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

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 µM) 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 × 103 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.

Development of an Indirect Quantitation Method to Assess Ichthyotoxic B-Type Prymnesins from Prymnesium parvum.

Harmful algal blooms of Prymnesium parvum have recurrently been associated with the killing of fish. The causative ichthyotoxic agents of this haptophyte are believed to be prymnesins, a group of supersized ladder-frame polyether compounds currently divided into three types. Here, the development of a quantitative method to assess the molar sum of prymnesins in water samples and in algal biomass is reported. The method is based on the derivatization of the primary amine group and subsequent fluorescence detection using external calibrants. The presence of prymnesins in the underivatized sample should be confirmed by liquid chromatography mass spectrometry. The method is currently only partly applicable to water samples due to the low amounts that are present. The growth and cellular toxin content of two B-type producing strains were monitored in batch cultures eventually limited by an elevated pH. The cellular toxin contents varied by a factor of ~2.5 throughout the growth cycle, with the highest amounts found in the exponential growth phase and the lowest in the stationary growth/death phases. The strain K-0081 contained ~5 times more toxin than K-0374. Further investigations showed that the majority of prymnesins were associated with the biomass (89% ± 7%). This study provides the basis for further investigations into the toxicity and production of prymnesins.

A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum.

Harmful blooms formed by planktonic microalgae (HABs) in both freshwater and coastal waters regularly lead to severe mortalities of fish and invertebrates causing substantial economic losses of marine products worldwide. The mixotrophic haptophyte Prymnesium parvum is one of the most important microalgae associated with fish kills. Here 26 strains of P. parvum with a wide geographical distribution were screened for the production of prymnesins, the suspected causative allelochemical toxins. All investigated strains produced prymnesins, indicating that the toxins play an important role for the organism. The prymnesins can be classified into three types based on the length of the carbon backbone of the compound and each algal strain produced only one of these types. Biogeographical mapping of the prymnesin distribution indicated a global distribution of each type. In addition, phylogenetic analyses based on internal transcribed spacer (ITS) sequences revealed monophyletic origin of all prymnesin types and clades could therefore be defined based on the toxic compound. It might be that evolution of new species within the P. parvum species complex is driven by changes in toxin type or that they are a result of it. Such a correlation between chemotype and phylotype has never been documented before for a harmful microalga. Chemotaxonomy and ITS-type classification may thus be used to further delimit the P. parvum species complex.

Metabolic engineering of yeast for fermentative production of flavonoids.

Yeast Saccharomyces cerevisiae was engineered for de novo production of six different flavonoids (naringenin, liquiritigenin, kaempferol, resokaempferol, quercetin, and fisetin) directly from glucose, without supplementation of expensive intermediates. This required reconstruction of long biosynthetic pathways, comprising up to eight heterologous genes from plants. The obtained titers of kaempferol 26.57±2.66mgL-1 and quercetin 20.38±2.57mgL-1 exceed the previously reported titers in yeast. This is also the first report of de novo biosynthesis of resokaempferol and fisetin in yeast. The work demonstrates the potential of flavonoid-producing yeast cell factories.

Quantifying intracellular metabolites in yeast using a matrix with minimal interference from naturally occurring analytes.

For quantification of intracellular metabolites, mass spectrometry combined with liquid chromatography, capillary electrophoresis, or gas chromatography is currently the method of choice, especially when combined with stable isotope-labeled internal standards (SIL-ISs). Due to the difficulties in finding a biological matrix free of intracellular metabolites, a standard addition based validation is needed. Here, we present an alternative by producing a matrix with minimal signal interferences on both the analytes and their SIL-ISs. The matrix was obtained by cultivating Saccharomyces cerevisiae in [(13)C6]glucose/nonlabeled glucose (50:50, w/w) growth medium. The areas of both (12)C6 and (13)C6 fractions of ATP in the matrix were measured to be 2% of the sum of the areas of all ATP isotopes detected. The matrix allowed for spiking of both the nonlabeled and SIL-ISs and more straightforward validation. The intra- and inter-day accuracy and precision were ⩾80% and ⩽20%, respectively. The methodology was used for quantification of nucleotides, coenzymes, and redox compounds from Saccharomyces cerevisiae. The determined energy charge ratio was 0.9, whereas the Mal-CoA/Ac-CoA ratio was 0.04. The analysis of the redox compounds was challenging due to the oxidation of NADH and NADPH, when dissolved in water or tributylamine. The oxidation was reduced by dissolving them in ammonium acetate solution (pH 8.0).