卵黄囊水肿24 h发育阻滞。斑马鱼对芴染毒最敏感指标为36 h心率异常,0 hpf胚胎心率值随染毒浓度增大而减小,并在8 mg·L-1达到抑制峰值,心率最大抑制率为41.3%。芴主要作用于斑马鱼胚胎心脏器官组织。     

Effects of the insect growth regulator Dimilin® (TH 6040) on larval development of two estuarine crabs

Effects of Dimilin^® (TH 6040), an insect growth regulator which interferes with the formation of the insect cuticle, were studied on the larval development of Rhithropanopeus harrisii (Gould) and Sesarma reticulatum (Say) (Crustacea: Brachyura). When larvae were exposed to 0.5 (R. harrisii only), 1, 3, 5, 7, and 10 ppb Dimilin from hatching to the first crab stage, survival in both species decreased in relation to increased concentrations of Dimilin. Survival of R. harrisii larvae wa significantly lower at 1 ppb and higher levels compared with control experiments, and in S. reticulatum a significant decrease in survival began at the 3 ppb level. At 10 ppb Dimilin, no larvae survived to the megalopa stage in either of the two species. The results indicate that early stage larvae of R. harrisii are more sensitive to Dimilin than those of S. reticulatum. When R. harrisii larvae were treated with 10 ppb Dimilin during the intermolt period of each of the 4 zoeal stages, nearly all larvae died during molting to the succeeding stage. First zoeal larvae of R. harrisii exposed to 10 ppb Dimilin at various days during the intermolt period were more sensitive to the compound late than early in the period. It is suggested that Dimilin also may interfere with the formation of the cuticle in crab larvae.     

Seasonal variation in the properties of muscle mitochondria from the tropical scallop Euvola (Pecten) ziczac

In scallops, gametogenesis leads to considerable transfer of energetic reserves from the adductor muscle to the gonads. During an annual cycle, the scallops are exposed to changes in temperature and food availability. As these changes may affect muscle metabolic capacities, we examined whether the properties of the mitochondria in the phasic adductor muscle were modified during the annual cycle of the scallop Euvola (Pecten) ziczac (L. 1758). During our study, temperature and chlorophyll a levels generally showed an inverse relationship: high temperatures and low chlorophyll a levels occurred from mid-April to early June. Lower temperatures and higher chlorophyll a levels were found from January to late March and from mid-June to mid-September. Throughout the annual cycle, the substrate preferences and the pH sensitivity of the isolated muscle mitochondria changed little, whereas the maximal oxidative capacities and respiratory control ratios (RCR) varied considerably. Consistently, the maximal capacities for substrate oxidation were 30 to 80% lower in mitochondria isolated in May than at other times in the year. The RCR values of mitochondrial oxidation of glutamate, glutamine and succinate varied throughout the year with lower values characterizing the mitochondria from scallops harvested in May and in certain cases in August. In May, adductor muscles had lower protein levels than at other times. These data suggest that the requirements of gametogenesis, coupled with␣the high temperatures and low food availability occurring during April and May, led to a mobilization of muscle proteins which concomitantly decreased the oxidative capacity of isolated mitochondria. Received: 29 November 1996 / Accepted: 5 December 1996     

Correlating gene expression with larval competence,and the effect of age and parentage on metamorphosis in the tropical abalone<Emphasis Type="Italic"> Haliotis asinina</Emphasis>

The non-geniculate crustose coralline alga (CCA) Mastophora pacifica can induce the metamorphosis of competent Haliotis asinina (Vetigastropoda) larvae. The ability to respond to this natural cue varies considerably with larval age, with a higher proportion of older larvae (e.g. 90 h) able to metamorphose in response to M. pacifica than younger larvae (e.g. 66 h). Here we document the variation in time to acquisition of competence within a larval age class. For example, after 18 h of exposure to M. pacifica, approximately 15 and 36% of 84 and 90-h-old H. asinina larvae had initiated metamorphosis, respectively. This age-dependent response to M. pacifica is also observed when different aged larvae are exposed to CCA for varying periods. A higher proportion of older larvae require shorter periods of exposure to CCA than younger larvae in order to initiate metamorphosis. In this experiment, as in the previous, a small proportion of young larvae were able to respond to brief periods of CCA exposure, suggesting that they had developed the same state of competency as the majority of their older counterparts. Comparisons of the proportions of larvae undergoing metamorphosis between families reveals that parentage also has a significant (P<0.05) affect on whether an individual will initiate metamorphosis at a given age. These familial differences are more pronounced when younger, largely pre-competent larvae (i.e. 66 h old) are exposed to M. pacifica, with proportions of larvae undergoing metamorphosis differing by as much as 10 fold between families. As these data suggest that variation in the rate of development of the competent state has a genetic basis, and as a first step towards identifying the molecular basis to this variation, we have identified numerous genes that are differentially expressed later in larval development using a differential display approach. Spatial expression analysis of these genes suggests that they may be directly involved in the acquisition of competence, or may play a functional role in the postlarva following metamorphosis.Communicated by M.S. Johnson, Crawley     

Ultrastructural study of the exoskeleton of the estuarine crab Rhithropanopeus harrisii: Effect of the insect growth regulator Dimilin® (diflubenzuron) on the formation of the larval cuticle

Ultrastructure of larval cuticle during the molt cycle of the estuarine crab Rhitropanopeus harrisii (Gould) (Crustacea: Brachyura) was studied in control larvae as well as in larvae exposed to 10 ppb of the insect growth regulator Dimilin^® (diflubenzuron). First zoeal larvae were used as test organisms. It has earlier been shown that 10 ppb Dimilin is lethal to zoeal larvae of R. harrisii, and nearly all exposed larvae died during molting to the next stage (Christiansen et al., 1978). Distinct differences in structure of the cuticle were found between the two groups of larvae. Both endocuticle and exocuticle appear to be deformed in Dimilin-treated larvae, whereas formation of epicuticle did not seem to be affected. The results indicate that Dimilin probably inhibits chitin synthesis in crab larvae as shown earlier by several authors for insect larvae.     

Vertical distribution of eggs and larvae ofEngraulis encrasicolus in stratified waters of the western Mediterranean

The vertical distribution of the eggs and larvae of the European anchovy (Engraulis encrasicolus) in the western Mediterranean Sea in June 1984 and Agust 1985 was analyzed based on multiple plankton tows carried out at varying depths and using nets equipped with opening and closing mechanisms. Hydrographic parameters such as temperature, salinity, and chlorophylla were recorded simultaneously. Maximum abundance of anchovy eggs and larvae always occurred above the thermocline, even when maximum chlorophylla concentrations were located below the thermocline. Larval distribution appeared to be associated with the availability of suitable food organisms. As in other clupeoid species, the anchovy larvae carried out vertical migrations related to the photoperiod.