EFFECTS OF STARVATION STRESS ON HEMOLYMPH PHYSIOLOGY AND INTESTINAL HEALTH OF PROCAMBARUS CLARKII

Procambarus clarkii is originated from the south-central United States and northern Mexico. In the 1930s, it was introduced to China as a freshwater economic crustacean via Japan because of its good economic benefits and a characteristic industrial chain. As the consumption hotspot of P. clarkii to grow, fundamental researches on the cultivation of P. clarkii were incredibly important. At present, researchers carried out a large number of analysis on the effects of starvation on the physiology, endocrine, and digestive systems of crustacea. To explore the effects of starvation stress on hemolymph physiological biochemical indexes, intestinal histology and intestinal flora of P. clarkii through histopathology, high-throughput sequencing and bioinformatics analysis techniques, P. clarkii was subjected to a 28-day starvation. Hemolymph and intestinal tract were collected at 0, 7, 14 and 28 days of starvation, respectively. Hemolymph was collected to assess the physiological status of P. clarkii by measuring conventional physiological and biochemical indexes; the intestinal tract was collected in 4% paraformaldehyde fixative for intestinal HE staining and TUNEL cell apoptosis detection; and intestinal contents were taken for 16S rRNA sequencing. The results of hemolymph biochemical analysis showed that alanine aminotransferase (ALT) did not change significantly; glucose (GLU) levels gradually decreased with the prolongation starvation (P<0.05). The levels of urea (UREA) and triglyceride (TG) first increased and then decreased (P<0.05), total protein (TP) and aspartate aminotransferase (AST) first decreased and then increased (P<0.05). Starvation stress caused significant changes in the intestinal morphology of P. clarkia: the intestinal cavity became wider, the intestinal villi were atrophied, the thickness of the muscle layer became thinner, and the tissue cell apoptosis index increased. High-throughput sequencing was performed on the 16S rRNA V3-V4 region of intestinal microbes. The Alpha diversity analysis showed that the highest diversity of intestinal microbes was on the day 7 of starvation. At the phylum level, the 12 samples were mainly anaerobic microorganisms such as Proteobacteria, Bacteroidetes, Firmicutes, etc. However, on the 7th day of starvation stress, the population abundance of anaerobic microorganisms (Bacteroidetes, Firmicutes, and Proteobacteria) decreased significantly; at the genetic level, the abundance of Bacteroides, Citrobacter, Streptococcus, Neisseria and Haemophilus increased significantly. The results indicated that P. clarkii may adapt to the process of starvation metabolism by changing the energy material utilization pathways and the intestinal microbiological composition structure.