Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD can be induced in salmon by exposure to freshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic differences between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions. Immunoblot analysis using polyclonal antisera, revealed differences in the antigen profiles of two cultured isolates of Neoparamoeba sp. when they were grown on agar versus in liquid medium. However, the antigen profiles of the two isolates were very similar when they were grown under the same culture conditions. Comparison of these antigen profiles with a preparation from parasites freshly isolated from infected gills revealed a very limited number of shared antigens. In addition monoclonal antibodies (mAbs) raised against surface antigens of cultured parasites were used in an indirect immunofluorescence assay to assess the expression of specific surface antigens of Neoparamoeba sp. after various periods in culture. Significant changes in antigen expression of freshly isolated parasites were observed after 15 days of in vitro culture. The use of mAb demonstrated progressive exposure/expression of individual antigens on the surface of the freshly isolated parasites during the period in culture.     

Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar)

Amoebic gill disease (AGD) is a potentially fatal disease of some marine fish. Two amphizoic amoebae Neoparamoeba pemaquidensis and Neoparamoeba branchiphila have been cultured from AGD-affected fish, yet it is not known if one or both are aetiological agents. Here, we PCR amplified the 18S rRNA gene of non-cultured, gill-derived (NCGD) amoebae from AGD-affected Atlantic salmon (Salmo salar) using N. pemaquidensis and N. branchiphila-specific oligonucleotides. Variability in PCR amplification led to comparisons of 18S rRNA and 28S rRNA gene sequences from NCGD and clonal cultured, gill-derived (CCGD) N. pemaquidensis and N. branchiphila. Phylogenetic analyses inferred from either 18S or 28S rRNA gene sequences unambiguously segregated a lineage consisting of NCGD amoebae from other members of the genus Neoparamoeba. Species-specific oligonucleotide probes that hybridise 18S rRNA were designed, validated and used to probe gill tissue from AGD-affected Atlantic salmon. The NCGD amoebae-specific probe bound AGD-associated amoebae while neither N. pemaquidensis nor N. branchiphila were associated with AGD-lesions. Together, these data indicate that NCGD amoebae are a new species, designated Neoparamoeba perurans n.sp. and this is the predominant aetiological agent of AGD of Atlantic salmon cultured in Tasmania, Australia.     

Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.)

The association between major histocompatibility (MH) polymorphism and the severity of infection by amoebic gill disease (AGD) was investigated across 30 full sibling families of Atlantic salmon. Individuals were challenged with AGD for 19days and then their severity of infection scored by histopathological examination of the gills. Fish were then genotyped for the MH class I (Sasa-UBA) and MH class II alpha (Sasa-DAA) genes using polymorphic repeats embedded within the 3' untranslated regions of the Sasa-UBA and Sasa-DAA genes. High variation in the severity of infection was observed across the sample material, ranging from 0% to 85% gill filaments infected. In total, seven Sasa-DAA-3UTR and ten Sasa-UBA-3UTR marker alleles were identified across the 30 families. A significant association between the marker allele Sasa-DAA-3UTR 239 and a reduction in AGD severity was detected. There was also a significant association found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes. While the associations between MH allele/genotypes and AGD severity reported herein may be statistically significant, the small sample sizes observed for some alleles and genotypes means these associations should be considered as suggestive and future research is required to verify their biological significance.     

Cardiovascular responses of three salmonid species affected with amoebic gill disease (AGD)

The cardiovascular effects of amoebic gill disease (AGD) were investigated immediately following surgery in three salmonid species; Atlantic salmon (Salmo salar L.), brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum). Fish, both naïve (control) and infected (AGD-affected) of each species, were fitted with dorsal aorta catheters and cardiac flow probes. Cardiac output and dorsal aortic pressures were then continuously measured over a 6-h period following surgery. Results showed that Atlantic salmon, brown trout and rainbow trout displayed similar dorsal aortic pressure, cardiac output, and systemic vascular resistance (mean dorsal aotic pressure divided by cardiac output) values. However, the only significant differences relating to disease status i.e. infected or control, were found in Atlantic salmon. Although no significant differences were seen in dorsal aortic pressure values, AGD-affected salmon displayed significantly elevated systemic vascular resistance at 4 and 6 h post surgery. Cardiac output was also approximately 35% lower in AGD-affected salmon compared to the non-affected control counterparts. These results comparatively examine cardiac function in response to AGD across three salmonid species and highlight species-specific cardiovascular responses that occur in association with disease. It is suggested that the apparent cardiac dysfunction seen in AGD-affected Atlantic salmon could, under stressful conditions, become exacerbated. Cardiac failure is therefore suggested to be a possible physiological mechanism by which AGD causes or contributes to mortality in Atlantic salmon.     

In vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD)

The objective of the present study was to evaluate the in vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD). The current treatment for AGD-affected Atlantic salmon involves bathing sea-caged fish in freshwater for a minimum of 3 h, a labour-intensive and costly exercise. Previous attempts to identify alternative treatments have suggested bithionol as an alternate therapeutic, but extensive in vitro efficacy testing has not yet been done. In vitro toxicity to Neoparamoeba spp. was examined using amoebae isolated from the gill of AGD-affected Atlantic salmon and exposing the parasites to freshwater, alumina (10 mg l(-1)), seawater, bithionol or bithionol sulphoxide at nominal concentrations of 0.1, 0.5, 1, 5 and 10 mg l(-1) in seawater. The numbers of viable amoebae were counted using the trypan blue exclusion method at 0, 24, 48 and 72 h. Both bithionol and bithionol sulphoxide demonstrated in vitro toxicity to Neoparamoeba spp. at all concentrations examined (0.1 to 10 mg l(-1) over 72 h), with a comparable toxicity to freshwater observed for both chemicals at concentrations > 5 mg l(-1) following a 72 h treatment. Freshwater remained the most effective treatment, with only 6% viable amoebae seen after 24 h and no viable amoebae observed after 48 h.     

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

Open field host selection and behavior by tamarisk beetles (Diorhabda spp.) (Coleoptera: Chrysomelidae) in biological control of exotic saltcedars (Tamarix spp.) and risks to non-target athel (T. aphylla) and native Frankenia spp.

Biological control of invasive saltcedars (Tamarix spp.) in the western U.S. by exotic tamarisk leaf beetles, Diorhabda spp., first released in 2001 after 15 years of development, has been successful. In Texas, beetles from Crete, Greece were first released in 2004 and are providing control. However, adults alight, feed and oviposit on athel (Tamarix aphylla), an evergreen tree used for shade and as a windbreak in the southwestern U.S. and México, and occasionally feed on native Frankenia spp. plants. The ability of tamarisk beetles to establish on these potential field hosts was investigated in the field. In no-choice tests in bagged branches, beetle species from Crete and Sfax, Tunisia produced 30–45% as many egg masses and 40–60% as many larvae on athel as on saltcedar. In uncaged choice tests in south Texas, adult, egg mass and larval densities were 10-fold higher on saltcedar than on adjacent athel trees after 2 weeks, and damage by the beetles was 2- to 10-fold greater on saltcedar. At a site near Big Spring, in west-central Texas, adults, egg masses and 1st and 2nd instar larvae were 2- to 8-fold more abundant on saltcedar than on athel planted within a mature saltcedar stand being defoliated by Crete beetles, and beetles were 200-fold or less abundant or not found at all on Frankenia. At a site near Lovelock, Nevada, damage by beetles of a species collected from Fukang, China was 12–78% higher on saltcedar than on athel planted among mature saltcedar trees undergoing defoliation. The results demonstrate that 50–90% reduced oviposition on athel and beetle dispersal patterns within resident saltcedar limit the ability of Diorhabda spp. to establish populations and have impact on athel in the field.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

小瓜虫在澳洲龙纹斑鳃器官上的分布及其影响

【目的】小瓜虫病是澳洲龙纹斑苗种阶段危害巨大的寄生虫病。探究小瓜虫在澳洲龙纹斑鳃器官上的分布及其影响可以丰富小瓜虫的致病性及病理学方面的研究内容,也可以为渔业生产中小瓜虫病的防治提供参考。【方法】采用光镜及扫描电镜技术确定病原,并观察小瓜虫在澳洲龙纹斑鳃丝、鳃小片及鳃盖上的分布情况及这些器官的变化情况。【结果】小瓜虫侵染澳洲龙纹斑的鳃器官后,分布在鳃丝、鳃小片及鳃盖的表面,上皮细胞之下及鳃小片之间,或是包裹在黏液细胞里,但侵染后期在鳃丝及鳃小片上的数量明显少于鳃盖。侵染后期,鳃丝、鳃小片及鳃盖出现一定程度的膨胀变形,黏液细胞分泌增多,鳃小片末端膨大变形甚至黏连融合,顶端充血呈球状或棒状。【结论】对于鳃部而言,侵染后期小瓜虫主要分布在澳洲龙纹斑的鳃盖上。小瓜虫主要通过侵染引发澳洲龙纹斑鳃部器官的变形膨大,使其丧失正常的功能,造成血液循环受阻、渗透压调节失衡,最终导致鱼体缺氧死亡。     

Characterization of the Caenorhabditis elegans UDP-galactopyranose mutase homolog glf-1 reveals an essential role for galactofuranose metabolism in nematode surface coat synthesis

Galactofuranose (Gal_f), the furanoic form of d-galactose produced by UDP-galactopyranose mutases (UGMs), is present in surface glycans of some prokaryotes and lower eukaryotes. Absence of the Gal_f biosynthetic pathway in vertebrates and its importance in several pathogens make UGMs attractive drug targets. Since the existence of Gal_f in nematodes has not been established, we investigated the role of the Caenorhabditis elegans UGM homolog glf-1 in worm development. glf-1 mutants display significant late embryonic and larval lethality, and other phenotypes indicative of defective surface coat synthesis, the glycan-rich outermost layer of the nematode cuticle. The glf homolog from the protozoan Leishmania major partially complements C. elegans glf-1. glf-1 mutants rescued by L. major glf, which behave as glf-1 hypomorphs, display resistance to infection by Microbacterium nematophilum, a pathogen of rhabditid nematodes thought to bind to surface coat glycans. To confirm the presence of Gal_f in C. elegans, we analyzed C. elegans nucleotide sugar pools using online electrospray ionization–mass spectrometry (ESI-MS). UDP-Gal_f was detected in wild-type animals while absent in glf-1 deletion mutants. Our data indicate that Gal_f likely has a pivotal role in maintenance of surface integrity in nematodes, supporting investigation of UGM as a drug target in parasitic species.     

Biological control of Tipula paludosa (Diptera: Nematocera) using entomopathogenic nematodes (Steinernema spp.) and Bacillus thuringiensis subsp. israelensis

Tipula paludosa (Diptera: Nematocera) is the major insect pest in grassland in Northwest Europe and has been accidentally introduced to North America. Oviposition occurs during late August and first instars hatch from September until mid-October. Laboratory and field trials were conducted to assess the control potential of entomopathogenic nematodes (EPN) (Steinernema carpocapsae and S. feltiae) and Bacillus thuringiensis subsp. israelensis (Bti) against T. paludosa and to investigate whether synergistic effects can be exploited by simultaneous application of nematodes and Bti. Results indicate that the early instars of the insect are most susceptible to nematodes and Bti. In the field the neonates prevail when temperatures tend to drop below 10 °C. S. carpocapsae, reaching >80% control, is more effective against young stages of T. paludosa than S. feltiae (<50%), but the potential of S. carpocapsae might be limited by temperatures below 12 °C. Mortality of T. paludosa caused by Bti was not affected by temperature even at 4 °C but the lethal time increased with decreasing temperatures. Synergistic effects of Bti and EPN against T. paludosa were observed in 3 out of 10 combinations in laboratory assays but not in a field trial. The potential of S. carpocapsae was demonstrated in field trials against early instars in October reaching an efficacy of >80% with 0.5 million nematodes m⁻² at soil temperatures ranging between 3 and 18 °C. Results with Bti were strongly influenced by the larval stage and concentration. Against early instars in autumn between 74 and 83% control was achieved with 13 kg ha⁻¹ Bti of 5,700 International Toxic Units (ITUs) and 20 kg ha⁻¹ of 3,000 ITUs. Applications in spring against third and fourth instars achieved between 0 and 32% reduction. The results indicate that application of Bti and nematodes will only be successful and economically feasible during the early instars and that the success of S. carpocapsae is dependent on temperatures >12 °C. Synergistic effects between S. carpocapsae and Bti require more detailed investigations in the field to determine maximal effect.     

Biology of Lilioceris spp. (Coleoptera: Chrysomelidae) and their parasitoids in Europe

The biology and parasitoid complex of the lily leaf beetle (LLB), Lilioceris lilii Scopoli, and two congeneric species were investigated in Europe, as part of a biological control program against the LLB in North America. Eggs, larvae, and adults of L. lilii were collected in several countries in Europe, on both cultivated and wild Lilium spp., and reared in the laboratory and under natural conditions. Parasitoids were obtained and their biologies were studied. Similar investigations were made in Switzerland on two closely related species Lilioceris tibialis (Villa) found on wild Lilium spp., and Lilioceris merdigera (L.) on several other Liliaceae. The three species are strictly univoltine. Adults overwinter and lay eggs on leaves in early spring. The three beetle species have four instars, which were characterized by their head capsule width. Pupation occurs in a cocoon in the soil. Adults emerge in late summer and start feeding before reaching overwintering sites. Egg and larval parasitoids were obtained. Eggs of L. lilii and L. merdigera were parasitized by the mymarid Anaphes sp., a multivoltine species that needs alternate hosts for overwintering. Larvae were heavily attacked by several parasitoids, among which the most abundant were three ichneumonids, Lemophagus pulcher (Szepligeti), L. errabundus (Gravenhorst), and Diaparsis jucunda (Holmgren), and the eulophid Tetrastichus setifer Thomson. All four parasitoid species were found in the three beetles and in most European regions, but strong variations were observed in their relative abundance among hosts and geographic regions. Three of the four main larval parasitoids are strictly univoltine, whereas L. pulcher has a partial second generation. Lemophagus spp. are frequently parasitized by the ichneumonid hyperparasitoid Mesochorus lilioceriphilus Schwenke. Further details of the biology of the parasitoids are described, and their potential as biological control agents is discussed.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Suitability of Aphis fabae, Myzus persicae (Homoptera: Aphididae) and Aleyrodes proletella (Homoptera: Aleyrodidae) as prey for Coccinella undecimpunctata (Coleoptera: Coccinellidae)

The suitability of Aphis fabae Scopoli, Myzus persicae Sulzer and Aleyrodes proletella L. as food sources for Coccinella undecimpunctata L. was evaluated by studying the impact of prey consumption on the predator’s population growth parameters and feeding parameters. Unlike A. proletella, A. fabae and M. persicae supported the development and reproduction of C. undecimpunctata. A. fabae and M. persicae were considered to be essential prey, whereas A. proletella was considered to be an alternative prey. Aphid species showed different degrees of suitability: M. persicae significantly decreased the pre-oviposition period and increased adult longevity, fecundity and fertility compared with A. fabae. Moreover, A. fabae represents a suitable diet for larval development, but is not a suitable food source for adult reproduction. The predator’s population growth parameters, R₀, r_m and λ were increased with M. persicae, whereas T decreased. We found that the 4th instar larvae were the most voracious, particularly when fed on M. persicae; nevertheless, with this prey daily weight gain and feeding efficiency of 4th instar larvae were similar to that of individuals fed with A. fabae.     

Temperature and host-plant effects on development and population growth of Mecinus janthinus (Coleoptera: Curculionidae), a biological control agent for invasive Linaria spp.

Mecinus janthinus Germar is a European stem-mining weevil that has been established in North America as a biological control agent against the invasive European weeds Linaria vulgaris P. Mill. and Linaria dalmatica (L.) P. Mill. (Scrophulariaceae). Establishment success and impact of the weevil have varied widely among sites. We investigated the hypothesis that some of this variation may be due to a lack of sufficient time for M. janthinus to develop to the adult (overwintering) stage in less favorable climates. Development time of M. janthinus was measured in L. vulgaris and L. dalmatica at four constant temperatures, and logistic regression was used to derive a model for the effect of temperature on development. Development rates were simulated using historic climate data for a site in central Alberta (where establishment was marginal on L. vulgaris) and one in southern British Columbia (where outbreaks occurred, resulting in heavy damage to L. dalmatica). The model showed that, on average, the British Columbia site had 50 more days available for the weevil to lay eggs that could reach the adult stage in time for overwintering than did the Alberta site. This may explain the more rapid population buildup at the British Columbia site. This model could be used to predict the climatic suitability of other areas for establishment of M. janthinus. An unexplained result was the very low survival rate of eggs laid in L. dalmatica under the same experimental conditions.     

Flamingomyces and Parvulago, new genera of marine smut fungi (Ustilaginomycotina)

Teliospore walls, teliospore germinations, hyphal septations, cellular interactions, and nucleotide sequences from the D1/D2 region of the nuLSU rRNA gene of the marine smut fungi Melanotaenium ruppiae and Ustilago marina were examined and compared with findings in other Ustilaginomycotina. The data show that Melanotaenium ruppiae belongs to the Urocystaceae and Ustilago marina to the Ustilaginaceae. Within the Urocystaceae, Melanotaenium ruppiae is morphologically similar to Melanustilospora and Vankya. However, according to the molecular results Melanotaenium ruppiae can neither be ascribed to Melanustilospora nor to Vankya. Therefore, the new genus Flamingomyces is proposed for Melanotaenium ruppiae. Ustilago marina differs from the other Ustilaginaceae in the mode of sporulation, which exclusively occurs at the base of the host plant culms. Accordingly, the new genus Parvulago is proposed for Ustilago marina.     

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.