A new trypan blue agar plate assay for in vivo evaluation of gut damage in Folsomia candida springtail

Abstract Although the springtail Folsomia candida is a representative soil test species and a widely used biological indicator, current toxic endpoints are limited and required to expand at the individual level. For instance, there is no simple and efficient method for evaluating gut barrier damage in Folsomia candida except for histopathological evaluation or electron microscopic analysis. Because the evaluation of intestinal tissue damage by environmental pollutants can be linked to the gut microbiome and digestive enzyme results, the present study proposes an efficient method for evaluating the gut damage in springtail using trypan blue dye to visualize adverse effects. The media, staining, and depuration periods were optimized, and the proposed method was validated using F. candida exposed to model chemicals (Cu and Cr). The optimal parameters were determined as 1) 0.1% trypan blue + 0.8% agar + 5% sugar plate for 15 min, followed by 2) depuration on an 0.8% agar + 10% sugar plate for 3 h. This new method is expected to provide a simple and cheap tool for evaluating gut damage in springtails compared with histopathological evaluation or electron microscopic analysis. Furthermore, this method can expand individual-level toxic endpoints for springtails.