Nycthemeral modulation of hemocyte dynamics across developmental stages in the terrestrial snail Cornu aspersum

In invertebrates, hemocytes are the principal effector cells of innate immunity, and a growing body of evidence indicates that the circulation of immune cells is under circadian and nycthemeral control across both vertebrate and invertebrate taxa. Yet whether such temporal organisation extends to terrestrial gastropods, increasingly used as comparative models in physiology and ecotoxicology through hemocyte-based biomarkers, remains poorly documented. This study investigated nycthemeral variations in hemocyte-related parameters in juvenile, sub-adult and adult Cornu aspersum maintained under controlled laboratory conditions. Snails were sampled at four time points over a 24 h cycle (06:00, 12:00, 18:00 and 24:00; n = 20 per developmental stage and sampling time). Circulating hemocyte concentration, hemocyte viability, proportions of hemocyte types I and II, and micronucleus frequency were assessed following standardised procedures. Significant temporal variations were observed in circulating hemocyte concentration, with lower values generally recorded during evening and nocturnal sampling periods (18:00 and 24:00). Similar nycthemeral patterns were observed across developmental stages, although they were less pronounced in juveniles. In contrast, hemocyte viability, hemocyte profile distribution and micronucleus frequency remained comparatively stable throughout the 24 h cycle, suggesting the absence of marked cytogenomic fluctuations associated with sampling time under the tested conditions. These findings indicate that nycthemeral physiological rhythms may substantially influence hemocyte circulation dynamics and hemolymph accessibility in terrestrial gastropods, while exerting limited effects on basal cellular integrity. The study highlights the importance of temporal standardisation in hemocyte-based biomarker protocols and supports the integration of chronobiological considerations into ecotoxicological and comparative physiological investigations involving terrestrial molluscs.