Proteomic and Lipidomic Plasma Evaluations Reveal Biomarkers for Domoic Acid Toxicosis in California Sea Lions

Abstract

Domoic acid is a neurotoxin secreted by the marine diatom genus Pseudo-nitzschia during toxic algal bloom events. California sea lions (Zalophus californianus) are exposed to domoic acid through the ingestion of fish that feed on toxic diatoms, resulting in domoic acid toxicosis (DAT), which can vary from mild to fatal. Sea lions with mild disease can be treated if toxicosis is detected early after exposure. Therefore, rapid diagnosis of DAT is essential but also challenging. In this work, we performed multiomics analyses, specifically proteomic and lipidomic, on blood samples from 31 California sea lions. Fourteen sea lions were diagnosed with DAT based on clinical signs and post-mortem histological examination of brain tissue, and 17 had no evidence of DAT. Proteomic analyses revealed 31 statistically significant proteins in the DAT individuals compared to the non-DAT individuals (adjusted p < 0.05). Of these proteins, 19 were decreased in the DAT group of which three were apolipoproteins that are known to transport lipids in the blood, prompting lipidomic analyses. In the lipidomic analyses, 331 lipid species were detected with high confidence and multidimensional separations, and 29 were found to be statistically significant (adjusted p < 0.05 and log2(FC) < -1 or >1) in the DAT versus non-DAT comparison. Of these, 28 were lower in the DAT individuals, while only 1 was higher. Furthermore, 15 of the 28 lower concentration lipids were triglycerides, illustrating their putative connection with the perturbed apolipoproteins and potential use in rapid DAT diagnoses.

Keywords: California Sea Lion; Domoic Acid; Ion Mobility Spectrometry; Lipidomics; Multiomics; Proteomics; Zalophus californianus.

Figure 1.

California sea lion domoic acid exposure routes. First, Pseudonitzschia accumulates domoic acid during algal blooms. The fish are then exposed due to their diet of zooplankton that feed on algal cells or the diatoms themselves. Finally, sea lions eat the contaminated fish, and depending on the amount and frequency, this exposure can cause toxicosis, which can be fatal.

Figure 2.

Proteomic and lipidomic sample workflows. A) For the proteomic workflow, a tryptic digest of sea lion was separated with nanoflow liquid chromatography, and data were acquired with a Fusion Lumos orbitrap mass spectrometer. Data was searched with FragPipe and analyzed in R. B) The lipidomic workflow consisted of a modified-Folch lipid extraction of the plasma, followed by LC-IMS-CID-MS analysis. Lipidomic data sets were processed using Skyline software with in-house libraries containing over 1000 lipids with LC retention time, CCS, and m/z values for each. Statistics and data visualization were performed by using MetaboAnalyst 6.0 and RStudio. Created in BioRender. Baker, E. (2024) https://BioRender.com/w66u385 and Baker, E. (2024) https://BioRender.com/x36i824.

Figure 3.

Eleven apolipoproteins detected in the sea lion proteomic analyses and illustrated in A) rank abundance and B) protein response graphs. Apolipoproteins are highlighted in either red or blue, indicating upregulation (red) or downregulation (blue), while all other detected proteins are shown in gray. The dashed line in B) represents a 1:1 reference, where proteins above are upregulated and below are downregulated. The statistically significant apolipoproteins are marked with asterisks.

Figure 4.

Lipids detected and dysregulated in the DAT vs non-DAT California sea lion plasma comparison. Blue data points show downregulated lipids, red show upregulated, and gray show those not statistically significant in the comparison. Of the significant lipids, 50% are triglycerides. The full list of significant lipids is included in Table S8.

Figure 5.

Saturation and chain length trends for detected and significant lipids in the DAT California sea lion plasma. A) The number of double bonds present in the fatty acyl chains of the significant and detected lipids, and B) variations in long chain fatty acids (13–20 carbons) or very long chain fatty acids (greater than 20 carbons). MCFA = medium chain fatty acids (6–12 carbons), LCFA = long chain fatty acids, and VLFA = very long chain fatty acids.