Cultured gill-derived Neoparamoeba pemaquidensis fails to elicit amoebic gill disease (AGD) in Atlantic salmon Salmo salar

Amoebic gill disease (AGD) affects the culture of Atlantic salmon Salmo salar in the southeast of Tasmania. The disease is characterised by the presence of epizoic Neoparamoeba spp. in association with hyperplastic gill tissue. Gill-associated amoebae trophozoites were positively selected by plastic adherence for culture in seawater, where they proliferated using heat-killed E. coli as a nutrient source. One isolate of gill-harvested amoebae designated NP251002 was morphologically consistent to N. pemaquidensis under light, fluorescence and transmission electron microscopy. Rabbit anti-N. pemaquidensis antiserum bound to NP251002, and N. pemaquidensis small subunit (SSU) ribosomal DNA (18S rDNA) was detected in NP251002 genomic DNA preparations using PCR. A high degree of similarity in the alignment of the NP251002 18S rDNA PCR amplicon sequence with reference isolates of N. pemaquidensis suggested conspecificity. While short-term culture (72 h) of gill-harvested amoebae does not affect the capacity of amoebae to induce AGD, Atlantic salmon challenged with NP251002 after the trophozoites had been 34 and 98 d in culture exhibited neither gross nor histological evidence of AGD. It is not known if NP251002 were avirulent at the time of isolation, had down-regulated putative virulence factors or virulence was inhibited by the culture conditions. Therefore, the time in culture could be a limiting factor in maintaining virulence using the culture technique described here.     

Effects of bacteria on the growth of an amoeba infecting the gills of turbot.

We analysed the influence of various bacteria on the in vitro growth of trophozoites of a Platyamoeba strain isolated from diseased gill tissues of cultured turbot. Little or no growth was shown by amoebae cultured in the presence of (1) the turbot-pathogenic bacteria Vibrio anguillarum, Aeromonas salmonicida or Streptococcus sp., (2) Pasteurella piscicida or Vibrio vulnificus (pathogenic for some fishes but not turbot), or (3) the non-pathogenic 'environmental' bacteria Vibrio campbelli, Vibrio fluvialis or Pseudomonas dondorofii. The only bacteria which were successfully utilized as food sources were Aeromonas hydrophila (pathogenic for some fishes but not turbot) and the non-pathogens Vibrio natriegens, Pseudomonas nautica and Escherichia coli. These results suggest that the colonization of the gills of cultured turbot by the epizoic amoeba Platyamoeba may be an indicator of faecal contamination.     

Influence of salmonid gill bacteria on development and severity of amoebic gill disease

The relationship between salmonid gill bacteria and Neoparamoeba sp., the aetiological agent of amoebic gill disease (AGD) was determined in vivo. Fish were divided into 4 groups and were subjected to following experimental infections: Group 1, amoebae only; Group 2, Staphylococcus sp. and amoebae; Group 3, Winogradskyella sp. and amoebae; Group 4, no treatment (control). Fish (Groups 1, 2 and 3) were exposed to potassium permanganate to remove the natural gill microflora prior to either bacterial or amoebae exposure. AGD severity was quantified by histological analysis of gill sections to determine the percentage of lesioned filaments and the number of affected lamellae within each lesion. All amoebae infected groups developed AGD, with fish in Group 3 showing significantly more filaments with lesions than other groups. Typically lesion size averaged between 2 to 4 interlamellar units in all AGD infected groups. The results suggest that the ability of Neoparamoeba sp. to infect filaments and cause lesions might be enhanced in the presence of Winogradskyella sp. The possibility is proposed that the prevalence of more severe AGD is due to the occurrence of Winogradskyella sp. at high concentrations on the gills.     

Respiratory pathogenesis of amoebic gill disease (AGD) in experimentally infected Atlantic salmon Salmo salar

The aim of this study was to investigate the respiratory responses of Atlantic salmon, Salmo salar, experimentally affected with amoebic gill disease (AGD). In Series I, arterial blood samples were taken over a 96 h period following amoebae addition to examine potential respiratory effects associated with initial exposure. No major significant treatment effects were found between fish exposed to amoebae and control (non-exposed) fish. Arterial pH (pHa) was seen to be significantly elevated at 48 h in AGD fish relative to the 0 h time point. To investigate the long-term respiratory effects associated with infection, fish were similarly exposed to amoebae and sampled over a 16 d period. As for Series I, caudal blood pH was significantly elevated by Day 2 (48 h) compared to the pre (Day 0)-time point, suggesting that initial exposure to amoebae and/or amoebae attachment may have induced an initial respiratory alkalosis via increased ventilation frequency and/or amplitude. From Day 7 onwards, and coinciding with a significant increase in the percentage of affected gill filaments, blood pH decreased significantly, possibly indicating the onset of the characteristic respiratory acidosis that has previously been described for experimentally AGD-affected Atlantic salmon. Although fish in this study showed up to 90% AGD-affected filaments, the corresponding respiratory results do not reflect a major acid-base disturbance. Therefore, the findings from the present study support the contention that, although AGD only affects the gill, AGD-associated mortality in Atlantic salmon may not be primarily associated with respiratory failure.     

大菱鲆幼鱼表型形态性状与体重之间的关系

随机选取1120尾3月龄大菱鲆幼鱼,测量其体长、全长、体高、头长、吻尖至鳃裂前缘长、尾柄长、尾柄高、体厚、体重等9个性状,计算各性状间的相关系数,采用通径分析方法计算以表型形态性状为自变量对体重作依变量的通径系数、决定系数及复相关指数, 对各性状的影响大小进行剖分,明确影响大菱鲆三月龄幼鱼体重的主要外部形态性状,为大菱鲆选育提供理论依据和理想的测度指标.结果表明:所测各表型性状与体重之间的相关系数均达到极其显著水平(P＜0.01＝;全长对体重的直接影响(R=0.702)最大, 对体重的决定程度(R2=51.84%)最高,是影响体重的主要因素;体高、体厚对体重的直接作用 (0.211 ,0.094)相对较小,主要通过体长的间接作用(0.5558,0.4342)影响体重.所选表型性状对体重的回归系数R2=0.911,表明所选性状是影响体重的主要性状.利用逐步回归分析方法建立以体长、体高、体厚为自变量估计体重的多元回归回归方程为:y=-6.216 1.294x1 0.518x2 1.293x3[动物学报 54(3):540-545,2008].     

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.     

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.     

Epizoic and parasitic rotifers

Many rotifer species live in close association with plants or other animals. Most of these associations are of a commensal or synoecious nature, some rotifer species having lost the ability to live independently. Few rotifers are true parasites, actually harming their hosts.The Seisonidae, Monogononta and Bdelloidea include epizoic and parasitic species. The most widely known are probably the parasites of colonial and filamentous algae (e.g. Volvox, Vaucheria). However, rotifers are also found on a wide range of invertebrates: colonial, sessile Protozoa; Porifera; Rotifera; Annelida; Bryozoa; Echinodermata; Mollusca, especially on the shells and egg masses of aquatic gastropods; Crustacea, including the lower forms (e.g. Daphnia, Asellus, Gammarus) and in the gill chambers of Astacus and Chasmagnathus; the aquatic larvae of insects. There appear to be few records of epizoic or parasitic rotifers among vertebrates, apart from Encentrum kozminskii on carp, Limnias ceratophylli on the Amazonian crocodile, Melanosuchus niger, and an unidentified Bdelloid apparently living as a pathogenic rotifer in Man.     

Neoparamoeba Page, 1987: light and electron microscopic observations on six strains of different origin

Although amoebic gill disease (AGD) has emerged as one of the most severe health problems in the fish industry, proof of the identity of AGD agents from various localities is still missing. Six strains of amoebae designated until recently as Paramoeba species (the agents of AGD) were studied in cultures by light and electron microscopy. Although they were isolated from gills of different hosts (Dicentrarchus labrax and Scophthalmus maximus) and from distant localities, their morphology was identical. The strains differed from Paramoeba eilhardi, the type species of the genus, in that they lacked the boat-shaped microscales on the cell surface but could be safely identified as belonging to the genus Neoparamoeba Page, 1987. Transmission electron microscopy revealed the presence of a symbiotic organism, Perkinsiella amoebae Hollande, 1980, in all strains under study. The only difference among the strains examined was found in the size of trophozoites, which could be attributed to the different origins of the strains, but until more refined diagnostic methods are available, in addition to N. pemaquidensis, the closely related species N. aestuarina also has to be taken into consideration as the agent of AGD.     

Searching for the sweet spot of amoebic gill disease of farmed Atlantic salmon: the potential role of glycan-lectin interactions in the adhesion of Neoparamoeba perurans

One of the first critical steps in the pathogenesis of amoebic gill disease (AGD) of farmed salmon is the adhesion of the causative amoeba to the host. The current study aimed to investigate the potential involvement of glycan-binding proteins expressed on the extracellular surface of Neoparamoeba perurans in gill tissue recognition and binding. The glycan-binding properties of the surface membrane of N. perurans and the carbohydrate binding profile of Atlantic salmon gill-derived epithelial cells were identified through the use of glycan and lectin microarrays, respectively. The occurrence of specific carbohydrate-mediated binding was then further assessed by in vitro attachment assays using microtitre plates pre-coated with the main glycan candidates. Adhesion assays were also performed in the presence of exogenous saccharides with the aim of blocking glycan-specific binding activity. Comparative analysis of the results from both lectin and glycan arrays showed significant overlap, as some glycans to which binding by the amoeba was seen were reflected as being present on the gill epithelial cells. The two main candidates proposed to be involved in amoeba attachment to the gills are mannobiose and N-acetylgalactosamine (GalNAc). Adhesion of amoebae significantly increased by 33.5 and 23% when cells were added to α1,3-Mannobiose-BSA and GalNAc-BSA coated plates. The observed increased in attachment was significantly reduced when the amoebae were incubated with exogenous glycans, further demonstrating the presence of mannobiose- and GalNAc-binding sites on the surfaces of the cells. We believe this study provides the first evidence for the presence of a highly specific carbohydrate recognition and binding system in N. perurans. These preliminary findings could be of extreme importance given that AGD is an external parasitic infestation and much of the current research on the development of alternative treatment strategies relies on either instant amoeba detachment or blocking parasite attachment.     

Diatoms prefer strangers: non-indigenous crayfish host completely different epizoic algal diatom communities from sympatric native species

Despite their wide distribution and ecological importance, almost no information is available about the role of freshwater crayfish as basibiont for epizoic algae. Moreover, studies on epizoic freshwater diatoms have been largely neglected. In this study, we examined the occurrence of diatoms on two sympatric species, the native white-clawed crayfish Austropotamobius pallipes and the non-indigenous signal crayfish Pacifastacus leniusculus, coexisting in the same stream in NW Italy. We detected that signal crayfish showed a far more productive epizoic algal community than white-clawed crayfish. Microscopical analysis confirmed that non-indigenous crayfish hosted rich and diversified diatom communities while virtually no diatom was found on the native. After analyzing different hypotheses, we suggested that this significant difference can be the result of diverse crayfish behavioral habits. Because of the lack of studies investigating epibiontic diatoms on freshwater crayfish, we performed a detailed characterization on the epizoic flora, including comparisons with natural epilithic communities. The exponential diffusion of non-indigenous freshwater crayfish is a subject of greatest interest. Increasing our knowledge on their role as basibionts is definitely necessary to better understand their ecological role, also considering their influence on primary producer community and their role as vectors of algal species of concern, such as Didymosphenia geminata.     

Evidence for Symbiont-induced Alteration of a Host's Gene Expression: Irreversible Loss of SAM Synthetase from Amoeba proteus

ABSTRACT. Symbiont-bearing xD amoebae no longer produce a 45-kDa cytoplasmic protein that functions as S-adenosylmethionine synthetase in symbiont-free D amoebae. The absence of the protein in xD amoebae is attributable to xD amoeba's failure to transcribe the corresponding gene as a result of harboring bacterial symbionts. However, xD amoebae have about half the level of enzyme activity found in D amoebae, indicating that they use an alternative source for the enzyme. xD amoebae originated from D amoebae by bacterial infection and now depend on their symbionts for survival. xD amoebae exhibit irreversible nucleolar abnormalities when their symbionts are removed, suggesting that X-bacteria supply the needed enzyme. A monoclonal antibody against the 45-kDa protein was produced and used as a probe in cloning its corresponding cDNA. The product of the cDNA was found to have S-adenosylmethionine synthetase activity. These results show how symbiotic X-bacteria may become essential cellular components of amoebae by supplementing a genetic defect for an amoeba's house-keeping gene that is brought about by an action of X-bacteria themselves. This is the first reported example in which symbionts alter the host's gene expression to block the production of an essential protein.     

滇西亚种树鼩体外寄生虫自然感染状况及分析

目的调查滇西亚种树鼩体外寄生虫的自然感染状况,为建立树鼩质量控制标准提供依据。方法对野生的滇西亚种树鼩和自繁F1代树鼩各60只分别用尸检法和活体法检测,随即采用拔毛或用透明胶带粘取腋下、耳根、腹股沟、肛门附近等处的毛发制片,置于体视镜和显微镜下观察虫体和虫卵。结果野生滇西亚种树鼩和自繁F1代树鼩体外寄生虫自然感染率分别为：虱子25%/6%、蚤5%/0、蜱1.6%/0、水蛭1.6%/0、虱子和蚤混合感染1.6%/0。结论野生树鼩体外寄生虫的自然感染多为虱子和蚤,其次还有蝉、水蛭,总感染率明显高于自繁F1代。使用灭虱灵对于驱除树鼩体外寄生虫具有较好的疗效。     

Bryozoan colonization of the marine isopod Glyptonotus antarcticus at Signy Island, Antarctica

Sixty specimens of the giant marine isopod Glyptonotus antarcticus Eights, collected from Borge Bay, Signy Island, Antarctica were examined for epizoans. Ten species of cheilostomatid bryozoans were found on the isopods. The purpose of the study was to quantify the prevalence, intensity, abundance, and spatial distribution of the bryozoans on the isopods. The proportion of isopods colonized was 42%. The larger isopods had both significantly more epizoic bryozoan colonies and species. The greatest density of bryozoans was on the fused pleon and telson. There was no significant difference between the dorsal and ventral abundance of bryozoan colonies. The diversity of epizoic bryozoans on the isopods is higher than on other host organisms from more stable environments. This may be because of active selection by settling larvae. The frequency of local substrata being scoured by ice is high around Signy Island, so there may be a selective advantage in colonizing a motile host. Accepted: 20 June 1998     

Seasonality, substrate perference and attachment sites of epizoic diatoms on cladoceran zooplankton

Cladocerans from three lakes in northwest Iowa were examinedfor attached diatoms over a period of 1 year to assess the seasonality,substrate preference and attachment site specificities of epizoicdiatoms. Over 90% of the cladocerans had attached diatoms immediatelyfollowing ice thaw, when the lakes were not yet thermally stratified,surface water temperatures were low(10–15°C)and watertransparency was at its annual high. Mean maximum density ofdiatoms on cladocerans averaged among lakes at this time was3000 cells animal^–1. The dominant epizoic diatom was Synedracyclopum Brutschy, which was found most often on large, pelagicDaphnia pulicaria and Daphnia galeata mendotae. Heavily infestedanimals had diatoms concentrated on the second antennae andanterior ventral margin of the carapace. Animals with <5epizoic diatoms were more likely to have cells attached to thepostabdomen or tail spine. In addition to living epizoically,S.cyclopum was found free living in the plankton, exhibitingtemporal fluctuations in densities similar to epizoic counterpartsHowever, the densities of the free-living cells seldom exceededepizoic densities, suggesting that S.cyclopum is adapted forgrowth on motile cladoceran substrates. ¹Present address: Savannah River Ecology Lab, Drawer E, Aiken,SC 29802, USA ²Present address: Department of Fisheries and Aquatic Sciences,7922 NW 71st St, Gainesville, FL 32606, USA     

Abundance of Naked Amoebae in Sediments of Hiroshima Bay, Seto Inland Sea of Japan

ABSTRACT The present paper provides the first data on naked amoebae from sediments of Hiroshima Bay. Three stations in the inner part of the bay were sampled over a three-month period. Abundance of naked amoebae ranged from 1,019 to 45,561 cells/g dry sediment. Results indicate: (i) surface sediment populations in most cases were higher than subsurface populations; (ii) there was some evidence of temporal variation with counts generally increasing from March to May: and (iii) the site located near Hiroshima City had fewer amoebae on several occasions than the other two sites. There was a negative exponential relationship between acid-volatile sulfide concentration and abundance of amoebae. Most amoebae were small with the average size ranging from 6.6–14 μm. Morphotype 1, amoebae that extend lobose pseudopodia or subpseudopodia during normal locomotion, were dominant (40–100% of enumerated amoebae). Morphotypes 2 and 3 (limax amoebae) were found in lower numbers than the other two morphotypes. The proportion of amoebae occupied by Morphotype 4 (fan-shaped or discoidal-flattened amoebae) was higher at a lower total abundance.     

Spatial distribution of gymnamoebae (Rhizopoda, Lobosea) in brackish-water sediments at the scale of centimeters and millimeters

In order to study micro-spatial distribution of amoebae, an intact slice of sandy sediment from the brackish-water Nivå Bay (Baltic Sea, The Sound), 40 × 24 mm in size and 2 mm in thickness was gently sectioned into cubes, 2 × 2 × 2 mm in size. Each cube was inoculated into enrichment media to reveal the biodiversity of amoebae. Seventeen species of amoebae were recovered. The 2-D map of amoebae species distribution in the slice, consisting of 240 2 × 2 mm cells was drawn and analyzed. Results show heterogeneous distribution of amoebae at the scale of centimeters and millimeters and confirm the idea of the presence of microhabitats, selectively occupied by amoebae species. Three types of distribution patterns were found: random, aggregated and equally spaced. Microelectrode studies indicated that amoebae distribution was not related to the dissolved oxygen content in the sediment. The studied slice of sediment contained several pronounced “hotspots” of amoebae biodiversity, where up to four species co-occur in the same area. Seven species of amoebae numbered 1–4 specimens in the studied slice (i.e. there was 0.5–2 cell ml^–1). Analysis of the amoebae distribution map shows the high probability of undersampling rare amoebae species during faunistic studies.     

Comparison of polyamine and S-adenosylmethionine contents of growing and encysted Acanthamoeba isolates

We have used High Performance Liquid Chromatography to determine metabolite characteristics of three recent isolates of Acanthamoeba which exhibit cultural characteristics consistent with those of established potential pathogens. Growing amoebae and dormant cysts of these isolates were explored in regard to their qualitative and quantitative intracellular levels of polyamine and S-adenosylmethionine metabolites. The polyamine found in the greatest concentration in the growing cells was 1,3-diaminopropane (DAP), followed by spermidine (SPD). A low level of putrescine was also found in the growing cells. These polyamines significantly decreased in concentration as the amoebae differentiated to cysts. N⁸-acetylspermidine and acetylspermine were found in both developmental stages while acetylcadaverine was found only in growing amoebae and N¹-acetylspermidine only in cysts. Acetylputrescine was present in both stages of two isolates but only in the growing amoebae of the third isolate. Spermine was not detected in any of the isolates.S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were present in growing amoebae but SAM was undetectable or barely detectable in cysts. SAH also decreased in concentration during encystation of two of the isolates to a level comparable to that of the other isolate.The developmental transition from growing amoebae to dormant cysts is characterized metabolically by a threshold adjustment in concentration of SAM, SAH and of the polyamines (esp., DAP and SPD).     

Epizoic mode of life in Brachionus rubens Ehrenberg as a deterrent against predation by Asplanchna intermedia Hudson

The planktonic rotifer Brachionus rubens has a propensity for an epizoic mode of life, and in nature is often found attached to cladocerans. In this way the rotifer avoids to a certain extent the adverse effects of interference competition with cladocerans. We test the hypothesis that the epizoic habit of B. rubens acts also as a deterrent against invertebrate predation. Using Asplanchna intermedia as predator, we followed the population growth patterns of B. rubens alone and in the presence of the host species Daphnia carinata and Ceriodaphnia rigaudi. In the absence of cladocerans, the prey was eliminated within three days, followed by extinction of the predator due to starvation. With D. carinata in the medium, the prey-predator system persisted much longer, with B. rubens reaching high population densities. With the smaller-sized C. rigaudi, allowing a significantly smaller fraction of B. rubens population to be epizoic, the system persisted longer than in the controls, but both the prey and predator eventually became extinct. We conclude that the epizoic habit of B. rubens, by acting as a prey refugium, helps a portion of the population to escape from predation, and facilitates its coexistence with Asplanchna intermedia.     

The rate of attachment of amoebae to the substratum: a study of nuclear-cytoplasmic relationships

The rate of attachment to the substratum was found to be a strain specific character of certain large free-living amoebae. Two strains, which differed significantly in this respect, were compared with regard to the rate of attachment of enucleated cytoplasm, nuclear-cytoplasmic hybrids and amoebae injected with cytoplasm from another strain. It was found that both the nucleus and the cytoplasm of the more rapidly attaching strain were able to transmit this character to amoebae of the more slowly attaching strain. The possible mechanisms underlying attachment and transmission of the rate of attachment are discussed.