Effect of biotic and abiotic factors on in vitro proliferation, encystment, and excystment of Pfiesteria piscicida

Pfiesteria spp. are mixotrophic armored dinoflagellates populating the Atlantic coastal waters of the United States. They have been a focus of intense research due to their reported association with several fish mortality events. We have now used a clonal culture of Pfiesteria piscicida and several new environmental isolates to describe growth characteristics, feeding, and factors contributing to the encystment and germination of the organism in both laboratory and environmental samples. We also discuss applied methods of detection of the different morphological forms of Pfiesteria in environmental samples. In summary, Pfiesteria, when grown with its algal prey, Rhodomonas sp., presents a typical growth curve with lag, exponential, and stationary phases, followed by encystment. The doubling time in exponential phase is about 12 h. The profiles of proliferation under a standard light cycle and in the dark were similar, although the peak cell densities were markedly lower when cells were grown in the dark. The addition of urea, chicken manure, and soil extracts did not enhance Pfiesteria proliferation, but crude unfiltered spent aquarium water did. Under conditions of food deprivation or cold (4 degrees C), Pfiesteria readily formed harvestable cysts that were further analyzed by PCR and scanning electron microscopy. The germination of Pfiesteria cysts in environmental sediment was enhanced by the presence of live fish: dinospores could be detected 13 to 15 days earlier and reached 5- to 10-times-higher peak cell densities with live fish than with artificial seawater or f/2 medium alone. The addition of ammonia, urea, nitrate, phosphate, or surprisingly, spent fish aquarium water had no effect.     

Characterization of Ichthyocidal Activity of Pfiesteria piscicida: Dependence on the Dinospore Cell Density

The ichthyocidal activity of Pfiesteria piscicida dinospores was examined in an aquarium bioassay format by exposing fish to either Pfiesteria-containing environmental sediments or clonal P. piscicida. The presence of Pfiesteria spp. and the complexity of the microbial assemblage in the bioassay were assessed by molecular approaches. Cell-free water from bioassays that yielded significant fish mortality failed to show ichthyocidal activity. Histopathological examination of moribund and dead fish failed to reveal the skin lesions reported elsewhere. Fish larvae within “cages” of variable mesh sizes were killed in those where the pore size exceeded that of Pfiesteria dinospores. In vitro exposure of fish larvae to clonal P. piscicida indicated that fish mortality was directly proportional to the dinospore cell density. Dinospores clustered around the mouth, eyes, and operculi, suggesting that fish health may be affected by their direct interaction with skin, gill epithelia, or mucous surfaces. Molecular fingerprinting revealed the presence of a very diverse microbial community of bacteria, protists, and fungi within bioassay aquaria containing environmental sediments. Some components of the microbial community were identified as potential fish pathogens, preventing the rigorous identification of Pfiesteria spp. as the only cause of fish death. In summary, our results strongly suggest (i) that this aquarium bioassay format, which has been extensively reported in the literature, is unsuitable to accurately assess the ichthyocidal activity of Pfiesteria spp. and (ii) that the ichthyocidal activity of Pfiesteria spp. is mostly due to direct interactions of the zoospores with fish skin and gill epithelia rather than to soluble factors.     

Formation and germination of temporary cysts of Cochlodinium polykrikoides Margalef (Dinophyceae) and their ecological role in dense blooms

While the initiation and development of dense bloom of Cochlodinium polykrikoides have been shown to be related to some environmental factors, little is known about the ecological role of the formation and germination of temporary cysts, nor of their significance for the rapid expansion of dense regional-scale blooms. This study examined the factors affecting the formation and germination of temporary cysts of C. polykrikoides, and provides details about the germination process. In the laboratory experiments, C. polykrikoides produced the chain-forming temporary cysts that are immobile and surrounded by a hyaline membrane. The encystment experiment indicated that darkness induces the formation of chain-forming temporary cysts, consistent with field observation of morphology and fluxes of temporary cysts. Germination occurred twice from a single four-celled temporary cysts within 24 h after exposure to light, and the germlings appeared as two-celled chain-forming vegetative cells. The germination behavior of temporary cysts of C. polykrikoides differs from that of other dinoflagellates, and this may be a survival strategy for the maintenance of population size during dense blooms.     

Host-specific plant signal and G-protein activator, mastoparan, trigger differentiation of zoospores of the phytopathogenic oomycete Aphanomyces cochlioides

We found that the gradient of a host-specific attractant, cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone) isolated from the roots of spinach triggered encystment followed by germination of zoospores of Aphanomyces cochlioidesat a concentration less than micromolar order. This compound did not affect the growth and reproduction of this phytopathogen up to 10^–6 M concentration in the culture medium. We also observed that mastoparan, an activator of heterotrimeric G-protein could inhibit the motility of zoospores and then strikingly effect encystment followed by 60–80% germination of cysts. Concomitant application of cochliophilin A and mastoparan showed stronger encystment followed by 100% germination of cysts. In addition, we have observed that chemicals interfering with phospholipase C activity (neomycin) and Ca²⁺ influx/release (EGTA and loperamide) suppress cochliophilin A or mastoparan induced encystment and germination. These results suggest that G-protein mediated signal transduction mechanism may be involved in the differentiation of the A. cochlioides zoospores. This is the first report on the differentiation of oomycete zoospores initiated by a host-specific plant signal or a G-protein activator.     

Toxigenic Pfiesteria species—Updates on biology,ecology, toxins,and impacts

The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3) × 10² to 10³ (rarely, 10⁴) flagellate cells mL⁻¹. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10 cells mL⁻¹), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates.     

Direct uptake of nitrogen by Pfiesteria piscicida and Pfiesteria shumwayae, and nitrogen nutritional preferences

The rates of uptake of a range of forms of nitrogenous nutrients were measured in cultures of Pfiesteria piscicida and Pfiesteria shumwayae maintained at varying physiological states. The measured rates of dissolved N uptake under some conditions approached the rates of N uptake that are achieved through phagotrophy. Rates of dissolved N uptake by P. piscicida contributed <10% of the cellular N of flagellated cells feeding on algae, but were equal to or greater than phagotrophic N acquisition in cells recently removed from fish cultures. Specific N uptake rates (V, h⁻¹) were higher for cells that were maintained on algal prey for long periods (months) than those that were grown with live fish. However, rates of N uptake on a cellular basis for cells grown on or recently removed from fish were comparable to those maintained on algal prey, likely reflecting differences in the sizes of cells of different physiological condition. Preferences for form of N generally followed a decreasing trend of amino acids > urea > NH₄⁺ > NO₃⁻. Nitrate consistently was not a preferred form of N. Although Pfiesteria spp. are often found in eutrophic environments, the relationship between Pfiesteria spp. and nutrient availability is likely to be primarily indirect, mediated through the production of various prey on which Pfiesteria spp. feed. These findings also confirm, however, that when dissolved N concentrations are elevated, they can contribute to the supplemental nutrition of these cells, and thus may provide a significant source of N to Pfiesteria spp. in nature.     

Generalist Life Cycle Aids Persistence of Alexandrium ostenfeldii (Dinophyceae) in Seasonal Coastal Habitats of the Baltic Sea

In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.     

HIGH ENCYSTMENT SUCCESS OF THE DINOFLAGELLATE SCRIPPSIELLA CF. LACHRYMOSA IN CULTURE EXPERIMENTS 1

Close to 100% encystment efficiency and a yield above 10⁵ cysts·mL ^{? 1} were routinely achieved in full strength f/2 medium‐based batch cultures (883 μM NO₃ ^? and 36 μM PO₄ ^{? 3}) of the marine dinoflagellate Scrippsiella cf. lachrymosa Lewis. Increases in cell density led to nutrient depletion in this enriched medium, which was the most likely cause for initiation of cyst formation. Lowering the concentration of either nutrient to 1/10 the initial levels decreased the encystment efficiency, whereas use of ammonium as the N source resulted in both low cell yield and low encystment efficiency. The mandatory dormancy period was ca. 60 days and was not affected by cold dark storage of the cysts. Cysts produced in the initial phase of sexual reproduction were relatively large (length 47 μm, width 31 μm) with a heavy calcareous cover. Cysts produced thereafter lacked apparent calcareous cover and were smaller (length 29 μm, width 19 μm). The decrease of cyst volume (by a factor of 0.24–0.4) suggested strong resource limitation during the course of encystment. However, after the mandatory dormancy period, germination success of the smaller cysts was higher (80%), compared with the larger cysts that had been produced initially (50%). Germling survival (74%) was independent of cyst type but was enhanced by higher nutrient concentration during incubation. The ratio of initial nutrient concentration in the medium to the cyst yield was used as a proxy to estimate the cellular nutrient quota. The conservative estimates of 9 pmol N·cyst ^{? 1} and 0.4 pmol P·cyst ^{? 1} obtained in this manner are at the low end of the range of previous published estimates for other dinoflagellate cysts. Given the high encystment observed in laboratory experiments, we have no reason to assume an inherently lower encystment success in dinoflagellate field populations. Our results do not challenge the low nutrient paradigm for dinoflagellate sexuality. We believe that the high encystment success and cyst yield of this particular species is at least partly due to its ability to achieve very high cell densities in cultures, which evidently leads to nutrient depletion even in f/2 medium.     

Protein storage in large peripheral vesicles in Phytophthora zoospores and its breakdown after cyst germination

The fate of large vesicles that line the periphery of Phytophthora cinnamomi zoospores was monitored by immunogold labeling during encystment and germination. Labeling was carried out using a monoclonal antibody, Lpv-1, directed against glycoprotein components of these vesicles. The results show that the vesicles are retained inside the zoospores during encystment, but their contents are degraded after germination. During initial stages of degradation, the large peripheral vesicles dilate to form small vacuoles containing fine electron-dense fibrillar material which is immunoreactive. Eventually, these small vacuoles fuse together to form large electron-lucent vacuoles which contain very little immunoreactive material. Immunoblot analysis of germinating cysts with Lpv-1 shows that the level of glycoprotein components in the vesicles declines dramatically following germination. It is proposed on the basis of this evidence that the large peripheral vesicles contain storage protein which acts as an endogenous supply of nitrogen reserves for the growing germ tube. Lpv-1 also labels peripheral vesicles in two other species of Phytophthora, P. parasitica var. nicotiana and P. nicotiana var. nicotiana. As in P. cinnamomi, these vesicles do not undergo exocytosis during encystment. The large peripheral vesicles thus appear to be analogous to protein bodies found in seeds of higher plants, and they may be a common feature of Phytophthora zoospores.     

Relative contribution of exotoxin and micropredation to icthyotoxicity of two strains of Pfiesteria shumwayae (Dinophyceae)

The mechanism by which Pfiesteria shumwayae (Glasgow and Burkholder) kills fish is controversial. Several studies have implicated a Pfiesteria-associated exotoxin in fish mortality while other studies indicate that physical attack of dinoflagellates on fish (micropredation) and not exotoxin is responsible. We examined the ichthyotoxicity of two strains of P. shumwayae (CAAE 101272 and CCMP 2089) in a bioassay system that exposed test fish to the dinoflagellates both with and without direct contact in the same aquarium at the same time. Dinoflagellate-free supernatants from both strains were also tested for toxicity. The results showed that direct contact between P. shumwayae and fish significantly enhanced fish mortality with both strains (P < 0.001). About 87.5% and 100% of fish died when exposed directly to CAAE 101272 and CCMP 2089, respectively. When protected from direct contact with Pfiesteria cells, 19% of the fish exposed to CAAE 101272 and 6% of those exposed to CCMP 2089 died. No deaths were observed in controls. Supernatant killed fish when obtained from cultures of CAAE 101272 but not when obtained from CCMP 2089.Analysis of variance showed that, for both strains, fish mortality in Pfiesteria-inoculated bioassays was significantly higher than control bioassays both with and without direct contact (P < 0.001). Differences between strains were not significant (P = 0.3). These results indicate that both strains are associated with exotoxin production. However, the dominant and most consistent mechanism of fish mortality observed in this study required physical contact between fish and Pfiesteria cells.     

A NEW LARVAL FISH BIOASSAY FOR TESTING THE PATHOGENICITY OF PFIESTERIA SPP. (DINOPHYCEAE)1

Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium‐format Pfiesteria bioassays. Therefore, we developed a sensitive 96‐h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six‐well tissue culture plates and 7‐day‐old larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%–100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL^?1. No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose‐response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae, generating a 96‐h LD₅₀ of 108 zoospores·mL^?1. Additionally, we applied the assay to evaluate a 38‐L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia (Oreochromis niloticus) in a 500‐mL assay system. Water from the fish‐killing 38‐L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell‐free fraction). After 96 h, the larval fish assay exhibited 50%–100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500‐mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity.     

几种培养方法对萼花臂尾轮虫(Brachionus calyciflorus)种群密度及其环境因子影响的比较研究

分别利用泥鳅 -藻 -轮虫和罗非鱼 -藻 -轮虫共生隔离培养的方法 ,研究萼花臂尾轮虫 ( Brachionuscalyciflorus)的种群密度和主要环境因子的变化。研究结果表明 :加入泥鳅的培养缸中 ,有 1 1 d轮虫数量超过 1 0 0 ind/ml,其中有 5d超过 2 0 0ind/ml,最高密度达 2 50 ind/ml;在罗非鱼的培养缸中 ,在整个实验过程中 ,种群密度超过 1 0 0 ind/ml以上的时间有 6d(第5～ 1 0天 ) ;单纯利用藻类培养轮虫时 ,种群密度较低 ,且维持时间较短 ,最高种群密度不足 1 0 0 ind/ml,利用藻类 -轮虫 -鱼类共存同一生态系统中 ,并使鱼类和轮虫隔离进行生态培养时 ,能提高轮虫种群密度 ,延长种群高峰期     

Factors regulating excystment of Alexandrium in Puget Sound,WA, USA

Factors regulating excystment of a toxic dinoflagellate in the genus Alexandrium were investigated in cysts from Puget Sound, Washington State, USA. Experiments were carried out in the laboratory using cysts collected from benthic seedbeds to determine if excystment is controlled by internal or environmental factors. The results suggest that the timing of germination is not tightly controlled by an endogenous clock, though there is a suggestion of a cyclical pattern. This was explored using cysts that had been stored under cold (4 °C), anoxic conditions in the dark and then incubated for 6 weeks at constant favorable environmental conditions. Excystment occurred during all months of the year, with variable excystment success ranging from 31–90%. When cysts were isolated directly from freshly collected sediments every month and incubated at the in situ bottom water temperature, a seasonal pattern in excystment was observed that was independent of temperature. This pattern may be consistent with secondary dormancy, an externally modulated pattern that prevents excystment during periods that are not favorable for sustained vegetative growth. However, observation over more annual cycles is required and the duration of the mandatory dormancy period of these cysts must be determined before the seasonality of germination can be fully characterized in Alexandrium from Puget Sound. Both temperature and light were found to be important environmental factors regulating excystment, with the highest rates of excystment observed for the warmest temperature treatment (20 °C) and in the light.     

The resistance of cysts of Stictodora lari (Trematoda: Heterophyidae) to encapsulation by cells of the fish host

Whereas glass beads are encapsulated by cells within 3 days after implantation into the abdominal cavity of Gambusia affinis, cysts of Stictodora lari in the same site are not encapsulated until 21–23 days after infection. The achievement of encystment in vitro demonstrated that the initial cyst wall is of parasite origin and fluorescent antibody methods showed that it does not mimic fish tissue in composition. Cysts formed in vivo have material, presumably of fish origin, associated with the cyst wall, as do living and fixed in vitro cysts following implantation.It is considered that cysts are not encapsulated for some weeks after infection because they are disguised as host tissue by material of fish origin associated with the cyst wall. An alternative explanation is proposed if the fish material does not have this functional role; the presence of spikes on the initial cyst wall may form an unsatisfactory substrate for the attachment of cells.     

Balamuthia mandrillaris: Role of galactose in encystment and identification of potential inhibitory targets

Balamuthia mandrillaris is a causative agent of granulomatous encephalitis that almost always proves fatal. A major concern during the course of therapy is that B. mandrillaris can transform into cysts. Cysts are highly resistant to physical and chemical conditions and present a problem in successful antimicrobial chemotherapy. However, the underlying mechanisms of B. mandrillaris transformation into cysts are not known. In this study, we examined the effects of exogenous sugars on B. mandrillaris encystment. The findings revealed that free exogenous galactose, but not other sugars, enhanced parasite differentiation into cysts, and apparently a galactose-binding protein is involved in B. mandrillaris encystment. Cytoskeletal re-arrangements and phosphatidylinositol 3-kinase (PI3K)-mediated pathways are involved in B. mandrillaris encystment based on inhibitor studies. Dual functionality of galactose-binding protein in B. mandrillaris pathogenesis and encystment is discussed further.     

ROLE OF ENCYSTMENT AND EXCYSTMENT OF PERIDINIUM BIPES F. OCCULATUM (DINOPHYCEAE) IN FRESH WATER RED TIDES IN LAKE KIZAKI,JAPAN1

The encystment flux of Peridinium bipes f. occulatum (Dinophyceae) was investigated with sediment traps from 1968 to 1990 in Lake Kazki. Cysts of P. bipes were formed throughout the blooms, Encystment flux of P. bipes in the pelagic zone was usually lower than those at shallow sites, and the density of P. bipes cysts in lake sediment was higher in the shallow region than in the pelagic zone. However, in the shallower region, The concentration of P. bipes cysts varied widely, possibly due to high rates of encystment and excystment. Peridinium bipes encystment occurred between 15° and 25° C in the laboratory, with very little cyst formation below 10°C. Though cyst formation was observed in continous darkness, the rate increased with irradiance. Under continuous darkness, no excystment was observed at any temperature from 5° to 25° C. Eighty-one percent of the cysts illuminated at 105 μE m^?2 s^?1 excysted after 13 days incubation at 15° C, and lower irradiances decreased germination success. Results from laboratory experiments suggest that light is a critical factor in the germination of P. bipes cysts. Bottom depth thus can have a significant effect on germination because cysts only can excyst from depths where light is sufficient. The shallow region of the lake is thus very important as a seed bed for P. bipes during early spring. Cyst deposited in deeper waters may not ever germinate unless they are resuspended and transported to shallow areas where light reaches the bottom.     

SEXUALITY AND CYST FORMATION IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS: CYST YIELD IN BATCH CULTURES1

Encystment of the toxic dinoflagellate Gonyaulax tamarensis Lebour (var. excavata) was monitored in batch cultures exposed to a variety of nutritional and environmental treatments. Limitation by nitrogen (as ammonium or nitrate) or phosphorus (as phosphate) resulted in cyst formation. When the initial concentration of limiting nutrient was varied, total cyst yield (mL^?1) was directly proportional to the cell yield at all but the highest nutrient concentrations (where encystment was minimal). Encystment efficiency was relatively constant (0.1–0.2 cysts · cell^?1) over a 5-fold range of cell densities, indicating that 20 to 40% of the vegetative populations successfully encysted. Cyst formation was negligible in nutrient-replete medium, even with a significant reduction in growth rate due to non-optimal light, temperature, or to high batch culture cell densities. Low light levels did decrease cyst yield once encystment was initiated by nutrient limitation, but this was probably linked to smaller motile cell yield and not to a specific inhibition of encystment. In contrast, encystment was more sensitive to temperature than was growth rate: optimal cyst production occurred over a relatively narrow temperature range and no cysts were formed at [Page missing]     

Life cycle and mode of infection of Leptolegnia chapmanii (Oomycetes) parasitizing Aedes aegypti

The life cycle and mode of infection of mosquito larvae by Leptolegnia chapmanii (Oomycetes: Saprolegniales) were determined. The life cycle is typical of saprolegniaceous fungi, as the species is dimorphic producing diplanetic biflagellate zoospores. Sexual reproduction is by means of gametangial contact and results in the production of a characteristic papillate oogonium containing a subcentric or eccentric oospore. L. chapmanii is capable of infecting Aedes aegypti larvae both by germination of encysted secondary zoospores on the exterior cuticle and by germination of ingested zoospore cysts in the larval midgut. Once the fungus is established in the host, the disease, a coelomomycosis, is fatal. The encystment pattern of secondary zoospores on the larval cuticle appcars preferential. Scanning electron microscopy indicates that mechanical pressure is not the sole force utilized by the fungus for cuticle penetration.