Authors
Contact Us

Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 assessment1,2,3,4. 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 biota1,5,6,7. Several effect-based methods use bivalve mollusks as test organisms in environmental monitoring and assessment8,9. 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 conditions8,9,10. Therefore, several environmental guidelines use bivalve species as standardized test species10,11.

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 induction12,13,14,15. Hemocytes, the immunocompetent hemolymphatic cells, are widely used to study the toxicological impacts of environmental pollutants in bivalve mollusks4,13,16,17. 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 burst18,19,20. Hemocytes are intrinsically motile cells21,22,23 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 molluscs21,25,26,27.

Despite the fundamental importance of motility in the physiology of hemocytes, very few studies have investigated the sensitivity of hemocyte motility to environmental chemical pollutants23,28,29,30. 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 edulis21,22,23,28, and already described in human immune cells31. Hemocyte motility has recently been demonstrated to be sensitive to chemical stressors23,28. 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.

Protocol

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.

Results

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 mic...

Discussion

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 cells21,22,23,37,38, therefore any ...

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
0.2 µm filter (diameter 25 mm)ABLUO labware
2.5 ml hypodermic syringe needdle 22GRays2522CM32labware
96-well flat-bottom polystyrene TC-treated microplateCorning3916labware
CaCl2.2H2OMerk (Sigma - Aldrich) C3881-1KGChemical
Chemotaxis and Migration Tool software (Ibidi GmbH)software
Cytation 5 Agilent BioTeckCytation 5Equipment: Cell imaging multimode reader
Dimethyl sulfoxide (DMSO) Merk (Sigma - Aldrich) 472301Solvent
Falcon 15 mL Tube Conical BottomCorning352196labware
H3BO3Merk (Sigma - Aldrich) B0394Chemical
Hemocytometer Fast read 102BiosigmaBVS100labware
HEPES (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid)Merk (Sigma - Aldrich) H3375-500GChemical
ImageJ softwareNIHsoftware
KBrMerk (Sigma - Aldrich) P9881Chemical
KClMerk 104936Chemical
L-glutamineMerk (Sigma - Aldrich) G7513Essential amino acid for cell culture medium
MgCl2·6H2OMerk (Sigma - Aldrich) M2670Chemical
MgSO4Merk (Sigma - Aldrich) M7506Chemical
Microscope Nikon Eclipse E600NikonEquipment: Cell imaging 
Na2SO4Riedel-de Haen31481Chemical
NaClMerk (Sigma - Aldrich) 31434-1KG-RChemical
NaFFluka71519 500gChemical
NaHCO3Merk (Sigma - Aldrich) S5761-1KGChemical
Neutral RedMerk (Sigma - Aldrich) N4638-1GVital cell dye
Penicillin/StreptomycinMerk (Sigma - Aldrich) P0781-100MLAntibiotics for cell culture
SrCl2·6H2OMerk (Sigma - Aldrich) 255521Chemical
Trypan blue Merk (Sigma - Aldrich) T8154Cell dye

References

  1. Di Paolo, C., et al. Bioassay battery interlaboratory investigation of emerging contaminants in spiked water extracts - Towards the implementation of bioanalytical monitoring tools in water quality assessment and monitoring. Water Res. 104, 473-484 (2016).
  2. Brack, W., et al. Effect-based methods are key. The European Collaborative Project SOLUTIONS recommends integrating effect-based methods for diagnosis and monitoring of water quality. Environ Sci Eur. 31 (1), 10 (2019).
  3. Lionetto, M. G., Caricato, R., Erroi, E., Giordano, M. E., Schettino, T. Potential application of carbonic anhydrase activity in bioassay and biomarker studies. Chem Ecol. 22 (sup1), S119-S125 (2006).
  4. Lionetto, M. G., Caricato, R., Giordano, M. E. Pollution biomarkers in the framework of marine biodiversity conservation: State of art and perspectives. Water. 13 (13), 1847 (2021).
  5. Connon, R. E., Geist, J., Werner, I. Effect-based tools for monitoring and predicting the ecotoxicological effects of chemicals in the aquatic environment. Sensors. 12 (9), 12741-12771 (2012).
  6. Fabbri, E., Valbonesi, P., Moon, T. W., León, V. M., Bellas, J. Pharmaceuticals in the marine environment: Occurrence, fate, and biological effects. Contaminants of Emerging Concern in the Marine Environment. Chapter 2, 11-71 (2023).
  7. Lionetto, F., et al. Autofluorescence of model polyethylene terephthalate nanoplastics for cell interaction studies. Nanomaterials. 12 (9), 1560 (2022).
  8. Damiens, G., Gnassia-Barelli, M., Loquès, F., Roméo, M., Salbert, V. Integrated biomarker response index as a useful tool for environmental assessment evaluated using transplanted mussels. Chemosphere. 66 (3), 574-583 (2007).
  9. Ogidi, O. I., Onwuagba, C. G., Richard-Nwachukwu, N., Izah, S. C., Ogwu, M. C., Hamidifar, H. Biomonitoring tools, techniques and approaches for environmental assessments. Biomonitoring of Pollutants in the Global South. , (2024).
  10. Rosner, A., et al. A broad-taxa approach as an important concept in ecotoxicological studies and pollution monitoring. Biol Rev. 99 (1), 131-176 (2024).
  11. Gagné, F., Burgeot, T. Bivalves in ecotoxicology. Encyclopedia of Aquatic Ecotoxicology. , 247-258 (2013).
  12. Caricato, R., et al. Carbonic anhydrase integrated into a multimarker approach for the detection of the stress status induced by pollution exposure in Mytilus galloprovincialis: A field case study. Sci Total Environ. 690, 140-150 (2019).
  13. Calisi, A., et al. Morphological and functional alterations in hemocytes of Mytilus galloprovincialis exposed in high-impact anthropogenic sites. Mar Environ Res. 188, 105988 (2023).
  14. Viarengo, A., Burlando, B., Evangelisti, V., Mozzone, S., Dondero, F., Garrigues, F., Barth, H., Walker, C. H., Narbonne, J. F. Sensitivity and specificity of metallothionein as a biomarker for aquatic environment biomonitoring. Biomarkers in Marine Organisms. , 29-43 (2001).
  15. Bocchetti, R., et al. Contaminant accumulation and biomarker responses in caged mussels, Mytilus galloprovincialis, to evaluate bioavailability and toxicological effects of remobilized chemicals during dredging and disposal operations in harbor areas. Aquat Toxicol. 89 (3), 257-266 (2008).
  16. Calisi, A., Lionetto, M. G., Caricato, R., Giordano, M. E., Schettino, T. Morphometric alterations in Mytilus galloprovincialis granulocytes: A new biomarker. Environ Toxicol Chem. 26 (6), 1435-1441 (2007).
  17. Burgos-Aceves, M. A., Faggio, C. An approach to the study of the immunity functions of bivalve haemocytes: Physiology and molecular aspects. Fish Shellfish Immunol. 67 (6), 513-517 (2017).
  18. Canesi, L., et al. Tyrosine kinase-mediated cell signaling in the activation of Mytilus hemocytes: Possible role of STAT-like proteins. Biol Cell. 95 (9), 603-613 (2003).
  19. Novas, A., Barcia, R., Ramos-Martínez, J. I. Nitric oxide production by hemocytes from Mytilus galloprovincialis shows seasonal variations. Fish Shellfish Immunol. 23 (4), 886-891 (2007).
  20. Pruzzo, C., Gallo, G., Canesi, L. Persistence of vibrios in marine bivalves: The role of interactions with hemolymph components. Environ Microbiol. 7 (5), 761-772 (2005).
  21. Le Foll, F., et al. Characterization of Mytilus edulis hemocyte subpopulations by single-cell time-lapse motility imaging. Fish Shellfish Immunol. 28 (2), 372-386 (2010).
  22. Rioult, D., Lebel, J. -. M., Le Foll, F. Cell tracking and velocimetric parameters analysis as an approach to assess activity of mussel (Mytilus edulis) hemocytes in vitro. Cytotechnology. 65 (5), 749-758 (2013).
  23. Udayan, G., Giordano, M. E., Pagliara, P., Lionetto, M. G. Motility of Mytilus galloprovincialis hemocytes: Sensitivity to paracetamol in vitro exposure. Aquat Toxicol. 265, 106779 (2023).
  24. Germain, R. N., Robey, E. A., Cahalan, M. D. A decade of imaging cellular motility and interaction dynamics in the immune system. Science. 336 (6089), 1676-1681 (2012).
  25. Ivanina, A., Falfushynska, H., Beniash, E., Piontkivska, H., Sokolova, I. Biomineralization-related specialization of hemocytes and mantle tissues of the Pacific oyster Crassostrea gigas. J Exp Biol. 220 (18), 3209-3221 (2017).
  26. Furukawa, F., et al. Hemocyte migration and expression of four Sox genes during wound healing in Pacific abalone, Haliotis discus hannai. Fish Shellfish Immunol. 117, 24-35 (2021).
  27. Lau, Y., Gambino, L., Santos, B., Espinosa, E., Allam, B. Regulation of oyster (Crassostrea virginica) hemocyte motility by the intracellular parasite Perkinsus marinus: A possible mechanism for host infection. Fish Shellfish Immunol. 78, 18-25 (2018).
  28. Gendre, H., et al. Modulation of hemocyte motility by chemical and biological stresses in Mytilus edulis and Dreissena polymorpha. Fish Shellfish Immunol. 139, 108919 (2023).
  29. Kaloyianni, M., et al. Oxidative effects of inorganic and organic contaminants on haemolymph of mussels. Comp Biochem Physiol C: Toxicol Pharmacol. 149 (4), 631-639 (2009).
  30. Sendra, M., et al. Nanoplastics: From tissue accumulation to cell translocation into Mytilus galloprovincialis hemocytes. Resilience of immune cells exposed to nanoplastics and nanoplastics plus Vibrio splendidus combination. J Hazard Mater. 388, 121788 (2020).
  31. Pizzagalli, D. U., Pulfer, A., Thelen, M., Krause, R., Gonzalez, S. F. In vivo motility patterns displayed by immune cells under inflammatory conditions. Front Immunol. 12, 804159 (2021).
  32. ASTM D1141-98. . Standard Practice for Preparation of Substitute Ocean Water. , (2021).
  33. Martínez-Gómez, C., Bignell, J., Lowe, D. Lysosomal membrane stability in mussels. ICES Techniques in Marine Environmental Sciences. 56, 1-41 (2015).
  34. UNEP/RAMOGE. Manual on the biomarkers recommended for the MED POL Biomonitoring Programme. UNEP. , (1999).
  35. Strober, W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 111, A3.B.1-A3.B.3 (2015).
  36. Asano, Y., Horn, E. . Instructions Chemotaxis and Migration Tool 2.0 Version 1.1. , (2013).
  37. Wilkinson, J. L., et al. Pharmaceutical pollution of the world's rivers. Proc Natl Acad Sci USA. 119 (8), e2113947119 (2022).
  38. Parisi, M. G., Pirrera, J., La Corte, C. Effects of organic mercury on Mytilus galloprovincialis hemocyte function and morphology. J Comp Physiol B. 191 (1), 143-158 (2021).
  39. Pollard, T. D., Borisy, G. G. Cellular motility driven by assembly and disassembly of actin filaments. Cell. 112 (4), 453-465 (2003).
  40. Bailly, M., Condeelis, J. Cell motility: Insights from the backstage. Nat Cell Biol. 4 (5), E292-E294 (2002).
  41. Blanchoin, L., Boujemaa-Paterski, R., Sykes, C., Plastino, J. Actin dynamics, architecture, and mechanics in cell motility. Physiol Rev. 94 (1), 235-263 (2014).
  42. Mosca, F., et al. Effects of high temperature and exposure to air on mussel (Mytilus galloprovincialis, Lmk 1819) hemocyte phagocytosis: Modulation of spreading and oxidative response. Tissue Cell. 45 (3), 198-203 (2013).
  43. Beaudry, A., et al. Effect of temperature on immunocompetence of the blue mussel (Mytilus edulis). J Xenobiot. 6 (1), 5889 (2016).
  44. Wu, F., et al. Combined effects of seawater acidification and high temperature on hemocyte parameters in the thick shell mussel Mytilus coruscus. Fish Shellfish Immunol. 56, 554-562 (2016).
  45. Schmeisser, S., et al. New approach methodologies in human regulatory toxicology - Not if, but how and when. Environ Int. 178, 108082 (2023).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

BiologyMytilus GalloprovincialisHemocytesCell MotilityImmune ResponseCell AdhesionDrug ExposurePollutant EffectsMicroplate AssayTime lapse MicroscopyCell Tracking3Rs CriteriaNon invasive Collection

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved