Age-based δ15N and δ13C values of otolith organic matter reveal trophic ecology in marine fishes

Fish inhabiting a similar environment confront space and resource sharing and thus develop diverse feeding strategies to adjust their trophic niches during the ontogeny to avoid competition. To explore the topic, we reconstructed the trophic trajectory of five Sciaenidae species by sequentially analysing the δ13C and δ15N values of otolith organic matter along the growth axis of the otolith. The isotope values were aligned to the age based on growth ring counting and 3D scanning of otolith morphology during organic matter extraction. The δ13C and δ15N values revealed the habitat shift and trophic levels of prey items, respectively. Sciaenidae fish use the benthic resources during their whole life and may move close to the estuary around 1.5-3 years old. They mainly prey on herbivores/zooplankton at the larval and early juvenile stage, but when they grow, there is minimisation of competition among age groups and species.

Methods
For the age-based trophic niche using stable isotope values recorded in otolith OM, we first adopted the ultrafiltration method because two stable isotope systems provide more trophic information. Then, we dissolve one of the paired otoliths layer by layer, extract the otolith OM in each layer and apply a 3D scanning technique to track the morphological change of each dissolution. Second, we estimate the fish age corresponding to each dissolved layer by image analysis. Third, we evaluate the habitat uses and trophic level of Sciaenidae fishes by comparing the stable isotope values of the particulate organic matter (POM).

Thirty-three fish samples of five Sciaenid species (Atrobucca nibe, Chrysochir aureus, Nibea albiflora, Pennahia macrocephalus, and Protonibea diacanthus) were collected off the western coast of Taiwan in 2020-2023. Muscle tissues and otoliths were collected from each sample. Muscle tissues were freeze-dried in which 0.7 mg dry powder was packed into tin capsules for further isotope analyses. The otoliths were washed with 0.2M NaOH, cleaned with ultrapure water three times, and dried in an oven at 50 ºC for 48 hrs. The packed muscle and otolith organic matter samples were analysed using Isotope Ratio Mass Spectrometry (Delta V Advantage, ThermoFisher Scientific, Germany) equipped with Elemental Analyser (Flash EA2000, ThermoFisher Scientific, Germany).

The otolith organic matter extraction follows the ultrafiltration using Amicon ultrafilters (Merck Millipore Ltd., Tullagreen, Carrigtwohill, Co.Cork, IRL).  3D scanned with GOM Scan 1 MV100 (Carl Zeiss GOM Metrology GmbH, Braunschweig, Germany) to monitor the morphological changes. The 3D images in a series of otolith dissolutions were overlaid to evaluate in which direction, the otolith shape changed the most using a compatible software (GOM Software 2021_Hotfix_1 Rev. 142421). The right otoliths from the second batch of samples were embedded in Epoxy Resin (Struers, Denmark), cut through the frontal plane using a low-speed diamond saw (IsoMet, Buelher, US), ground and polished until the core was observed at the surface.