The ecophysiology and bloom dynamics of Prymnesium spp.

Members of Prymnesium belong to the division Haptophyta, class Prymnesiophyceae, order Prymnesiales and family Prymnesiaceae. As most haptophytes, members of the genus Prymnesium are unicellular and planktonic. The most known of these species is the ichthyotoxic P. parvum, which may form nearly monospecific dense blooms in coastal and inland waters. This species possesses extraordinary plasticity concerning life survival strategies, and is specifically addressed in this review.Toxins produced by P. parvum have hemolytic properties, that not only kill fish but also co-existing plankton. These substances are allelopathic (when other algae are killed) and grazer deterrent (when grazers are killed). Allelopathy enables P. parvum to utilize inorganic nutrients present in the surrounding water without competition from other algal species; and by eliminating its grazers P. parvum reduces cell losses. The paralized microalgae and/or zooplankton, are therefter ingested by the P. parvum cells, a process called phagotrophy. P. parvum is also able of osmotrophy, i.e. utilization of dissolved organic matter. In this review, the cellular characteristics, life cycles, bloom formation, and factors affecting toxicity, allelopathy, phagotrophy, and osmotrophy of P. parvum are discussed.     

Beyond hydraulic flushing: Deep water mixing takes the harm out of a haptophyte algal bloom

Inflows are linked to water quality, food web dynamics, and the incidence of harmful algal blooms (HABs). It may be that inflows can be manipulated to mitigate some blooms and accelerate recovery of living natural resources, such as fisheries. Lack of available water, however, can limit this approach to management. Utilizing source water from deeper depths to displace surface waters, however, might effectively mimic aspects of inflow events, such as disrupting ecological processes and community succession through hydraulic displacement. We tested this notion by conducting in-lake mesocosm experiments with natural plankton communities plagued by Prymnesium parvum where we manipulated hydraulic flushing. We found that P. parvum cell densities were reduced by up to 69% and 53%, and ambient toxicity ameliorated, during pre-bloom and bloom development periods. Furthermore, other phytoplankton taxa and zooplankton benefited from these pulsed flushing events. In other words, hydraulic flushing with deep waters not only suppressed P. parvum bloom initiation and development, but also proved beneficial to other aspects of the lower food web. These observations provide the first demonstration that P. parvum bloom initiation may be interrupted, and bloom development may be arrested, through hydraulic manipulations.     

The effects of aeration on growth and toxicity of Prymnesium parvum grown with and without algal prey

We investigated the effects of aeration on growth and toxicity of the haptophyte Prymnesium parvum in the presence and absence of the algal prey Rhodomonas salina. Batch monocultures of P-limited P. parvum and N and P sufficient R. salina and mixed cultures of the two microalgae were grown with no, low (20) and high (100) ml min⁻¹ aeration for 18 days. Cell growth of P. parvum and R. salina and cell toxicity of P. parvum were studied over the experimental period. The highest specific growth rates of P. parvum were found at low aeration rates. R. salina in monocultures showed typical growth patterns, while R. salina numbers declined rapidly in the mixed cultures. Of the initial cell densities, 98–100% of the R. salina cells were lysed or ingested within 24 h of mixing with P. parvum cells. The maxima P. parvum biomasses were significantly higher in the mixed cultures than in the monocultures. Cell toxicity of P. parvum increased significantly in response to aeration rates and the highest levels were found in the high aeration condition. Availability of prey and resupply of inorganic nutrients decreased P. parvum cell toxicity. Our study suggests that P. parvum is tolerant and is able to grow over a broad range of aeration and associated turbulence effects though low aeration represents an optimal condition for growth. As P. parvum toxicity was higher in the high aeration treatment we suggest that the higher concentrations of oxygen cause more toxins to be produced, as these are oxygen rich compounds. We suggest that oxygenation and turbulence of surface waters caused by mixing may be involved in promoting high toxic P. parvum blooms in shallow lakes and coastal waters.     

GROWTH AND TOXICITY OF PRYMNESIUM PARVUM (HAPTOPHYTA) AS A FUNCTION OF SALINITY,LIGHT, AND TEMPERATURE1

The growth rate, stationary cell concentration, and toxicity of Prymnesium parvum N. Carter were measured using a strain isolated from Texas inland waters. We used a multifactor experimental approach with multiple regression analysis to determine the importance of environmental factors, including temperature, light, and salinity to these algal measurements. Exponential growth rate was unimodal in relation to temperature, salinity, and irradiance, with an estimated maximal growth of 0.94 d^?1 occurring at 27°C, 22 practical salinity units (psu), and 275 μmol photons·m^?2·s^?1. Stationary cell concentrations also had unimodal responses to temperature and salinity but increased with irradiance. Maximal cell concentrations were estimated to occur at 26°C and 22 psu. Both maximum growth rate and highest stationary cell concentrations were measured at levels of each factor resembling warm, estuarine conditions that differ from the conditions under which blooms occur in inland waters in the southwestern United States. Acute toxicity to fish was highest at the lowest salinity and temperature levels, conditions not optimal for exponential growth but similar to those under which blooms occur in inland waters. Our results imply that summer blooms could occur in inland waters of the southwestern United States. Generally, they have not, suggesting that factors other than those investigated in this research influence bloom dynamics.     

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.     

Recruitment of resting stages may induce blooms of Microcystis at low N:P ratios

Some species of cyanobacteria form resting stages at the sedimentsurface when environmental conditions become unfavourable. Asconditions turn more favourable, these resting stages hatchto the water phase, where the cells grow, reproduce, and sometimesform blooms. Since blooms of cyanobacteria have become an increasingthreat to inland and brackish waters, it is important to assessthe mechanisms and processes involved in the initiation of suchblooms. One such mechanism is recruitment from the sedimentsurface. Potential factors regulating the recruitment of restingstages include variations in nutrient concentrations and ratios,as well as variations in grazing. To investigate how the recruitmentof Microcystis responds to different levels of these factors,we performed an enclosure experiment (zooplankton abundanceswere regulated by predation from fish). We found that recruitmentand growth were most pronounced at the second highest nutrientconcentration (average concentrations were 498 µg l^-1of dissolved nitrogen and 134 µg l^-1 of total phosphorus),while no direct response to different grazing levels was detected.We also found that resting stages can be important for initiatingand sustaining blooms. The environmental conditions most importantin regulating the recruitment rate from resting stages correspondedto the requirements of the plankton cells, namely high nutrientaddition and low N:P ratio.     

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.     

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.     

Nitrogen Fixation During Blooms of Nodularia in Coastal Waters and Backwaters of the Arkona Sea (Baltic Sea) in 1974

Nitrogen fixation was investigated by means of the acetylene reduction method during the development of a water bloom of Nodularia in coastal waters of the Baltic Sea west of the island of Hiddensee and in backwaters showing different degrees of eutrophication. Depending on plankton density, the values found varied greatly. The maximum of nitrogen fixation values found in extremely dense water blooms under special conditions (Baltic Sea, 2250 μg N₂/l · h; Kleiner Jasmunder Bodden, 374 μg N₂/l · h) are up to 10³ times higher than from other parts of the Baltic Sea or from inland waters. The average nitrogenase activity determined for coastal water populations of the Baltic Sea is 2.15 pg N₂/heterocyst · h and that of the inmost backwaters 0.77 pg N₂/heterocyst · h. The relationship between N₂-fixation and nutrient content in water is discussed.     

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.     

Prorocentrum minimum (Pavillard) Schiller: A review of a harmful algal bloom species of growing worldwide importance

Prorocentrum minimum (Pavillard) Schiller, a common, neritic, bloom-forming dinoflagellate, is the cause of harmful blooms in many estuarine and coastal environments. Among harmful algal bloom species, P. minimum is important for the following reasons: it is widely distributed geographically in temperate and subtropical waters; it is potentially harmful to humans via shellfish poisoning; it has detrimental effects at both the organismal and environmental levels; blooms appear to be undergoing a geographical expansion over the past several decades; and, a relationship appears to exist between blooms of this species and increasing coastal eutrophication. Although shellfish toxicity with associated human impacts has been attributed to P. minimum blooms from a variety of coastal environments (Japan; France; Norway; Netherlands; New York, USA), only clones isolated from the Mediterranean coast of France, and shellfish exposed to P. minimum blooms in this area, have been shown to contain a water soluble neurotoxic component which killed mice. Detrimental ecosystem effects associated with blooms range from fish and zoobenthic mortalities to shellfish aquaculture mortalities, attributable to both indirect biomass effects (e.g., low dissolved oxygen) and toxic effects. P. minimum blooms generally occur under conditions of high temperatures and incident irradiances and low to moderate salinities in coastal and estuarine environments often characterized as eutrophic, although they have been found under widely varying salinities and temperatures if other factors are conducive for growth. The physiological flexibility of P. minimum in response to changing environmental parameters (e.g., light, temperature, salinity) as well as its ability to utilize both inorganic and organic nitrogen, phosphorus, and carbon nutrient sources, suggest that increasing blooms of this species are a response to increasing coastal eutrophication.     

Occurrences of Prorocentrum minimum (Pavillard) in México

Prorocentrum minimum is a planktonic dinoflagellate known to produce red tides that can be harmful. To recognize localities and understand occurrences of Prorocentrum minimum blooms in Mexico, published data of plankton from 1942 to present, as well as unpublished data from the authors, were reviewed. Studies and reports covered marine and coastal waters of México during different periods. Presence of P. minimum were reported in the Pacific coast, Gulf of California, Gulf of México, and the Caribbean, but blooms have been only reported since 1990. Thirteen bloom events were recorded. Six occurred in shrimp ponds and seven near aquaculture regions or coastal areas where intensive agriculture is practiced. Most of the blooms can be associated with damage to the surrounding marine biota either in aquaculture ponds or open waters. Direct toxicity has not been fully evaluated, but data suggest that low oxygen may not easily explain all of the damage. Interestingly, for yet unknown reasons, cells belonging to the triangular morphotype have seldom been reported in México.     

Rapid Quantification of the Toxic Alga Prymnesium parvum in Natural Samples by Use of a Specific Monoclonal Antibody and Solid-Phase Cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Use of Cell Culture To Assess Cryptosporidium parvum Survival Rates in Natural Groundwaters and Surface Waters

Bench-scale survival studies with Cryptosporidium parvum were conducted with representative aquifer and reservoir waters of Florida. C. parvum inactivation rates ranged from 0.0088 log₁₀/day at 5°C to −0.20 log₁₀/day at 30°C. Temperature, surface water or groundwater type, and the interaction of these factors had statistically significant effects on the survival of C. parvum.     

Golden alga presence and abundance are inversely related to salinity in a high-salinity river ecosystem,Pecos River,USA

Prymnesium parvum (golden alga, GA) is a toxigenic harmful alga native to marine ecosystems that has also affected brackish inland waters. The first toxic bloom of GA in the western hemisphere occurred in the Pecos River, one of the saltiest rivers in North America. Environmental factors (water quality) associated with GA occurrence in this basin, however, have not been examined. Water quality and GA presence and abundance were determined at eight sites in the Pecos River basin with or without prior history of toxic blooms. Sampling was conducted monthly from January 2012 to July 2013. Specific conductance (salinity) varied spatiotemporally between 4408 and 73,786 μS/cm. Results of graphical, principal component (PCA), and zero-inflated Poisson (ZIP) regression analyses indicated that the incidence and abundance of GA are reduced as salinity increases spatiotemporally. LOWESS regression and correlation analyses of archived data for specific conductance and GA abundance at one of the study sites retrospectively confirmed the negative association between these variables. Results of PCA also suggested that at <∼15,000 μS/cm, GA was present at a relatively wide range of nutrient (nitrogen and phosphorus) concentrations whereas at higher salinity, GA was observed only at mid-to-high nutrient levels. Generally consistent with earlier studies, results of ZIP regression indicated that GA presence is positively associated with organic phosphorus and in samples where GA is present, GA abundance is positively associated with organic nitrogen and negatively associated with inorganic nitrogen. This is the first report of an inverse relation between salinity and GA presence and abundance in riverine waters and of interaction effects of salinity and nutrients in the field. These observations contribute to a more complete understanding of environmental conditions that influence GA distribution in inland waters.     

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.     

Cryptosporidium parvum Infection in SCID Mice Infected with Only One Oocyst: qPCR Assessment of Parasite Replication in Tissues and Development of Digestive Cancer

Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10⁵ oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.     

Direct Detection of Vibrio cholerae and ctxA in Peruvian Coastal Water and Plankton by PCR

Seawater and plankton samples were collected over a period of 17 months from November 1998 to March 2000 along the coast of Peru. Total DNA was extracted from water and from plankton grouped by size into two fractions (64 μm to 202 μm and >202 μm). All samples were assayed for Vibrio cholerae, V. cholerae O1, V. cholerae O139, and ctxA by PCR. Of 50 samples collected and tested, 33 (66.0%) were positive for V. cholerae in at least one of the three fractions. Of these, 62.5% (n = 32) contained V. cholerae O1; ctxA was detected in 25% (n = 20) of the V. cholerae O1-positive samples. None were positive for V. cholerae O139. Thus, PCR was successfully employed in detecting toxigenic V. cholerae directly in seawater and plankton samples and provides evidence for an environmental reservoir for this pathogen in Peruvian coastal waters.     

Allelopathic effects of the Baltic cyanobacteria Nodularia spumdigena, Aphanizomenon flos-aquae and Anabaena lemmermannii on algal monocultures

Allelopathy, the release of extracellular compounds that inhibit the growth of other microorganisms, may be one factor contributing to the formation and/or maintenance of cyanobacterial blooms. We investigated the allelopathic effects of three cyanobacterial species (Nodularia spumigena, Aphanizomenon flos-aquae and Anabaena lemmermannii) that frequently form mass-occurrences in the Baltic Sea. We exposed monocultures of three phytoplankton species (Thalassiosira weissflogii, Rhodomonas sp. and Prymnesium parvum) to cell-free filtrates of the three cyanobacteria, and quantified allelopathic effects with cell counts. We also investigated the role of the growth phase of cyanobacteria in their allelopathy, by comparing the effects of an exponential and a stationary phase culture of N. spumigena. All tested cyanobacteria inhibited the growth of Rhodomonas sp., but none of them affected P. parvum. The effects on T. weissflogii were more variable, and they were amplified by repeated filtrate additions compared to a single filtrate addition. N. spumigena was more allelopathic in exponential than in stationary growth phase, whereas the culture filtrate was more hepatotoxic in stationary phase. Hepatotoxins were thus probably not involved in the allelopathic effects, which is also indicated by the allelopathic properties of the non-toxic A. flos-aquae and A. lemmermannii. The results demonstrate that the common Baltic cyanobacteria affect some coexisting phytoplankton species negatively. Allelopathy may therefore play a role in interspecific competition and contribute to cyanobacterial bloom maintenance.