Saxitoxin Modulates Immunological Parameters and Gene Transcription in Mytilus chilensis Hemocytes.

Allisson Astuya,Ambbar Aballay,Crisleri Carrera,Viviana Ulloa,Hélène Hégaret,Cristian Gallardo-Escárate,Gustavo Núñez-Acuña

doi:10.3390/ijms160715235

Abstract

Saxitoxin (STX) is a neurotoxin produced by dinoflagellates in diverse species, such as Alexandrium spp., and it causes paralytic shellfish poisoning (PSP) in humans after the ingestion of contaminated shellfish. Recent studies have suggested that the immune functions of bivalves could be affected by harmful algae and/or by their toxins. Herein, hemocytes are the main effector cells of the immune cellular response. In this study, we evaluated the response of hemocytes from the mussel Mytilus chilensis to STX exposure in a primary culture. Cell cultures were characterized according to size and complexity, while reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein diacetate (DCFH-DA) assay. Finally, phagocytic activity was measured using both flow cytometry and fluorescence microscopy assays. Additionally, gene transcription of candidate genes was evaluated by qPCR assays. The results evidenced that exposures to different concentrations of STX (1–100 nM) for 24 h did not affect cell viability, as determined by an MTT assay. However, when hemocytes were exposed for 4 or 16 h to STX (1–100 nM), there was a modulation of phagocytic activity and ROS production. Moreover, hemocytes exposed to 100 nM of STX for 4 or 16 h showed a significant increase in transcript levels of genes encoding for antioxidant enzymes (SOD, CAT), mitochondrial enzymes (COI, COIII, CYTB, ATP6, ND1) and ion channels (K+, Ca2+). Meanwhile, C-type lectin and toll-like receptor genes revealed a bi-phase transcriptional response after 16 and 24–48 h of exposure to STX. These results suggest that STX can negatively affect the immunocompetence of M. chilensis hemocytes, which were capable of responding to STX exposure in vitro by increasing the mRNA levels of antioxidant enzymes.

Highlights

Harmful algal blooms (HABs) are excessive accumulations of phytoplankton that produce biotoxins, which can adversely affect humans, animals and ecosystems
In vivo and in vitro studies have reported changes in cell morphology, increases in mortality, reductions in phagocytosis, changes in adhesion and reactive oxygen species (ROS) production in hemocytes after exposure to marine toxins derived from HABs, thereby indicating that the immune response to toxic microalgae could be directly related to toxin levels [16,17,18]
Hemocytes cultured from M. chilensis had a round-shape or a flattened polygonal shape with granules (Figure 1A), and these adhered to the substrate 3 h after seeding

Summary

Introduction

Harmful algal blooms (HABs) are excessive accumulations of phytoplankton that produce biotoxins, which can adversely affect humans, animals and ecosystems. In vivo and in vitro studies have reported changes in cell morphology, increases in mortality, reductions in phagocytosis, changes in adhesion and ROS production in hemocytes after exposure to marine toxins derived from HABs, thereby indicating that the immune response to toxic microalgae could be directly related to toxin levels [16,17,18]. In addition to cellular immune parameters, changes occur at the transcriptional level of stress response genes in hemocytes exposed to marine toxins [16,17,19]. A transcriptional response was observed in Mytilus chilensis exposed in vivo to saxitoxin (STX), through an increase in the mRNA levels of superoxide dismutase (SOD), catalase (CAT), ferritin and heat-shock protein genes, while, to a lesser extent, ependymin, fibrinogen-like, galectin and mytilin B genes evidenced differentiated expressions after toxin exposure [20]. The fact that this species is considered a sentinel species for biomonitoring and even with the high amount of knowledge regarding the “omics”

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Conclusion