Nutritional-status dependent effects of microplastics on activity and expression of alkaline phosphatase and alpha-amylase in Brachionus rotundiformis.

Tissue-nonspecific alkaline phosphatase (ALPL) and alpha-amylase (AMY) are essential in the immune and digestive systems, respectively. Microplastics (MPs) pose a risk to zooplankton which may be in a state of feeding, starvation, or subsequent refeeding. However, molecular characterization of both enzymes and the regulated mechanisms affected by nutritional statuses and MPs remain unclear in zooplankton. In the present study, four full-length genes encoding ALPL and two genes encoding AMY were cloned and characterized from an isolated marine rotifer, Brachionus rotundiformis, including alplA, alplB, alplC, alplD, amy2a, and amy2al. AMY activity and expression of amy2a and amy2al were reduced by starvation and recovered after refeeding compared with feeding. ALPL activity remained unchanged among different statuses, while alplA, alplB and alplD were down-regulated by starvation and refeeding compared with feeding. ALPL activity was not affected by exposure to 10, 100 and 1000 µg/L MPs in rotifers subjected to feeding, starvation and refeeding, whereas AMY activity was significantly enhanced by 1000 µg/L MPs in rotifers subjected to refeeding. Gene expression of the tested genes, except amy2a, was significantly responsive to MPs, especially in the feeding rotifers, depending on MPs concentrations and nutritional statuses. Two-way ANOVA confirmed that these changes were strongly associated with the interaction between MPs concentrations and nutritional statuses. The present study is the first to demonstrate a nutritional status-dependent impact of MPs on immune and digestive responses, and provides more sensitive molecular biomarkers for assessing MPs toxicity using the species as model animals.

Genome-wide identification of seven superoxide dismutase genes in the marine rotifer Brachionus rotundiformis and modulated expression and enzymatic activity in response to microplastics and nutritional status.

Microplastics (MPs) pollution has attracted worldwide attention. Superoxide dismutase (SOD) is a sensitive indicator for assessing the toxic effects of MPs in aquatic organisms. However, few studies have been performed to identify all genes encoding SOD in aquatic invertebrates. Especially, effects of MPs on SOD activity and expression in aquatic organisms under starvation or a subsequent refeeding status are unclear. In the present study, all full-length genes encoding SOD were cloned and characterized from the marine rotifer Brachionus rotundiformis, including CuZnSOD1, CuZnSOD2, CuZnSOD3, CuZnSOD4, CuZnSOD5, MnSOD1, and MnSOD2. The CuZnSOD1, CuZnSOD2 and MnSOD2 are homologous to SODs from vertebrates and the other SOD proteins are rotifer-specific according to the results from the phylogenetic tree. The conserved signature sequences and binding sites of Cu2+, Zn2+and Mn2+ were also identified in the seven SOD proteins. Compared with feeding, starvation down-regulated SOD activity and mRNA expression of CuZnSOD2, CuZnSOD4, CuZnSOD5, MnSOD1 and MnSOD2 while refeeding maintained SOD activity comparable to the feeding level and up-regulated CuZnSOD5 and MnSOD2. Intake of MPs by B. rotundiformis was observed by examining fluorescence signals from the fluorescently-labeled microplastics under different nutritional status. Exposure to MPs reduced rotifer density and increased malondialdehyde (MDA) content and SOD activity in the rotifers under the refeeding condition, but did not affect these indicators under the feeding and starvation conditions. However, mRNA expression of some tested genes was responsive to MPs in the fed, starved and refed rotifers. The present study for the first time demonstrated a nutritional status-dependent effect of MPs on oxidative stress response, and provided more sensitive molecular biomarkers for assessing the toxicity of MPs using B. rotundiformis as a model animal.