The Pineal Gland Is Required for Optimal Nighttime Immunity to Bacterial Infection in Larval Zebrafish

The immune response to bacterial infection demonstrates circadian rhythmicity, with heightened antibacterial immunity often observed during the daytime. This circadian-gated immune response is believed to have evolved to synchronize peak antibacterial immunity with the increased threat of infection when active. Using a larval zebrafish infection model, we have previously shown that light has a positive effect on antibacterial immunity, with enhanced survival and neutrophil recruitment observed when infections are delivered during the light phase. However, the specific contribution of the light-detecting pineal gland and its principal hormone, melatonin, to this circadian immune response has not been investigated. Here, we examined whether the pineal gland modulates the host response to bacterial infection in a time-of-day-dependent manner by targeted ablation of the pineal gland. To generate pineal gland-deficient larvae we created the transgenic zebrafish line Tg(exorh:YFP-NTR2.0) that restricts expression of the highly efficient nitroreductase variant NTR2.0 to pinealocytes, enabling metronidazole-mediated pinealectomy. We also analyzed larvae lacking the brain-specific homeobox (bsx) gene, that encodes a transcription factor essential for pineal gland development. Both approaches resulted in complete loss of the pineal gland, confirmed by live imaging, the absence of arylalkylamine N-acetyltransferase 2 (aanat2) expression and diminished melatonin production. Pineal gland-deficient larvae exhibited normal survival and neutrophil recruitment when infected during the light phase, indicating that daytime immune defenses can be maintained independently of the pineal gland. In contrast, infection of pineal gland-deficient larvae during the dark phase led to significantly reduced survival, which correlated with impaired neutrophil recruitment to the infection site. These findings demonstrate that the pineal gland plays an important role in supporting nighttime antibacterial immunity in larval zebrafish.