马尾松人工林土壤各粒径团聚体湿筛后的有机碳分配

选取25a、45a和65a马尾松人工林为研究对象,采用湿筛法对各粒径土壤团聚体分别湿筛。探究了马尾松人工林各粒径团聚体湿筛后的团聚体有机碳分配,以探讨各粒径团聚体湿筛后分配到同一粒级团聚体有机碳含量及其对团聚体水稳性的贡献差异。结果表明：种植年限增加显著降低土壤团聚体水稳性（P<0.05）;各粒径团聚体湿筛后分配的有机碳随粒级减小含量呈先降后增趋势,以保持原粒级团聚体有机碳（12.96-32.01 g/kg）含量最高,其次是<0.25 mm粒级（8.08-23.53 g/kg）。各粒径团聚体湿筛分配到同一粒级的有机碳以保持原粒级的含量最高（P<0.05）;土壤团聚体水稳性与各粒径团聚体湿筛后保持原粒径的有机碳呈显著或极显著正相关（P<0.05或0.01）,分配到越小的粒级正相关性越不显著。此外,团聚体水稳性与各粒径团聚体湿筛分配到同一粒级的有机碳呈正相关,以保持原粒级相关性最高（P<0.01或0.05）;回归方程及相关性系数表明,有机碳与保持原粒径团聚体呈显著呈或极显著正相关（P<0.05或0.01）,与消散到其他粒级的团聚体呈负相关或极显著负相关（P>0.05或<0.01）。本研究得出有机碳含量增加促进更大粒径团聚体形成。反之,促使大粒径团聚体向较小粒径团聚体转化。同一粒级团聚体间,保持原粒级团聚体比易转化形成更大粒级团聚体有更高的有机碳含量和更强的水稳性,这对团聚体的固碳提供了新的认识。     

利用饵料微藻表达抗菌肽及其初步应用

为了解抗菌肽在饵料微藻中表达后的抗菌特性,构建海洋微拟球藻(Nannochloropsis oceanica)、湖泊微拟球藻(N.limnetica)和三角褐指藻(Phaeodactylum tricornutum)的抗菌肽(源自虹鳟,Cath-1a)表达质粒,分别转化相应的微藻,检测转化子中抗菌肽的表达量和体外抑菌效果,将藻株作为鱼饲料添加剂喂食斑马鱼,初步分析了抗菌肽及藻体自身的岩藻黄素和多不饱和脂肪酸对鱼免疫系统的影响。结果表明,外源抗菌肽在3种微藻中均可以成功表达,体外抑菌试验表明,仅三角褐指藻对水产领域常见致病菌爱德华氏菌(Edwardsiella tarda)有一定的抑菌效果,然而抗菌肽的表达并未使3种藻株的体外抑菌性增加。添加藻粉对斑马鱼的生长无明显影响,通过检测鱼体肝脏中与抗氧化和免疫相关基因的表达水平及丙二醛的含量,表明添加藻粉可增强斑马鱼的抗氧化和抗炎症能力,表达抗菌肽(PtC组)能进一步提高斑马鱼的免疫力。另外,添加Pt6(富含岩藻黄素)藻粉组比添加PtC的抗炎效果更显著,表明三角褐指藻中的岩藻黄素和二十碳五烯酸对增强鱼的抗病能力具有潜在作用。     

世界自然遗产地陆生藻类物种多样性比较研究

藻类是真核生物进化、植物起源与进化、生物地理学研究的良好材料,研究藻类物种多样性对理解生命起源与早期进化,探明地球地质地貌演化有着非常重要的意义。以两世界自然遗产地施秉云台山白云岩喀斯特、赤水丹霞的陆生藻类为研究对象,通过G-F多样性指数、Jaccard相似性系数、Bray-Curtis矩阵比较分析了不同地质地貌的两世界自然遗产地陆生藻类的物种多样性及种组特征,结果如下：（1）两地陆生藻类丰富,均以蓝藻为优势,共有优势科为色球藻科Chroococcaceae、颤藻科Oscillatoriaceae,施秉云台山还具伪枝藻科Scytonemataceae、念珠藻科Nostocaceae优势,优势属为粘球藻属Gloeocapsa、色球藻属Chroococcus,赤水丹霞仅以粘球藻属Gloeocapsa为优势。（2）施秉云台山G-F多样性指数高于赤水丹霞。（3）两地陆生藻类泛热带成分明显,施秉云台山中国特有成分较赤水丹霞丰富;两地科、属、种相似性系数依次为：56.25%、44.05%、37.62%,科的相似性达到中度水平,与同纬度范围其它地区的Bray-Curtis矩阵聚类结果显示不同地质地貌的两地首先聚为一类。从陆生藻类多样性组成上,初步反映两世界自然遗产地的地质历史、地层岩性及地理位置的异同。     

通过同源重组在聚球藻7002中表达小鼠金属硫蛋白-Ⅰ的初步研究

用PCR方法从海洋单细胞蓝藻聚球藻7002（Synechococcus sp. PCC 7002）基因组DNA中扩增得到藻蓝蛋白β亚基基因（cpcβ）的上游序列（Pcpcβ）,及编码谷氨酰胺合成酶的glnA基因片段．以Pcpcβ作为启动子、以glnA基因片段作为整合平台,构建含有小鼠金属硫蛋白-Ⅰ（mMT-Ⅰ）cDNA的同源整合表达载体pKGC-MT.通过自然转化法将整合表达载体导入聚球藻7002中,经氨苄青霉素筛选,得到遗传性状稳定的转基因藻.PCR检测证明mMT-Ⅰ基因已整合到蓝藻基因组DNA上;蛋白质印迹表明mMT-Ⅰ已在蓝藻中表达;ELISA结果显示mMT-Ⅰ在蓝藻中的表达量约为800 μg/g.     

云南石蕊共生藻形态及分子系统学研究

该研究以2016年采自云南凤凰山的云南石蕊(Cladonia yunnan)为材料,在无菌条件下,采用微量微管吸法和直接法分离纯化培养云南石蕊共生藻,利用光学显微镜观察共生藻细胞的形态结构特征,并对该共生藻进行分子系统学分析。结果表明：(1)从云南石蕊中仅分离到1种共生藻,该藻细胞大小约2.5~6 μm,球形或椭球形,单细胞,不运动,色素体片状,无性繁殖,含有中央蛋白核和淀粉粒,且3~5个细胞包裹在粘液状的胶壳中,细胞核不可见。(2)该共生藻主要形态学特征与自由生活的胶球藻高度相似,根据采集地及形态特征将其命名为云南胶球藻(Coccomyxa sp. YN)。(3)基于对云南石蕊分子数据(rbcL, ITS)构建的云南石蕊共生藻的系统发育关系显示,云南胶球藻与胶球藻聚为一枝; ITS2 rRNA二级结构分析发现,云南胶球藻与胶球藻具有几乎一致的ITS2 rRNA二级结构。该研究首次揭示了胶球藻属(Coccomyxa)藻细胞与石蕊科地衣共生,且云南胶球藻与胶球藻具有高度一致的亲缘关系,而“地衣化”使两者细胞形态特征有明显差异,推测共生藻细胞可能是由自由生活的藻细胞进化而来。     

云冷杉红松林掘根倒木及其微立地对凋落叶分解速率及养分释放的影响

采用凋落物分解袋埋藏法,将采集的红松（Pinus koraiensis）、紫椴（Tilia amurensis）和色木槭（Acer mono）凋落叶按自然比例混合装入袋中,于2019年7月将其埋入云冷杉红松林中不同腐烂等级（Ⅱ、Ⅲ、Ⅳ级）的掘根倒木（UT）及其形成的坑底（PB）与丘面（MF）以及对照（包括林隙（FG）和林分（IS））等11种微立地下。8-10月每月从每种微立地取回3袋,处理后测定凋落叶的质量、碳、氮、磷含量。采用相关分析和方差分析的统计分析方法,探究不同微立地下凋落叶的质量、分解速率与养分元素之间的相关性和差异显著性。结果表明：（1）微立地和分解时间对凋落叶质量具有极显著影响（P<0.01）。凋落叶分解90天后,各微立地的凋落叶质量残留率排列为：PB （85.64%） > MF （83.09%） > FG （81.33%） > IS （80.93%） > UT （80.27%）;（2）各微立地的凋落叶分解速率排列为：k_IS=k_UT>k_FG>k_MF>k_PB;Olson指数模型能够较好的模拟各微立地的凋落叶分解动态;年分解速率k为0.61-1.42,分解50%和95%所需时间分别为0.49-1.14 a和2.10-4.92 a。（3）各微立地的凋落叶C残留率总体呈下降趋势,表现为释放模式;UT和MF微立地的N、P残留率持续下降,表现为释放;PB微立地的N、P变化模式分别为释放-富集-释放、富集-释放模式,释放均大于富集,最终表现为释放模式。微立地对凋落叶P释放具有极显著影响（P<0.01）,对N释放具有显著影响（P<0.05）;微立地与掘根倒木腐烂等级的交互作用对C、N释放均具有极显著的影响（P<0.01）。（4）凋落叶质量残留率与P的残留率在所有微立地中均存在显著或极显著正相关关系,与N的残留率仅在Ⅱ级UT、PB微立地、Ⅲ级UT微立地、Ⅳ级UT、PB和MF微立地以及FG对照中呈显著或极显著的正相关关系,与C的残留率仅在Ⅱ级UT、PB微立地、Ⅳ级PB、MF微立地中存在极显著或显著正相关关系,与C/N在Ⅱ级UT微立地、Ⅲ级UT、MF微立地和FG对照中呈显著或极显著负相关关系。可见,与不同腐烂等级的掘根倒木相比,掘根倒木形成的微立地环境对凋落叶分解和养分释放的影响较大。     

大陈岛海域浮游动物群落季节变化及其影响因素

为探究大陈岛海域浮游动物群落的季节变化,于2020年9月（夏季）、11月（秋季）和2021年1月（冬季）、4月（春季）分别对大陈岛海域的浮游动物及环境因子进行了4个航次的调查。结果共鉴定浮游动物90种,包括浮游幼体15类,其中夏季种类数最多（68种）,冬季最少（20种）,常见的优势种有：百陶箭虫（Sagitta bedoti）、微刺哲水蚤（Canthocalanus pauper）、中华哲水蚤（Calanus sinicus）等12种（Y>0.02）。浮游动物的年平均丰度和生物量分别为（153.40±214.73）个/m³、（411.93±561.76） mg/m³,二者存在明显的季节变化,平均丰度为春季（380.17±296.14）个/m³>夏季（135.30±112.59）个/m³>秋季（67.88±90.52）个/m³>冬季（25.30±19.11）个/m³;平均生物量为夏季（895.01±802.54） mg/m³>春季（623.39±358.73） mg/m³>秋季（91.08±82.36） mg/m³>冬季（45.96±84.95） mg/m³。多样性指数（H''）和均匀度指数（J''）的年平均值分别为1.71±0.96和0.53±0.20,均表现出夏秋季较高、冬春季较低的特征。聚类分析结果表明调查海域的浮游动物可划分为夏季类群、秋季类群、冬季类群和春季类群4组类群。Pearson相关性分析和冗余分析（RDA）结果表明,海水温度、盐度、叶绿素a浓度是影响大陈岛海域浮游动物群落特征的重要环境因素。此外,夏季大陈岛海域水母类浮游动物暴发的现象值得关注。研究结果将为大陈岛海域的生物多样性保护及渔业资源可持续开发利用提供可参考的数据资料。     

黄柏河流域不同区域浮游植物群落特征及总磷分析

黄柏河流域矿产资源丰富,磷矿开采导致的颗粒磷排放、地表径流汇聚沉积等会对流域内浮游植物的群落演替产生较大影响。为研究黄柏河流域磷矿开采后环境因子对浮游植物群落结构的影响,于2020年的春季（5月）、夏季（8月）和秋季（9月）分别对黄柏河流域10个监测断面水质和浮游植物采样。根据地区划分法和NMDS分析法将研究区分为磷矿开采影响区（A组）和非影响区（B组）,采用主坐标分析（PCoA）和聚类分析得到A组和B组的浮游植物群落结构具有明显差异,A组优势属为隐藻（Cryptomonas）、舟形藻（Navicula）和小环藻（Cyclotella）;B组优势属为隐藻（Cryptomonas）和针杆藻（Synedra）,且B组中物种数多于A组。采用典范对应分析（CCA）法分析了环境因子与浮游植物的关系。结果表明,溶解氧、水温、氨氮、氟化物、总磷对黄柏河流域的浮游植物影响较大,且A组浮游植物群落结构与总磷、溶解氧和水温呈负相关,与氟化物呈正相关;B组浮游植物群落结构与溶解氧、总磷呈负相关。研究结果可为该流域的生态环境保护和治理提供科学指导意义。     

核桃壳提取液用于单针藻Monoraphidium sp.QLZ-3培养产油脂

[目的] 研究核桃壳提取液（walnut shell extracts,WSE）对单针藻Monoraphidium sp.QLZ-3生长和油脂积累的影响。[方法] 向BG-11培养基中添加不同量的WSE（培养基中保留有BG-11中全部营养成分）。[结果] 结果显示,当BG-11培养基中的WSE含量为40%时,单针藻的生物量产率及油脂产率达到（534.70±4.07）mg/（L·d）和（296.35±15.36）mg/（L·d）,相比对照组分别提高了的14.82%和33.50%,蛋白质和碳水化合物含量分别有不同程度的上调和下调。与对照组相比,微藻中谷胱甘肽（glutathione,GSH）和超氧化物歧化酶（superoxide dismutase,SOD）含量与活性均上调。此外,WSE作用下,微藻对多酚的移除达到84.37%,同时上调了核酮糖1,5-二磷酸羧化酶基因（ribulose 1,5-bisphosphate carboxylase/oxygenase,rbcL）和乙酰辅酶A羧化酶（acetyl coenzyme A carboxylase,accD）基因的表达量。[结论] 研究表明,WSE联合BG-11可以提高微藻的生物量产率和油脂产率,降低微藻培养的原料成本,为核桃壳的资源化利用及微藻的工业化生产提供了一定的技术支撑。     

莱州湾小清河口近岸海域底栖生境健康评价

于2019年7月、8月和9月对莱州湾小清河口近岸海域的大型底栖动物进行调查，对该海域大型底栖动物群落的种类组成和群落结构等进行研究，利用丰度/生物量比较曲线法、AMBI指数法和M-AMBI指数法对研究海域大型底栖动物群落健康和底栖生境健康状况进行评价。研究结果表明，2019年7月、8月和9月共采集到大型底栖动物64种，其中多毛类24种，甲壳类19种，软体动物17种，其他类群4种。该海域的优势种为光滑河蓝蛤（Potamocorbula laevis）、彩虹明樱蛤（Moerella iridescens）、半褶织纹螺（Nassarius semiplicatus）、四角蛤蜊（Mactra veneriformis），寡鳃齿吻沙蚕（Nephthys oligobranchia）、中蚓虫（Mediomastus californiensis）、东方长眼虾（Ogyrides orientalis）、尖齿拳蟹（Philyra acutidens）和短角双眼钩虾（Ampelisca brevicornis），其中彩虹明樱蛤和寡鳃齿吻沙蚕在三次调查中均为优势物种，优势度明显。群落结构聚类分析和多维尺度分析表明，7月研究海域的大型底栖动物群落以10%的相似性可以分为3组，8月以18%相似性可以分为4组，9月以19%的相似性可以分为3组，三次调查的群落结构相似度均较低。丰度/生物量比较曲线法研究结果显示，底栖动物群落生物量优势显著，群落处于未干扰状态。AMBI指数及M-AMBI指数分析结果表明，莱州湾小清河口近岸海域底栖生境处于未干扰或者轻度干扰的状态，健康状况处于高等或者优良的状态。     

Use of the dinoflagellate <Emphasis Type="Italic">Karlodinium veneficum</Emphasis> as a sustainable source of biodiesel production

Microalgae are microscopic heterotrophic–autotrophic photosynthesizing organisms with enormous potential as a source of biofuel. Dinoflagellates, a class of microalgae, contain large amounts of high-quality lipids, the principal component of fatty acid methyl esters. The biotic characteristics of the dinoflagellate species Karlodinium veneficum include a growth rate of 0.14 day⁻¹, a wet biomass of 16.4 g/L, a growth period of approximately 30 days, and an approximate 97% increase in fatty acid content during the transition from exponential phase to stationary phase. These parameters make K. veneficum a suitable choice as a bioresource for biodiesel production. Similarly, two other species were also determined to be appropriate for biodiesel production: the Dinophyceae Alexandrium andersoni and the Raphidophyte Heterosigma akashiwo.     

Fast microzooplankton grazing on fast-growing,low-biomass phytoplankton: a case study in spring in Chesapeake Bay,Delaware Inland Bays and Delaware Bay

Dilution experiments were performed to examine the growth and grazing mortality rates of picophytoplankton (<2 μm), nanophytoplankton (2–20 μm), and microphytoplankton (>20 μm) at stations in the Chesapeake Bay (CB), the Delaware Inland Bays (DIB) and the Delaware Bay (DB), in early spring 2005. At station CB microphytoplankton, including chain-forming diatoms were dominant, and the microzooplankton assemblage was mainly composed of the tintinnid Tintinnopsis beroidea. At station DIB, the dominant species were microphytoplanktonic dinoflagellates, while the microzooplankton community was mainly composed of copepod nauplii and the oligotrich ciliate Strombidium sp. At station DB, nanophytoplankton were dominant components, and Strombidium and Tintinnopsis beroidea were the co-dominant microzooplankton. The growth rate and grazing mortality rate were 0.13–3.43 and 0.09–1.92 d⁻¹ for the different size fractionated phytoplankton. The microzooplankton ingested 73, 171, and 49% of standing stocks, and 95, 70, and 48% of potential primary productivity for total phytoplankton at station CB, DIB, and DB respectively. The carbon flux for total phytoplankton consumed by microzooplankton was 1224.11, 100.76, and 85.85 μg C l⁻¹ d⁻¹ at station CB, DIB, and DB, respectively. According to the grazing mortality rate, carbon consumption rate and carbon flux turn over rates, microzooplankton in study area mostly preferred to graze on picophytoplankton, which was faster growing but was lowest biomass component of the phytoplankton. The faster grazing on Fast-Growing-Low-Biomass (FGLB) phenomenon in coastal regions is explained as a resource partitioning strategy. This quite likely argues that although microzooplankton grazes strongly on phytoplankton in these regions, these microzooplankton grazers are passive. Handling editor: K. Martens     

Marine microalgae attack and feed on metazoans

Free-living microalgae from the dinoflagellate genus Karlodinium are known to form massive blooms in eutrophic coastal waters worldwide and are often associated with fish kills. Natural bloom populations, recently shown to consist of the two mixotrophic and toxic species Karlodinium armiger and Karlodinium veneficum have caused fast paralysis and mortality of finfish and copepods in the laboratory, and have been associated with reduced metazooplankton biomass in-situ. Here we show that a strain of K. armiger (K-0688) immobilises the common marine copepod Acartia tonsa in a density-dependent manner and collectively ingests the grazer to promote its own growth rate. In contrast, four strains of K. veneficum did not attack or affect the motility and survival of the copepods. Copepod immobilisation by the K. armiger strain was fast (within 15 min) and caused by attacks of swarming cells, likely through the transfer and action of a highly potent but uncharacterised neurotoxin. The copepods grazed and reproduced on a diet of K. armiger at densities below 1000, cells ml⁻¹, but above 3500 cells ml⁻¹ the mixotrophic dinoflagellates immobilised, fed on and killed the copepods. Switching the trophic role of the microalgae from prey to predator of copepods couples population growth to reduced grazing pressure, promoting the persistence of blooms at high densities. K. armiger also fed on three other metazoan organisms offered, suggesting that active predation by mixotrophic dinoflagellates may be directly involved in causing mortalities at several trophic levels in the marine food web.     

Growth,death, and photobiology of dinoflagellates (Dinophyceae) under bacterial-algicide control

Naturally occurring allelopathic compounds, specific to some phytoplankton, may be a good source of bio-control agents against microalgae responsible for harmful algal blooms (HABs). Global expansion of HABs has invigorated research into different approaches to control these algae, including the search for naturally derived algicidal compounds. Here, we investigated the effects of a filtrate from the algicidal marine bacterium Shewanella sp. IRI-160 on photochemical function of four cultured dinoflagellates, Karlodinium veneficum, Gyrodinium instriatum, Prorocentrum minimum, and Alexandrium tamarense. The filtrate (designated IRI-160AA) contains bioactive compound(s), which were recently shown to inhibit growth of several dinoflagellate species. Results of this study show that all dinoflagellates but P. minimum exhibited photosystem II (PSII) inhibition, loss of photosynthetic electron transport, and varying degrees of cellular mortality. Exposure assays over 24 h showed that PSII inhibition and loss of cell membrane integrity occurred simultaneously in G. instriatum, but not in K. veneficum, where PSII activity declined prior to losing outer-membrane integrity. In addition, PSII inhibition and population growth inhibition were dose-dependent in K. veneficum, with an average EC-50 of 7.9 % (v/v) IRI-160AA. Application of IRI-160AA induced significantly higher PSII inhibition and cell mortality in K. veneficum subjected to continuous darkness as compared to cells maintained with 12:12 h light/dark cycles, while no such dark effect was noted for G. instriatum. The marked differences in the rate and impact of this algicide suggest that multiple cellular targets and different cascades of cellular dysfunction occur across these dinoflagellates.     

Growth and competition of several harmful dinoflagellates under different nutrient and light conditions

Three near-shore type harmful dinoflagellates, Prorocentrum minimum, Prorocentrum donghaiense and Karlodinium veneficum, and one off-shore dinoflagellate, Karenia brevis, were grown in laboratory monocultures and mixed batch cultures. The dinoflagellate cultures were grown on treatments of two ambient nitrogen (N):phosphorus (P) ratios; two N substrates (nitrate and urea) and two light intensities. The microalgae Rhodomonas and Synechococcus were also added in separate treatments to the mixed culture treatments as potential food sources. All tested species grew well on both N substrates. In mixed culture, P. minimum outgrew K. veneficum, and P. donghaiense outgrew K. brevis in most treatments reaching higher growth rates and higher biomass. However, when a third algae, Rhodomonas, was added, the growth of P. minimum was inhibited relative to that of K. veneficum. In contrast, when grown with K. brevis, the growth rate of P. donghaiense was not significantly affected by the addition of potential prey. K. brevis had a longer growth phase, and kept growing after P. donghaiense reached stationary phase, suggesting better adaptation of K. brevis to low inorganic nutrient conditions. The growth of K. brevis was also significantly limited in the low light treatment. K. veneficum overgrew P. minimum in the presence of Rhodomonas, a potential nutrient source. The growth rates of both K. brevis and P. donghaiense were reduced with the presence of Synechococcus. In addition to nutrient competition, mixotrophy and allelopathy were likely mechanisms in determining the dominant species.     

Modulation of polyunsaturated fatty acids in mixotrophic Karlodinium veneficum (Dinophyceae) and its prey,Storeatula major (Cryptophyceae)1

We examined whether fatty acid (FA) composition changed when Karlodinium veneficum (D. Ballantine) J. Larsen (Dinophyceae) was grown phototrophically or mixotrophically on Storeatula major Butcher ex D. R. A. Hill (Cryptophyceae). We hypothesized that the FA composition of mixotrophic K. veneficum would not change relative to the FA composition of phototrophic K. veneficum. As in other phototrophic dinoflagellates, octadecapentaenoic acid (18:5n3) represented 9% to 20% of total FA in K. veneficum and was enriched within chloroplast‐associated galactolipid classes. The 18:5n3 content showed a highly significant positive correlation (r² = 0.95) with chl a content and a highly significant negative correlation with growth rate (r² = 0.88). A previously undescribed chloroplast galactolipid molecular species, digalactosyldiacylglycerol (DGDG; 18:5n3/18:5n3), was a dominant structural lipid in K. veneficum. Docosahexaenoic acid (22:6n3) represented 14% to 19% of total K. veneficum FA and was enriched within phospholipids. In the prey S. major, 18:5n3 was not present, but octadecatetraenoic acid (18:4n3) and α‐linolenic acid (18:3n3) represented approximately 50% of total FA and were enriched within chloroplast‐associated galactolipid classes. Eicosapentaenoic acid (20:5n3) and 22:6n3 represented approximately 18% of total FA in S. major and were enriched within phospholipids. The FA profile of mixotrophic K. veneficum, compared to phototrophic K. veneficum, showed elevated levels of 18:3n3, 18:4n3, and 20:5n3, and lower but persistent levels of 18:5n3. Production to ingestion (P:I) ratios >1 for major polyunsaturated fatty acids (PUFAs) indicated that direct assimilation from prey under balanced growth could not support rates of PUFA production in mixotrophic K. veneficum. These data suggest that the plastid plays a continuing and essential role in lipid metabolism during mixotrophic growth.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Karlotoxin mediates grazing by Oxyrrhis marina on strains of Karlodinium veneficum

Karlodinium veneficum is a common member of temperate, coastal phytoplankton assemblages that occasionally forms blooms associated with fish kills. Here, we tested the hypothesis that the cytotoxic and ichthyotoxic compounds produced by K. veneficum, karlotoxins, can have anti-grazing properties against the heterotrophic dinoflagellate, Oxyrrhis marina. The sterol composition of O. marina (>80% cholesterol) renders it sensitive to karlotoxin, and does not vary substantially when fed different algal diets even for prey that are resistant to karlotoxin. At in situ bloom concentrations (10⁴–10⁵ K. veneficum ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 55% that observed on the non-toxic K. veneficum strain MD5. At lower prey concentrations typical of in situ non-bloom levels (<10³ cells ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 70–80% of rates on non-toxic strain MD5. Growth of O. marina was significantly suppressed when fed the toxic strain of K. veneficum. Experiments with mixed prey cultures, where non-toxic strain MD5 was fluorescently stained, showed that the presence of toxic strain CCMP 2064 inhibited grazing of O. marina on the co-occurring non-toxic strain MD5. Exogenous addition of a sub-lethal dose (100 ng ml⁻¹) of purified karlotoxin inhibited grazing of O. marina by approximately 50% on the non-toxic K. veneficum strain MD5 or the cryptophyte S. major. These results identify karlotoxin as an anti-grazing compound for those grazers with appropriate sterol composition (i.e., desmethyl sterols). This strategy is likely to be an important mechanism whereby growth of K. veneficum is uncoupled from losses due to grazing, allowing it to form ichthyotoxic blooms in situ.     

CHARACTERIZATION OF NW MEDITERRANEAN KARLODINIUM SPP. (DINOPHYCEAE) STRAINS USING MORPHOLOGICAL,MOLECULAR, CHEMICAL,AND PHYSIOLOGICAL METHODOLOGIES1

Recurrent fish kills in the Spanish Alfacs Bay (NW Mediterranean) have been detected during winter seasons since 1994, and were attributed to an unarmored, ichthyotoxic, dinoflagellate, initially identified as Gyrodinium corsicum Paulmier, Berland, Billard, & Nezan. Several strains were isolated from the bay and their clonal cultures were compared by combined techniques, including light and electron microscopy, internal transcribed spacer and 5.8S rDNA nucleotide sequencing, and HPLC pigment analyses, together with studies of their photochemical performance, growth rates, and toxicity. Using phylogenetic analyses, all strains were identified as members of the genus Karlodinium, but they were separated into two genetically distinct groups. These groups, identified as Karlodinium veneficum (Ballantine) J. Larsen and K. armiger Bergholtz, Daugbjerg et. Moestrup, were also supported by the other techniques used. Detailed analyses of fine structural characteristics (including plug‐like structures in amphiesma and a possible layer of semi‐opaque material beneath the outer membrane) allowed discrimination of the mentioned two species. Specific differences in pigment patterns coincided with that expected for low‐ (K. veneficum) and high‐light (K. armiger) adapted relatives. The higher photosynthetic efficiency of K. veneficum and the longer reactivation times of the PSII reaction centers observed for K. armiger were in agreement with this hypothesis. The two species differed in toxicity, but the strains used always induced mortality when incubated with bivalves, rotifers, and finfish. Compared with K. armiger, strains of K. veneficum yielded higher cell densities, but had lower growth rates.     

Biochemical characteristics support the recently described species Karlodinium zhouanum (Gymnodiniales,Dinophyceae)

The small athecate dinoflagellate Karlodinium zhouanum is a species recently described in the coastal waters of China. K. zhouanum is morphologically similar to Karlodinium veneficum, a typical ichthyotoxic blooming karlotoxin‐producing species, and it is impossible to distinguish between these two species based on light microscopy. In this study, strains of K. zhouanum isolated from the East China Sea were studied. By analyzing toxins, toxicity, lipid characteristics and typical molecular and physiological traits of this species, K. zhouanum was shown to be nontoxic to brine shrimp and widely spread over the coastal waters of China. No karlotoxin‐like toxin was detected by liquid chromatography‐mass spectrometry (LC–MS). Instead of gymnodinosterol, the critical sterol in toxic K. veneficum, 27(nor)‐24S‐4α‐Methyl‐5α‐ergosta‐8(14)‐en‐3β‐ol ( NEE ) was dominant in K. zhouanum, while gymnodinosterol was absent. These sterol characteristics may provide not only support for the species separation between toxic and nontoxic species of Karlodinium but also environmental survey tools to differentiate the contribution of nontoxic Karlodinium strains, which has been unclear until now.