Presence and diversity of free-living amoebae and their potential application as water quality indicators.

Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.

Modelling dynamics between free-living amoebae and bacteria.

Free-living amoebae (FLA) serve as hosts for a variety of endosymbionts, which are microorganisms that reside and multiply within the FLA. Some of these endosymbionts pose a pathogenic threat to humans, animals, or both. The symbiotic relationship with FLA not only offers these microorganisms protection but also enhances their survival outside their hosts and assists in their dispersal across diverse habitats, thereby escalating disease transmission. This review is intended to offer an exhaustive overview of the existing mathematical models that have been applied to understand the dynamics of FLA, especially concerning their interactions with bacteria. An extensive literature review was conducted across Google Scholar, PubMed, and Scopus databases to identify mathematical models that describe the dynamics of interactions between FLA and bacteria, as published in peer-reviewed scientific journals. The literature search revealed several FLA-bacteria model systems, including Pseudomonas aeruginosa, Pasteurella multocida, and Legionella spp. Although the published mathematical models account for significant system dynamics such as predator-prey relationships and non-linear growth rates, they generally overlook spatial and temporal heterogeneity in environmental conditions, such as temperature, and population diversity. Future mathematical models will need to incorporate these factors to enhance our understanding of FLA-bacteria dynamics and to provide valuable insights for future risk assessment and disease control measures.

Metabarcoding of protozoa and helminth in black-necked cranes: a high prevalence of parasites and free-living amoebae.

Parasites and free-living amoebae (FLA) are common pathogens that pose threats to wildlife and humans. The black-necked crane (Grus nigricollis) is a near-threatened species and there is a shortage of research on its parasite diversity. Our study aimed to use noninvasive methods to detect intestinal parasites and pathogenic FLA in G. nigricollis using high-throughput sequencing (HTS) based on the 18S rDNA V9 region. A total of 38 fresh fecal samples were collected in Dashanbao, China, during the overwintering period (early-, middle I-, middle II-, and late-winter). Based on the 18S data, eight genera of parasites were identified, including three protozoan parasites: Eimeria sp. (92.1%) was the dominant parasite, followed by Tetratrichomonas sp. (36.8%) and Theileria sp. (2.6%). Five genera of helminths were found: Echinostoma sp. (100%), Posthodiplostomum sp. (50.0%), Euryhelmis sp. (26.3%), Eucoleus sp. (50.0%), and Halomonhystera sp. (2.6%). Additionally, eight genera of FLA were detected, including the known pathogens Acanthamoeba spp. (n = 13) and Allovahlkampfia spp. (n = 3). Specific PCRs were used to further identify the species of some parasites and FLA. Furthermore, the 18S data indicated significant changes in the relative abundance and genus diversity of the protozoan parasites and FLA among the four periods. These results underscore the importance of long-term monitoring of pathogens in black-necked cranes to protect this near-endangered species.

Title: Métabarcoding des protozoaires et des helminthes chez les grues à cou noir : forte prévalence de parasites et d'amibes libres. Abstract: Les parasites et les amibes libres sont des agents pathogènes courants qui constituent une menace pour la faune et les humains. La grue à cou noir (Grus nigricollis) est une espèce quasi menacée et les recherches sur sa diversité parasitaire sont insuffisantes. Notre étude visait à utiliser des méthodes non invasives pour détecter les parasites intestinaux et les amibes libres pathogènes chez G. nigricollis en utilisant le séquençage à haut débit basé sur la région V9 de l'ADNr 18S. Au total, 38 échantillons de matières fécales fraîches ont été collectés à Dashanbao, en Chine, au cours de la période d'hivernage (début, milieu I, milieu II et fin de l'hiver). Sur la base des données 18S, huit genres de parasites ont été identifiés, dont trois parasites protozoaires : Eimeria sp. (92,1 %) était le parasite dominant, suivi de Tetratrichomonas sp. (36,8 %) et Theileria sp. (2,6 %). Cinq genres d'helminthes ont été trouvés : Echinostoma sp. (100 %), Posthodiplostomum sp. (50,0 %), Euryhelmis sp. (26,3 %), Eucoleus sp. (50,0 %) et Halomonhystera sp. (2,6 %). De plus, huit genres d'amibes libres ont été détectés, y compris les agents pathogènes connus Acanthamoeba spp. (n = 13) et Allovahlkampfia spp. (n = 3). Des PCR spécifiques ont été utilisées pour identifier davantage les espèces de certains parasites et amibes libres. En outre, les données 18S ont indiqué des changements significatifs dans l'abondance relative et la diversité des genres des parasites protozoaires et des amibes au cours des quatre périodes. Ces résultats soulignent l'importance de la surveillance à long terme des agents pathogènes chez les grues à cou noir pour protéger cette espèce quasi menacée.

In the social amoeba Dictyostelium discoideum, shortened stalks may limit obligate cheater success even when exploitable partners are available.

Cooperation is widespread across life, but its existence can be threatened by exploitation. The rise of obligate social cheaters that are incapable of contributing to a necessary cooperative function can lead to the loss of that function. In the social amoeba Dictyostelium discoideum, obligate social cheaters cannot form dead stalk cells and in chimeras instead form living spore cells. This gives them a competitive advantage within chimeras. However, obligate cheaters of this kind have thus far not been found in nature, probably because they are often enough in clonal populations that they need to retain the ability to produce stalks. In this study we discovered an additional cost to obligate cheaters. Even when there are wild-type cells to parasitize, the chimeric fruiting bodies that result have shorter stalks and these are disadvantaged in spore dispersal. The inability of obligate cheaters to form fruiting bodies when they are on their own combined with the lower functionality of fruiting bodies when they are not represent limits on obligate social cheating as a strategy.

Prestalk-like positioning of de-differentiated cells in the social amoeba Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum switches between solitary growth and social fruitification depending on nutrient availability. Under starvation, cells aggregate and form fruiting bodies consisting of spores and altruistic stalk cells. Once cells socially committed, they complete fruitification, even if a new source of nutrients becomes available. This social commitment is puzzling because it hinders individual cells from resuming solitary growth quickly. One idea posits that traits that facilitate premature de-commitment are hindered from being selected. We studied outcomes of the premature de-commitment through forced refeeding. Our results show that when refed cells interacted with non-refed cells, some of them became solitary, whereas a fraction was redirected to the altruistic stalk, regardless of their original fate. The refed cells exhibited reduced cohesiveness and were sorted out during morphogenesis. Our findings provide an insight into a division of labor of the social amoeba, in which less cohesive individuals become altruists.

Size dependent uptake and trophic transfer of polystyrene microplastics in unicellular freshwater eukaryotes.

Microplastics (MP) have become a well-known and widely investigated environmental pollutant. Despite the huge amount of new studies investigating the potential threat posed by MP, the possible uptake and trophic transfer in lower trophic levels of freshwater ecosystems remains understudied. This study aims to investigate the internalization and potential trophic transfer of fluorescent polystyrene (PS) beads (0.5 µm, 3.6 × 108 particles/mL; 6 µm, 2.1 × 105 particles/mL) and fragments (<30 µm, 5 × 103 particles/mL) in three unicellular eukaryotes. This study focuses on the size-dependent uptake of MP by two freshwater Ciliophora, Tetrahymena pyriformis, Paramecium caudatum and one Amoebozoa, Amoeba proteus, serving also as predator for experiments on potential trophic transfer. Size-dependent uptake of MP in all three unicellular eukaryotes was shown. P. caudatum is able to take up MP fragments up to 27.7 µm, while T. pyriformis ingests particles up to 10 µm. In A. proteus, small MP (PS0.5µm and PS6µm) were taken up via pinocytosis and were detected in the cytoplasm for up to 14 days after exposure. Large PS-MP (PS<30µm) were detected in A. proteus only after predation on MP-fed Ciliophora. These results indicate that A. proteus ingests larger MP via predation on Ciliophora (PS<30µm), which would not be taken up otherwise. This study shows trophic transfer of MP at the base of the aquatic food web and serves as basis to study the impact of MP in freshwater ecosystems.

Immunogens in Balamuthia mandrillaris: a proteomic exploration.

Balamuthia mandrillaris is the causative agent of granulomatous amoebic encephalitis, a rare and often fatal infection affecting the central nervous system. The amoeba is isolated from diverse environmental sources and can cause severe infections in both immunocompromised and immunocompetent individuals. Given the limited understanding of B. mandrillaris, our research aimed to explore its protein profile, identifying potential immunogens crucial for early granulomatous amoebic encephalitis diagnosis. Cultures of B. mandrillaris and other amoebas were grown under axenic conditions, and total amoebic extracts were obtained. Proteomic analyses, including two-dimensional electrophoresis and mass spectrometry, were performed. A 50-kDa band showed a robust recognition of antibodies from immunized BALB/c mice; peptides contained in this band were matched with elongation factor-1 alpha, which emerged as a putative key immunogen. Besides, lectin blotting revealed the presence of glycoproteins in B. mandrillaris, and confocal microscopy demonstrated the focal distribution of the 50-kDa band throughout trophozoites. Cumulatively, these observations suggest the participation of the 50-kDa band in adhesion and recognition mechanisms. Thus, these collective findings demonstrate some protein characteristics of B. mandrillaris, opening avenues for understanding its pathogenicity and developing diagnostic and therapeutic strategies.

Constant-pH Simulations with the Polarizable Atomic Multipole AMOEBA Force Field.

Accurately predicting protein behavior across diverse pH environments remains a significant challenge in biomolecular simulations. Existing constant-pH molecular dynamics (CpHMD) algorithms are limited to fixed-charge force fields, hindering their application to biomolecular systems described by permanent atomic multipoles or induced dipoles. This work overcomes these limitations by introducing the first polarizable CpHMD algorithm in the context of the Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field. Additionally, our implementation in the open-source Force Field X (FFX) software has the unique ability to handle titration state changes for crystalline systems including flexible support for all 230 space groups. The evaluation of constant-pH molecular dynamics (CpHMD) with the AMOEBA force field was performed on 11 crystalline peptide systems that span the titrating amino acids (Asp, Glu, His, Lys, and Cys). Titration states were correctly predicted for 15 out of the 16 amino acids present in the 11 systems, including for the coordination of Zn2+ by cysteines. The lone exception was for a HIS-ALA peptide where CpHMD predicted both neutral histidine tautomers to be equally populated, whereas the experimental model did not consider multiple conformers and diffraction data are unavailable for rerefinement. This work demonstrates the promise polarizable CpHMD simulations for pKa predictions, the study of biochemical mechanisms such as the catalytic triad of proteases, and for improved protein-ligand binding affinity accuracy in the context of pharmaceutical lead optimization.

A Small Genome amidst the Giants: Evidence of Genome Reduction in a Small Tubulinid Free-Living Amoeba.

This study investigates the genomic characteristics of Echinamoeba silvestris, a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance in various fields, genomic data for this clade have been scarce. E. silvestris presents the smallest free-living amoeba genome within Tubulinea and Amoebozoa to date. Comparative analysis reveals intriguing parallels with parasitic lineages in terms of genome size and predicted gene numbers, emphasizing the need to understand the consequences of reduced genomes in free-living amoebae. Functional categorization of predicted genes in E. silvestris shows similar percentages of ortholog groups to other amoebae in various categories, but a distinctive feature is the extensive gene contraction in orphan (ORFan) genes and those involved in biological processes. Notably, among the few genes that underwent expansion, none are related to cellular components, suggesting adaptive processes that streamline biological processes and cellular components for efficiency and energy conservation. Additionally, our investigation into noncoding and repetitive elements sheds light on the evolution of genome size in amoebae, with E. silvestris distinguished by low percentage of repetitive elements. Furthermore, the analysis reveals that E. silvestris has the lowest mean number of introns per gene among the species studied, providing further support for its observed compact genome. Overall, this research underscores the diversity within Tubulinea, highlights knowledge gaps in Amoebozoa genomics, and positions E. silvestris as a valuable addition to genomic data sets, prompting further exploration of complexities in Amoebozoa diversity and genome evolution.

Adaptation to an amoeba host drives selection of virulence-associated traits and genetic variation in saprotrophic Candida albicans.

Amoebae are micropredators that play an important role in controlling fungal populations in ecosystems. However, the interaction between fungi and their amoebic predators suggests that the pressure from predatory selection can significantly influence the development of fungal virulence and evolutionary processes. Thus, the purpose of this study was to investigate the adaptation of saprotrophic Candida albicans strains during their interactions with Acanthamoeba castellanii. We conducted a comprehensive analysis of survival after co-culture by colony counting of the yeast cells and examining yeast cell phenotypic and genetic characteristics. Our results indicated that exposure to amoebae enhanced the survival capacity of environmental C. albicans and induced visible morphological alterations in C. albicans, particularly by an increase in filamentation. These observed phenotypic changes were closely related to concurrent genetic variations. Notably, mutations in genes encoding transcriptional repressors (TUP1 and SSN6), recognized for their negative regulation of filamentous growth, were exclusively identified in amoeba-passaged isolates, and absent in unexposed isolates. Furthermore, these adaptations increased the exposed isolates' fitness against various stressors, simultaneously enhancing virulence factors and demonstrating an increased ability to invade A549 lung human epithelial cells. These observations indicate that the sustained survival of C. albicans under ongoing amoebic predation involved a key role of mutation events in microevolution to modulate the ability of these isolates to change phenotype and increase their virulence factors, demonstrating an enhanced potential to survive in diverse environmental niches.

Costs of being a diet generalist for the protist predator Dictyostelium discoideum.

Consumers range from specialists that feed on few resources to generalists that feed on many. Generalism has the clear advantage of having more resources to exploit, but the costs that limit generalism are less clear. We explore two understudied costs of generalism in a generalist amoeba predator, Dictyostelium discoideum, feeding on naturally co-occurring bacterial prey. Both involve costs of combining prey that are suitable on their own. First, amoebas exhibit a reduction in growth rate when they switched to one species of prey bacteria from another compared to controls that experience only the second prey. The effect was consistent across all six tested species of bacteria. These switching costs typically disappear within a day, indicating adjustment to new prey bacteria. This suggests that these costs are physiological. Second, amoebas usually grow more slowly on mixtures of prey bacteria compared to the expectation based on their growth on single prey. There were clear mixing costs in three of the six tested prey mixtures, and none showed significant mixing benefits. These results support the idea that, although amoebas can consume a variety of prey, they must use partially different methods and thus must pay costs to handle multiple prey, either sequentially or simultaneously.

DNA nanomachines reveal an adaptive energy mode in confinement-induced amoeboid migration powered by polarized mitochondrial distribution.

Energy metabolism is highly interdependent with adaptive cell migration in vivo. Mechanical confinement is a critical physical cue that induces switchable migration modes of the mesenchymal-to-amoeboid transition (MAT). However, the energy states in distinct migration modes, especially amoeboid-like stable bleb (A2) movement, remain unclear. In this report, we developed multivalent DNA framework-based nanomachines to explore strategical mitochondrial trafficking and differential ATP levels during cell migration in mechanically heterogeneous microenvironments. Through single-particle tracking and metabolomic analysis, we revealed that fast A2-moving cells driven by biomimetic confinement recruited back-end positioning of mitochondria for powering highly polarized cytoskeletal networks, preferentially adopting an energy-saving mode compared with a mesenchymal mode of cell migration. We present a versatile DNA nanotool for cellular energy exploration and highlight that adaptive energy strategies coordinately support switchable migration modes for facilitating efficient metastatic escape, offering a unique perspective for therapeutic interventions in cancer metastasis.

Global prevalence of potentially pathogenic free-living amoebae in sewage and sewage-related environments-systematic review with meta-analysis.

Free-living amoebae (FLA) include amphizoic microorganisms important in public health, widely isolated from air, water, and soil. However, its occurrence in sewage-related environments still needs to be systematically documented. This study summarizes the occurrence of FLA in sewage-related environments through a systematic review with meta-analysis. A total of 1983 scientific article were retrieved from different databases, of which 35 were selected and analyzed using a random effects forest plot model with a 95% confidence interval (IC). The pooled overall prevalence of FLA in sewage across 12 countries was 68.96% (95% IC = 58.5-79.42). Subgroup analysis indicates high prevalence in all environments analyzed, including sewage water from the sewage treatment plant (81.19%), treated sewage water (75.57%), sewage-contaminated water (67.70%), sediment contaminated by sewage (48.91%), and sewage water (47.84%). Prevalence values of Acanthamoeba spp., Hartmanella/Vermamoeba spp., and Naegleria spp. are 47.48%, 28.24%, and 16.69%, respectively. Analyzing the species level, the distribution is as follows: Acanthamoeba palestinensis (88%), A. castellanii (23.74%), A. astronyxis (19.18%), A. polyphaga (13.59%), A. culbertsoni (12.5%), A. stevensoni (8.33%), A. tubiashi (4.35%) and A. hatchetti (1.1%), Naegleria fowleri (28.4%), N. gruberi (25%), N. clarki (8.33%), N. australiensis (4.89%) and N. italica (4.29%), Hartmannella/Vermamoeba exundans (40%) and H.V. vermiform (32.61%). Overall, our findings indicate a high risk associated with sewage-related environments, as the prevalence of FLA, including pathogenic strains, is high, even in treated sewage water. The findings of this study may be valuable both for risk remediation actions against amoebic infections and for future research endeavors.

Metagenomics insights into microbiome and antibiotic resistance genes from free living amoeba in chlorinated wastewater effluents.

Free living amoeba (FLA) are among the organisms commonly found in wastewater and are well-established hosts for diverse microbial communities. Despite its clinical significance, there is little knowledge on the FLA microbiome and resistome, with previous studies relying mostly on conventional approaches. In this study we comprehensively analyzed the microbiome, antibiotic resistome and virulence factors (VFs) within FLA isolated from final treated effluents of two wastewater treatment plants (WWTPs) using shotgun metagenomics. Acanthamoeba has been identified as the most common FLA, followed by Entamoeba. The bacterial diversity showed no significant difference (p > 0.05) in FLA microbiomes obtained from the two WWTPs. At phylum level, the most dominant taxa were Proteobacteria, followed by Firmicutes and Actinobacteria. The most abundant genera identified were Enterobacter followed by Citrobacter, Paenibacillus, and Cupriavidus. The latter three genera are reported here for the first time in Acanthamoeba. In total, we identified 43 types of ARG conferring resistance to cephalosporins, phenicol, streptomycin, trimethoprim, quinolones, cephalosporins, tigecycline, rifamycin, and kanamycin. Similarly, a variety of VFs in FLA metagenomes were detected which included flagellar proteins, Type IV pili twitching motility proteins (pilH and rpoN), alginate biosynthesis genes AlgI, AlgG, AlgD and AlgW and Type VI secretion system proteins and general secretion pathway proteins (tssM, tssA, tssL, tssK, tssJ, fha, tssG, tssF, tssC and tssB, gspC, gspE, gspD, gspF, gspG, gspH, gspI, gspJ, gspK, and gspM). To the best of our knowledge, this is the first study of its kind to examine both the microbiomes and resistome in FLA, as well as their potential pathogenicity in treated effluents. Additionally, this study showed that FLA can host a variety of potentially pathogenic bacteria including Paenibacillus, and Cupriavidus that had not previously been reported, indicating that their relationship may play a role in the spread and persistence of antibiotic resistant bacteria (ARBs) and antibiotic resistance genes (ARGs) as well as the evolution of novel pathogens.

How good are global DNA-based environmental surveys for detecting all protist diversity? Arcellinida as an example of biased representation.

Metabarcoding approaches targeting microeukaryotes have deeply changed our vision of protist environmental diversity. The public repository EukBank consists of 18S v4 metabarcodes from 12,672 samples worldwide. To estimate how far this database provides a reasonable overview of all eukaryotic diversity, we used Arcellinida (lobose testate amoebae) as a case study. We hypothesised that (1) this approach would allow the discovery of unexpected diversity, but also that (2) some groups would be underrepresented because of primer/sequencing biases. Most of the Arcellinida sequences appeared in freshwater and soil, but their abundance and diversity appeared underrepresented. Moreover, 84% of ASVs belonged to the suborder Phryganellina, a supposedly species-poor clade, whereas the best-documented suborder (Glutinoconcha, 600 described species) was only marginally represented. We explored some possible causes of these biases. Mismatches in the primer-binding site seem to play a minor role. Excessive length of the target region could explain some of these biases, but not all. There must be some other unknown factors involved. Altogether, while metabarcoding based on ribosomal genes remains a good first approach to document microbial eukaryotic clades, alternative approaches based on other genes or sequencing techniques must be considered for an unbiased picture of the diversity of some groups.

Development of nucleic acid lateral flow immunoassay for molecular detection of Entamoeba moshkovskii and Entamoeba dispar in stool samples.

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

Isolation and habitat analysis of Balamuthia mandrillaris from soil.

Balamuthia mandrillaris is a free-living amoeba that causes meningoencephalitis in mammals. Over 200 cases of infection were reported worldwide, with a fatality rate of over 95%. A clear route of infection was unknown for a long time until a girl died of granulomatous amoebic encephalitis (GAE) in California, USA, in 2003 due to infection with B. mandrillaris detected in a potted plant. Since then, epidemiological studies were conducted worldwide to detect B. mandrillaris in soil and other environmental samples. We previously reported the isolation of B. mandrillaris from the soil in Japan; however, the existing B. mandrillaris culture method with BM3 medium and COS-7 cells was unsuccessful. Therefore, in this study, we aimed to conduct soil analysis to determine the growth conditions of B. mandrillaris. B. mandrillaris-positive soils were defined as soils from which B. mandrillaris was isolated and environmental DNA was PCR-positive. Soils inhabited by B. mandrillaris were alkaline, with high electrical conductivity and characteristics of nutrient-rich soils of loam and clay loam. The results of this study suggest a possible reason for the high prevalence of GAE caused by B. mandrillaris among individuals employed in agriculture-related occupations.

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.

Biomonitoring tool for New Zealand peatlands: Testate amoebae and vascular plants as promising bioindicators.

In the last two centuries, a high proportion of peatlands have been lost or severely degraded across the world. The value of peatlands is now well-recognised for biodiversity conservation, flood management, and carbon mitigation, with peatland restoration now central to many government policies for climate action. A challenge, however, is to determine 'natural' and 'disturbed' conditions of peatlands to establish realistic baselines for assessing degradation and setting restoration targets. This requires a tool or set of tools that can rapidly and reliably capture peatland condition across space and time. Our aim was to develop such a tool based on combined analysis of plant and testate amoebae; a group of shelled protists commonly used as indicators of ecological change in peatlands. The value of testate amoebae is well established in Northern Hemisphere Sphagnum-dominated peatlands; however, relatively little work has been undertaken for Southern Hemisphere peat forming systems. Here we provide the first assessment and comparison of the bioindicator value of testate amoebae and vascular plants in the context of Southern Hemisphere peatlands. Our results further demonstrate the unique ecohydrological dynamics at play in New Zealand peat forming systems that set them apart from Northern Hemisphere peatlands. Our results show that plant and testate amoeba communities provided valuable information on peatland condition at different scales, we found that testate amoebae tracked changes in the abiotic variables (depth to water table, pH, and conductivity) more closely than vascular plants. Our results further demonstrate that functional traits of testate amoebae showed promising relationships with disturbance. Amoeba test compression, aperture position and test size were linked to changes in hydrology driven by fluctuations in ground water tables; however, trait responses manifested differently in ombrotrophic and minerotrophic peatlands. Overall, testate amoebae provide a promising bioindicator for tracking degradation in New Zealand peatlands and a potential additional tool to assess peatland condition.

Molecular identification, phylogenetic analysis and histopathological study of pathogenic free-living amoebae isolated from discus fish (Symphysodon aequifasciatus) in Iran: 2020-2022.

Free-living amoebae (FLA) are capable of inhabiting diverse reservoirs independently, without relying on a host organism, hence their designation as "free-living". The majority of amoebae that infect freshwater or marine fish are amphizoic, or free-living forms that may colonize fish under particular circumstances. Symphysodon aequifasciatus, commonly referred to as the discus, is widely recognized as a popular ornamental fish species. The primary objective of the present study was to determine the presence of pathogenic free-living amoebae (FLA) in samples of discus fish. Fish exhibiting clinical signs, sourced from various fish farms, were transferred to the ornamental fish clinic. The skin, gills, and intestinal mucosa of the fish were collected and subjected to culturing on plates containing a 1% non-nutrient agar medium. The detection of FLA was conducted through morphological, histopathological and molecular methods. The construction of the phylogenetic tree for Acanthamoeba genotypes was achieved using the maximum likelihood approach. The molecular sequence analysis revealed that all cultures that tested positive for FLA were T4 genotype of Acanthamoeba and Acanthamoeba sp. The examination of gill samples using histopathological methods demonstrated the presence of lamellar epithelial hyperplasia, significant fusion of secondary lamellae, and infiltration of inflammatory cells. A multitude of cysts, varying in shape from circular to elliptical, were observed within the gills. The occurrence of interlamellar vesicles and amoeboid organisms could be observed within the epithelial tissue of the gills. In the current study, presence of the Acanthamoeba T4 genotype on the skin and gills of discus fish exhibiting signs of illness in freshwater ornamental fish farms was identified. This observation suggests the potential of a transmission of amoebic infection from ornamental fish to humans, thereby highlighting the need for further investigation into this infection among ornamental fish maintained as pets, as well as individuals who interact with them and their environment.