Is immune response of freshwater insects triggered by microplastics ingestion? A case study using the aquatic midge Chironomus riparius

, Silva Carlos, Beleza Fernandes Sónia, Campos Diana, Soares Amadeu, Silva Ana Luisa, Pestana João, Gravato Carlos.

The activation of insects' immune system due to ingestion of microplastics (MPs) has been evidenced by the upregulation of specific genes. Yet, the pathway through which MP elicit such responses is poorly understood. The activation of phenoloxidase (PO) is one of the main responses involved in the innate immunity of insects when facing parasites and pathogens, and similar process may occur in the presence of MPs. Therefore, this study aims to address the activity of PO and total PO (PO+proPO) activities in larvae of Chironomus riparius, allowing to estimate if de novo synthesis of prophenoloxidase (proPO) occurs. Larvae were exposed 48 h to irregularly-shaped (size range 32-63 µm) polyethylene microplastics (PE-MPs) present in sediments (1.25; 5; to 20 g kg-1). The ingestion of PE-MPs by larvae triggered a significant increase of PO activity (+25 and +28% in larvae exposed to 5 and to 20 g kg-1, respectively), whereas total PO activity increased +48% in larvae exposed to 20 g kg-1. Results also showed an increased activity of PO showing that particles can induce the innate immune system of larvae. Moreover, the induction of total PO activity on larvae exposed to 20 g kg-1 PE-MPs suggests de novo synthesis of the inactive form of the enzyme (proPO). Since the size of the PE-MPs ingested by larvae are too large to cross biological barriers, the activation of the immune response is probably linked to an inflammatory response due to their accumulation and retention inside the gut. This research work provides the first evidence on the direct activation of innate immune system of an insect after ingestion of MPs. Thanks are due to FCT/MCTES for the financial support to CESAM (UIDP/50017/2020+UIDB/50017/2020, through national funds) and the research project ComPET (POCI-01-0145-FEDER-030361, funded by FEDER, through COMPETE2020-POCI and by national funds (OE)).

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