Variation in the abundance of Pseudo-nitzschia and domoic acid with surf zone type

Most harmful algal blooms (HAB) originate away from the shore and, for them to endanger human health, they must be first transported to shore after which they must enter the surf zone where they can be feed upon by filter feeders. The last step in this sequence, entrance into the surf zone, depends on surf zone hydrodynamics. During two 30-day periods, we sampled Pseudo-nitzschia and particulate domoic acid (pDA) in and offshore of a more dissipative surf zone at Sand City, California (2010) and sampled Pseudo-nitzschia in and out of reflective surf zones at a beach and rocky shores at Carmel River State Beach, California (2011). At Sand City, we measured domoic acid in sand crabs, Emerita analoga. In the more dissipative surf zone, concentrations of Pseudo-nitzschia and pDA were an order of magnitude higher in samples from a rip current than in samples collected just seaward of the surf zone and were 1000 times more abundant than in samples from the shoals separating rip currents. Domoic acid was present in all the Emerita samples and varied directly with the concentration of pDA and Pseudo-nitzschia in the rip current. In the more reflective surf zones, Pseudo-nitzschia concentrations were 1–2 orders of magnitude lower than in samples from 125 and 20 m from shore. Surf zone hydrodynamics affects the ingress of Pseudo-nitzschia into surf zones and the exposure of intertidal organisms to HABs on the inner shelf.     

62 An updated checklist of the seaweeds and seagrasses of oregon

In order to control harmful algal blooms, many biological approaches have been tried. Specially, there have recently been discussions concerning the roles of bacteria in algal bloom dynamics. Then, algicidal bacteria are expected as an agent considerate for harmful algal blooms control. Development of these organisms as biological control agents involves isolation from environmental samples. With the aim of develop eco-technology controlling water blooms in fresh waters, we isolated the diatom-lysing bacteria from the sediments of Lake Seokchon and Pal¡¯tang River-Reservoir. A soft agar-overlay technique was used to isolate the diatom lytic bacteria. The SK-02 showed a diatom lytic activity against Stephanodiscus hantzschii . Taxonomic identification including 16S rDNA base sequencing, and phylogenetic analysis indicated that the isolate SK-02 had a 99.20% homology in its 16S rDNA base sequence with Pseudomonas putida . The nature of these diatom-lying components is still under investigation. These results suggest that the indigenous bacteria isolated from the sediments may have a potential in the application and development of eco-technology controlling harmful water blooms in the fresh water environments.     

60 Identification of diatom‐lytic bacterium sk‐02 and its capability to degrade stephanodiscus hantzschii

In order to control harmful algal blooms, many biological approaches have been tried. Specially, there have recently been discussions concerning the roles of bacteria in algal bloom dynamics. Then, algicidal bacteria are expected as an agent considerate for harmful algal blooms control. Development of these organisms as biological control agents involves isolation from environmental samples. With the aim of develop eco‐technology controlling water blooms in fresh waters, we isolated the diatom‐lysing bacteria from the sediments of Lake Seokchon and Pal¡¯tang River‐Reservoir. A soft agar‐overlay technique was used to isolate the diatom lytic bacteria. The SK‐02 showed a diatom lytic activity against Stephanodiscus hantzschii. Taxonomic identification including 16S rDNA base sequencing, and phylogenetic analysis indicated that the isolate SK‐02 had a 99.20% homology in its 16S rDNA base sequence with Pseudomonas putida. The nature of these diatom‐lying components is still under investigation. These results suggest that the indigenous bacteria isolated from the sediments may have a potential in the application and development of eco‐technology controlling harmful water blooms in the fresh water environments.     

The potential role of anthropogenically derived nitrogen in the growth of harmful algae in California,USA

Cultural eutrophication is frequently invoked as one factor in the global increase in harmful algal blooms, but is difficult to definitively prove due to the myriad of factors influencing coastal phytoplankton bloom development. To assess whether eutrophication could be a factor in the development of harmful algal blooms in California (USA), we review the ecophysiological potential for urea uptake by Pseudo-nitzschia australis (Bacillariophyceae), Heterosigma akashiwo (Raphidophyceae), and Lingulodinium polyedrum (Dinophyceae), all of which have been found at bloom concentrations and/or exhibited noxious effects in recent years in California coastal waters. We include new measurements from a large (Chlorophyll a > 500 mg m⁻³) red tide event dominated by Akashiwo sanguinea (Dinophyceae) in Monterey Bay, CA during September 2006. All of these phytoplankton are capable of using nitrate, ammonium, and urea, although their preference for these nitrogenous substrates varies. Using published data and recent coastal time series measurements conducted in Monterey Bay and San Francisco Bay, CA, we show that urea, presumably from coastal eutrophication, was present in California waters at measurable concentrations during past harmful algal bloom events. Based on these observations, we suggest that urea uptake could potentially sustain these harmful algae, and that urea, which is seldom measured as part of coastal monitoring programs, may be associated with these harmful algal events in California.     

Remote,subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

First record of the dynamics of domoic acid producing Pseudo-nitzschia spp. in Indonesian waters as a function of environmental variability

Within the past few decades, harmful algal blooms (HABs) have occurred frequently in Indonesian waters, resulting in environmental degradation, economic loss and human health problems. So far, HAB related studies mainly addressed ecological traits and species distribution, yet toxin measurements were virtually absent for Indonesian waters. The aim of the present study was to explore variability of the potentially toxic marine diatom genus Pseudo-nitzschia, as well as its neurotoxin domoic acid as a function of environmental conditions in Ambon Bay, eastern Indonesia. Weekly phytoplankton samples, oceanographic (CTD, nutrients) and meteorological (precipitation, wind) parameters were analyzed at 5 stations in the bay during the dry and wet seasons of 2018. Liquid chromatography – tandem mass spectrometry (LC–MS/MS) was used to detect particulate DA (pDA). Vegetative cells of Pseudo-nitzschia spp. and pDA were found in 98.6% and 51.4% of the samples, respectively. pDA levels were low, yet detected throughout the campaign, implying that Ambon Bay might potentially be subject to amnesic shellfish poisoning. The highest levels of both Pseudo-nitzschia spp. cell abundance and pDA were found in the wet season, showing a strong positive correlation between both parameters, compared to the dry season, (r = 0.87 and r = 0.66 (p < 0.01), respectively). Statistical analyses revealed that temperature and mixed layer depth positively correlated with Pseudo-nitzschia spp. and pDA during the dry season, while ammonium showed positive correlations in both seasons. This study represents the first successful investigation of the presence and variability of Pseudo-nitzschia spp. and its neurotoxin DA in Indonesian waters.     

Concurrent exposure of bottlenose dolphins (Tursiops truncatus) to multiple algal toxins in Sarasota Bay, Florida, USA

Sentinel species such as bottlenose dolphins (Tursiops truncatus) can be impacted by large-scale mortality events due to exposure to marine algal toxins. In the Sarasota Bay region (Gulf of Mexico, Florida, USA), the bottlenose dolphin population is frequently exposed to harmful algal blooms (HABs) of Karenia brevis and the neurotoxic brevetoxins (PbTx; BTX) produced by this dinoflagellate. Live dolphins sampled during capture-release health assessments performed in this region tested positive for two HAB toxins; brevetoxin and domoic acid (DA). Over a ten-year study period (2000–2009) we have determined that bottlenose dolphins are exposed to brevetoxin and/or DA on a nearly annual basis (i.e., DA: 2004, 2005, 2006, 2008, 2009; brevetoxin: 2000, 2004, 2005, 2008, 2009) with 36% of all animals testing positive for brevetoxin (n = 118) and 53% positive for DA (n = 83) with several individuals (14%) testing positive for both neurotoxins in at least one tissue/fluid. To date there have been no previously published reports of DA in southwestern Florida marine mammals, however the May 2008 health assessment coincided with a Pseudo-nitzschia pseudodelicatissima bloom that was the likely source of DA observed in seawater and live dolphin samples. Concurrently, both DA and brevetoxin were observed in common prey fish. Although no Pseudo-nitzschia bloom was identified the following year, DA was identified in seawater, fish, sediment, snails, and dolphins. DA concentrations in feces were positively correlated with hematologic parameters including an increase in total white blood cell (p = 0.001) and eosinophil (p<0.001) counts. Our findings demonstrate that dolphins within Sarasota Bay are commonly exposed to two algal toxins, and provide the impetus to further explore the potential long-term impacts on bottlenose dolphin health.     

CONFIRMATION OF DOMOIC ACID PRODUCTION BY PSEUDONITZSCHIA AUSTRALIS (BACILLARIOPHYCEAE) CULTURES1

Single done isolates of Pseudonitzschia australis Frenguelli (= Nitzschia pseudoseriata Hasle) isolated from a toxic bloom in Monterey Bay, California produced domoic acid in culture. Although long-term historical records do not indicate previous blooms of this species on the Pacific coast, this is probably because it has been often misidentified as Nitzschia seriata Hasle; previous evidence for toxicity is lacking. Hydrographic data suggest that areas such as Monterey Bay might be “hot spots” for domoic acid-producing blooms.     

127 Climate Variability and Phytoplankton Production in PNW Coast Estuaries

The west coast of North America receives strong forcing from climate modes such as El Niño-Southern Oscillation and the Pacific Decadal Oscillation. Estuaries are poised at a sensitive interface because estuarine biota and habitat will be affected by variability in properties and processes associated with the ocean, the watershed, and the local weather. Observations from the Washington coast and Willapa Bay illustrate these three arenas of influence. Variation in ocean upwelling and ocean thermocline depth associated with the 1997–98 El Niño versus the 1999 La Niña affected temperature and nutrient availability in Willapa Bay. Variation in river flow associated with the 2000–01 drought affected estuarine circulation and residence time. And, variation in prevailing wind direction and/or cloudiness was highly correlated with phytoplankton biomass. This situation is responsible for the complexity of understanding climate impacts on estuarine systems. In order to help evaluate which mechanisms, remote oceanic processes or local watershed/estuarine characteristics, most affected Willapa Bay's phytoplankton production, several phytoplankton species were used as indicators of water mass origin and compared with primary productivity data to assess whether phytoplankton blooms were dominated by endemic or imported species. Our analysis resolved that the highest primary production (and the appearance of Pseudo-nitzschia spp.) was associated with oceanic intrusions of phytoplankton biomass into Willapa Bay. This result underscores the dominant role that variation in ocean and climate play in controlling Pacific Northwest estuarine production. However, while the largest blooms were oceanic in origin, numerous medium-sized production events were from either oceanic or estuarine sources, indicating a complex picture.     

Understanding the blob bloom: Warming increases toxicity and abundance of the harmful bloom diatom Pseudo-nitzschia in California coastal waters

The toxic diatom genus Pseudo-nitzschia produces environmentally damaging harmful algal blooms (HABs) along the U.S. west coast and elsewhere, and a recent ocean warming event coincided with toxic blooms of record extent. This study examined the effects of temperature on growth, domoic acid toxin production, and competitive dominance of two Pseudo-nitzschia species from Southern California. Growth rates of cultured P. australis were maximal at 23 °C (∼0.8 d⁻¹), similar to the maximum temperature recorded during the 2014–2015 warming anomaly, and decreased to ∼0.1 d⁻¹ by 30 °C. In contrast, cellular domoic acid concentrations only became detectable at 23 °C, and increased to maximum levels at 30 °C. In two incubation experiments using natural Southern California phytoplankton communities, warming also increased the relative abundance of another potentially toxic local species, P. delicatissima. These results suggest that both the toxicity and the competitive success of particular Pseudo-nitzschia spp. can be positively correlated with temperature, and therefore there is a need to determine whether harmful blooms of this diatom genus may be increasingly prevalent in a warmer future coastal ocean.     

Adsorptive removal of harmful algal species <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> directly from aqueous solution using polyethylenimine coated polysulfone-biomass composite fiber

In recent times, the treatment of harmful algal blooms (HABs) became an important environmental issue to preserve and remediate water resources globally. In the present study, the adsorptive removal of harmful algal species Microcystis aeruginosa directly from an aqueous medium was attempted. Waste biomass (Escherichia coli) was immobilized using polysulfone and coated using the cationic polymer polyethylenimine (PEI) to generate PEI-coated polysulfone-biomass composite fiber (PEI-PSBF). The density of M. aeruginosa in an aqueous medium (BG11) was significantly decreased by treatment with PEI-PSBF. additionally, analysis using FE-SEM, confirmed that the removal of M. aeruginosa algal cells by PEI-PSBF was caused by the adsorption mechanism. According to the profiles of phosphorus for the algal cell growth in M. aeruginosa cultivating samples, we found that the adsorbed M. aeruginosa onto the PEI-PSBF lost their biological activity compared to the non-treated M. aeruginosa cells.     

Detection of several harmful algal species by sandwich hybridization integrated with a nuclease protection assay

The frequent occurrence of harmful algal blooms (HABs) is a pressing topic in marine research. An integrated sandwich hybridization and nuclease protection assay was established to qualitatively and quantitatively detect 12 harmful algal species. This method demonstrated good reliability, specificity and accuracy for analyzing samples from individual and mixed cultures, as well as field collection, and cell volumes were positively correlated to the slopes of calibration curves. The lowest quantitative detection limits were those concentrations observed during blooms; thus, this technique provides an efficient alternative to microscopy for rapid identification and quantitation of harmful algal species and could be routinely used to monitor phytoplankton in field surveys.     

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

Harmful algal bloom forecast system for SW Ireland. Part II: Are operational oceanographic models useful in a HAB warning system

This study investigated the application of a three-dimensional physical hydrodynamic model in a harmful algal bloom forecast system for Bantry Bay, southwest Ireland. Modelled oceanographic conditions were studied and used to help understand observed changes in the chemical and biological patterns from the national biotoxins and phytoplankton monitoring program. The study focused on two toxic events in 2013. An upwelling event was predicted by the model prior to the appearance and population increase of potentially toxic diatoms, Pseudo-nitzschia, and associated domoic acid in shellfish. A downwelling episode was provided as a forecast in the model prior to the arrival of a Dinophysis bloom and detection of its associated biotoxins in Bay shellfish. The modelled forecast products developed included expected surface, mid-depth and bottom current pathways at the mouth of the Bay and on the adjacent shelf. The rate and direction of water volume flow at the mouth and mid-bay sections were produced by the model to examine predicted upwelling and downwelling pulses. The model also calculated the evolution of water properties (temperature, salinity and density) with depth along the Bay axis and on the adjacent continental shelf. Direct measurements of water properties at a fixed point, mid-bay, were comparable to model calculations. The operational model for southwest Ireland produces a reliable 3-day physical hydrodynamic forecast of the dominant regional physical processes that result in water exchange events between Bantry Bay and its adjacent shelf. While simulated physical hydrodynamics were provided as a 3-day forecast, the upwelling and downwelling signals from the model, closely linked to toxic HAB episodes, were evident up to 10 days prior to the contamination of shellfish in the Bay.     

Monitoring of harmful algal blooms in the era of diminishing resources: A case study of the U.S. West Coast

Spatial and temporal decorrelation scales in phytoplankton bloom magnitudes are reviewed with the goal of informing the design of efficient and informative observing networks for monitoring of potentially harmful algal blooms (HABs) along the U.S. West Coast. Our analysis of historic MODIS Fluorescent Line Height data shows that, unlike several previous studies, seasonal timing of phytoplankton blooms off the U.S. West Coast propagates from South to North. In situ data show that temporal decorrelation scales are shortest off Northern California (∼4 days) and longest in the Southern California Bight (∼17 days). In a cross-shore direction, we find that variability in the near-shore biomass is decoupled from biomass further offshore (∼2–4 km).Our review of the cloud cover gap statistics suggests that satellite data provide reasonably inexpensive information about bloom events, particularly at seasonal to inter-annual scales, but is insufficient to capture many event-scale blooms. Absent adequate satellite data, in situ monitoring becomes essential. Existing networks of automated monitoring sites from piers and moorings off the California coast is insufficient to describe regional variability in blooms, but is likely informative of bloom magnitudes in the immediate proximity of observation stations. We suggest that a more effective network will have a combination of shore-based stations and a few (5–10) offshore moorings. Shore-based stations should be located in proximity to sensitive HAB targets. This would provide monitoring of existing conditions and guide decision-making about beach closures and aquaculture management practices. Offshore stations can serve as proxy for regional conditions and can be used to issue early warnings of potential HAB conditions developing in a specific region.     

Novel algicidal evidence of a bacterium Bacillus sp. LP-10 killing Phaeocystis globosa,a harmful algal bloom causing species

While searching for effective bio-agents to control harmful algal blooms (HABs), the bacterial strain LP-10, which has strong algicidal activity against Phaeocystis globosa (Prymnesiophyceae), was isolated from surface seawater samples taken from the East China Sea. 16S rDNA sequence analysis and morphological characteristics revealed the strain LP-10 belonged to the genus Bacillus. The lytic effect of Bacillus sp. LP-10 against P. globosa was both concentration- and time-dependent. Algicidal activities of different growth stages of the bacterial culture varied significantly. The lytic effect of different parts of the bacterial cultures indicated that the algal cells were lysed by algicidal active compounds in the cell-free filtrate. Analysis of the properties of the active compounds showed that they had a molecular weight of less than 1000 Da and that the active compounds were stable between −80 and 121 °C. The algicidal range assay indicated that five other algal species were also suppressed by strain LP-10, including: Alexandrium catenella, A. tamarense, A. minutum, Prorocentrum micans and Asterionella japonica. Our results suggested that the algicidal bacterium Bacillus sp. LP-10 could be a potential bio-agent to control the blooms of harmful algal species.     

Discoloration of microalgal blooms as a bioindicating parameter in the Bahia de Mazatlán, México

In order to verify whether harmful algal blooms increased since 1979, a long-term project for the study of harmful algal blooms in Mazatlán Bay began 25 years ago (1979-2003). Observations were done daily and results indicate five trend types: (I) years without discoloration related to El Ni?o events; (II) years with days of sudden occurrence of discoloration, related to the release of accumulated energy after El Ni?o events; (III) years with similar number of discoloration events (days); (IV) years with a progressive decrease of the occurrence (days) of discoloration events; (V) years with gradual increase of discoloration events. The annual mean average of discoloration is of 25.3 days. Ninety-six discoloration events were recorded during year 2000, and represent the highest number of events in the last 25 years. Discoloration events are more common in March (174 days), harmful and toxic blooms included. This indicates continuity in the occurrence of the highest content of microalgae, and therefore, it is concluded that they play an important role for a better understanding of the environmental deterioration undergone by the coastal zone due to an increase of harmful and toxic microalgae blooms.     

43 Testeria gen. nov. (dinophyceae) from manatee cay belize, Central America

Saxitoxin and domoic acid sequestration by bivalve molluscs occurs periodically along the Oregon coast, presumably as a result of harmful algal blooms (HABs). Since 1958 and more continuously since 1979, the Oregon Shellfish Program (OSP) has assayed toxin levels in these molluscs as part of a monitoring program for paralytic (PSP) and amnesic (ASP) shellfish poisoning. We have created a working data base for all PSP sampling by the OSP between 1958 and 2001 and have examined the data for spatial and temporal trends in the appearance of toxin in shellfish, amount of toxin, and apparent duration of toxic events. In this report, we examine the data from the five stations with the longest record of continuous sampling (1979–2000) for evidence of correlation with El Niño events, upwelling, and/or a pattern of increasing frequency or intensity of toxic events. We also compare the pattern of appearance of toxin at open coast stations with the timing of first appearance of toxin in shellfish at adjacent estuarine stations. This is an important analysis because, in Oregon, shellfish closures due to PSP occur frequently in mussel beds on the open coast and the source of toxin-producing organisms is unknown.     

41 Incidence of paralytic shellfish toxin in bivalve mollusc tissue from the oregon coast

Saxitoxin and domoic acid sequestration by bivalve molluscs occurs periodically along the Oregon coast, presumably as a result of harmful algal blooms (HABs). Since 1958 and more continuously since 1979, the Oregon Shellfish Program (OSP) has assayed toxin levels in these molluscs as part of a monitoring program for paralytic (PSP) and amnesic (ASP) shellfish poisoning. We have created a working data base for all PSP sampling by the OSP between 1958 and 2001 and have examined the data for spatial and temporal trends in the appearance of toxin in shellfish, amount of toxin, and apparent duration of toxic events. In this report, we examine the data from the five stations with the longest record of continuous sampling (1979–2000) for evidence of correlation with El Niño events, upwelling, and/or a pattern of increasing frequency or intensity of toxic events. We also compare the pattern of appearance of toxin at open coast stations with the timing of first appearance of toxin in shellfish at adjacent estuarine stations. This is an important analysis because, in Oregon, shellfish closures due to PSP occur frequently in mussel beds on the open coast and the source of toxin‐producing organisms is unknown.     

Dinoflagellate cysts from surface sediments of Saldanha Bay, South Africa: an indication of the potential risk of harmful algal blooms

The distribution and abundance of dinoflagellate cysts from recent coastal sediments in Saldanha Bay, was investigated, and compared to the cyst assemblages of the adjacent coastal upwelling system as reflected in the sediments off Lambert's Bay on the southern Namaqua shelf. Twenty-two cyst types were identified from three sample sites off Lambert's Bay with recorded abundances between 1726 and 1863 cysts ml⁻¹ wet sediment. At least 21 distinctive cyst types were identified from 32 sample sites within Saldanha Bay. Cyst abundance in Saldanha Bay was relatively low, averaging 116 cysts ml⁻¹ wet sediment. The region off Lambert's Bay is especially susceptible to the formation of harmful algal blooms attributed to high biomass dinoflagellate blooms. Owing to these blooms and the retentive circulation characteristics of this area, cyst formation and deposition is high. Blooms can be advected into Saldanha Bay, but their development and duration in the Bay is restricted by the system of exchange that operates between the Bay and the coastal upwelling system, in that there is a net export of surface waters from the Bay. Consequently, fewer cysts are formed and deposited within the Bay thereby reducing the likelihood of in situ bloom development initiated from the excystment of cysts.