INTER- AND INTRASPECIFIC GENETIC VARIATION IN TWELVE PRYMAESIUM (HAPTOPHYCEAE) CLONES1

The haptophytes Prymnesium parvum Carter and Prymnesium patelliferum Green, Hibberd, and Pienaar are two closely related species, which can only be distinguished by minor differences in the morphology of their organic body scales. The two Prymnesium species are reported to coexist at several locations, including the Sands-fjord system in southwestern Norway. Comparisons of physiology and toxicity within the two species have failed to reveal differences that can add to the small morphological distinctions used to separate them. To investigate the genetic relationship between the two species, we compared the sequence of the first internal transcribed spacer region (ITS1)and length variation in one intron separating calmodulin genes for four P. parvum strains and eight P. patelliferum strains. Both the ITS1 sequence and the banding patterns obtained by PCR amplification of one intron in the calmodulin genes indicated that the Prymnesium isolates are related by their geographic origin instead of 4 their species affiliation. The results indicate that P. parvum and P. patelliferum are so closely related that they could be considered one species. Alternatively, we discuss the possibility that the two species might be joined in a heteromorphic haploid-diploid life cycle, as is now widely reported for other haptophycean algae.     

THE AUTOFLUORESCENT FLAGELLUM: A NEW PHYLOGENETIC ENIGMA1

Epifluorescence microscopy reveals the presence of fluorescence in the living cells of at least three classes of flagellates. In Ochromonas cells, the fluorescence is blue-green in color and is found only in the short flagellum, both in the flagellar swelling and throughout the length of the flagellum. As recognized by the locale and color of the flagellar fluorescence, the same fluorescence is observed in only certain other heterokont algal groups but is also found in one of the two isokont flagella of the prymnesiophyte Prymnesium parvum.     

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.     

Removal of Prymnesium parvum (Haptophyceae) and its toxins using clay minerals

Laboratory experiments were conducted to examine the ability of several clay minerals from Sweden to remove the fish-killing microalga, Prymnesium parvum Carter, from suspension. In their commercial form (i.e. after incineration at 400 °C), seawater slurries (salinity = 26) of the three minerals tested were generally ineffective at removing P. parvum from culture within a range of 0.01 to 0.50 g/L, and after 2.5 h of flocculation and settling. Dry bentonite (SWE1) displayed the highest removal efficiency (RE) at 17.5%, with 0.50 g/L. Illite (SWE3) averaged only 7.5% RE between 0.10 to 0.50 g/L, while kaolinite (SWE2) kept the cells suspended instead of removing them. Brief mixing of the clay-cell suspension after SWE1 addition improved RE by a factor of 2.5 (i.e. 49% at 0.50 g/L), relative to no mixing. The addition of polyaluminum chloride (PAC, at 5 ppm) to 0.50 g/L SWE1 also improved RE to 50% relative to SWE1 alone, but only minor improvements in RE were seen with SWE2 and SWE2 combined with PAC. In further experiments, P. parvum grown in NP-replete conditions were removed in greater numbers than cells in N- or P-limited cultures, at 0.10–0.25 g/L of SWE1 and 5 ppm PAC. With 0.50 g/L, RE converged at 40% for all three culture conditions. The toxin concentration of NP-replete cultures decreased from 24.2 to 9.2 μg/mL (60% toxin RE) with 0.10–0.50 g/L SWE1 treatment and 5 ppm PAC. A strong correlation was found between cell and toxin RE (r²=0.995). For N-limited cultures, toxin RE ranged between 21 and 87% with the same clay/PAC concentrations, although the correlation between cell and toxin removal was more moderate (r²=0.746) than for NP-replete conditions. Interestingly, the toxin concentration within the clay-cell pellet increased dramatically after treatment, suggesting that clay addition may stimulate toxin production in N-stressed cells. For P-limited cultures, toxin concentration also decreased following clay/PAC treatment (i.e. 36% toxin RE), but toxin removal was poorly correlated to cell removal (r²=0.462). To determine whether incineration affected SWE1’s removal ability, a sample of its wet, unprocessed form was tested. The RE of wet bentonite (SWE4) was slightly better than that of SWE1 (31% versus 17%, respectively, at 0.50 g/L), but when 5 ppm PAC was added, RE increased from 10 to 64% with 0.05 g/L of SWE4, and increased further to 77% with 0.50 g/L. There were no significant differences in RE among NP-replete, N-limited and P-limited cultures using PAC-treated SWE4. Finally, RE varied with P. parvum concentration, reaching a maximum level at the lowest cell concentration (1×10³ cells/mL): 100% RE with 0.10 and 0.50 g/L SWE4 + 5 ppm PAC. RE dropped as cell concentration increased to 1×10⁴ and 5×10⁴ cells/mL, but rose again when concentration increased to 1×10⁵ cells/mL, the concentration used routinely for the removal experiments above. Based on these results, SWE4 with PAC was the most effective mineral sample against P. parvum. Overall, these studies demonstrated that clay flocculation can be effective at removing P. parvum and its toxins only under certain treatment conditions with respect to cell concentration, clay type and concentration, and physiological status.     

STIMULATING EFFECT OF ANABAENA SP. (CYANOBACTERIA) EXUDATE ON PRYMNESIUM PARVUM (HAPTOPHYTA)1

Prymnesium parvum blooms have become more frequent in the south‐central United States, leading to significant ecological and economic impacts. Allelopathic effects from cyanobacteria were suggested as a mechanism that might limit the development of P. parvum blooms. This research focused on the effects of cultured cyanobacteria, Anabaena sp., on P. parvum. Over a 6‐d period, daily additions of filtrate from the senescent Anabaena culture were made to P. parvum cultures growing in log phase. All treatments, including several types of controls, showed reductions in P. parvum biomass over the course of the experiment, but the treatments receiving Anabaena filtrate were reduced to a lesser degree, suggesting that filtrate from the senescent cyanobacteria culture was beneficial to P. parvum in some way. This unexpected outcome may have resulted from stimulation of heterotrophic bacteria by the addition of Anabaena filtrate, which likely contained exudates rich in dissolved organic carbon compounds. P. parvum was then able to supplement its nutritional requirements for growth by feeding on the elevated bacteria population. These findings coupled to previous observations suggest that interactions between cyanobacteria and P. parvum in natural environments are complex, where both allelopathic and growth‐stimulating interactions are possible.     

STEROLS OF SOME MARINE PRYMNESIOPHYCEAE

Sterols were identified in six marine prymnesiophyte isolates, some of which appear to have value as bivalve food. The principal sterol in Pleurochrysis carterae (Milford #961) and an unidentified prymnesiophyte (CCMP1215) was 24-methylcholesta-5,22-dienol, a common sterol in prymnesiophytes. Isolates CCMP594, CCMP609, and CCMP459 contained either 24-ethylcholesta-5,22-dienol or 24-ethylcholest-22-enol as the major sterol. In addition, Pavlova pinguis (CCMP609) and Pavlova sp. (CCMP459) contained the unusual dihydroxysterols 24-methylpavlovol and 24-ethylpavlovol, which have been found only in members of the Pavlovales. Prymnesium parvum contained cholesterol without traces of other sterols. Compared to the other isolates, the quantity of sterols was extremely low in P. parvum.     

Microcladia exserta sp. nov. (Ceramiaceae,Rhodophyta) from the East coast of South Africa

Microcladia exserta, a new species of the red algal genus Microcladia Greville (Ceramiaceae, Ceramiales), is described from the Natal coast of South Africa. This small, creeping alga, an epiphyte on the coralline alga Amphiroa anceps (Lamarck) Decaisne, is distinguished from other species in the genus by the following combination of characteristics: the prostrate habit, the exerted position of the tetrasporangia, and the presence in the cortex of numerous and conspicuous vesicle cells. Evidence is presented to demonstrate that the criterion of an erect habit in Microcladia vs. a prostrate habit in Herpochondria used to separate these genera is not sound.     

Two new species of Isochrysis with remarks on the genus Ruttnera

Two marine strains of Chrysophyceae have been studied with the aid of light and electron microscopy. The benthic stages show definite similarities with two marine Gloeochrysis spp. (G. maritima and G. litoralis) described by Anand (1937), but the swarmers do not agree with the definition of this genus. The reason why they also cannot be assigned to the genus Ruttnera is discussed and their inclusion within the Isochrysidales is justified by the observation of haptophycean scales on the motile cells. The names Isochrysis maritima sp. nov. and Isochrysis litoralis sp. nov. are proposed in view of the fact that these strains might prove to be the species observed by Anand.     

Fish kills related toPrymnesium parvum N. Carter (Haptophyta) in the People's Republic of China

Prymnesium parvum has been known to cause mass mortality of fish in PR of China since 1963. It usually occurs in brackish waters and inland high-mineral waters. The fish-breeding industry (mainly species of carp) in these regions of the PRC has been threatened by this microalga. Electron microscopic examination of isolates from Dalian and Tianjin revealed that the isolates wereP. parvum, based on specific scale patterns and two kinds of scales. The symptoms of the poisoned fish and the control of this toxic alga are also discussed. The addition of ammonium sulfate, copper sulfate, mud, reduced salinity and organic fertilizer to fish ponds has been partially successful in controlling blooms of this toxic alga. Adding 50–70 kg ha^–1 day^–1 manure (dry weight) to the fish pond to inhibitP. parvum from becoming the dominant species in the fish pond is recommended. A reduction in salinity to less than 2 is the easiest way to save freshwater fish from being poisoned byP. parvum. Use of ammonium sulfate is an efficient, economical and safer method to controlP. parvum than copper sulfate or mud.