**Background:** Hypertidal mudflats (tidal ranges >6 m) in the Bay of Fundy are ecologically critical habitats that support high primary productivity, carbon sequestration, and migratory shorebird populations. Protists (microbial eukaryotes) are key players in biogeochemical cycles and food webs, yet their diversity and dynamics in these extreme intertidal environments remain poorly understood. This study aimed to quantify spatial and seasonal variation in protist community composition and trophic functional structure in Bay of Fundy mudflats.
**Methods:** Sediment cores were collected from two mudflats—Grande Anse and Pecks Cove—five times from June to October 2019 (late spring to early fall). Four sediment depths were analyzed: surface (0–5 mm), 1 cm, 4 cm, and 7 cm. Environmental DNA was extracted and the V4 region of the 18S rRNA gene was sequenced on the Illumina MiSeq platform. After quality filtering and denoising, 562,941 quality-filtered reads and 2118 protist amplicon sequence variants (ASVs) were retained. Taxonomic assignment used the PR² database, and ASVs were assigned to trophic functional groups. Statistical analyses included PERMANOVA, PERMDISP, SIMPER, RELATE, and BEST routines.
**Key Results:** Cercozoa (37% of reads), Bacillariophyta (18%), and Dinophyceae (15%) formed the core eukaryotic microbiome, collectively dominating across all spatial and temporal scales. Cercozoa was the richest and most abundant taxon, with the class Thecofilosea (60% of cercozoan reads) and the Protaspa-lineage (7% of total protist reads, 100% prevalence) being particularly dominant. Among Bacillariophyta, polar centric Mediophyceae comprised 58% of diatom reads, with Thalassiosira spp. (7% of total protist reads, 100% prevalence) as the dominant genus. Dinophyceae was dominated by Gonyaulacales (43% of dinoflagellate reads), primarily Alexandrium fundyense (7% of total protist reads, 90% prevalence). Community composition varied significantly among sites (27.4% of total variation), seasons (7.8%), and depths (9.8%), with significant two-way and three-way interactions collectively accounting for 32.6% of variation. At Grande Anse, seasonal variability peaked at the sediment surface, and depth-related differences were largest in late spring (surface vs. 1 cm: 45% dissimilarity) compared to other seasons (17–26% dissimilarity). At Pecks Cove, communities displayed a 'depth decay' pattern. Trophic functional analysis showed consumers dominated (45% of reads), followed by phototrophs (30%) and parasites (10%). Eukaryvores (43% of consumer reads) were dominated by Cercozoa (75%), while phototrophs were primarily Bacillariophyta (60%), Dinophyceae (25%), and Chlorophyta (14%). Parasites were equally split between invertebrate parasites (Apicomplexa, primarily gregarines) and protist parasites (Syndiniales, Group I Clade 4). Sediment physico-chemical properties (pH, oxygen, temperature) decreased with depth and showed seasonality, but correlations with community composition were weak.
**Clinical Implications:** This study does not have direct clinical implications. However, the detection of toxigenic Alexandrium fundyense (a saxitoxin producer) in sediments, confirmed by PCR detection of the sxtA4 gene, has implications for human and ecosystem health through potential harmful algal blooms. The findings establish a baseline for understanding microbial food web dynamics in hypertidal systems, which may inform coastal ecosystem management and conservation efforts, particularly regarding migratory shorebird habitat quality and harmful algal bloom monitoring.