Amoebozoan testate amoebae illuminate the diversity of heterotrophs and the complexity of ecosystems throughout geological time.

Heterotrophic protists are vital in Earth's ecosystems, influencing carbon and nutrient cycles and occupying key positions in food webs as microbial predators. Fossils and molecular data suggest the emergence of predatory microeukaryotes and the transition to a eukaryote-rich marine environment by 800 million years ago (Ma). Neoproterozoic vase-shaped microfossils (VSMs) linked to Arcellinida testate amoebae represent the oldest evidence of heterotrophic microeukaryotes. This study explores the phylogenetic relationship and divergence times of modern Arcellinida and related taxa using a relaxed molecular clock approach. We estimate the origin of nodes leading to extant members of the Arcellinida Order to have happened during the latest Mesoproterozoic and Neoproterozoic (1054 to 661 Ma), while the divergence of extant infraorders postdates the Silurian. Our results demonstrate that at least one major heterotrophic eukaryote lineage originated during the Neoproterozoic. A putative radiation of eukaryotic groups (e.g., Arcellinida) during the early-Neoproterozoic sustained by favorable ecological and environmental conditions may have contributed to eukaryotic life endurance during the Cryogenian severe ice ages. Moreover, we infer that Arcellinida most likely already inhabited terrestrial habitats during the Neoproterozoic, coexisting with terrestrial Fungi and green algae, before land plant radiation. The most recent extant Arcellinida groups diverged during the Silurian Period, alongside other taxa within Fungi and flowering plants. These findings shed light on heterotrophic microeukaryotes' evolutionary history and ecological significance in Earth's ecosystems, using testate amoebae as a proxy.

Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity.

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.

Conservation and Variability of Meiosis Across the Eukaryotes.

Comparisons among a variety of eukaryotes have revealed considerable variability in the structures and processes involved in their meiosis. Nevertheless, conventional forms of meiosis occur in all major groups of eukaryotes, including early-branching protists. This finding confirms that meiosis originated in the common ancestor of all eukaryotes and suggests that primordial meiosis may have had many characteristics in common with conventional extant meiosis. However, it is possible that the synaptonemal complex and the delicate crossover control related to its presence were later acquisitions. Later still, modifications to meiotic processes occurred within different groups of eukaryotes. Better knowledge on the spectrum of derived and uncommon forms of meiosis will improve our understanding of many still mysterious aspects of the meiotic process and help to explain the evolutionary basis of functional adaptations to the meiotic program.

Thin film structural color is widespread in slime molds (Myxomycetes, Amoebozoa).

Brilliant colors in nature arise from the interference of light with periodic nanostructures resulting in structural color. While such biological photonic structures have long attracted interest in insects and plants, they are little known in other groups of organisms. Unexpected in the kingdom of Amoebozoa, which assembles unicellular organisms, structural colors were observed in myxomycetes, an evolutionary group of amoebae forming macroscopic, fungal-like structures. Previous work related the sparkling appearance of Diachea leucopodia to thin film interference. Using optical and ultrastructural characterization, we here investigated the occurrence of structural color across 22 species representing two major evolutionary clades of myxomycetes including 14 genera. All investigated species showed thin film interference at the peridium, producing colors with hues distributed throughout the visible range that were altered by pigmentary absorption. A white reflective layer of densely packed calcium-rich shells is observed in a compound peridium in Metatrichia vesparium, whose formation and function are still unknown. These results raise interesting questions on the biological relevance of thin film structural colors in myxomycetes, suggesting they may be a by-product of their reproductive cycle.

The record of Vannella species (Vannellidae, Discosea, Amoebozoa) from freshwater sources in Dakahlyia Governorate, Egypt.

The River Nile is the main source of fresh water in Egypt, where its water is used for irrigation, drinking, fisheries, industrial uses, and recreation. For sustainable utilization of the River Nile and its branches in the Nile Delta region, it is necessary to monitor regular investigation for the biodiversity of protozoan fauna in the Damietta branch and other freshwater canals in Dakahlyia Governorate. Water samples were collected monthly from different water sources, for 1 year, and examined for protozoans, using phase-contrast microscopy and recorded video films, The genus Vannella Bovee 1965 is recorded for the first time in four freshwater localities: Demietta branch of the River Nile, Mansouria Canal, Bouhia Canal, and Bahr El-Saghir Canal. A detailed morphological description with a brief report of their locomotion has been given for four morphologically different Vannella species. The locomotive form of Vannella sp.1 has a long pointed posterior tail and 2 lateral posterior processes. Such a tail was absent in other Vannella species. Vannella sp.2 is unique among other recorded species, where its locomotive form possesses a long posterior rounded tail region and a frontal hyaloplasm provided with a wavy surface that forms several lobes and finger-like processes during locomotion. In addition, the hyaloplasm produces several transverse waves that vary in thickness and density. The floating form of Vannella sp.2 is of a radial type and has comparatively long hyaline pointed and spiral pseudopodia. The process of transformation of locomotive form to floating form in Vannella sp.2 has been followed up using several recorded video films. The locomotive form of Vannella sp.3 is bear-shaped, while that of Vannella sp.4 has variable shapes from semicircular to rectangular and sometimes fan-shaped. During movement in vivo, locomotive cells of all Vannella species, except Vannella sp.1, move in nearly a straight line, but there were variations in their rate of locomotion. Vannella sp.4 recorded the highest rate (6.8 µm/s), followed by Vannella sp.2 (4.5 µm/s), Vannella sp.3 (2.4 µm/s), and finally Vannella sp.1 (1.0 µm/s). Molecular studies and transmission electron microscope examinations are still needed to confirm the precise identity of each Vannella species.

Living in the cracks: Two novel genera of Variosea (Amoebozoa) discovered on an urban sidewalk.

Biological soil crusts represent a rich habitat for diverse and complex eukaryotic microbial communities. A unique but extremely common habitat is the urban sidewalk and its cracks that collect detritus. While these habitats are ubiquitous across the globe, little to no work has been conducted to characterize protists found there. Amoeboid protists are major predators of bacteria and other microbial eukaryotes in these microhabitats and therefore play a substantial ecological role. From sidewalk crack soil crusts, we have isolated three naked amoebae with finely tapered subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a holistic approach including light, electron, and fluorescence microscopy as well as phylogenetics using the ribosomal small subunit rRNA gene and phylogenomics using 230 nuclear genes, we find that these amoeboid organisms fail to match any previously described eukaryote genus. However, we determined the amoebae belong to the amoebozoan lineage Variosea based on phylogenetics. The molecular analyses place our isolates in two novel genera forming a grade at the base of the variosean group Protosteliida. These three novel varioseans among two novel genera and species are herein named "Kanabo kenzan" and "Parakanabo toge."

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.

Phylogenetic placement of the protosteloid amoeba Microglomus paxillus identifies another case of sporocarpic fruiting in Discosea (Amoebozoa).

Protosteloid amoebae are a paraphyletic assemblage of amoeboid protists found exclusively in the eukaryotic assemblage Amoebozoa. These amoebae can facultatively form a dispersal structure known as a fruiting body, or more specifically, a sporocarp, from a single amoeboid cell. Sporocarps consist of one to a few spores atop a noncellular stalk. Protosteloid amoebae are known in two out of three well-established major assemblages of Amoebozoa. Amoebae with a protosteloid life cycle are known in the major Amoebozoa lineages Discosea and Evosea but not in Tubulinea. To date, only one genus, which is monotypic, lacks sequence data and, therefore, remains phylogenetically homeless. To further clarify the evolutionary milieu of sporocarpic fruiting we used single-cell transcriptomics to obtain data from individual sporocarps of isolates of the protosteloid amoeba Microglomus paxillus. Our phylogenomic analyses using 229 protein coding markers suggest that M. paxillus is a member of the Discosea lineage of Amoebozoa most closely related to Mycamoeba gemmipara. Due to the hypervariable nature of the SSU rRNA sequence we were unable to further resolve the phylogenetic position of M. paxillus in taxon rich datasets using only this marker. Regardless, our results widen the known distribution of sporocarpy in Discosea and stimulate the debate between a single or multiple origins of sporocarpic fruiting in Amoebozoa.

Genetic structure of the protist Physarum albescens (Amoebozoa) revealed by multiple markers and genotyping by sequencing.

Myxomycetes are terrestrial protists with many presumably cosmopolitan species dispersing via airborne spores. A truly cosmopolitan species would suffer from outbreeding depression hampering local adaptation, while locally adapted species with limited distribution would be at a higher risk of extinction in changing environments. Here, we investigate intraspecific genetic diversity and phylogeography of Physarum albescens over the entire Northern Hemisphere. We sequenced 324 field collections of fruit bodies for 1-3 genetic markers (SSU, EF1A, COI) and analysed 98 specimens with genotyping by sequencing. The structure of the three-gene phylogeny, SNP-based phylogeny, phylogenetic networks, and the observed recombination pattern of three independently inherited gene markers can be best explained by the presence of at least 18 reproductively isolated groups, which can be seen as cryptic species. In all intensively sampled regions and in many localities, members of several phylogroups coexisted. Some phylogroups were found to be abundant in only one region and completely absent in other well-studied regions, and thus may represent regional endemics. Our results demonstrate that the widely distributed myxomycete species Ph. albescens represents a complex of at least 18 cryptic species, and some of these seem to have a limited geographical distribution. In addition, the presence of groups of presumably clonal specimens suggests that sexual and asexual reproduction coexist in natural populations of myxomycetes.

Deconstructing Difflugia: The tangled evolution of lobose testate amoebae shells (Amoebozoa: Arcellinida) illustrates the importance of convergent evolution in protist phylogeny.

Protists, the micro-eukaryotes that are neither plants, animals nor fungi build up the greatest part of eukaryotic diversity on Earth. Yet, their evolutionary histories and patterns are still mostly ignored, and their complexity overlooked. Protists are often assumed to keep stable morphologies for long periods of time (morphological stasis). In this work, we test this paradigm taking Arcellinida testate amoebae as a model. We build a taxon-rich phylogeny based on two mitochondrial (COI and NADH) and one nuclear (SSU) gene, and reconstruct morphological evolution among clades. In addition, we prove the existence of mitochondrial mRNA editing for the COI gene. The trees show a lack of conservatism of shell outlines within the main clades, as well as a widespread occurrence of morphological convergences between far-related taxa. Our results refute, therefore, a widespread morphological stasis, which may be an artefact resulting from low taxon coverage. As a corollary, we also revise the groups systematics, notably by emending the large and highly polyphyletic genus Difflugia. These results lead, amongst others, to the erection of a new infraorder Cylindrothecina, as well as two new genera Cylindrifflugia and Golemanskia.

Phylogeny and evolution of morphological structures in a highly diverse lineage of fruiting-body-forming amoebae, order Trichiales (Myxomycetes, Amoebozoa).

Early phylogenetic studies refuted most previous assumptions concerning the evolution of the morphological traits in the fruiting bodies of the order Trichiales and did not detect discernible evolutionary patterns, yet they were based on a limited number of species. We infer a new Trichiales phylogeny based on three independently inherited genetic regions (nuclear and mitochondrial), with a fair taxonomic sampling encompassing its broad diversity. Besides, we study the evolutionary history of some key morphological characters. According to the new phylogeny, most fruiting body traits in Trichiales systematics do not represent exclusive synapomorphies or autapomorphies for most monophyletic groups. Instead, the evolution of the features derived from the peridium, stalk, capillitium, and spores showed intricate patterns, and character state transitions occurred rather within- than between clades. Thus, we should consider other evolutionary scenarios instead of assuming the homology of some characters. According to these results, we propose a new classification of Trichiales, including the creation of a new genus, Gulielmina, the resurrection of the family Dictydiaethaliaceae and the genus Ophiotheca, and the proposal of 13 new combinations for species of the genera Arcyria (1), Hemitrichia (2), Ophiotheca (2), Oligonema (4), Gulielmina (3), and Perichaena (1).

IPANEMAP: integrative probing analysis of nucleic acids empowered by multiple accessibility profiles.

The manual production of reliable RNA structure models from chemical probing experiments benefits from the integration of information derived from multiple protocols and reagents. However, the interpretation of multiple probing profiles remains a complex task, hindering the quality and reproducibility of modeling efforts. We introduce IPANEMAP, the first automated method for the modeling of RNA structure from multiple probing reactivity profiles. Input profiles can result from experiments based on diverse protocols, reagents, or collection of variants, and are jointly analyzed to predict the dominant conformations of an RNA. IPANEMAP combines sampling, clustering and multi-optimization, to produce secondary structure models that are both stable and well-supported by experimental evidences. The analysis of multiple reactivity profiles, both publicly available and produced in our study, demonstrates the good performances of IPANEMAP, even in a mono probing setting. It confirms the potential of integrating multiple sources of probing data, informing the design of informative probing assays.

Vannella mustalahtiana sp. nov. (Amoebozoa, Vannellida) and rainbow trout nodular gill disease (NGD) in Russia.

An outbreak of nodular gill disease (NGD) in farmed rainbow trout Oncorhynchus mykiss (Walbaum, 1792) was recorded in Ladoga Lake (Karelia, north-western Russia) in Spring 2020. The disease was characterised by typical clinical signs including asphyxiation, distended opercula, loss of activity and swimming upside down under the water surface. Maximum monthly mortality was 15.2%. The histological examination of the gills showed deformation and clubbing of lamellae, epithelial hypertrophy and hyperplasia, lamellar fusion and fusion of filaments. Granulomas were located within the epithelial layer and/or rose above its surface. Light microscopic in vivo observations of the mucus smears from the affected gills revealed numerous amoeboid protists demonstrating a flattened body when adhering to the substratum, and blunt, radiating pseudopodia when afloat. Based on these morphological characters, these amoebae could be assigned to the Discosea (Amoebozoa), and analyses of their small subunit rRNA gene sequences showed that they belonged to the genus Vannella Bovee, 1965. The results reported herein support the designation of a new species, V. mustalahtiana sp. nov. Despite having been isolated from the gills of a freshwater fish, the species belongs to a clade of Vannella comprising mostly species isolated from marine and brackish water habitats. These findings may be essential for the aetiology and treatment of the disease.

Balamuthia spinosa n. sp. (Amoebozoa, Discosea) from the brackish-water sediments of Nivå Bay (Baltic Sea, The Sound) - a novel potential vector of Legionella pneumophila in the environment.

We have found a new free-living amoeba species named Balamuthia spinosa n. sp. (Amoebozoa, Discosea) in the bottom sediments of the brackish-water Nivå Bay (Baltic Sea, The Sound). This species resembles members of the genus Stygamoeba morphologically and was (mis)identified as belonging to this genus during the initial investigation. However, SSU rRNA gene data show that it robustly groups with Balamuthia mandrillaris sequence among Acanthopodida and represents a new species of the genus Balamuthia. Fragments of Legionella pneumophila genome were found among the NGS contigs obtained from B. spinosa n. sp., suggesting that this species may be a vector of Legionella in the environment. We discuss a remarkable morphological and ultrastructural similarity between the genus Balamuthia and the genus Stygamoeba. In addition, our phylogenetic analysis based on the SSU rRNA gene sequences revealed a close relationship between the genera Stygamoeba and Vermistella. It is one more confirmation of the order Stygamoebida, which was formed basing on the morphological evidence. The position of these branches close to Thecamoebida clade is congruent with current phylogenomic data.

Evolutionary Analysis of Dipeptidyl Peptidase I.

Human dipeptidyl peptidase I (DPPI) belongs to the family of papain-like cysteine peptidases. Its distinctive features are the unique exclusion domain which enables the eponymous activity and homotetramerization of DPPI, and its dependence on chloride ions for enzymatic activity. The oligomeric state of DPPI is unique in this family of predominantly monomeric peptidases. However, a distant DPPI ortholog from Plasmodium falciparum has been shown to be monomeric, indicating that the oligomeric state of DPPI varies between lineages. The aim of this work was to study the evolution of DPPI, with particular attention to the structural features that determine its characteristic enzymatic activity and preferences, and to reconstruct the evolution of its oligomerization. We analyzed fifty-seven selected sequences of DPPI and confirmed its presence in three lineages, namely, Amorphea (including animals and Amoebozoa), Alveolates and the metamonad Giardia. The amino acid residues that bind the chloride ion are highly conserved in all species, indicating that the dependence on chloride ions for activity is an evolutionarily conserved feature of DPPI. The number of N-glycosylation sites is significantly increased in animals, particularly vertebrates. Analysis of homology models and subunit contacts suggests that oligomerization is likely restricted to DPPIs in the Amorphea group.

A morphological and molecular reinvestigation of Janickina pigmentifera (Grassi, 1881) Chatton 1953 - an amoebozoan parasite of arrow-worms (Chaetognatha).

Amoebozoan parasites of arrow-worms (Chaetognatha) were isolated from their hosts living in plankton of the Bay of Villefranche (Mediterranean Sea). Based on the light microscopic characters, the amoebae were identified as Janickina pigmentifera (Grassi, 1881) by their limax locomotive form and due to the presence of the intracellular symbiont, Perkinsela amoebae, surrounded by a layer of pigment granules. Sequences of the 18S rRNA gene of both J. pigmentifera and its symbiont were obtained for the first time. The molecular phylogenetic analyses of 18S rRNA gene placed J. pigmentifera within the genus Neoparamoeba, a taxon also characterized by the presence of a symbiont, known as Perkinsela amoebae-like organism (PLO). The 18S rRNA gene sequence of P. amoebae from J. pigmentifera grouped with the sequences of 18S rRNA genes of PLOs from Neoparamoeba branchiphila and Neoparamoeba invadens. The first photo documentation of the light microscopic features of J. pigmentifera, such as locomotive form, the morphology of the nucleus and P. amoebae have been provided. The new results support the affinity of J. pigmentifera with the family Paramoebidae suggested previously based on the presence of PLO. In contrast to Janickina, typical members of Paramoebidae (Neoparamoeba and Paramoeba) have a flattened, dactylopodial locomotive form. This discrepancy in morphology can be explained by the obligate parasitic lifestyle of Janickina.

Superficially described and ignored for 92 years, rediscovered and emended: Apodera angatakere (Amoebozoa: Arcellinida: Hyalospheniformes) is a new flagship testate amoeba taxon from Aotearoa (New Zealand).

Eukaryotic microbial diversity is known to be extensive but remains largely undescribed and uncharted. While much of this unknown diversity is composed of inconspicuous flagellates and parasites, larger and morphologically distinct protists are regularly discovered, most notably from poorly studied regions. Here we report a new flagship species of hyalospheniid (Amoebozoa; Arcellinida; Hyalospheniformes) testate amoeba from New Zealand and an unusual story of overlooked description under a preoccupied name and subsequent oversight for nearly one century. Through a process involving The Maori Language Commission, we named the species Apodera angatakere, meaning "a shell with a keel." This species resembles Apodera vas but differs by the presence of a distinctive hollow keel. Cytochrome Oxidase Subunit 1 (COI) sequence data show that this species forms a distinct clade nested within genus Apodera. This conspicuous species is so far known only from New Zealand and is restricted to peatlands. It is one of the few examples of endemic microorganisms from this biodiversity hotspot and biogeographer's paradise. As over 90% of New Zealand's peatlands have been lost since European colonization and much of the remaining surfaces are threatened, Apodera angatakere could be a flagship species not only for microbial biogeography but also for island biodiversity conservation.

The amoebae of Idionectes vortex (Cutosea, Amoebozoa): Motility, cytoskeleton architecture and extracellular scales.

The Cutosea represent a deep-branching lineage within the phylum Amoebozoa that is still relatively poorly explored. Currently, there are four cutosean representatives known - the monotypic genera Armaparvus, Idionectes, Sapocribrum, and Squamamoeba - with marked genetic distances. Idionectes vortex is the deepest-branching species and differs markedly from the other Cutosea in ecology, life history, and most importantly, in its ability to form a flagellated swarmer with an exceptional swimming mechanism. As far as we know, the other Cutosea lack flagella and rather represent small, marine amoebae with a characteristic cell coat. The present paper focuses on the amoeboid life history stage of the algivorous amoeboflagellate Idionectes vortex to provide data for a first in-depth comparison with other Cutosea and to document structural specialties. The amoeboid stage of Idionectes is mainly associated with the specific feeding process, that is, the interaction with algal prey cells and phagocytosis of protoplast material. Yet, the present data from time-lapse microscopy, cytochemical stainings, and electron microscopy demonstrate clear similarities with the other cutosean species concerning amoeboid locomotion and cell coat ultrastructure. Furthermore, Idionectes amoebae exhibit a well-developed microtubular cytoskeleton, and an unusual basal apparatus that seems to undergo marked changes during the life history of this exceptional amoebozoan.

Immersion challenge of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. did not increase the severity of Neoparamoeba perurans-induced amoebic gill disease (AGD).

Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans-induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post-exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development.

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.