Discovery and characterization of the α-amylases cDNAs from Enchytraeus albidus shed light on the evolution of "Enchytraeus-Eisenia type" Amy homologs in Annelida.

Although enchytraeids have gained popularity in scientific research, fundamental questions regarding their feeding ecology and biology remain largely unexplored. This study investigates α-amylases, major digestive enzymes responsible for hydrolyzing starch and similar polysaccharides into sugars, in Enchytraeus albidus. Genetic data related to α-amylases is currently lacking for the family Enchytraeidae but also for the entire Annelida. To detect and identify coding sequences of the expressed α-amylase genes in COI-monohaplotype culture (PL-A strain) of E. albidus, we used classical "gene fishing" and transcriptomic approaches. We also compared coding sequence variants of α-amylase retrieved from transcriptomic data related to freeze-tolerant strains. Our results reveal that E. albidus possesses two distinct α-amylase genes (Amy I and Amy II) that are homologs to earthworm Eisenia fetida Ef-Amy genes. Different strains of E. albidus possess distinctive alleles of α-amylases with unique SNP patterns specific to a particular strain. Unlike Amy II, Amy I seems to be a highly polymorphic and multicopy gene. The domain architecture of the putative Amy proteins was found the same as for classical animal α-amylases with ABC-domains. A characteristic feature of Amy II is the lack of GHGA motif in the flexible loop region, similarly to many insect amylases. We identified "Enchytraeus-Eisenia type" α-amylase homologs in other clitellates and polychaetes, indicating the ancestral origin of Amy I/II proteins in Annelida. This study provides the first insight into the endogenous non-proteolytic digestive enzyme genes in potworms, discusses the evolution of Amy α-amylases in Annelida, and explores phylogenetic implications.