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

The article proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on the motility of Mytilus galloprovincialis hemocytes. The method aims to contribute to the development of more ethical and sensitive toxicological and ecotoxicological exposure tests.

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate immunity. During the early stages of the immune response, hemocytes actively migrate to the site of infection. This inherent motility is a fundamental characteristic of these cells. It represents a key cellular function that integrates multiple processes, such as cell adhesion, cell signaling, cytoskeletal dynamics, and changes in cell volume. Therefore, alterations in cell motility following exposure to drugs or pollutants can serve as a useful toxicological endpoint. Despite the fundamental role of cell motility in cellular physiology, it has been poorly investigated from a toxicological perspective. This work proposes 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. We developed a cell motility assay on hemocytes adhering to the bottom of a 96-well polystyrene microplate. Following exposure to increasing concentrations of drugs, cell trajectories, and velocities were quantified by cell tracking under time-lapse microscopy, allowing us to measure the effects on hemocyte motility. Due to the ease of hemocyte collection from the animals in a relatively non-invasive manner, the proposed method offers an alternative test for screening the effects and mechanisms of action of pollutants and drugs. It aligns with the 3Rs (Replacement, Reduction, and Refinement) criteria, addressing ethical concerns and contributing to the reduction of vertebrate in vivo animal testing.

Effect-based methods, such as in vitro and in vivo bioassays, represent innovative tools for the detection of the effects of environmental chemical pollutants in living organisms and for their use as tools in environmental monitoring and risk assessment^1,2,3,4. They complement the classical analytical chemical approach by overcoming some of its limitations. For instance, effect-based methods can assess the bioavailability of pollutants, their impact on organism health, and the combined toxicological effects of mixtures. These combined effects may not be predictable based solely on chemical analysis⁵.

In recent years, the ecotoxicology of pollutants of emerging concern (emerging pollutants) represents a field where effect-based methods can be useful tools for detecting exposure and assessing the impact on the biota^1,5,6,7. Several effect-based methods use bivalve mollusks as test organisms in environmental monitoring and assessment^8,9. Some characteristics make these organisms suitable for ecotoxicological studies, such as their wide distribution, their filter-feeding nature, their sessile lifestyle, the capability of bioaccumulation of a wide range of environmental pollutants and to develop detectable responses to pollutants, the possibility of working with different life stages, and to maintain under laboratory conditions⁷. They are highly sensitive to pollution exposure and show a variety of responses to toxic contaminants depending on species, life stage, and environmental conditions^8,9,10. Therefore, several environmental guidelines use bivalve species as standardized test species^10,11.

Among the bivalve mollusks, the widespread Mytilus galloprovincialis is one of the most used species in the ecotoxicological field due to its ability to develop early detectable responses to chemical pollution exposure, including metallothionein induction, antioxidant enzyme alteration, lysosomal membrane destabilization, lipid peroxidation, lipofuscin accumulation, increased micronuclei frequency, carbonic anhydrase induction^12,13,14,15. Hemocytes, the immunocompetent hemolymphatic cells, are widely used to study the toxicological impacts of environmental pollutants in bivalve mollusks^4,13,16,17. These cells are crucial to the organism's immune response, carrying out several important functions of cell-mediated innate immunity. These include the elimination of microbes through phagocytosis and various cytotoxic reactions, such as the release of lysosomal enzymes, anti-microbial peptides, and the production of oxygen metabolites during the respiratory burst^18,19,20. Hemocytes are intrinsically motile cells^21,22,23 able to migrate to the site of infection during the early stage of the organism's immune response. In general, motility is a fundamental feature that characterizes all immune cells since it enables the immunosurveillance of these cells to protect the body²⁴. Research across various molluscan species demonstrates that hemocyte motility is a critical component of their immune response, wound healing, and interaction with pathogens. This motility is regulated by specific molecular pathways, highlighting the complexity and specialization of hemocyte functions in molluscs^21,25,26,27.

Despite the fundamental importance of motility in the physiology of hemocytes, very few studies have investigated the sensitivity of hemocyte motility to environmental chemical pollutants^23,28,29,30. Recently, our group characterized the spontaneous movement of Mytilus galloprovincialis hemocytes in a tissue culture-treated polystyrene 96-well microplate and examined the sensitivity of hemocyte motility to in vitro exposure to paracetamol²³. M. galloprovincialis hemocytes showed a random-like cell movement based on lamellipodia and fast shape changes, as previously found in another mussel species, Mytilus edulis^21,22,23,28, and already described in human immune cells³¹. Hemocyte motility has recently been demonstrated to be sensitive to chemical stressors^23,28. Based on these previous findings, this work proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants based on evaluating the motility of M. galloprovincialis hemocytes and its alterations, through velocimetric analysis of cell motility (quantification of mean velocity, migrated distance, Euclidean distance, and directness). The method offers the possibility to in vitro screen the toxicity of several substances either in short-term assays (lasting 1-4 h) or prolonged exposure assays, lasting 24-48 h.

All experiments were performed under the Italian Animal Welfare legislation (D.L.26/2014) that implemented the European Committee Council Directive (2010/63 EEC). Mytilus galloprovincialis is a filter-feeding bivalve, commonly known as the Mediterranean mussel. It is native to the Mediterranean Sea and the Atlantic coast of southern Europe. It was introduced and is widespread in Western North America, Asia, and South Africa. It is an important commercial fishery species in several parts of the world. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Preparation of artificial seawater (ASTM) or filtered natural seawater

1. For the preparation of artificial seawater ASW according to ASTM D1141-98 (2021)³², dissolve the following salts in distilled water (g for 1 L): 27.65 NaCl, 0.133 NaHCO₃, 3.33 MgCl₂· 6H₂O, 2.66 Na₂SO₄, 0.733 CaCl₂ · 2H₂O, 0.466 KCl, 0.067 KBr, 0.02 H₃BO₃, 0.013 SrCl₂·6H₂O, 0.0019 NaF. The ASW solution has a pH of 7.80.
2. For the preparation of filtered natural seawater (FSW), filter natural seawater 37 PSU, collected from a site not impacted by anthropogenic activities with 0.2 µm filters.

2. Animal acclimation

1. Acclimatize adult mussel specimens (Mytilus galloprovincialis Lam.) for 24 h in static tanks (in starvation) containing aerated ASW or FSW (1 L/animal) at 15 °C in a thermostatic room before experimental use.

3. Reagent preparation for hemocyte motility assessment

1. Prepare filtered seawater (FSW) as above or standard physiological saline (SPS). Dissolve HEPES (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid) 4.77 g, NaCl 25.48 g, MgSO₄ 13.06 g, KCl 0.75 g, CaCl₂ 1.47 g in approximately 800 mL of distilled water, then made up to 1 L by the addition of more distilled water. Adjust the pH to 7.36 with 1 M NaOH.
2. Prepare a Neutral Red stock solution of 20 mg/mL in Dimethyl sulfoxide (DMSO).
3. Prepare Neutral Red staining solution: Add 5 µL of a Neutral Red stock solution to 995 µL of FSW or SPS.
4. Prepare FSW or SPS supplemented with 2 mM of L-glutamine, 40 IU/mL penicillin G, and 100 µg/mL streptomycin as follows: Add 100 µL of 2 mM L-glutamine and 100 µL of Penicillin/Streptomycin mix to 10 mL of FSW/SPS.
5. Prepare 0.4% Trypan blue in FSW or SPS.
   NOTE: Neutral Red stock solution was prepared according to Martínez-Gómez et al.³³. The Neutral Red stock solution lasts for about 2-3 weeks when stored at 4 °C. Neutral Red staining solution and 0.4% Trypan blue should be prepared just before the assay.

4. Hemolymph sampling

1. Prefill a syringe with 0.5 mL of filtered seawater FSW or standard physiological saline SPS (at 15 °C).
2. Slightly and carefully prise apart the valves along the ventral surface with a scalpel. Maintain the scalpel in position or use a pipette tip. Allow the needle of a hypodermic syringe to enter the space between the valves.
3. Gently collect 0.5 mL of hemolymph from the posterior adductor muscle with the prefilled syringe according to UNEP/RAMOGE³⁴.
4. Remove the needle from the syringe and transfer the contents of the syringe into a microtube.
5. Collect the hemolymph from 3-4 mussels and pool together the hemolymph samples in a tube at 15 °C using the same ratio between individuals.
   NOTE: The prefilling of the syringe is used to immediately dilute the hemolymph 1:1 during sampling. The dilution of the hemolymph with FSW or SPS in a 50:50 ratio is commonly used to prevent hemocyte aggregation, according to Martínez-Gómez et al.³³.

5. Hemocyte plating and culture

1. Count the cells with a hemocytometer.
2. Dilute the hemolymph at the concentration of 1 × 10⁶ cells/mL with FSW or SPS.
3. Evaluate the viability of hemocytes in suspension by Trypan blue staining: Add 100 µL of 0.4% Trypan blue dissolved in FSW or SPS to 100 µL of hemocyte suspension. Incubate for 5 min at 15 °C.
4. Next, add a drop of suspension in a hemocytometer and count the unstained (viable) and stained (nonviable) cells under microscopy observation.
5. Assess the hemocyte viability as the percentage of viable cells calculated on the total number of counted cells³⁵.
6. Use the following formula for cell viability assessment³⁵:
   Viable cells (%) = [number of viable cells/total number of cells (viable + nonviable)] x 100
7. Ensure that the hemocyte viability should be higher than 95% to proceed further in the protocol.
8. Add 50 µL of diluted hemolymph into each well of a cell culture 96-well flat-bottom polystyrene TC-treated microplate. Cover the plate with its lid.
9. Allow hemocytes to adhere to the bottom of the wells for 30 min at 15 °C to form a monolayer.
10. Remove the excess hemolymph by gently aspirating with a micropipette.

6. Short-term assay

1. For short-term assays (within 1-4 h), immediately incubate the cells adherent to the bottom of the wells with 100 µL of the substance of interest solved in FSW or SPS at different concentrations at 15 °C. Use at least three wells for three replicate determinations of each experimental condition.
2. After incubation, assess cell motility following step 8.

7. Prolonged exposure assay

1. For prolonged exposures, i.e., 24-48 h exposure assays, incubate the cultured adherent cells with increasing concentrations of the test substance dissolved in the culture medium (FSW or ASW) supplemented with 2 mM L-glutamine 40 IU/mL penicillin G and 100 µg/mL streptomycin) at 15°C. Use at least three wells for three replicate determinations of each experimental condition.
2. After incubation, assess cell motility following step 8.

8. Cell motility assessment by time-lapse microscopy

1. Insert the multiwell plate containing the adherent living hemocytes into a multimode reader.
2. Assess the motility of the adherent living cells by real-time imaging on the plate through time-lapse microscopy: capture images of the same region of interest (ROI) (dimension of the ROI used: 720µm x 720µm) in each well at a rate of 1 image every 1 min for 10 min using the multimode reader under 20x magnification and color brightfield visualization.
3. For better visualization of the hemocytes, stain the cells with the vital dye Neutral Red just before time-lapse microscopy as follows:
   1. Remove the incubation medium from each well.
   2. Add 100 µL of staining solution of  Neutral Red dissolved in standard physiological saline containing the substance of interest into the wells of the plate containing the adherent cells.
   3. Incubate the cells at 15 °C for 15 min. Wash out the excess dye.
   4. Add 100 µL of the substance of interest solved in FSW or SPS. Visualize the cells for motility assessment.
      NOTE: The use of a multimode reader allows the simultaneous acquisition of images from a high number of wells, allowing simultaneous motility detection of all experimental conditions and replicates contained in the well. The Neutral red dye is retained inside the lysosomes, which appear as little orange spots in the cytoplasm, while the nucleus appears as a translucent hole since the acidophilus vital dye does not stain it. The image acquisition was done for 10 min at a room temperature of 20 °C.

9. Cell tracking and velocimetric parameters calculation

1. Analyze the frames of the same region of interest acquired under time-lapse microscopy by Image J for each well.
2. Save the images in a folder with no spaces in the name. The images can be either in JPEG or PNG format.
3. Open ImageJ. Click on File > Import > Image Sequence.
4. Perform cell tracking on single cells using the Manual Tracking plug-in of ImageJ. The positions of individual cells are marked in consecutive images, allowing for the tracking of positional changes over time.
5. Analyze at least 40 cells per well, excluding cells that escape the recorded field from the analysis.
6. Import Datasets from the ImageJ plug-in, "Manual Tracking", into the freely available Chemotaxis and Migration Tool software.
7. Quantify velocimetric parameters such as mean velocity, Euclidean distance, migrated distance, and directness using the Chemotaxis and Migration Tool software according to the detailed instructions reported in the freely available User guide of the software³⁶or the steps provided below:
   1. Import datasets from the ImageJ plug-in "Manual Tracking" into the Chemotaxis and Migration Tool software.
   2. Select the desired number of slices (e.g., the number of images used for tracking).
   3. Calibrate the software by setting the x/y pixel size and the time interval. The x/y calibration indicates the edge length of a pixel in µm, while the time interval represents the duration between each slice.
   4. After adjusting the values and parameters, click on Apply Settings.
   5. Plot trajectories and export as image. Click on the Measured values button to see the measured values. Save it.
   6. Click on the Statistics button and save the velocity and directness of the track series.
8. Compare the velocimetric parameters calculated for control cells with those of hemocytes exposed to the test substance at different concentrations.
   NOTE: Migrated distance (measured in µm) refers to the length of the migration path during the observation period (10 min). Euclidean distance (also measured in µm) represents the straight-line distance between the starting point and the endpoint of the cell. Mean velocity (expressed in µm min^-1) is calculated as the ratio of migrated distance to the duration of cell tracking for each cell. Directness is defined as the ratio of Euclidean distance to the accumulated distance for each trajectory.

The study introduces a novel in vitro method for quickly and sensitively assessing the toxicity and ecotoxicity of pollutants, utilizing the motility of Mytilus galloprovincialis hemocytes. Figure 1A-C shows representative time-lapse imaging of hemocytes after 30 min attachment to the bottom of the well. The cells in the figure were stained with Neutral Red just before the motility assessment. Cell movements were monitored using optical microscopy at a rate of one image every minute for 10 min, under 20x magnification, with a multimode reader. Figure 1A-C displays three of the 11 frames captured from the same region of interest (ROI) at 0 min, 5 min, and 10 min. The same cells are numbered in all three figures to highlight their movement, and the numbering remains consistent across the figures to clearly illustrate their trajectories.

The cells show single-cell movements based on lamellipodia activity and fast shape changes. The cells were labeled with the vital dye Neutral red to improve their visualization during tracking. Neutral red is an amphiphilic and weak cationic dye that can easily permeate the cell membrane. Once inside the cells, the dye becomes trapped in the lysosomes due to protonation. Mussel hemocytes, particularly granulocytes, show a well-developed lysosomal system; therefore, they are well visualized by Neutral Red staining, with the cytoplasm rich in several red labeled lysosomes and the nucleus (not marked by the dye) appearing as a translucent hole.

Through the application of the protocol, two main hemocyte morphotypes can be observed according to Udayan et al.²³: (1) spread cells with a bipolar bean shape and sometimes an amoeboid outline, and smaller round-star-shaped cells (cells n = 3 and 10). In both cell types, movement was characterized by continuous shape changes, with ongoing extension and retraction of pseudopodia and filopodia.

The protocol application allows the successful assessment of hemocyte motility by constructing trajectory plots, as reported in Figure 2, and calculating the main velocimetric parameters such as mean velocity and directness. These corresponded respectively to 6.46 ± 0.2 µm/min and 0.56 ± 0.02 (mean ± SEM) for spread cells and 5.18 ± 0.34 µm/min and 0.37 ± 0.03 for small cells, respectively under basal physiological conditions after 30 min attachment to the bottom of the well. Therefore, the protocol allows the classification of different hemocyte populations based on velocimetric parameters.

Through the application of the protocol, alterations in the velocimetric parameter of hemocyte motility can be detected following exposure to drugs or pollutants. Figure 3A-D shows representative results of the in vitro exposure of mussel hemocytes to paracetamol, which is one of the pharmaceuticals released into the environment (PiE) with the highest concentration and the highest detection frequencies in rivers all over the world³¹.

According to Udayan et al.²³, in control cells, a significant increase in velocity with time was observed in both cell morphotypes, suggesting activation of hemocytes during the 24 h culture, presumably evocative of the inflammatory response experimentally induced by withdrawal and plating in the culture environment. When the cells were exposed to the drug, the motility activation of the hemocytes was significantly reduced in spread cells (Figure 3A) after 24 h but not in small round cells (Figure 3C), characterized by a lower mean basal velocity (as assessed at time 0). In addition, the protocol allowed the detection of cell trajectory alterations following drug exposure, as indicated by the significant increase in the directness of small cells after 24 h and the decrease in the directness after 1 h exposure in spread cells (Figure 3C,D).

Figure 1: Representative time-lapse imaging of neutral red-stained hemocytes after 30 min of attachment to the bottom of the well. The cells were stained with the vital dye Neutral Red just prior to motility assessment. Cell movements were monitored using optical microscopy at a rate of one image every minute for 10 min under 20x magnification. For brevity, panels A, B, and C display three of the 11 frames captured from the same region of interest (ROI) at 0 min, 5 min, and 10 min. The same cells are numbered in all three figures to illustrate their movement, and arrows indicate the time intervals. Scale bars represent 100 µm. Please click here to view a larger version of this figure.

Figure 2: Representative trajectory plot of M. galloprovincialis hemocytes. The trajectories shown come from a single well. The trajectories of 25 cells are shown. Please click here to view a larger version of this figure.

Figure 3: Effect of paracetamol exposure (24 h) on velocity and directness of spread cells (A,B) and small round star-shaped cells (C,D). Data are expressed as mean ± SEM. The statistical significance of data was assessed by one-way ANOVA and Tukey's post-test. *p < 0.05 vs. control (p < 0.05). Please click here to view a larger version of this figure.

The protocol described in this work represents a novel in vitro method suitable for the rapid and sensitive assessment of the toxicity of drugs and pollutants based on evaluating the motility of M. galloprovincialis hemocytes and its alterations. Motility is a peculiar aspect of the immune function of these cells^{21,22,23,37,38}, therefore any alteration in cell motility may portend alterations in the immune function of these cells. Motility is an essential cellular function that integrates several cellular processes, such as cell adhesion, cell signaling, cytoskeleton activity, intracellular signaling, and cell volume regulation, and requires the integration and coordination of complex biochemical and biomechanical signals^39,40,41. Each of these processes or the components of each process can be potential targets of the toxic action of drugs and pollutants. Therefore, alterations in motility either in the cell velocity and/or trajectory represent sensitive endpoints integrating the effects on multiple cellular targets.

The protocol is suitable for the in vitro assessment of the effects of test substances either in short-term assays (lasting 1-4 h) or prolonged exposure assays, lasting 24-48 h, enabling rapid screening of pollutants and drugs for potential toxic effects. By using a 96-well plate, the method facilitates simultaneous testing of multiple substances or varying concentrations of a single substance, thus saving time and reagents. In addition, the protocol can also be adapted to be used with other equipment, such as a phase contrast microscope or fluorescent microscope. In this case, cells could be imaged without any stain or using fluorescent vital dyes.

While the method offers novel and sensitive alternatives for toxicological screening, some limitations of the method have to be considered for broader applications. First, a critical aspect of the protocol application is temperature. It is known that mussel hemocytes are sensitive to temperature changes. The effects of temperature on hemocytes are complex, with high and low temperatures negatively impacting their immune functions^42,43,44. As observed during the method setup, the critical phase concerning the effect of temperature on cell motility is the preliminary phase of cell harvesting, adhesion, and cultivation on the substrate, which needs to follow the acclimatization temperature of the organisms. Temperature increases of a few degrees during this first phase above the acclimation temperature can be critical for acquiring the spread cell shape and motility. Therefore, a temperature of 15 °C is recommended for hemocyte culturing. The time-lapse image acquisition for motility assessment was done for 10 min at a room temperature of 20 °C. Based on preliminary observation during the method setup, the exposure of cultured cells for this time interval at 20 °C does not significantly alter hemocyte motility. Another limitation of the method involves the manual tracking of hemocytes, which may introduce user-dependent variability and require significant time and attention to detail. However, the use of the vital dye neutral red improves the visualization of the cells and, in turn, cell tracking through the visualization of the well-developed lysosomal system. The nucleus is not stained by the dye, and it appears as an empty hole that is easily recognizable and easily pointable during cell manual tracking. Another limitation of the method is related to its in vitro nature. While the in vitro condition provides a controlled environment for assessing motility, it may not fully replicate the complex interactions and responses that occur in vivo, particularly within whole-organism immune responses.

In recent years, new approaches and methodologies have come into common use in life sciences, aiming to improve outcomes and reduce reliance on animal testing⁴⁵. The protocol offers an alternative sensitive test for in vitro screening of the effects and mechanisms of action of pollutants and drugs to rapidly screen the toxicological effects of chemical contaminants present, either individually or in mixtures, in environmental matrices. It focuses on a new endpoint, the motility of immune system cells, which can provide an integrated response to the toxicological effects exerted by drugs and pollutants on various cellular targets. In this sense, it expands the range of in vitro tests available in the literature, also from the perspective of an integrated multi-assay approach for application in environmental biomonitoring. Some characteristics of the protocol align it with the 3R's (Replacement, Reduction, and Refinement) criteria, contributing to the reduction of vertebrate in vivo animal testing and to the development of more ethical and efficient toxicological and ecotoxicological exposure toxicity assay. These characteristics include the easy hemolymph collection from mussels in a lowly invasive way, the small amount of hemolymph required for testing, and the use of a multiwell plate, which allows simultaneous exposure of multiple toxicant concentrations and replicates offering significant savings in time and reagents.

The authors have nothing to disclose.

This research was funded by the project "Dipartimento di Eccellenza" awarded to DiSTeBA by the Italian Ministry of University and Research, CUP: F85D18000130001, and by NBFC (National Biodiversity Future Center) funded by European Union NextGenerationEU, PNRR, project n. CN00000033. We also thank BIOforIU infrastructure at the Department of Biological and Environmental Sciences and Technologies of the University of Salento.