Ontogenesis of coelomocyte subsets and immune-related genes during Eisenia andrei embryogenesis

Earthworm coelomocytes (e.g. amoebocytes and eleocytes) participate in diverse physiological processes from nutrition storage to immune defense. While the morphology and functional attributes of adult coelomocytes have been extensively studied, their origin and ontogeny are largely unexplored during earthworm development. To uncover this knowledge gap, we examined the emergence, distribution, and molecular characteristics of amoebocytes vs. eleocytes during embryogenesis (E1-E4 stages) of Eisenia andrei earthworms. Applying light-microscopy and flow cytometry, we observed a sequential manifestation of coelomocyte subsets: amoebocytes predominated in E3 stage, whereas eleocytes became more abundant in E4; although amoebocytes remain prevalent overall. In parallel, the expression dynamics of key earthworm immune-related genes evidenced characteristic differences. The transcripts of evolutionarily conserved TLR and MyD88 displayed constitutive expression throughout all embryonic stages (E1-E4). Conversely, LBP/BPI gene expression decreased, while CCF mRNA level increased during Eisenia embryogenesis. Moreover, antimicrobial effectors (lysenin and lysozyme) were progressively increased from E1 to E4 stages. Our novel experimental data shed more light at molecular and cellular levels into the development and maturation of immune cell populations during earthworm embryogenesis.