Identification of amoebae implicated in the life cycle of Pfiesteria and Pfiesteria-like dinoflagellates

This study was undertaken to assess whether amoebae commonly found in mesohaline environments are in fact stages in the life cycles of Pfiesteria and Pfiesteria-like dinoflagellates. Primary isolations of amoebae and dinoflagellates were made from water and sediment samples from five tributaries of the Chesapeake Bay. Additional amoebae were also cloned from bioassay aquaria where fish mortality was attributed to Pfiesteria. Electron microscopy and small subunit (SSU) rRNA gene sequence analysis of these isolates clearly demonstrated that the commonly depicted amoeboid form of Pfiisteria is very likely a species of Korotnevella and is unrelated to Pfiesteria or Pfiesteria-like dinoflagellates. We have determined that the Pfiesteria and Pfiesteria-like dinoflagellates examined in this study undergo a typical homothallic life cycle without amoeboid stages. Furthermore, we have demonstrated that cloned amoebae sharing morphological characteristics described for stages in the life cycle of Pfiesteria do not transform into dinozoites. The strict clonal isolation and cultivation techniques used in this study substantially support the conclusion that the amoebae and some of the flagellates depicted in the life cycle of Pfiesteria are environmental contaminants of the Pfiesteria culture system and that the Ambush Predator Hypothesis needs to be rigorously reevaluated.     

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.     

Pfiesteria piscicida and Pfiesteria shumwayae in coastal waters of Long Island, New York, USA

Water and sediment samples were collected during summer and early fall 1999–2004 from coastal waters of New York State, USA, to test for the presence of Pfiesteria piscicida and Pfiesteria shumwayae. Physical and chemical conditions were characterized, and real-time polymerase chain reaction assays were conducted. Both species were relatively common and found at most sites at least once, and the frequency of positive assays was higher in sediments than in the water column. In a subset of the data from Suffolk County, Long Island, the presence of Pfiesteria was related to high chlorophyll a and relatively high nutrient concentrations. Partial SSU rDNA sequences of four PCR amplicons generated using P. shumwayae primers indicated two sequences: three were identical to GenBank P. shumwayae entries, but one showed enough sequence difference (15 positions in a 454 bp amplicon) to suggest a possible new species. Three isolates were tested for toxicity, and one was found to kill fish in bioassays. Despite the widespread presence of both Pfiesteria species and demonstration of potential to harm fish, no blooms of these dinoflagellates have been observed, nor has there been evidence of Pfiesteria-related fish or human health problems in these waters, likely related to colder temperatures than optimal for Pfiesteria species.     

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.     

THE PHYLOGENETIC RELATIONSHIP OF PFIESTERIA PISCICIDA, CRYPTOPERIDINIOPSOID SP. AMYLOODINOUM OCELLATUM AND A PFIESTERIA-LIKE DINOFLAGELLATE TO OTHER DINOFLAGELLATES AND APICOMPLEXANS

The taxonomic relationship between heterotrophic and parasitic dinoflagellates has not been studied extensively at the molecular level. In order to investigate these taxonomic relationships, we sequenced the small subunit (SSU) ribosomal RNA gene of Pfiesteria piscicida (Steidinger et Burkholder), a Pfiesteria -like dinoflagellate, Cryptoperidiniopsoid sp., and Amyloodinium ocellatum (Brown) and submitted those sequences to GenBank. Pfiesteria piscicida and Cryptoperidiniopsoid sp. are heterotrophic dinoflagellates, purportedly pathogenic to fish, and A. ocellatum, a major fish pathogen, has caused extensive economic losses in both the aquarium and aquaculture industries. The pathogenicity of the Pfiesteria -like dinoflagellate is unknown at this time, but its growth characteristics and in vitro food preferences are similar to those of P. piscicda. The SSU sequences of these species were aligned with the other full-length dinoflagellate sequences, as well as those of representative apicomplexans and Perkinsus species, the groups most closely related to dinoflagellates. Phylogenetic analyses indicate that Cryptoperidiniopsoid sp., P. piscicida, and the Pfiesteria -like dinoflagellate are closely related and group into the class Blastodiniphyceae, as does A. ocellatum. None of the species examined were closely related to the apicomplexans or to Perkinsus marinus, the parasite that causes "Dermo disease" in oysters. The overall phylogenetic analyses largely supported the current class and subclass groupings within the dinoflagellates.     

Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

Detection of Pfiesteria spp. by PCR in surface sediments collected from Chesapeake Bay tributaries (Maryland)

In 1997 blooms of Pfiesteria piscicida occurred in association with fish kills and human health problems in tributaries of the Chesapeake Bay (Maryland) and the scientific and media response resulted in large economic losses in seafood sales and tourism. These events prompted the Maryland Department of Natural Resources (MDNR) to begin monitoring for Pfiesteria spp. in water column samples. Real-time PCR assays targeted to the 18S rRNA gene were developed by our laboratories and utilized in conjunction with traditional microscopy and fish kill bioassays for detection of these organisms in estuarine water samples. This monitoring strategy aided in determining temporal and spatial distribution of motile forms of Pfiesteria spp. (i.e. zoospores), but did not assess resting stages of the dinoflagellates’ life cycle. To address this area, a 3-year study was designed using real-time PCR assays for analysis of surface sediment samples collected from several Chesapeake Bay tributaries. These samples were tested with the real-time PCR assays previously developed by our laboratories. The data reported herein suggest a strong positive association between presence of Pfiesteria spp. in the sediment and water column, based on long-term water column monitoring data. P. piscicida is detected more commonly in Maryland's estuarine waters than Pfiesteria shumwayae and sediment ‘cyst beds’ may exist for these organisms.     

Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 μg of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

The dinoflagellates Pfiesteria shumwayae and Luciella masanensis cause fish kills in recirculation fish farms in Denmark

Fish kills in two geographically separate fish farms in northern Denmark in 2012, one using marine, the other brackish water ‘Recirculation Aquaculture Systems’ (RAS), were found to be caused by Pfiesteria shumwayae and Luciella masanensis, two species of dinoflagellates belonging to the family Pfiesteriaceae. There were no other harmful algae present in either of the aquaculture plants. Serious fish kills in the US have been attributed to Pfiesteria during the past 20 years, but this type of mortality has not been documented elsewhere. L. masanensis, described recently from Korea and USA, has not been previously reported to be the source of fish kills. In the marine farm, the affected fish was rainbow trout, in the brackish water farm pikeperch. Light microscopy is presently insufficient to discriminate between the approx. 20 species of the family Pfiesteriaceae described. Identification of the two algal species was therefore based on molecular sequencing of nuclear-encoded LSU rDNA, confirmed by scanning electron microscopy and, eventually, also by examination of the very thin amphiesmal plates of the flagellates by calcofluor-stained cells in a fluorescence microscope.Although the two fish farms differed in light and salinity conditions, both farms used re-circulating water in closed circuit systems. The dinoflagellates were examined in detail and shown to feed on organic material such as live, damaged nematodes, as described for the single pfiesteriacean flagellate known from freshwater, Tyrannodinium edax. Algal cells were observed to attach to their prey by an attachment filament and subsequently used a peduncle to suck up the food. Fish farms utilizing water recirculation technology are gaining popularity due to their reduced effect on the environment. The two cases from Denmark are apparently the first RAS farms in which serious fish kills have been reported. In the marine farm (Luciella) fish mortality increased dramatically despite treatment of the water with peracetic acid and chloramine-T. The plant was temporarily closed down pending investigation into the cause of mortality and subsequently to determine a method of management to control the dinoflagellate and avoid future fish kills. In the brackish water farm (Pfiesteria), water was treated with chloramine-T, which caused the dinoflagellates to disappear temporarily from the water column, apparently forming temporary cysts. The treatment was repeated after a few days to a week, when the temporary cysts appeared to germinate and the dinoflagellates reappeared in the water column.     

Comparative culture and toxicity studies between the toxic dinoflagellate Pfiesteria piscicida and a morphologically similar cryptoperidiniopsoid dinoflagellate

A series of fish bioassays using cultures of the toxic dinoflagellate, Pfiesteria piscicida and a cryptoperidiniopsoid dinoflagellate indicated various degrees of toxicity for Pfiesteria piscicida and no toxicity by the cryptoperidiniopsoid. P. piscicida maintained toxicity in the presence of live fish, and this toxicity was perpetuated following a series of inoculations to other culture vessels. Differences in the onset and magnitude of the fish deaths occurred, requiring 16 days for the initial fish death when using P. piscicida from a culture that had previously been maintained on algal cells, to kills within hours when using a culture that had recently (previous day) killed fish. Autopsies of moribund fish from the test and control fish bioassays indicated a general lack of bacterial infection, which ensued following death of other autopsied fish. Moreover, bacterial comparisons of waters in the fish bioassay and control fish cultures indicated that similar bacterial concentrations were present. Neither oxygen or ammonia levels were determined to be factors in the fish death. Life stages of a cryptoperidiniopsoid dinoflagellate from Virginia estuaries were also identified, including motile zoospore, gametes, planozygote, amoebae, and cyst stages. The cryptoperidiniopsioid did not initiate fish deaths in bioassays conducted over a 14-week period at zoospore concentrations of ca. 700-800 cells ml(-1). Elemental X-ray analysis of the scales from cysts of this dinoflagellate and P. piscicida indicate that they both contain silicon. Overall, the data from this study demonstrate that the cryptoperidiniopsoid possesses several similar life stages and feeding patterns as P. piscicida, but was not toxic to fish.     

Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates

Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.     

FIRST REPORT OF GYMNODINIUM PULCHELLUM (DINOPHYCEAE) IN NORTH AMERICA AND ASSOCIATED FISH KILLS IN THE INDIAN RIVER, FLORIDA

Fish and invertebrate kills were reported from September to October 1996 in the Indian River, Florida, coincident with blooms of the dinoflagellate Gymnodinium pulchellum Larsen 1994. This is the first report of a bloom of this species in the Americas. Fish and invertebrate species affected were common snook ( Centropomus undecimalis ), striped mullet ( Mugil cephalus ), hardhead catfish ( Arius felis ), red drum ( Sciaenops ocellatus ), sheepshead ( Archosargus probatocephalus ), black drum ( Pogonias cromis ), blue crab ( Callinectes sapidus ), and shrimp ( Penaeus spp.). However, Gymnodinium pulchellum has previously caused fish kills in Japan and Australia. Examination of archived phytoplankton samples from a fish kill reported in the same area of the Indian River in August 1990 confirmed the presence of high concentrations of G. pulchellum. Fish kills associated with Alexandrium monilatum and potentially Pfiesteria -like species in the Indian River also are discussed. Scanning electron microscopy provided additional morphological detail on this distinct but little-known dinoflagellate.     

Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 microM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 microM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.     

Characterisation of Pseudomonas aeruginosa isolated from freshwater culture systems

Members of the genus Pseudomonas are important phytopathogens and agents of human infections, while other strains and species exhibit bioremediation and biocontrol activities. Species-specific detection of Pseudomonas species in the environment may help to gain a more complete understanding of the ecological significance of these microorganisms. The objective of present study was comparative analysis of biochemically and PCR based confirmed 10 isolates of Pseudomonas aeruginosa (6 from fish intestine and 4 from pond sediment). PCR-ribotyping and PAGE revealed that there was extensive heterogeneity at the genetic and protein levels. Both genetic and phenotypic heterogeneity were more in the sediment isolates compared to the fish isolates. SDS-PAGE clearly demonstrated the differences between fish and sediment isolates as evident from the higher range of protein profiling. In antibiotic sentivity test no habitat specific antibiogram was obtained. Zinc adversely affected the DNA of all the isolates to be amplified by PCR as DNA banding pattern was different from normal DNA in stressed DNA. Thus stress, particularly, zinc may interfere monitoring of Pseudomonas by PCR.     

Human health effects of exposure to Pfiesteria piscicida: a review

Since its identification, the dinoflagellate Pfiesteria piscicida has been implicated in fish kills and fish disease in the southeastern United States. Adverse health effects have been reported in researchers working with the organism and in watermen following exposure to a fish kill in Maryland. A bioactive secretion is postulated as the cause of these effects but has not yet been isolated and chemically characterized. The biology and toxicology of this organism remain the topic of debate and research.     

Susceptibility of two fishes (Oreochromis niloticus and Cyprinodon variegatus) to Pfiesteria shumwayae and its associated toxin: Influence of salinity

Researchers examining the mechanisms of ichthyotoxicity of Pfiesteria shumwayae have come to different conclusions about the role of toxin in this process. Some attribute fish mortality solely to direct attack by these pedunculate dinoflagellates on exposed fish tissue while others have provided evidence for a role of a soluble toxin. Detection of toxin, especially in low concentrations, is a function of the sensitivity of the selected bioassay methods and the various groups addressing this question have utilized different methods. One notable difference in fish bioassay methods utilized to detect Pfiesteria-associated toxin (PfTx) is the species of fish tested. Studies that have not detected PfTx in bioassays generally have used Cyprinodon variegatus (sheepshead minnow) as the test fish while those that have detected toxin generally used Oreochromis spp. (Tilapia). In this study response of these two fish species was compared to determine their relative sensitivity to physical attack by P. shumwayae and to PfTx. The results indicate that Oreochromis niloticus is more susceptible to P. shumwayae and its associated toxin than C. variegatus and implicate differences in the ability these species to osmoregulate as a contributing factor for this phenomenon. Salinity stress enhanced susceptibility of O. niloticus to PfTx and thus improved the sensitivity of the bioassay. The observation that salinity stress enhances toxicity to O. niloticus provides additional information regarding the mechanism of PfTx toxicity although the conditions utilized are not representative of the natural habitat of these freshwater fish.     

Trophic Controls on Stage Transformations of a Toxic Ambush-Predator Dinoflagellate1

ABSTRACT. The toxic dinoflagellate, Pfiesteria piscicida, was recently implicated as the causative agent for about 50% of the major fish kills occurring over a three-year period in the Albemarle-Pamlico Estuarine System of the southeastern USA. Transformations between life-history stages of this dinoflagellate are controlled by the availability of fresh fish secretions or fish tissues, and secondarily influenced by the availability of alternate prey including bacteria, algae, microfauna, and mammalian tissues. Toxic zoospores of P. piscicida subdue fish by excreting lethal neurotoxins that narcotize the prey, disrupt its osmoregulatory system, and attack its nervous system. While prey are dying, the zoospores feed upon bits of fish tissue and complete the sexual phase of the dinoflagellate life cycle. Other stages in the complex life cycle of P. piscidia include cryptic forms of filose, rhizopodial, and lobose amoebae that can form within minutes from toxic zoospores, gametes, or planozygotes. These cryptic amoebae feed upon fish carcasses and other prey and, thus far, have proven less vulnerable to microbial predators than flagellated life-history stages. Lobose amoebae that develop from toxic zoospores and planozygotes during colder periods have also shown ambush behavior toward live fish. In the presence of abundant flagellated algal prey, amoeboid stages produce nontoxic zoospores that can become toxic and form gametes when they detect what is presumed to be a threshold level of a stimulatory substance(s) derived from live fish. The diverse amoeboid stages of this fish “ambush-predator” and at least one other Pfiesteria-like species are ubiquitous and abundant in brackish waters along the western Atlantic and Gulf Coasts, indicating a need to re-evaluate the role of dinoflagellates in the microbial food webs of turbid nutrient-enriched estuaries.     

Detection and quantification of Pfiesteria piscicida by using the mitochondrial cytochrome b gene

Mitochondrial cytochrome b was isolated from the dinoflagellate Pfiesteria piscicida, and the utility of the gene for species identification was examined. One of the primer sets designed was shown to be highly specific for P. piscicida. A time step PCR protocol was used to demonstrate the potential of this primer set for quantification of this species.     

The restoration of fish ponds in agricultural landscapes

Fish ponds in central and eastern Europe are artificial lakes of less than 1 ha to several 100 ha in size. Some of them were created around 900 years ago. These fish ponds form an important part of the hydrological system in the landscape; they also serve as water purification systems and sediment traps, provide habitat for many plant and animal species, as well as being used for recreation purposes—but above all, they are dedicated to fish production. The original oligo- or mesotrophic character of these fish ponds has been altered to one of eutrophic or even hypertrophic conditions due to the nature of the intensive management for high fish production, as well as agriculture practices within the catchment area. It can also be due to other uses of fish ponds, such as wastewater recipients. A thick layer of black anaerobic sediment invariably develops and serves as an uncontrolled internal source of nutrients. In order to stop the development of water blooms and restore the lake to reasonable healthy conditions, both the external and internal loading has to be reduced. An example of such a fish pond restoration within the Czech Republic is described. The phosphorus budget of the Vajgar fish pond (40 ha, S Bohemia) was evaluated and the dry matter, organic matter, nutrients, heavy metals and other metals content were estimated in the vertical profile of the fish pond's sediment. A precision suction dredger was designed, constructed, and used for the selective removal of 330 000 m³ black sediment from the Vajgar fish pond. The sediment was pumped to settling ponds at a distance of 2.5 km. Later, the suction dredger was used for sediment removal from other fish ponds. In one case, 40 000 m³ of eutrophic sediment was directly applied on an agriculture field. In another case, sediment containing oil products was decontaminated by using a biocatalyst and bacterial culture. Sediment removal from Vajgar fish pond resulted in a temporary negative phosphorus budget (lower concentrations in the outflow than the inflow). The temporary absence of cyanobacteria (Microcystis sp.) blooms was the result of the removal of the black sediment layer. The improvement of water quality after sediment removal was, however, temporary. A more holistic approach, aimed at the reduction of matter losses from the fish pond's catchment area, is therefore being adopted. The principles of such an holistic approach are described.     

Occurrence of stones, sediment and fish scales in stomach contents of demersal fish of the Rockall Trough

The occurrence of stones, sediment and fish scales was determined among the stomach contents of some 5000 fish. These fish comprised representatives of more than 70 different demersal species, sampled at 250 m bathymetric intervals, from the Rockall Trough, northeastern Atlantic Ocean, at depths ranging from 500 to 2900 m. Stones only occurred in stomachs of fish caught at 500–1000 m depth, their incidence in stomachs with food present being 4.6% at 500m, 1.1% at 750 m and 1.3% at 1000m depth. Sediment occurred in 9%, fish scales in 7%, of stomachs with contents. Sediment and scales co-occurred in the stomachs of primarily benthopelagic feeding fish. The sediment, however, was associated with the less dominant epibenthic components of the diets while the scales occurred with the more common benthopelagic components. The co-occurrence of sediment, scales and benthopelagic prey may arise through the former items having longer residence times in the stomachs than, for example, epibenthic prey. There is no evidence of increased occurrence of sediment (and its associated meiofauna) in the diets of fish on the lower slope at depths of 1500–2900 m in the Rockall Trough.