A Toxicological and Ecotoxicological Assay Based on Mussel (Mytilus galloprovincialis) Hemocytes Motility

The scope of our research is to develop a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants based on evaluating the hemocyte motility of Mytilus galloprovincialis as model organism. Effect-based methods, such as in vitro and in vivo bio assays, represent innovative tools for detecting the effects of environmental chemical pollutants on living organisms. These methods can serve as tools in environmental biomonitoring and risk assessment.

This research addresses a significant gap in toxicology by focusing on using hemocyte motility as new endpoint to assess the effect of pollutants on mussels. Although hemocyte motility is crucial for immune responses, its sensitivity to pollutant exposure is understudied in bivalves. Our finding advance research in toxicology and ecotoxicology in several ways.

By offering a new endpoint for toxicity screening, supporting ethical research by minimizing vertebrate testing, and advancing applicability in ecotoxicology. This approach can have a broad application in environmental biomonitoring and could be adapted for other immune cells in ecotoxicology. To begin, pre-fill a syringe with 0.5 milliliters of standard physiological saline tempered to 15 degrees Celsius.

Position the acclimatized adult mussel specimen for the hemolymph collection. Using a scalpel, slightly and carefully prise apart the valves along the ventral surface. Maintain the scalpel in position or use a pipette tip to keep the space open.

Then, insert the needle of a hypodermic syringe into the space between the valves. Gently collect 0.5 milliliters of hemolymph from the posterior adductor muscle using the prefilled syringe. Now, remove the needle from the syringe and transfer the contents of the syringe into a microtube.

Pool the samples collected from three to four mussels together in a tube maintaining the temperature of 15 degrees Celsius. After assessing the hemocyte viability, to culture them, add 50 microliters of diluted hemolymph into the wells of a 96-well, flat-bottom, polystyrene, TC-treated microplate, and cover the plate with its lid. Let the hemocytes adhere to the bottom of the wells for 30 minutes at 15 degrees Celsius to form a monolayer.

Then, using a micropipette, gently aspirate excess hemolymph from the wells. To perform short-term assays within one to four hours, immediately add 100 microliters of the substance of interest dissolved in physiological saline. For prolonged exposures of 24 to 48 hours, incubate the cultured adherent cells with the test substance dissolved in the supplemented culture medium at 15 degrees Celsius.

To begin, obtain the cultured hemocytes treated with the substance of interest. For enhanced visualization of hemocytes, remove the incubation medium from each well, and stain the cells with 100 microliters of neutral red vital dye staining solution before time-lapse microscopy. Incubate the cells at 15 degrees Celsius in a climatic chamber for 15 minutes.

After incubation, wash out the excess dye, and add 100 microliters of the substance of interest dissolved in standard physiological saline. Insert the multi-well plate containing the treated cells into a multi-mode reader. Perform real-time imaging on the plate through time-lapse microscopy under 20X magnification and bright-field visualization.

For cell tracking and calculating velocimetric parameters, obtain the individual frames using Gen5 software, and save the images in a folder with no spaces in the name. In ImageJ, navigate to File, followed by Import and Image Sequence. Perform cell tracking on single cells using the manual tracking plugin.

Import datasets from the ImageJ manual tracking plugin into the Chemotaxis and Migration Tool software. Select the desired number of slices for tracking. Calibrate the software with the X/Y pixel size and time interval settings.

After setting the parameters, click on Apply settings. Plot trajectories and export as an image. Click on the Measured values button to view the results, and save the data.

Then, click on the Statistics button and save the velocity and directness data for the track series. Compare the calculated velocimetric parameters of control cells with those of hemocytes exposed to the test substance at varying concentrations. Hemocyte motility was assessed using time-lapse imaging, showing individual cell movements over 10 minutes, with consistent tracking of numbered cells across three time points of zero, five, and 10 minutes.

Two morphotypes of hemocytes, spreading cells and smaller round star-shaped cells, were observed. Both types showed continuous shape changes due to lamellipodia and pseudopodia activity. Velocity and directness plots indicated that, under basal physiological conditions, spread cells moved faster compared to smaller cells.

Paracetamol exposure significantly reduced the motility of spread cells after 24 hours, while small cells showed no such reduction. The directness of small cells increased significantly after 24 hours of paracetamol exposure, while spread cells showed decreased directness after just one hour.