Mode of Action of Prymnesium parvum Ichthyotoxin

SYNOPSIS. The mechanism of Prymnesium parvum ichthyotoxicity was studied in immersed minnows ( Gambusia affinis ) in the presence of various synergists (especially polyamines and invert soaps) and under varying conditions of pH and salinity. The effect of immersion in toxin-synergist solution on permeability of gill tissues to trypan blue and to radioactive substances, e.g., I¹²⁵ and radio-iodinated albumin, was established. Fish sensitivity to various toxic substances was increased after immersion for short periods in suitable toxinsynergist solutions. This sensitivity, as well as the increased permeability of gill tissue, was transient and disappeared when fish were stored in tap water for several hours after the toxin-synergist treatment. Good correlation was found between the conditions (synergist quality and concentration, pH, salinity) leading to P. parvum ichthyotoxicity and conditions instrumental in increased gill permeability and toxin sensitivity. The primary site of damage is concluded to be the gill membrane.     

Growth and Colony Formation of the Phytoflagellate Prymnesium parvum Carter on Solid Media

SYNOPSIS. Colonies of Prymnesium parvum from single cells were obtained in an atmosphere of high humidity, in soft-agar overlayers on solid medium. Light intensity above 60 foot candles at the surface of the agar plates inhibited colonial development. Such colonies were used to obtain pure lines of the phytoflagellate.
A method for direct demonstration of hemolysin formation on agar plates was developed. The occurrence of hemolytic activity by P. parvum colonies was light-dependent.     

Growth and Toxigenesis of the Chrysomonad Prymnesium parvum as a Function of Salinity*

SYNOPSIS The euryhaline chrysomonad Prymnesium parvum (Carter) was grown in artificial (ASW) and natural sea water (NSW) media at salinities 1.3-33.3 ‰, and constant illumination. Low salinities (< 10‰) increased the doubling time (DT) and induced higher levels of protein and nucleic acids. DT was lowest for cells in ASW at 25 ‰ and NSW 17.9 ‰. The protein content was more variable. RNA content was lowest for cells at ca. 10 ‰. The DNA content was lowest for cells in 10‰ ASW and 11‰ NSW. Generally, the cells grew faster in NSW but had less protein and RNA than cells in ASW. The highest hemolytic content was in cells grown at 22.8 ‰ ASW or NSW. Glycerol enhanced growth rate and toxin synthesis within 24 hr of addition to the cells. Hemolysis was inhibited by high pH's. A membrane fraction containing ca. 20% of the hemolysin was isolated by density gradient centrifugation.     

Nitrogen uptake kinetics of Prymnesium parvum (Haptophyte)

The uptake rates of different nitrogen (N) forms (NO₃⁻, urea, and the amino acids glycine and glutamic acid) by N-deficient, laboratory-grown cells of the mixotrophic haptophyte, Prymnesium parvum, were measured and the preference by the cells for the different forms determined. Cellular N uptake rates (ρ_cell, fmol N cell⁻¹ h⁻¹) were measured using ¹⁵N-labeled N substrates. P. parvum showed high preference for the tested amino acids, in particular glutamic acid, over urea and NO₃⁻ under the culture nutrient conditions. However, extrapolating these rates to Baltic Seawater summer conditions, P. parvum would be expected to show higher uptake rates of NO₃⁻ and the amino acids relative to urea because of the difference in average concentrations of these substrates. A high uptake rate of glutamic acid at low substrate concentrations suggests that this substrate is likely used through extracellular enzymes. Nitrate, urea and glycine, on the other hand, showed a non-saturating uptake over the tested substrate concentration (1–40 μM-N for NO₃⁻ and urea, 0.5–10 μM-N for glycine), indicating slower membrane-transport rates for these substrates.     

Computer-determined rate constants of hemolysis induced by Prymnesium parvum toxin

Rates of hemolysis of rabbit erythrocyte suspensions induced by P. parvum (prymnesin) have been measured colorimetrically at 25.5°C and pH 5.5. The data have been treated previously as consecutive first-order rate processes associated with the prolytic and lytic periods from which two specific rate constants have been obtained, k′ and kψ, respectively. These constants have been related to those obtained by a computer-generated fit of the rate data (absorbance A_t, as a function of time t) with the rate equation $\text{Y =}\text{D}\text{[1 +}\text{exp}\text{((X ? B)}\text{C)}\text{]}\text{+ E}$. Here Y equals A_t, X = time, t; D is equal to a spread factor, A_i ? A_∞; C is the slope of the curve at the inflection point; B is the midpoint time value, i.e., the time at which $\text{A}{}_{\mathrm{t}}\text{=}\text{D}\text{2}$; E is termed the off-set constant and is equal to A_∞. Of these constants, B is directly related to the length of the prolytic period, and C^?1 is directly related to the specific first-order rate constant for hemolysis, k_ψ.     

Photoinactivation of Ichthyrotoxin from Axenic Cultures of Prymnesium parvum Carter

The ichthyotoxin of Prymnesium parvum is inactivated by visible (400 to 510 mμ) as well as by ultraviolet light (255 mμ). The changes in the absorption spectrum of purified (pigment-free) ichthyotoxin during this process indicate that different mechanisms may be involved in the inactivation by visible or ultraviolet light. Photoinactivation is not affected by the presence of cells, cell pigments, oxygen, or glutathione.     

Ethionine and Methionine Metabolism by the Chrysomonad Flagellate Prymnesium parvum

SYNOPSIS. Ethionine or methionine can serve as sole nitrogen source for growth of Prymnesium parvum. Both amino acids are taken up as such at a ratio of 2 : 1 methionine/ethionine. Ethionine is totally de-ethylated in the cell, while methionine is probably only partially de-methylated. The homocysteine moiety of both amino acids is similarly metabolised to form cysteine or re-methylated to form methionine. De-ethylation of ethionine seems how P. parvum avoids its antimetabolic effect     

Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR

Aims: The ichthyotoxic species Prymnesium parvum (Haptophyceae) is difficult to quantify in a microscopy‐based monitoring programme, because the cells are very small, fragile and their morphology can be distorted by the use of fixatives. In the attempt to overcome these problems, a real‐time PCR‐based method for the rapid and sensitive identification and quantification of P. parvum was developed. Methods and Results: A quantitative real‐time PCR assay was optimized with primers designed on the internal transcribed spacer 2 rDNA region of P. parvum. This PCR assay was specific, showing no amplification of DNA extracted from closely related species, and sensitive. Moreover, this method was able to detect and reliably quantify P. parvum cells in preserved environmental samples artificially spiked with known amounts of cultured cells. Conclusions: Considering the specificity, sensitivity and applicability to preserved environmental samples, this method may be a useful tool for the monitoring of this toxic species. Significance and Impact of the Study: The real‐time PCR method described in this study may represent a progress towards the rapid detection and quantification of P. parvum cells in water‐monitoring programmes, allowing the early application of strategies to control bloom events, such as the use of clay minerals.     

Behavioural differences underlie toxicity and predation variation in blooms of Prymnesium parvum

Much of the evolutionary ecology of toxic algal blooms (TABs) remains unclear, including the role of algal toxins in the adaptive ‘strategies’ of TAB-forming species. Most eukaryotic TABs are caused by mixotrophs that augment autotrophy with organic nutrient sources, including competing algae (intraguild predation). We leverage the standing diversity of TABs formed by the toxic, invasive mixotroph Prymnesium parvum to identify cell-level behaviours involved in toxin-assisted predation using direct observations as well as comparisons between genetically distinct low- and high-toxicity isolates. Our results suggest that P. parvum toxins are primarily delivered at close range and promote subsequent prey capture/consumption. Surprisingly, we find opposite chemotactic preferences for organic (prey-derived) and inorganic nutrients between differentially toxic isolates, respectively, suggesting behavioural integration of toxicity and phagotrophy. Variation in toxicity may, therefore, reflect broader phenotypic integration of key traits that ultimately contribute to the remarkable flexibility, diversity, and success of invasive populations.     

Effect of Illumination on Ichthyotoxin in an Axenic Culture of Prymnesium parvum Carter

SYNOPSIS. Cultures of Prymnesium parvum subjected to constant illumination failed to produce ichthyotoxin. On the other hand cultures subjected to alternate periods of light and darkness showed a gradually rising ichthyotoxic activity during the dark period reaching a maximum after about 7 hours.