Nitrogen and Sulfur Assimilation in Fish Otoliths Evaluated by Stable Isotope Labeling and NanoSIMS Analysis

Stable nitrogen (δ¹⁵N) and sulfur (δ³⁴S) isotopes within the metabolically inert organic matrix of teleost otoliths provide high-fidelity archives for reconstructing animal life histories. However, low organic content limits the spatial resolution of conventional bulk isotopic analysis, leaving biomineralization kinetics and the temporal integration of these dietary signals poorly quantified. We utilized nanoscale secondary ion mass spectrometry (NanoSIMS) to overcome these limitations and determine intracrystalline isotopic dynamics.

Isotopic pulses were established by feeding juvenile Mozambique tilapia (Oreochromis mossambicus) ¹⁵N-enriched algae and diets supplemented with L-methionine-³⁴S, utilizing Alizarin Red S fluorescent marking for precise temporal referencing. High-resolution and in situ NanoSIMS analysis was applied to quantify the temporal distribution of the labeling signals within the otolith organic matrix. A first-order kinetic model was used to estimate isotopic dynamics half-lives (t_50%).

NanoSIMS analysis revealed rapid isotopic changes across both assimilation and depuration phases (t_50% = 0.5-13.9 days). Both δ¹⁵N and δ³⁴S exhibited statistically indistinguishable kinetics, reflecting synchronized metabolic routing where dietary signals are deposited directly into the otolith without being buffered by internal tissue reserves. Crucially, the dietary matrix modulated assimilation; despite comparable dietary δ³⁴S proportions, otolith sulfur incorporation and signal attenuation were significantly faster in fish fed algae-based diets than in those fed eel-meal-based diets. This demonstrates the preferential utilization of exogenous labeled methionine when the base diet is naturally deficient in this essential amino acid.

High-resolution NanoSIMS profiling demonstrates that the otolith organic matrix archives dietary signals governed by rapid assimilation pathways rather than strict thermodynamic equilibrium. This analytical approach establishes a crucial mechanistic basis and experimental framework for utilizing intracrystalline organic isotopes as precise indicators in trophic ecology.