Virulence of Paramoeba invadens Jones (Amoebida,Paramoebidae) from Monoxenic and Polyxenic Culture1

Paramoeba invadens is a pathogenic marine amoeba responsible for mass mortalities of sea urchins (Strongylocentrotus droebachiensis) along the Atlantic coast of Nova Scotia in the early 1980's. The amoeba has been maintained in vivo in S. droebachiensis for five years in the laboratory without observable loss of virulence. Paramoeba invadens was cultured polyxenically (on mixed marine bacteria) and monoxenically (on a single strain of Pseudomonas nautica) on non-nutrient agar for 58 weeks and 19 weeks respectively. Pathogenicity tests showed some loss of virulence in monoxenic culture after 15 weeks and in polyxenic culture after 58 weeks. Polyxenic culture is recommended for long-term culture of P. invadens although periodic passage of the amoeba through the sea urchin host may be required to maintain virulence for periods exceeding one year.     

Uncommon southwest swells trigger sea urchin disease outbreaks in Eastern Atlantic archipelagos

Recurrent sea urchin mass mortality has recently affected eastern Atlantic populations of the barren‐forming sea urchin Diadema africanum. This new episode of die‐off affords the opportunity to determine common meteorological and oceanographic conditions that may promote disease outbreaks. The population dynamics of this sea urchin species are well known—urchin barrens have persisted for many decades along most of the coastlines off the archipelagos of Madeira, Selvages, and the Canary Islands, where they limit macroalgae biomass growth. However, this new and explosive mortality event decimated the sea urchin population by 93% on Tenerife and La Palma Islands. Two severe episodes of southwestern rough sea that led to winter storms, in February 2010 (Xynthia) and February 2018 (Emma), preceded both mass mortality events. The autumn and winter months of those years were anomalous and characterized by swells with an average wave height above 2 m that hit the south and southwest sides of the islands. The amoeba Paramoeba brachiphila was the only pathogen isolated this time from the moribund and dead sea urchins, suggesting that the amoeba was the primary cause of the mortality. This new sea urchin die‐off event supports the “killer‐storm” hypothesis that has been already described for western Atlantic coasts. These anomalous southwest storms during winters generate pronounced underwater sediment movement and large‐scale vertical mixing, detected in local tide gauge, which may promote paramoebiasis. This study presents valuable insights about climate‐mediated changes in disease frequency and its impacts on the future of coastal marine ecosystems in the Atlantic.     

Effect of Temperature and Prey Availability on Growth of Paramoeba invadens in Monoxenic Culture

Paramoeba invadens Jones 1985 is a pathogenic marine amoeba responsible for mass mortalities of sea urchins (Strongylocentrotus droebachiensis) of Nova Scotia between 1980 and 1983. A direct relationship between temperature and sea urchin paramoebiasis has been shown in previous laboratory and field studies. This study examined the effect of prey availability and temperature on the growth of P. invadens in monoxenic culture (with the marine bacterium Pseudomonas nautica). At 15°C, the specific growth rate of P. invadens increased with bacterial prey concentration and was highest at 10⁸ bacterial cells ml⁻¹. Growth rate of P. invadens was maximal at 15 to 20°C (which corresponds to annual sea temperature maxima in the natural environment) and the minimum generation time was 19.41 h at 20°C. At 10 and 12°C, generation times were 91.18 and 73.39 h, respectively; at 2 and 5°C, there was no growth. P. invadens did not survive in monoxenic culture at 27°C. Growth rates of P. invadens in vitro were positively correlated with time to morbidity of infected S. droebachiensis.     

Phylogeny of Neoparamoeba strains isolated from marine fish and invertebrates as inferred from SSU rDNA sequences

ABSTRACT: We characterised 9 strains selected from primary isolates referable to Paramoeba/Neoparamoeba spp. Based on ultrastructural study, 5 strains isolated from fish (amoebic gill disease [AGD]-affected Atlantic salmon and dead southern bluefin tuna), 1 strain from netting of a floating sea cage and 3 strains isolated from invertebrates (sea urchins and crab) were assigned to the genus Neoparamoeba Page, 1987. Phylogenetic analyses based on SSU rDNA sequences revealed affiliations of newly introduced and previously analysed Neoparamoeba strains. Three strains from the invertebrates and 2 out of 3 strains from gills of southern bluefin tunas were members of the N. branchiphila clade, while the remaining, fish-isolated strains, as well as the fish cage strain, clustered within the clade of N. pemaquidensis. These findings and previous reports point to the possibility that N. pemaquidensis and N. branchiphila can affect both fish and invertebrates. A new potential fish host, southern bluefin tuna, was included in the list of farmed fish endangered by N. branchiphila. The sequence of P. eilhardi (Culture Collection of Algae and Protozoa [CCAP] strain 1560/2) appeared in all analyses among sequences of strain representatives of Neoparamoeba species, in a position well supported by bootstrap value, Bremer index and Bayesian posterior probability. Our research shows that isolation of additional strains from invertebrates and further analyses of relations between molecular data and morphological characters of the genera Paramoeba and Neoparamoeba are required. This complexity needs to be considered when attempting to define molecular markers for identification of Paramoeba/Neoparamoeba species in tissues of fish and invertebrates.     

Paramoeba invadens n. sp. (Amoebida,Paramoebidae), a Pathogenic Amoeba from the Sea Urchin,Strongylocentrotus droebachiensis,in Eastern Canada1

Paramoeba invadens n. sp. is described from sea urchin tissues and from culture. Amoebae are 20–40 μm in length with an irregular hyaline region producing short subpseudopodia. One parasome per cell is present. The parasome is bipolar with basophilic, Feulgen-positive poles and a Feulgen-negative median segment. The fine structure of the parasome resembles those in other species of Paramoeba and the surface of the amoeba is plain with no hairs or scales. Amoebae dislodged from tissues often adopt a semifloating form; floating forms have been seen in culture. Amoebae circulate within lumina of the water vascular system and can migrate readily into or out of tissues.     

Microbiological study of the body wall lesions of the echinoid Tripneustes gratilla

The microbiota of the body wall lesions of the echinoid Tripneustes gratilla, initiated by the grazing action of the parasitic gastropod Vexilla vexillum, was investigated with a pluridisciplinary approach. Parasitised sea urchins showed body wall lesions strongly infected by bacteria that progressed through the test and reached the coelomic cavity after ca. 1 mo. We report here on the bacterial community observed in lesions of echinoids collected in situ and on the bacteria that successively appeared during laboratory experiments. Two Alphaproteobacteria, a CFB (Cytophaga-Flavobacterium-Bacteroides) bacterium, 3 Vibrio species and Exiguobacterium aestuarii were identified in the field-collected lesions by 16S rDNA sequencing. The last 4 bacteria were cultured and each induced the disease when inoculated on scalpel-made wounds, with 100% of the individuals infected within 2 d. Scalpel-induced scarifications tended to heal within 3 wk, while gastropod-induced lesions evolved into disease, suggesting a role of Vexilla vexillum in the development of the infection. Denaturing gradient gel electrophoresis (DGGE) and sequencing suggest that (1) bacteria associated with healthy integument were not present in the lesions and were thus not responsible for their infection, (2) Alphaproteobacteria with close phylogenetic affiliation with other bacteria involved in several diseases affecting marine invertebrates were present, and (3) these Alphaproteobacteria were present from the beginning of the infection and appeared earlier in the infection than other bacteria such as CFB bacteria.     

Utilization of purple and red sea urchins (Strongylocentrotus purpuratus Stimpson and S. franciscanus Agassiz) as food by the white sea urchin (Lytechinus anamesus Clark) in the field and laboratory

White sea urchins (Lytechinus anamesus Clark) attacked purple (Strongylocentrotus purpuratus Stimpson) and red (S. franciscanus Agassiz) sea urchins at Anacapa Island, California. Densities of white urchins were highest in the deep algal crust-dominated community where up to 6% of purple and 25% of red urchins were being attacked by white urchins. Up to 9% of Lytechinus anamesus in an area were actively eating stronglylocentrotids and usually, more than one white urchin was involved in the attack. In areas with low densities of white urchins, no strongylocentrotids were being attacked.After 36 h in the laboratory, there was no difference in the number of white urchins attacking injured or healthy purple urchins in each of the three experimental densities of white urchins. However, both injured and healthy urchins were attacked by more white urchins in high density. When given a choice between injured purple urchins or fresh kelp, white urchins overwhelmingly chose kelp. Data suggest that white urchins utilize other urchin species as an alternative source of food when more preferred food is absent, but will switch to preferred food should it become available.     

Survival of polioviruses and echoviruses in Acanthamoeba castellanii cultivated in vitro

Cultures of Acanthamoeba castellanii, kept at room temperature in sterile Bacto-Casitone (Difco) medium, were artificially infected with vaccination poliovirus strains type 1 and type 3 and with echovirus type 4 and echovirus type 30. No remarkable virus cumulation was observed on the surface or inside amoeba cells during the observation period of 21 days. Amoeba-adsorbed viruses were impossible to remove by repeated washings. Virus neutralization with specific antisera showed that enteroviruses were most probably present only on amoeba surfaces. In contrast to amoeba-free virus suspension, echoviruses bound to amoebae and their cell pulp persisted even after 52 to 75 days. However, the tested amoeba species played only the role of a solids-like carrier in this survival of echoviruses.     

Overgrazing of Kelp Beds Along the Coast of Norway

The aim of this study was to better understand the down-grazing of kelp beds by sea urchins (Strongylocentrotus droebachiensis) along the coast of Norway. Barren grounds were first observed in sheltered areas along the coast of the counties of Trø ndelag, Nordland and Troms in 1974. In the 1980s, the barren grounds spread to areas more heavily exposed to waves. In the 1990s, the kelp beds were re-established in some localities in southern Trø ndelag, initially in wave-exposed areas. In the northernmost parts of Norway, i.e. the counties of Troms and Finnmark, the barren ground areas may still be increasing. Crabs (Cancer pagurus) and common eiders are the most common predators on urchins. Predation on sea urchins in kelp beds is probably not among the factors that limit the sea urchin populations. Along the coast of Nordland and further north, sea urchins are infected by nematodes, resulting in a low, but significant increase in their mortality. No re-growth of kelp beds has been found in the most infected areas. In the late 1960s and the early 1970s, a high occurrence of echinoderm larvae was observed in deeper waters. This was a period with cold water, which may have caused high recruitment of sea urchins. The bet-hedging life strategy of sea urchins may account for the sudden increase in the size of the populations. In the present paper I propose the hypothesis that higher individual growth rates and higher mortality rates in the south than in the north may explain the decrease in the populations, which may in turn account for the re-growth of kelp in the southern areas.     

In Vitro Propagation of the Protozoan Perkinsus Marinus, A Pathogen of the Eastern Oyster, Crassostrea Virginica

ABSTRACT. Perkinsus marinus , a pathogen of eastern oysters ( Crassostrea virginica ), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus -1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P. marinus in naturally infected oysters. In addition, cultured cells incubated in fluid thioglycollate medium produced enlarged cells (prezoosporangia) that stained blue-black in Lugol's solution, a response characteristic to Perkinsus spp. and used in routine diagnosis. Polyclonal antibodies raised against P. marinus prezoosporangia reacted positively to Perkinsus -1. Finally, the cultured cells infected susceptible oysters and reisolation of Perkinsus -1 cells was possible from the hearts of experimentally infected oysters. the culture medium contained most of the known constituents of cell-free hemolymph of oysters. the success achieved in culturing P. marinus will allow further investigations aimed at reducing mortalities caused by this important oyster pathogen and at addressing many unanswered questions about its biology and pathobiology.     

Increased macroalgal abundance following mass mortalities of sea urchins (Strongylocentrotus droebachiensis) along the Atlantic coast of Nova Scotia

Summary Recurrent outbreaks of disease between 1980 and 1983 caused catastrophic mortality of sea urchins (>260,000 t fresh weight) along 280 km (straight line distance) of the Atlantic coast of Nova Scotia. The complete elimination of sea urchins and concomitant development of fleshy macroalgal communities have occurred along different parts of this coast in different years. Macroalgal communities in areas where sea urchins died off 1, 3 and 4 years previously are compared to existing sea urchin-dominated barren grounds and to a mature kelp bed without sea urchins. Changes in macroalgal cover and species composition, and increases in biomass, density and size of kelp (Laminaria) species, characterize the succession from barren grounds to 3- and 4-year-old kelp beds. The greatest change occurred between one and three years following sea urchin mass mortality. Within 3 years, kelp beds attained a level of biomass (7.6 kg m^-2) comparable to that of mature beds. Recovery of sea urchin populations via recruitment of planktonic larvae has been slow and spatially variable. Large-scale reciprocal fluctuations in kelp and sea urchin biomass may characterize the trajectory of a dynamic system which cycles between two alternate community states: kelp beds and sea urchin-dominated barren grounds. Periodic decimation of sea urchin populations by disease may be an important mechanism underlying this cyclicity.     

Epidermal lesions and mortality caused by vibriosis in deep-sea Bahamian echinoids: a laboratory study

When significant mortality of the bathyal spatangoid echinoid Paleopneustes cristatus occurred under laboratory conditions, we investigated the cause and course of the disease by culturing and identifying internal pathogens, then experimentally infecting healthy urchins with isolates of the suspected disease organism. The pathogen was determined to be the gram-negative halophilic bacterium Vibrio alginolyticus. This species was also recovered from frozen post-challenge specimens of P. cristatus and from moribund individuals of Archaeopneustes hystrix, another spatangoid reared under similar in vitro conditions. This is the first experimental study of bacterial disease in any deep-sea invertebrate.     

Effect of temperature on virus eradication and growth of infected tissue cultures

Cucumber mosaic (CMV) and alfalfa mosaic (AlfMV) viruses were eradicated or their concentrations greatly diminished when infected meristem tips were grown in static or shake culture at 32 or 34 ^oC. Similar cultures grown at 22 ^oC remained infected. Tobacco mosaic virus (TMV) was not eradicated from shake cultures at 32 ^oC and the concentration of TMV in such cultures at 32 ^oC was sometimes higher than at 22 ^oC. The concentration of CMV and AlfMV was considerably less in cultured tissues than in infected leaf material, but that of TMV was similar. Cultures infected with CMV or TMV grew more slowly than healthy cultures. Growth of AlfMV infected and healthy cultures was similar.     

Synopsis of infectious diseases and parasites of commercially exploited shellfish

The development of shellfish-based industries and the concomitant increase in demand for the introduction and transfer of different shellfish species and stocks has increased the risks of spreading their parasites and diseases around the world. To avoid the accidental introduction of infectious disease agents, information on known parasites and diseases must be readily available. Since the number of recognized infectious agents and facts on known diseases is continuously increasing, it is necessary to update the current state of knowledge. Thus, published and new accounts of viruses, bacteria, protozoa, fungi, metazoa, and infectious diseases of unknown aetiology in commercially exploited shellfish (molluscs, echinoderms, and crustaceans) are summarised. The summaries were devised to be of use to regulatory agencies, diagnosticians, researchers, and students who may require information on this diverse subject. The information is organised according to the host that is normally infected (i.e. oysters, mussels, clams, cockles, scallops, abalone, sea urchins, sea stars, lobsters, shrimp, prawns, crabs, and freshwater crayfish). Each of the 169 summaries includes the common or widely accepted name of the parasite or disease agent, and the scientific name (where known) or taxonomic affiliation. In addition, geographic distribution, host species infected (both naturally and experimentally), impact on host health, diagnostic techniques including illustrations for many of the diseases, known methods of control, and appropriate references are provided.     

Spine skeleton morphogenesis during regeneration in clypeasteroid and camarodont sea urchins

The process of skeleton morphogenesis is described for broken and totally removed spines in clypeasteroid (hollow spine) and camarodont (solid spine) sea urchins. Spine regeneration after total spine removal is completed in 40–45 days in clypeasteroids and in 60–70 days in camarodont sea urchins. Along with common stages of formation of longitudinal ribs in both hollow and solid spines, fundamental differences were found between the initial stages of reparative growth of the spine shaft. The spine shaft is formed from a single median process in clypeasteroids and from many simultaneously growing processes in camarodont sea urchins. Reparative morphogenesis of totally removed and partly broken spines in clypeasteroid sea urchins and totally removed spines in camarodont sea urchins leads to the formation of a skeletal structure identical to the intact spine. However, during the regeneration of broken camarodont spines, lateral growth is markedly retarded. As a result, the regenerated part of the spine shaft has a smaller diameter when the initial spine length is achieved. A hypothesis is proposed on a paedomorphic origin of spines in the clypeasteroid sea urchins on the basis of the juvenile stage of definitive spines in the camarodont sea urchins.     

In vitro cultured Neoparamoeba perurans causes amoebic gill disease in Atlantic salmon and fulfils Koch's postulates

Amoebic gill disease (AGD) in marine farmed Atlantic salmon is of growing concern worldwide and remains a significant health issue for salmon growers in Australia. Until now the aetiological agent, Neoparamoeba perurans, has not been amenable to in vitro culture and therefore Koch's postulates could not be fulfilled. The inability to culture the amoeba has been a limiting factor in the progression of research into AGD and required the maintenance of an on-going laboratory-based infection to supply infective material. Culture methods using malt yeast agar with sea water overlaid and subculturing every 3-4 days have resulted in the establishment of a clonal culture of N. perurans, designated clone 4. Identity of the amoeba was confirmed by PCR. After 70 days in culture clone 4 infected Atlantic salmon, causing AGD, and was re-isolated from the infected fish. Diagnosis was confirmed by histology and the infectious agent identified by PCR and in situ hybridisation using oligonucleotide primers and probes previously developed and specific to N. perurans. This study has fulfilled Koch's postulates for N. perurans as a causative agent of AGD and illustrates its free-living and parasitic nature.     

Select light spectra affect gonad color in the sea urchin Lytechinus variegatus

It is suggested that gonad color in sea urchins depends upon the in vivo accumulation and metabolism of red and yellow carotenoid pigments. We hypothesized that differential light exposure could affect carotenoid deposition and, hence, gonad color in sea urchins. We therefore performed two experiments to determine whether light spectra affect the gonad color of Lytechinus variegatus. In the first experiment, urchins were fed a formulated feed supplemented with or without β-carotene and held beneath three lighting regimes designed to emit differing wavelengths of the visible spectrum. After 12 weeks, urchins were dissected and gonad color (CIE L*a*b*) was measured with a Pantone Capsure RM200. Actinic light significantly increased the value of a* (red) in gonad color. Color in the orange and yellow spectra in the gonads increased in individuals fed the β-carotene supplemented diet. In the second experiment, we cultured urchins for nine weeks under lamps specialized to emit UV radiation. All urchins in this experiment received diets supplemented with β-carotene. There was no significant difference in harvested gonad color between these treatments. These data suggest that light quality and dietary carotenoids affect carotenoid deposition in the gonads.     

Adhesion,Morphology, and Locomotion of Paramoeba pemaquidensis Page (Amoebida,Paramoebidae): Effects of Substrate Charge Density and External Cations1

Morphology and locomotive behavior in the marine amoeba, Paramoeba pemaquidensis Page, was examined under different environmental conditions. Paramoeba requires a minimum surface negative charge density for adhesion of amoebae to substrata. Once adhesion to the substratum has been attained, however, surface negative charge density has no effect on morphology or locomotive rate. Divalent cations are not required for adhesion, but external calcium is required for normal locomotion. In the presence of calcium, Paramoeba often assumes a locomotive form with a broad, well-developed anterior hyaline region and truncate posterior region. Locomotive forms vary from those with only a well-developed hyaline region (Flabellula-like) to forms with long digitiform sub-pseudopodia (Vexillifera-like), with intermediate morphotypes. Locomotive rates decrease and anteroposterior polarity disappears in the presence of living or heat-killed bacteria, indicating that phagocytosis temporarily interferes with locomotion and alters form.     

EPSTEIN-BARR病毒血清抗体阳性健康人外周血体外培养“自发性”转化的研究

对35名Epstein-Barr病毒血清抗体阳性健康人的外周血淋巴细胞进行了体外培养,观察B淋巴细胞感染了EB病毒后所导致“自发性”转化的情况。由于在培养基中加入了免疫抑制剂环胞菌素A,使“自发性”转化的发生率由26.7％提高到74.3％,说明了在血清抗体阳性健康人的外周血液中,EB病毒感染的B淋巴细胞的数量远比既往文献报道的高。     

Dispersion and phagokinesis in the echinoid Evechinus chloroticus (Val.)

Movement in the echinometrid sea urchin Evechinus chloroticus (Val.) was investigated in relation to the availability of a preferred food species, the laminarian alga Ecklonia radiata (C. Ag.). Patterns of microhabitat occupancy, dispersion, and movement were described in two habitats: Coralline Flats, dominated by encrusting coralline algae; and Sediment Flats, occupied by turfing corallines, Ecklonia and the mussel Modiolus areolatus (Gould). In both habitats, sea urchins were positively associated with encrusting coralline algae, despite the fact that encrusting corallines occupied a minor proportion of the substratum in the Sediment Flat habitat. Sea urchins were significantly clumped in both habitat types, and more so in sites within the Sediment Flats habitat. Movement was not directional in either habitat, but the magnitude of movement was almost twice as high in sites from the Coralline Flats habitat, where kelp was absent, as in the Sediment Flats sites, where kelp was present. Behaviour in response to the presence of Ecklonia in both habitats was investigated experimentally. In both habitats, urchins exposed to a “drift” kelp plant at a distance of 1–2 m exhibited net movement approximately twice as great as that shown by control urchins, not exposed to a kelp plant. Despite the relatively small distances the urchins were from the kelp, there was no evidence of directionality in movement. We conclude from the present study that further understanding of the feeding behaviour of urchins requires that a distinction be made between attached and drift algae, and between directionality and magnitude of movement, and that the interaction of these and other factors are best investigated under field conditions.