Zebrafish larval nitrogen excretion is flexible and resilient to loss of rhesus glycoproteins

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Abstract Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na + and H + (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb -crispants. Effects of high external ammonia (HEA, 500 µM NH 4 Cl) and 10 mM HEPES-buffering were further characterized in rhcgb -crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb- crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste. Summary statement We targeted zebrafish ammonia transporters rhbg and rhcgb with CRISPR-Cas9 mutagenesis to characterize larval nitrogen excretion and found high resilience to disruptions of ammonia excretion, including a switch to urea excretion.

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