Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

The ability of Neoparamoeba perurans to bind to and digest non-fish-derived mucin: Insights into the amoeba's mechanism of action to overcome gill mucus production.

Amoebic gill disease (AGD) is caused by the marine amoeba Neoparamoeba perurans, a facultative parasite. Despite the significant impact this disease has on production of Atlantic salmon worldwide, the mechanisms involved in host-parasite interaction remains unknown. Excessive gill mucus secretion is reported as a host defence mechanism to prevent microbial colonization in the gill epithelium. Despite this response, N. perurans still attaches and proliferates. The present study aimed to investigate the interaction between N. perurans and mucin, the most abundant component in mucus. An in vitro adhesion assay using bovine submaxillary mucin (BSM) demonstrated that amoeba binding to mucin-coated substrate was significantly higher than to the BSA control. This binding interaction is likely glycan-mediated as pre-incubation with galactose, galactosamine, N-acetylgalactosamine and fucose reduced mucin adhesion to control levels. The ability of N. perurans to secrete proteases that target mucin was also investigated. Protease activity was detected in the amoeba culture media in the presence of BSM, but not when protease inhibitor was added. Mucin degradation was visually assessed on protein gels. This study provides preliminary evidence that N. perurans has developed mechanisms to interact with and evade mucus by binding to mucin glycan receptors and secreting proteases with mucolytic activity.

The sexual side of parasitic protists.

Much of the vast evolutionary landscape occupied by Eukaryotes is dominated by protists. Though parasitism has arisen in many lineages, there are three main groups of parasitic protists of relevance to human and livestock health: the Apicomplexa, including the malaria parasite Plasmodium and coccidian pathogens of livestock such as Eimeria; the excavate flagellates, encompassing a diverse range of protist pathogens including trypanosomes, Leishmania, Giardia and Trichomonas; and the Amoebozoa, including pathogenic amoebae such as Entamoeba. These three groups represent separate, deep branches of the eukaryote tree, underlining their divergent evolutionary histories. Here, I explore what is known about sex in these three main groups of parasitic protists.

Method for cryopreservation of Paramoeba perurans.

Paramoeba perurans causes amoebic gill disease (AGD), which is a major problem in aquaculture worldwide. The parasite can be cultured in vitro, but to this date, no method for long-term storage of the clones exists. In this study, we describe a method for cryopreservation of Paramoeba perurans. The method was successfully employed on four out the five clones we tested. The thawing success rate, that is the percentage of successfully thawed vials relative to the total number of vials that were thawed, differed for the clones and ranged from 25% to 100%. The age of the clones seemed to have a negative impact on the ability to survive cryopreservation.

Resistance of the oyster pathogen Vibrio tasmaniensis LGP32 against grazing by Vannella sp. marine amoeba involves Vsm and CopA virulence factors.

Vibrios are ubiquitous in marine environments and opportunistically colonize a broad range of hosts. Strains of Vibrio tasmaniensis present in oyster farms can thrive in oysters during juvenile mortality events and behave as facultative intracellular pathogen of oyster haemocytes. Herein, we wondered whether V. tasmaniensis LGP32 resistance to phagocytosis is specific to oyster immune cells or contributes to resistance to other phagocytes, like marine amoebae. To address this question, we developed an integrative study, from the first description of amoeba diversity in oyster farms to the characterization of LGP32 interactions with amoebae. An isolate of the Vannella genus, Vannella sp. AP1411, which was collected from oyster farms, is ubiquitous, and belongs to one clade of Vannella that could be found associated with Vibrionaceae. LGP32 was shown to be resistant to grazing by Vannella sp. AP1411 and this phenotype depends on some previously identified virulence factors: secreted metalloprotease Vsm and copper efflux p-ATPase CopA, which act at different steps during amoeba-vibrio interactions, whereas some other virulence factors were not involved. Altogether, our work indicates that some virulence factors can be involved in multi-host interactions of V. tasmaniensis ranging from protozoans to metazoans, potentially favouring their opportunistic behaviour.

Responses of unicellular predators to cope with the phototoxicity of photosynthetic prey.

Feeding on unicellular photosynthetic organisms by unicellular eukaryotes is the base of the aquatic food chain and evolutionarily led to the establishment of photosynthetic endosymbionts/organelles. Photosynthesis generates reactive oxygen species and damages cells; thus, photosynthetic organisms possess several mechanisms to cope with the stress. Here, we demonstrate that photosynthetic prey also exposes unicellular amoebozoan and excavates predators to photosynthetic oxidative stress. Upon illumination, there is a commonality in transcriptomic changes among evolutionarily distant organisms feeding on photosynthetic prey. One of the genes commonly upregulated is a horizontally transferred homolog of algal and plant genes for chlorophyll degradation/detoxification. In addition, the predators reduce their phagocytic uptake while accelerating digestion of photosynthetic prey upon illumination, reducing the number of photosynthetic cells inside the predator cells, as this also occurs in facultative endosymbiotic associations upon certain stresses. Thus, some mechanisms in predators observed here probably have been necessary for evolution of endosymbiotic associations.

Survival and Growth in vitro of Paramoeba perurans Populations Cultured Under Different Salinities and Temperatures.

Growth rates of Paramoeba perurans cultures under different temperature and salinity conditions were investigated in vitro over a 15day period. Optimal population growth, under the experimental conditions, was observed at 15°C and a salinity of 35‰, with amoebae populations doubling every 14h. Positive P. perurans populations growth was observed at 15°C between salinities of above 20‰ and 50‰, and at 8°C, 11°C and 18°C at salinities between 25‰ and 50‰, 50‰ being the maximum salinity tested. Amoebae numbers were sustained at 4°C. Therefore, lower temperature and salinity thresholds for P. perurans population growth lie between 4 to 8°C, and salinities of 20 to 25‰, respectively. Upper limits were not determined in this study. The populations remained relatively stable at 4°C and 2°C at permissive salinities with respect to numbers of viable amoebae over the 15day exposure period.

The origins and evolution of macropinocytosis.

In macropinocytosis, cells take up micrometre-sized droplets of medium into internal vesicles. These vesicles are acidified and fused to lysosomes, their contents digested and useful compounds extracted. Indigestible contents can be exocytosed. Macropinocytosis has been known for approaching 100 years and is described in both metazoa and amoebae, but not in plants or fungi. Its evolutionary origin goes back to at least the common ancestor of the amoebozoa and opisthokonts, with apparent secondary loss from fungi. The primary function of macropinocytosis in amoebae and some cancer cells is feeding, but the conserved processing pathway for macropinosomes, which involves shrinkage and the retrieval of membrane to the cell surface, has been adapted in immune cells for antigen presentation. Macropinocytic cups are large actin-driven processes, closely related to phagocytic cups and pseudopods and appear to be organized around a conserved signalling patch of PIP3, active Ras and active Rac that directs actin polymerization to its periphery. Patches can form spontaneously and must be sustained by excitable kinetics with strong cooperation from the actin cytoskeleton. Growth-factor signalling shares core components with macropinocytosis, based around phosphatidylinositol 3-kinase (PI3-kinase), and we suggest that it evolved to take control of ancient feeding structures through a coupled growth factor receptor. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

Testate Amoebae in the 407-Million-Year-Old Rhynie Chert.

The Lower Devonian Rhynie chert is justly famous for the clear glimpse it offers of early terrestrial ecosystems [1]. Seven species of stem- and crown-group vascular plants have been described from Rhynie, many preserved in growth position [2], as well as 14 species of invertebrate animals, all arthropods [3] save for a single nematode population [4]. While these shed welcome light on early tracheophytes and land animals, modern terrestrial ecosystems additionally contain a diversity of microscopic organisms that are key to ecosystem function, including fungi, protists, and bacteria. Fungi ranging from mycorrhizae to saprophytes are well preserved in Rhynie rocks ([5] and references therein), and oomycetes are also present [5]. Both green algae (charophytes) and cyanobacteria have also been documented locally [6, 7, 8]. To date, however, phagotrophic protists have not been observed in Rhynie cherts, even though such organisms contribute importantly to carbon, nitrogen, and silica cycling in modern terrestrial communities [9]. Here, we report a population of organic tests described as Palaeoleptochlamys hassii gen. nov., sp. nov. from a pond along the Rhynie alluvial plain, which we interpret as arcellinid amoebozoans. These fossils expand the ecological dimensions of the Rhynie biota and support the hypothesis that arcellinids transitioned from marine through freshwater environments to colonize soil ecosystems in synchrony with early vascular plants.

Reinvestigation of Phryganella paradoxa (Arcellinida, Amoebozoa) Penard 1902.

A major drawback in testate amoeba research is a general lack of scientific studies combining molecular approaches and classical laboratory experiments. We isolated five yet uncultured testate amoebae of the genus Phryganella Penard, 1902 from three different rivers and one pond in Germany. Based on established cultures we show their morphology, which we studied by light and electron microscopy, and present their unique feeding mode on abundant and common pennate diatoms like Nitzschia spp. and Synedra spp., whose frustules are bent and frequently, but not always, broken during the feeding process. We further obtained the first SSU rDNA sequences of strains of the family Phryganellidae, all of which contain introns. We used the sequences to confirm the taxonomic placement of the Phryganellidae in the Arcellinida (Amoebozoa), branching as a sister group to the Cryptodifflugiidae.

Lyophilisation as a simple and safe method for long-term storage of free-living amoebae at ambient temperature.

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. As some species or strains of these FLA are pathogenic for humans and animals, they represent objects of medical and parasitological research worldwide. Storage of valuable FLA strains in laboratories is often time- and energy-consuming and expensive. The shipment of such strains as frozen stocks is cumbersome and challenging in terms of cooling requirements as well as of transport regulations. To overcome these difficulties and challenges in maintenance and transport, we present a new method to generate lyophilised samples of non-cyst-forming FLA (Ripella (Vannella) spp.) and cyst-forming FLA (Acanthamoeba spp.) strains which guarantees a simple mechanism for long-term storage at ambient temperature, as well as easy handling and/or shipment. The survival rate of all FLA lyophilisates after short-term storage (2 months) was comparable to the survival rate of freeze cultures of the respective strains. Furthermore, the viability of Acanthamoeba spp. cysts after storage for 29 months was 20 to 40% following lyophilisation and rehydration, with strain variation.

Repeated sublethal freshwater exposures reduce the amoebic gill disease parasite, Neoparamoeba perurans, on Atlantic salmon.

Freshwater bathing is one of the main treatment options available against amoebic gill disease (AGD) affecting multiple fish hosts in mariculture systems. Prevailing freshwater treatments are designed to be long enough to kill Neoparamoeba perurans, the ectoparasite causing AGD, which may select for freshwater tolerance. Here, we tested whether using shorter, sublethal freshwater treatment durations are a viable alternative to lethal ones for N. perurans (2-4 hr). Under in vitro conditions, gill-isolated N. perurans attached to plastic substrate in sea water lifted off after ≥2 min in freshwater, but survival was not impacted until 60 min. In an in vivo experiment, AGD-affected Atlantic salmon Salmo salar subjected daily to 30 min (sublethal to N. perurans) and 120 min (lethal to N. perurans) freshwater treatments for 6 days consistently reduced N. perurans cell numbers on gills (based on qPCR analysis) compared to daily 3 min freshwater or seawater treatments for 6 days. Our results suggest that targeting cell detachment rather than cell death with repeated freshwater treatments of shorter duration than typical baths could be used in AGD management. However, the consequences of modifying the intensity of freshwater treatment regimes on freshwater tolerance evolution in N. perurans populations require careful consideration.

Effect of repeated exposure to AQUI-S® on the viability and growth of Neoparamoeba perurans.

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28-day period to AQUI-S® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post-smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non-attached amoeba in the culture was assessed with a vital stain. AQUI-S® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.

Genetic Evidence for Sexuality in Cochliopodium (Amoebozoa).

Microbial eukaryotes, including amoeboids, display diverse and complex life cycles that may or may not involve sexual reproduction. A recent comprehensive gene inventory study concluded that the Amoebozoa are ancestrally sexual. However, the detection of sex genes in some lineages known for their potentially sexual life cycle was very low. Particularly, the genus Cochliopodium, known to undergo a process of cell fusion, karyogamy, and subsequent fission previously described as parasexual, had no meiosis genes detected. This is likely due to low data representation, given the extensive nuclear fusion observed in the genus. In this study, we generate large amounts of transcriptome data for 2 species of Cochliopodium, known for their high frequency of cellular and nuclear fusion, in order to study the genetic basis of the complex life cycle observed in the genus. We inventory 60 sex-related genes, including 11 meiosis-specific genes, and 31 genes involved in fusion and karyogamy. We find a much higher detection of sex-related genes, including 5 meiosis-specific genes not previously detected in Cochliopodium, in this large transcriptome data. The expressed genes form a near-complete recombination machinery, indicating that Cochliopodium is an actively recombining sexual lineage. We also find 9 fusion-related genes in Cochliopodium, although no conserved fusion-specific genes were detected in the transcriptomes. Cochliopodium thus likely uses lineage specific genes for the fusion and depolyploidization processes. Our results demonstrate that Cochliopodium possess the genetic toolkit for recombination, while the mechanism involving fusion and genome reduction remains to be elucidated.

Vannella pentlandii n. sp., (Amoebozoa, Discosea, Vannellida) a small, cyst-forming soil amoeba.

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii, instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 µm in diameter, are the smallest so far described in genus Vannella. Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others.

Local and systemic humoral immune response in farmed Atlantic salmon (Salmo salar L.) under a natural amoebic gill disease outbreak.

Amoebic gill disease (AGD), caused by the protozoan parasite Neoparamoeba perurans, is one of the most significant infectious diseases for Atlantic salmon (Salmo salar L.) mariculture. The present study investigated the humoral immune response (both local in gill mucus and systemic in serum) of farmed Atlantic salmon naturally infected with N. perurans in commercial sea pens, at two different stages of the disease and after freshwater treatment. Parameters analysed included activity of immune related enzymes (i.e. lysozyme, peroxidase, protease, anti-protease, esterase, alkaline phosphatase), IgM levels, and the terminal carbohydrate profile in the gill mucus. Overall, greater variations between groups were noted in the immune parameters determined in gill mucus than the equivalent in the serum. In gill mucus, IgM levels and peroxidase, lysozyme, esterase and protease activities were decreased in fish showing longer exposure time to the infection and higher disease severity, then showed a sequential increase after treatment. Results obtained highlight the capacity of gills to elicit a local response to the infection, indicate an impaired immune response at the later stages of the disease, and show partial reestablishment of the host immune status after freshwater treatment. In addition to providing data on the humoral response to AGD, this study increases knowledge on gill mucosal humoral immunity, since some of the parameters were analysed for the first time in gill mucus.

Leaf Litter Chemistry Drives the Structure and Composition of Soil Testate Amoeba Communities in a Tropical Montane Rainforest of the Ecuadorian Andes.

We investigated the role of leaf litter chemistry and richness in affecting testate amoeba communities of tropical rainforest in the Ecuadorian Andes. Litterbags containing leaf litter from four dominating tree species (Clusia sp., Myrcia pubescens, Graffenrieda emarginata, and Cecropia andina) with richness 1, 2, and 4 species were established and exposed in the field for 12 months at 2000 m a.s.l. Chemical elements and compounds of leaf litter were analyzed before exposure. At the end of exposure, microbial biomass and litter mass loss were measured, and living testate amoeba species number, density, biomass, and community composition were determined. In total, 125 testate amoeba species colonized the litter in litterbags. The results suggest that high litter nitrogen and low lignin concentrations are indicators of high litter quality for testate amoebae density and species richness. Their species number and density significantly declined in the order 1 > 4 > 2 leaf litter species and varied with leaf litter chemistry being at a maximum in high-quality single leaf litter species and low in low-quality leaf litter. Further, the addition of litter of high-quality to low-quality litter increased testate amoebae biomass and density; however, the values did not exceed the ones in single high-quality litter treatments. Moreover, the structure of testate amoeba communities varied with litter chemistry, with Fe, Na, lignin, and litter C-to-N ratio being of major importance, and indicating that litter chemistry reflects habitat quality for testate amoebae. Overall, the data show that leaf litter chemistry overrides leaf litter richness in structuring testate amoeba communities.