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Sofie Bjørnholt Binzer:
Chemical ecology of fish-killing microalgae

Date: 30-09-2019    Supervisor: Per Juel Hansen

Some microalgal species are able to form high biomass blooms in coastal areas that negatively affect the marine environments. These blooms can cause mortality of the marine fauna at all trophic levels, including fish and shellfish. This thesis concerns members of two important harmful algal genera, Karlodinium and Prymnesium, and their toxins. Karlodinium armiger, known from recurrent annual harmful blooms in the Mediterranean, produces karmitoxin, a toxin that resembles karlotoxins, produced by the closely related K. veneficum. The toxic effects of K. armiger cultures on different life stages of blue mussels were studied using different algal concentrations. The effect on mussels was severe, and adult mussels rejected the K. armiger cells and refused to ingest them. The mussels died within 24 h of exposure to ecologically relevant algal concentrations and a clear dose-response relationship was observed. The mussel embryos and larvae were more sensitive and died at lower algal concentrations than the adults. Micropredation of K. armiger on the early life stages of mussels was observed. The effects on fish larvae were different, as swarming behavior or attachment of K. armiger cells in high numbers on fish larvae was not observed, even at high K. armiger concentrations. The toxic effects of K. armiger cultures on juvenile fish and fish larvae were also investigated and doseresponse relationships were established.

A method for quantitation of karmitoxin in culture samples was developed which allowed for the first measurements of quantified toxin concentrations during laboratory experiments. Purified karmitoxin lysed rainbow trout gill cells, and caused mortality in both fish larvae and copepods in a dose-dependent manor. Purified karmitoxin induced physical damages to fish larvae similar to what was caused by live K. armiger cells. However, twice the amount of pure toxin was needed to induce similar toxicity as was observed with live cells. Although a loss of karmitoxin of twenty percent was observed during the experimental exposure, this could not explain the discrepancy. Other factors may as well influence the toxicity of live cultures. It is possible that there are other unknown toxins at play, in addition to karmitoxin, or that live cells facilitate toxin transfer towards the fish larvae and thus, increase the toxicity of a live culture. To study the toxic mechanism of K. armiger further, a K. armiger culture was treated with HP-20 resin, which absorbs the extracellular karmitoxin. Approximately 37% of the total karmitoxin concentration in the algal culture was removed by HP-20, without a reduction in cell concentration. This coincided with decreased toxicity towards fish larvae; there was high mortality of fish larvae in the untreated controls, whereas the fish larvae which were exposed to cultures treated with HP-20 were immobilized, but survived the 12 h exposure. Altogether, these results suggest that karmitoxin released by K. armiger to the surrounding water constitutes the main toxic mechanism in fish kills by K. armiger.

Three fish representatives expressed different sensitivities towards K. armiger and karmitoxin in these studies. Juvenile rainbow trout were around six times more sensitive than fish larvae when exposed to live K. armiger culture, and gill cells were about three times more sensitive than fish larvae when exposed to purified toxins. These differences are important to keep in mind when interpreting mortality data of different fish substitutes exposed to K. armiger and possibly also other microalgae that produce broad targeting toxins.