Unlocking the hidden anatomy: synchrotron micro-tomography of the stomach, midgut, and organs in Penaeus vannamei and the potential route of Enterocytozoon hepatopaenaei (EHP) infection.

Due to the structural complexity of the crustacean digestive system, this study characterizes the three-dimensional (3D) morphology and histology of the juvenile Penaeus vannamei alimentary tract using synchrotron radiation X-ray tomographic microscopy (SR-XTM) and H&E staining. SR-XTM volumetric imaging effectively reveals the structural interconnections among the pyloric stomach (PS), hepatopancreas (HP), and midgut (MG). Histological analysis further distinguishes the regional processing of fine and coarse chyme, which are segregated by a pair of lateral pyloric valves (LPVs). These valves function as mechanical barriers to prevent mixing of coarse and fine chyme within the anterior MG. Furthermore, this study identifies a secondary communication pathway, a sub-LPV channel, that enables a direct connection between the MG and the HP. In situ hybridization of EHP-specific transcripts identifies significant Enterocytozoon hepatopenaei (EHP) infection in cells lining the primary hepatopancreatic chamber and proximal regions of the secondary and tertiary HP tubules. Given that the gastric sieve restricts pathogen transmission by particle size, our findings suggest the sub-LPV channel serves as a critical alternative route for the oral transmission of EHP spores. This study provides a foundational structural framework for understanding shrimp digestive anatomy and host-pathogen internalization.