Protocol for Cellular Sexual Staging of Female Euphausiids Using Fluorescent Imaging and Quantitative Morphometrics

This protocol describes a standardized laboratory method for determining the sexual development stage (SDS) of female euphausiids through fluorescent staining of ovarian tissue, digital imaging, machine-learning–based image segmentation, and quantitative morphometric analysis. The workflow was developed and validated using Antarctic krill (Euphausia superba) and applies the ovarian developmental framework described by Cuzin-Roudy & Amsler (1991). Oocyte diameter and area measurements derived from fluorescence microscopy are used to assign individuals to SDS categories based on established developmental thresholds. This procedure is designed for use with formalin-preserved or fresh specimens. It is not suitable for specimens preserved in ethanol or stored frozen, as these preservation methods compromise tissue integrity and nuclear staining quality.The protocol includes:Ovarian dissection from preserved specimensNuclear staining with DAPIFluorescence microscopyPixel classification using ilastikObject identification and morphometric quantification using CellProfilerAssignment of SDS based on maximum oocyte size per individualAlthough optimized for Euphausia superba, this workflow can be adapted to other euphausiid species provided that species-specific oocyte size thresholds are established.