Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

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.     

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.     

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.     

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.     

Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity, as well as the temporal stability of both the intracellular and the extracellular lytic compounds of P. parvum. We show high strain variation in toxicities after 3 h incubation with LC₅₀s ranging from 24 to 223 × 10³ cells ml⁻¹. Most importantly we prove the necessity of testing physico-chemical properties of P. parvum toxins before attempting to isolate and characterize them. The extracellular toxin in the supernatant is highly unstable, and it loses significant lytic effects after 3 days despite storage at −20 °C and after only 24 h stored at 4 °C. However, when stored at −80 °C, lytic activity is more easily maintained. Reducing oxidation by storing the supernatant with no headspace in the vials significantly slowed loss of activity when stored at 4 °C. We show that the lytic activity of the intracellular toxins, when released by sonication, is not as high as the extracellular toxins, however the stability of the intracellular toxins when kept as a cell pellet at −20 °C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge of how to handle and store the toxins from P. parvum so as to maintain biologically relevant toxicity.     

Identification of toxic fatty acid amides isolated from the harmful alga Prymnesium parvum carter

The golden alga Prymnesium parvum has been implicated in fish and aquatic animal kills globally for over a century. In addition to widespread ecological impacts through the loss of entire fish populations within lakes, an economic burden is also felt by state and local agencies due to year class losses of fish raised for stocking lakes as well as loss of fishing and recreational use of the affected water. Multiple compounds have been implicated in P. parvum toxicity, but the unequivocal identification and characterization of all P. parvum toxins remained to be accomplished. To unambiguously characterize these toxins, we analyzed laboratory-cultured cells exposed to limited nitrogen and phosphorus concentrations, uni-algal wild cells collected from an ichthytoxic bloom event at Lake Wichita, TX, and the water from both cultured and field-collected algae. A bioassay-guided fractionation process was employed to chemically isolate P. parvum toxins using both mammalian cells and larval fish. The results of these assays revealed that there was a distinct similarity in the toxic compounds characterized as seven primary fatty acid amides (myristamide, palmitamide, linoleamide, oleamide, elaidamide, stearamide, and erucamide) and one hydroxamic acid (linoleyl hydroxamic acid). These compounds display cytotoxic and ichthytoxic activity and have not yet been reported in P. parvum toxicity or in the toxicity of harmful algal species.     

Comparison of the efficiency of urea,aqueous ammonia and ammonium sulphate as nitrogen fertilizers

Summary Nitrogen-15 labelled urea, aqueous NH₃ and (NH₄)₂SO₄ were applied to soils contained in pots. The fertilizers were injected in 5 cm³ of solution, 3.5 cm below the soil surface, to simulate a fertilizer band in the field. Ryegrass (Lolium perenne) was planted, and several cuttings and roots were harvested. Efficiency was determined as the recovery of fertilizer-N in the plant tissues and soil.Total recovery varied from 94 to 100%. There was no significant difference between the total recovery of the 3 fertilizer forms, although recovery in the soil component was lower for (NH₄)₂SO₄ than for urea or NH₃. There was a significant difference in total recovery between soils due to the soil component. Only small amounts of¹⁵N were not recovered, whereas laboratory experiments reported elsewhere had demonstrated that substantial gaseous losses of N as N₂, N₂O and NO +NO₂ occurred in these soils during nitrification of added NH₃ fertilizer.     

Distribution pattern of sulphate reducing and thiosulphate oxidizing bacterial populations in fish ponds of differing farming managements

The population size of SRB and TOB was enumerated at bimonthly intervals from water and sediment samples of six fish ponds (polyculture, monoculture and traditional systems) over a period of two and a half years. Density of SRB and TOB was maximal in polyculture and minimal in traditional systems of fish farming. Using sine and cosine waves of periodic functions or polynomial equations of high order, seasonal changes in the numbers of SRB and TOB were represented; the former occurred in largest numbers in summer while the latter in winter. Step wise multiple regression analysis revealed that organic carbon, organic matter, phosphate and different forms of N were important attributes to the seasonal changes of SRB and TOB in these fish ponds.     

Behavioral control of the efficiency of pharmacological anesthesia in fish

An original behavioral test was used to study the effect of opioid substances on the thresholds of nociceptive responses to pain stimuli—a series of electric impulses applied to nerve endings of the caudal fin—in the common carp (Cyprinus carpio). The substances tested included tramadol (μ-agonist of opioid receptors), DADLE (δ-agonist), and U-50488 (κ-agonist) injected intramuscularly in concentrations 10–100 nmol/g of body weight. Raised thresholds of sensitivity to the pain stimulus were observed in the studied fish 5 to 15 min after the injection. The degree of analgesia and the rate of its increase varied depending on the dose. The total duration of analgesia was 40 to 90 min and depended on the concentration of the injected substance. It was observed in some experiments that the analgesic effect of tramadol (the most efficient of the analgesics used) could last longer than 4 h. The analgesic effect of opioids was not detected in experiments where they were applied together with naloxone, an antagonist of opioids. Decreased motor response to pain stimuli after injections of analgesics was not caused by the immobilization of the animal, because the tested fish individuals released into an aquarium demonstrated normal swimming and their usual behavior. We concluded that the systems of opioid nociceptive regulation function similarly in fish and land vertebrates. This regulation can play an important role in defense behavior and in other behaviors in fish.     

Prymnesium parvum invasion success into coastal bays of the Gulf of Mexico: Galveston Bay case study

The toxic haptophyte Prymnesium parvum regularly forms fish-killing blooms in inland brackish water bodies in the south-central USA. Along the Texas coast smaller blooms have occurred in isolated areas. There appears to be an increasing risk that harmful P. parvum blooms will propagate into open coastal waters with implementation of future water plans. These plans will include increased interbasin water transfers from the Brazos River, regularly impacted by P. parvum blooms, to the San Jacinto-Brazos Coastal Basin, which ultimately flows into Galveston Bay (GB). Persisting source populations of P. parvum in inland waters elevates this risk. Thus, there is a need for an increased understanding of how P. parvum might perform in coastal waters, such as those found in GB. Here, two in-field experiments were conducted to investigate the influence of various plankton size-fractions of GB water on inoculated P. parvum during fall and winter, periods when blooms are typically initiating and developing inland. Stationary- and log-growth phase P. parvum were used to represent high and low toxicity initial conditions. Results revealed that P. parvum could grow in GB waters and cause acute mortality to silverside minnows (Menidia beryllina). Depending on season and growth phase, however, P. parvum growth and toxicity varied in different size fractions. During the fall, P. parvum inoculated from stationary-, but not log-growth phase culture, was negatively affected by bacteria-sized particles. During the winter, bacteria and nanoplankton together had a negative effect on P. parvum inoculated from stationary- and, to a lesser degree, log-growth phase cultures. Intermediate- and large-sized grazers when combined with bacteria and nanoplankton had complex relationships with inoculated P. parvum, sometimes stimulating and sometimes suppressing population growth. Toxicity to fish occurred in almost all plankton size fractions. The inclusion of progressively larger sized plankton fractions resulted in trends of decreased toxicity in treatments inoculated with stationary-, but not log-growth phase P. parvum in the fall. In the winter, however, inclusion of larger sized plankton fractions resulted in trends of increased toxicity to fish in treatments inoculated with both stationary- and log-growth phase P. parvum. This study indicates that understanding P. parvum population dynamics in open waters of estuaries and bays will be challenging, as there appears to be complex relationships with naturally occurring components of the plankton. The observations that P. parvum is able to grow to high population density and produce fish-killing levels of toxins underscores the need for advanced risk assessment studies, especially in light of water use plans that will result in P. parvum invasions of greater size.     

Aspects of the ecology of Prymnesium parvum (Haptophyta) and water chemistry in the Norfolk Broads, England

SUMMARY. Prymnesium parvum , an ichthyotoxic phytoplankter, has been recorded, at times abundantly, in the River Thurne, Norfolk, and its associated Broads. Its occurrence has been apparently more frequent and its population sizes probably larger since the late 1960s than previously and fish mortalities due to it now occur almost annually.
The Thurne system is brackish and may have become more so, due to exploitation by drainage pumps, of a saline water table, in recent years. Evidence for this is conflicting, but in any case an increase in salinity is unlikely to have made increased Prymnesium growth more likely. Eutrophication of some Broads in the system is most likely to have increased the populations of Prymnesium since the late 1960s and data are presented on the present water chemistry of the system for comparison with previous records, and on current phytoplankton and Prymnesium crops in different parts of it.
P. parvum has been isolated from the system as a unialgal culture and compared in morphology, salinity tolerance and ichthyotoxicity with a strain of P. parvum from Israel. The Broads strain differs slightly in size and pigmentation, but not in salinity tolerance. In culture it produces more ichthyotoxin than the Israeli strain.     

The effect of calcium carbonate and potassium sulphate on the efficiency of ammonium as tested by barley seedlings

Summary Using the Neubauer technique and the slope ratio, it was found that efficiency of (NH₄)₂SO₄ in the presence of 12.5 or 25.0 g CaCO₃ per pot relative to its efficiency without CaCO₃ was 0.6 and 0.47, respectively.Application of 4 or 6 mg K/pot, the efficiency of (NH₄)₂SO₄ was 0.56 and 0.33, respectively, relative, to its efficiency without K application.The Dean's a value was found to vary with percent CaCO₃ or K₂SO₄ application.