Seabirds in crisis: Plastic ingestion induces proteomic signatures of multiorgan failure and neurodegeneration

Abstract

Understanding plastics' harmful impacts on wildlife would benefit from the application of hypothesis agnostic testing commonly used in medical research to detect declines in population health. Adopting a data-driven, proteomic approach, we assessed changes in 745 proteins in a free-living nonmodel organism with differing levels of plastic exposure. Seabird chicks heavily affected by plastic ingestion demonstrated a range of negative health consequences: Intracellular components that should not be found in the blood were frequently detected, indicative of cell lysis. Secreted proteins were less abundant, indicating that the stomach, liver, and kidneys are not functioning as normal. Alarmingly, these signatures included evidence of neurodegeneration in <90-day-old seabird chicks with high levels of ingested plastic. The proteomic signatures reflect the effects of plastic distal to the site of exposure (i.e., the stomach). Notably, metrics commonly used to assess condition in wildlife (such as body mass) do not provide an accurate description of health or the impacts of plastic ingestion.

Fig. 1.. Plastic ingestion induces substantial differences in the plasma proteome.

(A) Scaled PCA of the proteomic data revealed clear distinction between the proteomes of sable shearwater A. carneipes chicks that have ingested low (n = 13) or high (n = 18) quantities of plastic. (B) The volcano plot shows the result of t test analysis of the proteomic data and highlights the changes in relative abundance of secreted and intracellular proteins in seabird chicks heavily affected by plastic ingestion. The line indicates the threshold for significance (FDR-adjusted P < 0.05), and the proteins of interest that are labeled are BDNF, ALB, GAPDH, HMGB1, and PGA5.

Fig. 2.. Proteomics demonstrate that plastics cause a decrease in organ-secreted proteins and an increase in intracellular proteins in the plasma of sable shearwater chicks (A. carneipes).

(A to C) Volcano plots display the log fold change in protein abundance between chicks with low (n = 13) and high (n = 18) levels of ingested plastic, indicating that many of the intracellular and secreted proteins from the (A) brain, (B) liver, and (C) kidney and stomach are either more or less relatively abundant between the groups. t tests were used to determine significance with the dashed line denoting the significant threshold (FRD-adjusted P < 0.05). Proteins of interest include GKN2, GPX3, and PGA5. (D and E) Heatmaps displaying the standardized relative abundance of secreted and intracellular proteins in the (D) brain and (E) liver between low and high levels of plastic ingestion. Protein abundance was normalized by calculating Z-scores. Columns of the heatmaps correspond to individual chicks. (F) Total mass of ingested plastic for each chick corresponding to the heatmap, with scale square root transformation applied.

Fig. 3.. Plastic ingestion induces changes to gene regulation.

qPCR on RNA extracted from the (A and B) liver (n = 36) and (C and D) proventriculus (n = 13) of euthanized sable shearwater (A. carneipes) chicks with low and high levels of plastic exposure reveals up-regulation of antioxidant pathways. Genes of interest include albumin (ALB), superoxide dismutase (SOD1), and gastrokine-2 (GKN2). Relative C_t values of target genes of interest were calculated using the delta-delta (ΔΔC_t) method against the reference housekeeping gene. Hmbs was selected as the housekeeping gene. Gene expression differences between low and high plastic ingestion rates were analyzed with general linear models. Data were Box-Cox transformed and tested for equal variance. *P < 0.05 and ***P < 0.001.

Fig. 4.. Proteomic analysis on hippocampal brain tissue (n = 10) from sable shearwater (A. carneipes) chicks reveals a positive correlation between neuroplasticity-associated pathway enrichment and high levels of ingested plastic.

Proteins of interest include (A) MAP1A, (B) PDGFRβ, (C) GAP43, and (D) SOD1. Linear models were used to assess a dose-dependent relationship between protein relative abundance and ingested plastic load (g), following Box-Cox transformation and testing for homogeneity of variance to ensure that model assumptions were met. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.. Plastic ingestion induces proteomic signatures distal to the site of exposure.

Anatomical diagram of sable shearwater (A. carneipes) exposed to low (n = 13) and high (n = 18) quantities of ingested plastics. Proteomics on the plasma of chicks exposed to high quantities of ingested plastic revealed loss of brain, liver, and kidney function and stomach integrity disruption. Proteins of interest include BDNF, GPLD1 (glycosylphosphatidylinositol-specific phospholipase D1), GPX3, and PGA5.

Fig. 6.. Four hundred three pieces of plastic removed from sable shearwater A. carneipes 90-day-old chick.

On the left are five plastic items (0.073 g) removed during lavage, and on the right are the 398 items (41.812 g) that remained inside the stomach after lavage. The 398 items could not be removed during lavage as the large volume of plastic created a blockage. Instead, these items were recorded during necropsy after the bird was deceased. Pumice (128 pieces, 6.819 g) and squid beaks (not counted) also ingested by the bird are displayed in the petri dishes. The image was captured by J. Gilligan. This is the largest count record of plastic ingestion published for this species.