Polymorphism-driven transcriptomic changes in anthelmintic metabolism pathways of Anisakis simplex s.s. L3 larvae.

BACKGROUND: Helminth infections continue to pose major challenges in human and veterinary medicine, with additional complications arising from the emergence of anthelmintic resistance. Anisakis simplex sensu stricto (A. simplex s.s.), a zoonotic nematode transmitted through the consumption of fish, is of growing concern due to its allergenic potential and clinical relevance. However, the molecular mechanisms underlying the parasite's response to anthelmintic treatment remain poorly defined. METHODS: Third-stage larvae (L3) of A. simplex s.s. were exposed to three widely used anthelmintics: albendazole (ALB), ivermectin (IVC) and pyrantel (PYR). High-throughput RNA sequencing was combined with differential gene expression, multivariate alternative splicing analysis (Replicate Multivariate Analysis of Transcript Splicing [rMATS] v3.2.5 computational tool) and single nucleotide variant (SNV) profiling with Oxford Nanopore sequencing. Drug-specific effects were assessed across protein-coding genes, long non-coding RNAs (lncRNAs) and splicing events. RESULTS: Distinct transcriptomic features, including splicing and sequence variants, were observed across treatments, with ALB primarily altering the expression of cuticle-associated genes, IVC inducing extensive alternative splicing in immune-related pathways and PYR exposure linked to widespread SNVs in neuronal projection and metabolic genes. Significant splicing events included exon skipping in the trehalase gene (ALB) and combined skipped exon/alternative 5' splice site events in moesin/ezrin/radixin-like protein 1 (IVC). A stop/splice-region SNV in trehalose phosphatase was detected with PYR exposure, highlighting coordinated disruption of the trehalose metabolism pathway. Across treatments, 68, 83 and 95 protein-coding genes with allelic variation were identified for ALB, PYR and IVC, respectively, including genes involved in detoxification, oxidative stress, cytoskeletal remodeling and transcriptional regulation. CONCLUSIONS: Our findings reveal complex, drug-specific regulatory mechanisms in A. simplex, encompassing transcriptional remodeling, alternative splicing and functional SNVs. Novel modulation of trehalose metabolism and cytoskeletal genes, alongside potential roles for ABC transporters and RNA-binding proteins, suggests diverse adaptive strategies underlying anthelmintic tolerance. This study represents the first integrated transcriptomic and variant-level analysis of Anisakis under drug pressure and provides new insights into molecular resistance mechanisms in marine nematodes, with implications for therapeutic innovation and monitoring strategies.