This study investigates immune priming effects associated with granulocytes in crickets through a comprehensive analysis. Kaplan-Meier survival analysis reveals a significant contrast in survival rates, with the heat-killed Bacillus thuringiensis (Bt)-primed group (B-1) exhibiting an impressive ~80% survival rate compared to the PBS buffer-primed group (A-1) with only ~10% survival 60 hours post live Bt infection. Hemocyte analysis underscores elevated hemocyte counts, particularly in granulocytes of the B-1 group, suggesting a correlation between heat-killed Bt priming and heightened immune activation. Microscopy techniques further explore granulocyte morphology, unveiling distinctive immune responses in the B-1 group characterized by prolonged immune activation, heightened granulocyte activity, phagocytosis, and extracellular trap formation, contributing to enhanced survival rates. In particular, after 24 hours of injecting live Bt, most granulocytes in the A-1 group exhibited ETosis (death with release of extracellular traps), while in the B-1 group, the majority of granulocytes were observed to maintain highly activated extracellular traps, sustaining the immune response. Gene expression analysis supports these findings, revealing differential regulation of immune-related genes such as antibacterial humoral response, detection of bacterial lipopeptides, and cellular response to bacteria lipopeptides. Additionally, groups A (preinjected with heat-killed Bt), B (preinjected with heat-killed E. coli), and C (preinjected with PBS) were re- injected with live Bt and evaluated for survival rates over two- and nine-day intervals. Two days later, only group C displayed low survival rates. After injecting live Bt 9 days later, group B surprisingly showed a similarly low survival rate, while group A exhibited a high survival rate of ~60% after 60 hours, with actively moving and healthy crickets. In conclusion, this research provides valuable insights into both short-term and long-term immune priming effects in crickets, contributing to our understanding of invertebrate immunity with potential applications in public health.
Overall design: We divided 1,800 crickets into 8 groups and conducted the experiments with 75 crickets per group, 3 repetitions (8ⅹ75ⅹ3=1,800 crickets). In this study, immune priming was induced using two groups, A and B. PBS buffer and heat-killed Bacillus thuringiensis (Bt) were utilized for this purpose, resulting in the induction of immune priming. Two days later, live Bt was injected again to create groups A-1 and B-1. Subsequently, the immune priming effect was assessed by examining survival rates, the immune activation of granulocytes, and conducting RNA-Seq analysis for both the A-1 and B-1 groups. Next, group C, D, and E was the first injection of PBS (control), heat-killed E. coli, and heat-killed Bt. After 2 days, live Bt was injected into all groups and they were named group C-1, D-1, and E-1. 9 days after the first injection, we injected live Bt into group C, D, and E (called C-2, D-2, and E-2).
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