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Karmitoxin production by Karlodinium armiger and the effects of K. armiger and karmitoxin towards fish

机译：Karmitoxin通过Karlodinium Armiger产生的K.Acriger和Karmitoxin对鱼类的影响

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摘要

The dinoflagellate Karlodinium armiger has a huge impact on wild and caged fish during blooms in coastal waters. Recently, a new toxin, karmitoxin, was chemically characterized from K. armiger and a quantification method was established, thereby allowing investigations of the fish killing mechanism. K. armiger is not able to grow in standard growth media that are based on nitrate as a nitrogen source, and successful cultures of this species have only been achieved in mixotrophic cultures after addition of a prey source. Here we show that addition of ammonium (up to 50 mu M) to the growth media is a good alternative, as K. armiger batch cultures achieve growth rates, which are comparable to growth rates reached in mixotrophic cultures. Karmitoxin production (1.9 and 2.9 pg cell(-1) d(-1)) and cellular karmitoxin content (8.72 +/- 0.25 pg cell(-1) and 7.14 +/- 0.29 pg cell(-1)) were in the same range, though significantly different, in prey-fed cultures and monocultures supplied with ammonium, respectively. Net production of karmitoxin stopped when the K. armiger cultures reached stationary growth phase, indicating no accumulation of karmitoxin in cells or growth media. Toxicity tests towards sheepshead minnow fish larvae indicated rapid death of the fish larvae when exposed to high K. armiger cell concentrations (LT50 of 2.06 h at 44.9 x 10(3) cells mL(-1) cultivated with ammonium). Purified toxins caused the same physical damage to fish larvae as living K. armiger cultures. An exposure of purified karmitoxin to fish larvae and rainbow trout gill cells indicated that the fish larvae were about three times less sensitive than gill cells. When comparing the effect of purified toxins with the effect of whole K. armiger cultures, twice the toxin concentration of the purified toxins was needed to cause the same effect. Although a loss of karmitoxin of twenty percent was observed during the incubation, this could not explain the apparent discrepancy. Other factors, like a direct effect of the K. armiger cells on the fish larvae or other, yet unknown toxins may influence the effect of whole cell cultures. To study the effects of released karmitoxin, fish larvae were exposed to a K. armiger culture that was treated with HP-20 resin, which adsorbs extracellular karmitoxin. The 24 h HP-20 treatment resulted in a K. armiger culture that had 37% less total karmitoxin, without a reduction in cell concentration, and a reduced toxic effect was observed in the HP-20 treated culture, as compared to non-treated controls. Fish larvae that were exposed to HP-20 treated culture were immobilized, but survived during the 12 h exposure, whereas the exposure to non-treated culture led to high mortality of the fish larvae. Direct observations under the microscope revealed no evidence of micropredation of K. armiger on the fish larvae during any of the exposures. Thus, the results presented here, indicate that released karmitoxin is the main cause for fish kills by K. armiger. Finally, we found that juvenile rainbow trout were six times more sensitive than fish larvae towards K. armiger, indicating that juvenile fish are more sensitive to K. armiger in bloom situations than early larval stages.
机译：Dinoflagellate Karlodinium Armiger对沿海水域的盛开期间对野生和笼中的鱼产生了巨大影响。最近，建立了一种新的毒素，Karmitoxin从CAR armiger和定量方法进行了化学表征，从而允许对鱼类杀伤机制的研究。 K. Armiger无法在基于硝酸盐作为氮源的标准生长培养基中生长，并且在加入猎物源后，在混合营养培养物中才能实现该物种的成功培养物。在这里，我们显示加入生长介质的铵（最多50μm）是一种良好的替代方案，因为K.Armiger分批培养物实现生长率，这与在混纺培养物中达到的生长速率相当。 Karmitoxin生产（1.9和2.9 pg细胞（-1）d（-1））和细胞Karmitoxin含量（8.72 +/- 0.25 pg细胞（-1）和7.14 +/- 0.29 pg细胞（-1））相同的范围，虽然显着不同，但分别在用铵提供的急性培养和单一栽培中。 Karmitoxin的净生产停止当K.Armiger培养达到固定生长阶段，表明在细胞或生长培养基中没有Karmitoxin的积累。在羊皮鱼类幼虫的毒性试验表明，当暴露于高K. armiger细胞浓度（在44.9×10（3）细胞ml（-1）含有铵）的高K. armiger细胞浓度（LT50，为2.06小时的LT50），表明了鱼类幼虫的快速死亡。纯化的毒素导致鱼类幼虫与生活K. Armiger文化相同的物理损伤。将纯化的Karmitoxin暴露于鱼类幼虫和虹鳟鱼鳃细胞表明鱼幼虫比鳃细胞敏感的约三倍。当比较纯化毒素与全K armiger培养物的效果的影响时，需要纯化毒素的毒素浓度的两倍以引起相同的效果。虽然在孵化期间观察到了20％的Karmitoxin的丧失，但这不能解释表观差异。其他因素，如K. Armiger细胞对鱼类幼虫或其他的直接效果，但未知的毒素可能会影响全细胞培养物的效果。为研究释放的Karmitoxin的影响，将鱼类幼虫暴露于用HP-20树脂处理的K. Armiger培养物，其吸附细胞外Karmitoxin。 24小时HP-20治疗导致K.Armiger培养物，其总Karmitoxin的总少于37％，而不会降低细胞浓度，并且与未处理相比，在HP-20处理的培养物中观察到降低的毒性效果控制。暴露于HP-20处理培养物的鱼类幼虫被固定，但在12小时暴露过程中存活，而暴露于未处理的培养物导致鱼类幼虫的高死亡率。显微镜下的直接观察显示在任何曝光期间没有在鱼类幼虫上的K.Amabriger微量化的证据。因此，这里呈现的结果表明释放的Karmitoxin是K. Armiger鱼类杀死的主要原因。最后，我们发现少年虹鳟鱼比鱼类幼虫六倍敏感，表明少年鱼对盛开的情况比早期幼虫阶段更敏感。

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  • 来源
    《Harmful Algae》 |2020年第11期|101905.1-101905.13|共13页
  • 作者

    Binzer Sofie Bjornholt; Varga Elisabeth; Andersen Aaron John Christian; Svenssen Daniel Killerup; de Medeiros Livia Soman; Rasmussen Silas Anselm; Larsen Thomas Ostenfeld; Hansen Per Juel;

  • 作者单位

    Univ Copenhagen Marine Biol Sect Strandpromenaden 5 DK-3000 Helsingor Denmark;

    Tech Univ Denmark Dept Biotechnol & Biomed Soltofts Plads 221 DK-2800 Lyngby Denmark|Univ Vienna Fac Chem Dept Food Chem & Toxicol Wahringer Str 38 A-1090 Vienna Austria;

    Tech Univ Denmark Dept Biotechnol & Biomed Soltofts Plads 221 DK-2800 Lyngby Denmark|Tech Univ Denmark Natl Food Inst DK-2800 Lyngby Denmark;

    Tech Univ Denmark Dept Biotechnol & Biomed Soltofts Plads 221 DK-2800 Lyngby Denmark|Univ Coll Absalon Ctr Engn & Sci J Hagemann Petersens Alle 4 DK-4400 Kalundborg Denmark;

    Tech Univ Denmark Dept Biotechnol & Biomed Soltofts Plads 221 DK-2800 Lyngby Denmark|Univ Fed Sao Paulo UNIFESP Dept Chem Rua Sao Nicolau 210 BR-09913030 Diadema SP Brazil;

    Tech Univ Denmark Dept Biotechnol & Biomed Soltofts Plads 221 DK-2800 Lyngby Denmark;

    Tech Univ Denmark Dept Biotechnol & Biomed Soltofts Plads 221 DK-2800 Lyngby Denmark;

    Univ Copenhagen Marine Biol Sect Strandpromenaden 5 DK-3000 Helsingor Denmark;

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  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    Harmful dinoflagellate; Phycotoxin; Released toxin; Fish mortality; Toxic mechanism; Bioassay;

    机译：有害恐龙;植物毒素;释放毒素;鱼死亡率;有毒机制;生物测定;

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