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.     

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.     

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.     

Metabolic effects of amoebic gill disease (AGD) and chloramine-T exposure in seawater-acclimated Atlantic salmon Salmo salar

Our aim was to determine possible metabolic effects amoebic gill disease (AGD) on Atlantic salmon Salmo salar. Standard (R(S)) and routine (R(ROU)) metabolic rates were evaluated by continually measuring oxygen consumption in 2 independent tanks of fish (18.69 +/- 1.01 kg m(-3), mean +/- SE). Active metabolic rate (R(ACT)) and metabolic scope (R(ACT) - R(S)) were assessed using a chasing protocol and determined at 3 time periods: (1) pre-infection, (2) 3 d post-infection, and (3) 2 d post-treatment. On Day 3 of the study, the fish were infected with amoebae isolated from the gills of AGD-affected salmon (2300 cells l(-1)). No significant elevations in R(ACT) or metabolic scope were detected 3 d post-infection and 2 d post-treatment; however, significant elevations in R(S) and R(ROU) were detected 3 d post-infection and 2 d post-treatment. Assessment of R(ROU) data, especially for the light period, also indicated a rise in oxygen consumption rate over the course of the experiment. Treatment of AGD-affected Atlantic salmon with chloramine-T (CL-T) appeared to briefly mitigate the rise in R(S), as there was a 30% drop (though non-significant) in R(S) following treatment. Despite this, R(S) continued the upward trend 1 d following treatment. These results suggest that over the course of AGD development, R(S) in Atlantic salmon increases. Therefore, considering the physical conditions which constrain R(ACT), we expect that metabolic scope would become compromised in fish more heavily affected with AGD. Treatment with CL-T shows promise for mitigating the respiratory effects of AGD and potentially minimising the loss of metabolic scope.     

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.     

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.     

Development of an In Vitro Model System for Studying Bacterially Expressed dsRNA-Mediated Knockdown in Neoparamoeba genus

RNA interference (RNAi) has been extensively used to study gene function in non-model organisms and has the potential to identify parasite target molecules in order to develop alternative treatment strategies. This technology could assist in further development of preventive methods against amoebic gill disease (AGD), the main health problem affecting the Atlantic salmon aquaculture industry in Tasmania (Australia) and now a significant emerging issue in Europe. Using β-actin and EF1-α as candidate genes, we investigated the feasibility of gene knockdown by double-stranded RNA (dsRNA) in Neoparamoeba pemaquidensis, the non-infective strain closely related to the causative agent of AGD, Neoparamoeba perurans. Bacterially expressed dsRNA targeting the selected target genes was administered by soaking (2, 20 and 50 μg/mL) and a time course sampling regime performed. Quantitative real-time PCR analysis showed that candidate genes were successfully downregulated with silencing efficiency and duration both target and dose-dependent. Additionally, β-actin deficient trophozoites unexpectedly transformed into a cyst-like stage, which has not been previously reported in this species. An effective RNAi model system for N. pemaquidensis was validated in the current study. Such findings will greatly facilitate further application of RNAi in the aetiological agent of AGD. To our knowledge, this is the first time that RNAi-mediated technology has been successfully employed in a member of the Neoparamoeba genus.     

Shotgun metagenomics and microscopy indicate diverse cyanophytes,other bacteria,and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria)

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.     

Thecamoeba cosmophorea n. sp. (Amoebozoa,Discosea, Thecamoebida) — An example of sibling species within the genus Thecamoeba

A new species Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) was isolated from leaf litter collected in the surroundings of Saint-Petersburg (Russia). This species resembles T. quadrilineata in light-microscopic morphology, but has certain morphological differences and significantly differs in 18S rRNA gene sequence. We performed a direct comparison of this newly isolated species with the Thecamoeba strain isolated from leaf litter in East Siberia (Russia) and identified as T. quadrilineata both at the morphological and the molecular level. There is no type strain of T. quadrilineata, and the type material on this species is represented with the stained slide by F.C. Page that he designated as neotype in 1977. The sequence of the 18S rRNA gene of this species deposited in the GenBank belongs to the isolate identified as T. quadrilineata by Rolf Michel in 1998 and deposited as CCAP 1583/10 strain. Hence we cannot be entirely sure that morphological and molecular data on T. quadrilineata belong to the same amoebae species. The use of molecular data for reliable species differentiation is getting obligate even within the amoebae genus Thecamoeba, which until recently was believed to be among few genera of naked lobose amoebae allowing morphological identification of species.     

Acid–base and respiratory effects of confinement in Atlantic salmon affected with amoebic gill disease

Amoebic gill disease (AGD) in cultured salmonids causes severe multifocal hyperplastic lesions in the gills with the potential to influence respiratory and acid–base physiology. Atlantic salmon Salmo salar affected with AGD were surgically implanted with dorsal aortic catheters and, following recovery, were confined for 5 min ( n  = 16) or left undisturbed ( n  = 8). Confinement caused an acute extracellular acidosis that was corrected in 6 h amongst surviving fish. There was a gradual increase in plasma lactate concentrations that peaked at 1 h post-confinement then declined by 9 h recovery. In a second experiment, AGD-affected fish were confined then recovered either in a tank of static water ( n  = 9) or while being forced to swim at 1·5 body lengths s⁻¹ ( n  = 6). There was no significant difference between fish recovered by swimming and those in static water in terms of recovery of the acute extracellular acidosis and lactate accumulations coincident with exhaustive exercise. Confinement severely compromised the survival of AGD-affected Atlantic salmon, although survivors appeared to recover similarly to other studies. Forced swimming of AGD-affected Atlantic salmon following confinement did not facilitate recovery and is unlikely to be a useful strategy for mitigating the effects of stressful episodes such as crowding and fish movement and commercial handling.     

Concentration effects of Winogradskyella sp. on the incidence and severity of amoebic gill disease

To study the concentration effects of the bacterium Winogradskyella sp. on amoebic gill disease (AGD), Atlantic salmon Salmo salar were pre-exposed to 2 different doses (10(8) or 10(10) cells 1(-1)) of Winogradskyella sp. before being challenged with Neoparamoeba spp. Exposure of fish to Winogradskyella sp. caused a significant increase in the percentage of AGD-affected filaments compared with controls challenged with Neoparamoeba only; however, these percentages did not increase significantly with an increase in bacterial concentration. The results show that the presence of Winogradskyella sp. on salmonid gills can increase the severity of AGD.     

Spirotrichonymphea (Parabasalia) symbionts of the termite Paraneotermes simplicicornis

The desert dampwood termite Paraneotermes simplicicornis harbors several species of obligately symbiotic protists that support its nutrition by fermenting lignocellulose. Among them are three morphotypes with the dexiotropic spiraling flagellar bands characteristic of Spirotrichonymphea (Parabasalia). The largest morphotype, characterized by an elongated cell apex with axial columella and internally positioned spiraling flagellar bands, was previously described as Spirotrichonympha polygyra. A smaller morphotype, with similarly internalized flagellar bands but a more rounded posterior without a protruding axostyle, was previously reported but not named. The smallest morphotype has surface flagellar bands and can attach to other protist cells by its apex. In this study, we combine light microscopy of live specimens and 18S rRNA gene sequencing of individually isolated cells to better understand the diversity of symbionts in P. simplicicornis. We found that S. polygyra branches distantly from true Spirotrichonympha, which are associated with Reticulitermes termites. Thus, we propose the new genus Cuppa to accommodate C. polygyra n. comb. (type species) and the similar but smaller morphotype Cuppa taenia n. sp. The undescribed smallest morphotype can be excluded from all previously described Spirotrichonymphea genera by molecular and behavioral evidence, so we propose Fraterculus simplicicornis n. gen., n. sp., to accommodate this organism.     

Phylogenetic Analysis of the Spider Mite Sub-Family Tetranychinae (Acari: Tetranychidae) Based on the Mitochondrial COI Gene and the 18S and the 5′ End of the 28S rRNA Genes Indicates That Several Genera Are Polyphyletic

The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp). As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered.     

Epizoic amoebae from the gills of turbot Scophthalmus maximus

Species of amoebae belonging to the genera Platyamoeba Page, 1969, Vannella Bovee, 1965 and Flabellula Schaeffer, 1926 were found to accompany Paramoeba sp., the agent of amoebic gill disease (AGD), in clinically diseased turbots. The same community of epizoic gymnamoebae was found on the gills of turbots which revealed no gill abnormalities but slight behavioral signs indicative of suboptimal health status. The assemblage of the above-mentioned free-living amoebae capable of colonizing gill tissue of turbots was supplemented with species recognized in samples fixed from primary isolates for transmission electron microscopy. The pathogenic potential of epizoic gill amoebae in turbots is discussed.     

Genetic diversity and population connectivity of the Asian green mussel Perna viridis in South China Sea,inferred from mitochondria DNA markers

The Asian green mussel Perna viridis is ecologically and economically important in the coastal regions of China. In order to characterize the genetic diversity and population connectivity of P. viridis in South China Sea, a 664 bp region of mitochondrial COI gene and a 293 bp region of 16S rRNA gene were sequenced and analyzed for 78 and 92 individuals from four populations in South China Sea, respectively. A total of 15 haplotypes were defined by 14 variable nucleotide sites in COI gene, and 7 haplotypes by 6 variable nucleotide sites in 16S rRNA gene. High haplotype diversity and low nucleotide diversity were observed in COI gene, while moderate haplotype diversity and low nucleotide diversity were observed in 16S rRNA gene. Pairwise F_ST values of COI gene were all negative and those of 16S rRNA gene ranged from −0.01409 to 0.10289. The results showed that no significant genetic divergence (or shallow genetic structure) and high levels of population connectivity among the four populations of P. viridis in South China Sea.     

Global phylogeny and new classification of the Rapaninae (Gastropoda: Muricidae), dominant molluscan predators on tropical rocky seashores

The monophyly of the muricid subfamily Rapaninae has recently been confirmed with molecular techniques, but its composition and the relationships among its constituent genera remain unclear. We use four genes (28S rRNA, 12S rRNA, 16S rRNA and cytochrome c oxidase subunit I, COI) to construct a Bayesian phylogeny of 80 rapanine species (73% of the approximately 109 currently accepted), representing 27 of the 31 nominal genera. This is the most complete phylogeny of this taxonomically confusing subfamily yet produced. We propose a revised phylogenetic classification of the Rapaninae, assigning the recognized species to 28 genera. Most of the morphologically-defined rapanine genera are considered valid, including Purpura, Drupa, Thais and Nassa, but many of them are here restricted or redefined so that they are monophyletic. In particular the familiar genus Thais is narrowly restricted to a single species. Many groups previously accepted as subgenera, including Mancinella, Vasula, Thalessa and Thaisella, are here accorded full generic rank. We describe one new genus, Indothais. While we do not formally alter species-level taxonomy, we show molecular evidence for two cryptic species and several instances of probable species synonymy. We estimate the age of diversification of the Rapaninae as Late Cretaceous (75.9 Ma) and of many of its genera as Miocene.     

Species diversity of Onchidiidae (Eupulmonata: Heterobranchia) on the mainland of China based on molecular data

The identification of members of the Onchidiidae is based on morphological characters; this is often time-consuming and can be inconclusive. In order to explore the species diversity of onchidiids in China, we provide a phylogeny constructed using partial sequences of two mitochondrial genes (16S rRNA and COI) and one nuclear ribosomal RNA gene (28S rRNA) from 32 samples comprising five genera. The topology, using both Bayesian and Maximum Likelihood inference methods, showed that the taxa clustered in two main groups of six species, one of which included Platevindex mortoni, Platevindex sp. and Onchidium ‘struma’; the other included Paraoncidium reevesii, Onchidella sp. and Peronia verruculata. It is clear that COI will be useful in discriminating onchidiid species-group taxa.     

Diversity and Distribution of Freshwater Testate Amoebae (Protozoa) Along Latitudinal and Trophic Gradients in China

Freshwater microbial diversity is subject to multiple stressors in the Anthropocene epoch. However, the effects of climate changes and human activities on freshwater protozoa remain poorly understood. In this study, the diversity and distribution of testate amoebae from the surface sediments were investigated in 51 Chinese lakes and reservoirs along two gradients, latitude and trophic status. A total of 169 taxa belonging to 24 genera were identified, and the most diverse and dominant genera were Difflugia (78 taxa), Centropyxis (26 taxa) and Arcella (12 taxa). Our analysis revealed that biomass of testate amoebae decreased significantly along the latitudinal gradient, while Shannon-Wiener indices and species richness presented an opposite trend (P?P?相似文献