Variable performance of metamorphosis success indicators in an in vitro culture of freshwater mussel glochidia

Abstract

The in vitro cultivation of parasitic larvae (glochidia) is becoming an important constituent of culture methods of freshwater mussels (order Unionida) worldwide. Despite rapid methodological progress, published methods for the vast majority of freshwater mussel species do not exist. In the present study, we tested the performance of current approaches to evaluate in vitro culture success quantification. A full factorial design was used to culture Anodonta anatina glochidia in vitro in separate media (M199 and DMEM), antibiotic concentrations and postincubation treatments (dilution methods). The glochidia metamorphosis success (the proportion of successfully transformed glochidia) in each treatment was quantified at two different time points, and the postlarval growth (8 days) of the juveniles was also estimated. The results showed a significant effect of medium type on metamorphosis success at the first time point (day 13) (M199 = 60.98 ± 20.20%, DMEM = 36.10 ± 23.61%; generalized mixed effect models, GLMMs, p < .001) but not at the second (day 21). Similarly, the dilution approach showed a significant variation in metamorphosis success only at the first quantification time point (water and medium = 33.97 ± 24.16%, water only = 63.11 ± 16.03%; GLMM p < .001). In contrast, the postlarval growth quantification showed an increased growth increment in the juveniles in the M199 medium (M199: 65.90 ± 24.20 μm, DMEM = 52.90 ± 19.84 μm; linear mixed effect model p < .001). The study shows that with current methods, the metamorphosis success assessment of in vitro culture protocols can be influenced by the effects of assessment time and dilution approach, two aspects that are not well standardized in the current methodology. Greater emphasis should be placed on the optimization of indicators with direct links to the final criteria, such as postlarval growth or physiology-based parameters. The optimization of metamorphosis success and viability indicators can be an important step towards the increased efficacy of current juvenile production by in vitro techniques and the development of new protocols.