Primary Cultures Of Hemocytes Research Articles

Protection of Pacific white shrimp (Penaeus vannamei) against white spot syndrome virus infection by nitric oxide-generating compound S-nitrosoglutathione

Nitric oxide (NO), a highly reactive free radical generated endogenously by nitric oxide synthase (NOS), plays crucial roles in immune defense against a broad range of pathogens including viruses, bacteria, fungi, and protozoans. However, the regulation and effect of NO during viral infection in invertebrates remain largely unexplored. White spot syndrome virus (WSSV) is one of the most severe pathogens affecting the global shrimp farming industry. Our study shows that NO production in Pacific white shrimp Penaeus vannamei decreases during WSSV infection. Reducing NO production using the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly promotes WSSV replication in primary culture of hemocytes isolated from P. vannamei. This effect is partically reversed by L-arginine, a native substrate for NOS, indicating that L-NAME's immunity-reducing action is NO-dependent. In vitro studies also reveal inhibition of WSSV replication in the presence of three NO donors: S-nitrosoglutathione (GSNO), S-nitro-N-acetylpenicillamine (SNAP), and diethylenetriamine nitric oxide adduct (DETA/NO). The involvement of NO in anti-WSSV protection is further demonstrated by an in vivo study where dietary supplementation with GSNO at non-toxic doses increases NO levels and reduces viral load and mortality in WSSV-challenged shrimp. Our results contribute to understanding the antiviral activity of NO in invertebrates and provide a theoretical basis for developing NO-based therapeutic strategies to control WSSV infection.

A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.•A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.•The preparation method of shrimp cell growth medium is successfully established.•Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.

Gene expression in primary hemocyte culture of the Pacific white shrimp Penaeus vannamei infected with different white spot syndrome virus (WSSV) strains

Five previously analyzed white spot syndrome virus (WSSV) strains from northwest Mexico, differing in their genome architecture as well as in virulence, were selected (high virulence JP and LG strains; moderate virulence GVE and DIV strains; and low virulence LC10 strain) to evaluate pathogenesis response in vitro. Expression of phagocytosis-activating protein PAP, manganese superoxide dismutase MnSOD and peroxiredoxin PRX, and two genes of immediate-early expression (IE1 and WSSV304) were measured by qPCR in a primary hemocyte cell culture from Penaeus vannamei at 1, 3, 6, 12, and 24 h post-infection (hpi). PAP expression was significantly higher at 1 and 3 hpi, and JP and LC10 strains induced the highest expression. The response of MnSOD was high at 1 hpi, and a significant increase in PRX expression was detected at 3 hpi, probably due to the occurrence of an oxidative burst; expression levels of MnSOD and PRX were significantly higher at 1 and 3 hpi, respectively, induced by the LG strain (high virulence), suggesting an acute response. In general, expression of most immune-related - genes decreased after the initial hours of infection. Expression levels of IE1 and WSSV304 were exceptionally high at 1 hpi in almost all five WSSV analyzed strains, confirming their efficient mechanism for replication and viral fitness. The results of this study do not show an accurate link between the genome size and WSSV virulence of the strains, albeit the strain with the smallest genome showed the highest virulence. All strains induced an early immune response in heterogeneous ways.

Zebrafish U6 promoter driving short‐hairpin RNA expression for PmRab7 knockdown to inhibit yellow head virus infection in shrimp hemocytes

AbstractRNA interference (RNAi) was investigated as potential antiviral strategy to mitigate losses in shrimp aquaculture. With this aim, an effective short‐hairpin RNA (shRNA) expressed intracellularly from bacterial vectors incorporating eukaryotic promoters offers an alternative to an injected synthetic small‐interfering RNA (siRNA) or long double‐stranded RNA (dsRNA). The vector‐based RNAi designed to contain a U6 snRNA polymerase III promoter sequence from zebrafish (Danio rerio) for driving shRNA was previously introduced to shrimp cell extract and was able to express the shRNA. Here, four DNA plasmids containing putative zebrafish U6 promoter to drive shRNA against PmRab7‐specific sequence were used to transfect primary hemocyte culture. The cells were subsequently infected by Yellow head virus (YHV). As results, when analyzed by RT‐PCR at 24 hr post‐transfection, Penaeus monodon Rab7 (PmRab7) mRNA transcription was inhibited most significantly by the pshPmRab7‐2 construct. YHV replication in primary shrimp hemocyte cultures was shown to be inhibited substantially by this PmRab7 gene‐specific hpRNA construct. Transfection of pshPmRab7‐2 construct also reduced YHV replication most effectively when analyzed similarly between 24 hr until 72 hr post‐infection. These results demonstrate a potential application of DNA‐based shRNA construct as effective molecule for antiviral therapy in shrimp.

Cellular Immune Response of an Endemic Lake Baikal Amphipod to Indigenous Pseudomonas sp.

Studies of invertebrates have shown that the internal environment of crustaceans is not always sterile in normal conditions, and in many species, it can be populated by microorganisms even in the absence of any visible pathological processes in the body. This observation raises the question of whether genetically modified indigenous hemolymph microorganisms can be used for biotechnological purposes inside the crustacean either as local producers of some compounds or as sensors to physiological parameters. In this study, we tested the ability of the bacteria isolated from the hemolymph of the amphipod Eulimnogammarus verrucosus to hide from the cellular immune response of the host as the most important feature for their potential long-term application in vivo. 16S rDNA amplicon sequencing revealed five common bacterial genera in all analyzed samples of the amphipod hemolymph, among which Pseudomonas is most easily subjected to genome modification and, thus, the most prospective for biotechnological application. Cultivation of Pseudomonas gave us a number of strains undoubtedly derived from the amphipod hemolymph, and one of them (belonging to the Pseudomonas fluorescens group) was chosen for further tests. The primary culture of amphipod hemocytes was used to analyze the immunogenicity of the strain and showed a pronounced reaction of the immune cells to a high amount of the bacteria within six hours. This result indicates that modulation of cellular immune response to metabolically active bacterial cells is not mandatory for the survival and wide distribution of these microorganisms in the hemolymph of numerous amphipod individuals.

In vitro and in vivo immunologic potentiation of herb extracts on shrimp (Litopenaeus vannamei)

In vitro and in vivo effects of Astragalus polysaccharide (APS), chlorogenic acid (CGA) and berberine (BBR) on shrimp (Litopenaeus vannamei) were studied. In vitro test showed that the combination of APS and BBR and the combination of APS and CGA have strong immune enhancement effects and no lysosomal membrane damage on hemocyte. Then, feeding experiment was proceeded to optimize the concentrations of compound herbal extracts. Four diets containing G1-G4(0.5 g kg −1 APS + 0.5 g kg −1 BBR, 1.0 g kg −1 APS +1.0 g kg −1 BBR, 0.5 g kg −1 APS +0.5 g kg −1 CGA, 1.0 g kg −1 APS + 1.0 g kg −1 CGA) associated with the control group (common diet) were compared and determined their biomolecule damage to hepatopancreas including DNA damage, lipid peroxidation and protein carbonyl. The results indicated that G3 (0.5 g kg −1 APS +0.5 g kg −1 CGA) showed higher total hemocyte counts, phagocytic activities, antibacterial activities and bacteriolytic activities during 6 days feeding, and without biomolecule damages after 6 days post-withdrawal. Therefore, the appropriate immunostimulants formula in this study was the combination of 0.5 g kg −1 APS and 0.5 g kg−1 CGA, which was used for 6 days followed by 6 days post-withdrawal. Additionally, our study provides new support for screening composite immunostimulants formula by using primary shrimp hemocyte culture.

Effective suppression of yellow head virus replication in Penaeus monodon hemocytes using constitutive expression vector for long-hairpin RNA (lhRNA)

Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called “pLVX-lhRdRp”, against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24–72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.

In vitro effects of glyphosate-based herbicides and related adjuvants on primary culture of hemocytes from Haliotis tuberculata

Glyphosate-based herbicides are among the most produced and widely-used herbicides. Studies have shown that commercial formulations and adjuvants may be more toxic to non-target organisms than the active ingredients alone, but the mechanisms of action of these chemicals remain unclear. The aim of this study was to investigate the in vitro effects of glyphosate, a commercial formulation and adjuvant alone using primary culture of hemocytes from the European abalone Haliotis tuberculata, a commonly farmed shellfish. Glyphosate was found to have negligible effects on viability, phagocytic activities and lysosome stability even with very high doses (i.e. 100 mg L−1). By contrast, greater effects on viability were observed for the commercial formulation and adjuvant alone, with EC50 values of 41.42 mg L−1 and 1.85 mg L−1, respectively. These results demonstrate that the toxic sublethal effects (i.e. phagocytic activity and destabilization of lysosomal membranes) of formulated glyphosate came from adjuvants and suggest they may be related to cell and organelle membrane destabilization.

In vitro toxicity testing in hemocytes of the marine mussel Mytilus galloprovincialis (L.) to uncover mechanisms of action of the water accommodated fraction (WAF) of a naphthenic North Sea crude oil without and with dispersant

Dispersants used in oil spills could result toxic to marine organisms and could influence the toxicity of oil compounds. The aim of this work was to uncover the mechanisms of action of the water accommodated fraction (WAF) of a naphthenic North Sea crude oil produced at 10, 15 and 20 °C without and with the dispersant Finasol OSR52 (WAF and WAFD, respectively) using hemocytes of the marine mussel Mytilus galloprovincialis. Primary cultures of hemocytes were exposed in glass-coated microplates to different WAF or WAFD dilutions (0.25, 2.5, 25, 50 and 100%) and to the dispersant alone at the same concentrations present in the WAFD dilutions (1.25, 12.5, 125, 250 and 500 mg/L). Of the two in vitro approaches tested, the second one was selected which involved exposure of hemocytes for 4 h to unfiltered WAF, WAFD and dispersant dilutions without cell culture media. WAF decreased hemocytes viability only at the highest dilution whereas WAFD and the dispersant alone were cytotoxic at the three highest concentrations. Temperature of production of WAF, WAFD and dispersant did not influence their cytotoxicity to hemocytes. WAF increased ROS production and MXR transport activity in hemocytes. Exposure to WAFD and dispersant increased ROS production, provoked plasma membrane and actin cytoskeleton disruption and decreased phagocytic activity. In conclusion, the dispersant tested was toxic to mussel hemocytes and it greatly increased the toxicity of WAFD. The present data could be useful for the environmental risk assessment of oil spills and their remediation strategies in the marine environment.

Omim][BF4]-mediated toxicity in mussel hemocytes includes its interaction with cellular membrane proteins

The current study is based on the increasing demand for the assessment of ionic liquid (IL)-mediated aquatic toxicity. Specifically, although a lot of studies have been performed so far, investigating IL-mediated adverse effects on numerous aquatic organisms, little is known about their mode of action. Given that the use of in vitro models is considered as a reliable tool for determining the mediated biological effects, the modulation of specific biochemical pathways and the onset of various forms of damage with great precision and reproducibility, mixed primary cultures of mussel Mytilus galloprovincialis hemocytes were used for investigating whether 1-octyl-3-methylimidazolium tetrafluoroborate ([omim][BF4]) mediated toxicity is related to its interaction with cellular membrane proteins. Specifically, [omim][BF4]-mediated cytotoxic, oxidative and genotoxic effects were investigated in mussel hemocytes before and after pre-treatment of cells with non-toxic concentration of guanidine hydrochloride (1 mM GndHCl). The results showed that [omim][BF4] at concentrations ranging from 0.7 to 1.75 μM can induce cytotoxic (almost <50% reduction of cell viability), oxidative (increased levels of O2•− production and lipid peroxidation by-products) and genotoxic (increased levels of DNA damage) effects, while cells pre-treated with 1 mM GndHCl showed a significant attenuation of IL’s toxic potency in all cases. According to the latter, the current study showed that [omim][BF4]-mediated toxicity could be related not only to its well-known interaction with membrane lipid bilayers, but also to its interference with membrane proteins. Using GndHCl, a chaotropic agent that disrupts the hydrogen bonding network and the stability of membrane proteins via its interference with the intramolecular interactions mediated by non-covalent forces on cellular membranes, it was firstly shown that altering the membrane integrity as well as the native state of cellular membrane proteins, by weakening the hydrophobic effect, could attenuate the possible interaction of [omim][BF4] with cellular membranes and the concomitant induction of protein-based intracellular processes, commonly linked with the induction of severe cellular damage.

Synthesis methods influence characteristics, behaviour and toxicity of bare CuO NPs compared to bulk CuO and ionic Cu after in vitro exposure of Ruditapes philippinarum hemocytes

Copper oxide (CuO) nanoparticles (NPs) are increasingly investigated, developed and produced for a wide range of industrial and consumer products. Notwithstanding their promising novel applications, concern has been raised that their increased use and disposal could consequently increase their release into marine systems and potentially affect species within. To date the understanding of factors and mechanisms of CuO (nano-) toxicity to marine invertebrates is still limited. Hence, we studied the characteristics and behaviour of two commercially available CuO NPs of similar size, but produced employing distinct synthesis methods, under various environmentally and experimentally relevant conditions. In addition, cell viability and DNA damage, as well as gene expression of detoxification, oxidative stress, inflammatory response, DNA damage repair and cell death mediator markers were studied in primary cultures of hemocytes from the marine clam Ruditapes philippinarum and, where applicable, compared to bulk CuO and ionic Cu (as CuSO4) behaviour and effects. We found that the synthesis method can influence particle characteristics and behaviour, as well as the toxicity of CuO NPs to Ruditapes philippinarum hemocytes. Our results further indicate that under the tested conditions aggregating behaviour influences the toxicity of CuO NPs by influencing their rate of extra- and intracellular dissolution. In addition, gene expression analysis identified similar transcriptional de-regulation for all tested copper treatments for the here measured suite of genes. Finally, our work highlights various differences in the aggregation and dissolution kinetics of CuO particles under environmental (marine) and cell culture exposure conditions that need consideration when extrapolating in vitro findings.

Investigating the establishment of primary cultures of hemocytes from Mytilus edulis.

Anthropogenic influences on the environment have been become a focal point for many social and political endeavors. With an ever-increasing rate of new contaminants being introduced into the environment every year, regulatory policies have begun to shift to prevention rather than mitigation. However, current in vivo testing strategies, in addition to ethical considerations, are too expensive and time consuming to adequately screen potential contaminants within a realistic timeframe. As a result, in vitro testing on cell cultures has been identified as an ideal alternative testing strategy for emerging contaminants. In the context of ecotoxicology, in vitro testing has had limited use particularly with marine invertebrates like the marine mussel Mytilus edulis mainly due to difficulties in establishing longer term cell cultures and cell lines. The aim of this study was to define an optimal technique (extraction and maintenance) for establishing a primary cell culture on M. edulis hemocytes that could be used for screening contaminants.

The feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants

An analysis of the immune activity of herbal extract in vivo and in vitro assays was performed to explore the feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants. In vitro experiment was conducted by adding herbal extract into the medium of primary cultured hemocytes and incubating for 24h. In vivo experiment was carried out by feeding shrimp with diets incorporated with herbal extract for 21 days. Challenge test can be used to directly reflect comprehensive immune capacity of organisms. Four immune indexes, total hemocyte counts (THC and lysozyme, phenoloxidase (PO), and phagocytic activity were examined both in vitro and in vivo. The results showed that effects of herbal extract on THC or lysozyme activity were different between in vitro and in vivo. On the contrary, effects of herbal extract on PO activity and phagocytic activity in vitro and in vivo showed a strong correlation. Herbal extract, which enhanced PO activity and phagocytic activity in vitro, had ultimate immune-promoting function in vivo. Therefore, it was feasible to use primary hemocyte culture to screen herbal immunostimulants by determining the effects of herbal immunostimulants on PO and phagocytic activities.

A simple in vitro method to evaluate the toxicity of functional additives used in shrimp aquaculture

To mitigate the economic losses provoked by disease outbreaks, shrimp producers employ therapeutic additives. However, important issues such as the toxicity of these products on shrimp are not always considered. In vivo toxicity assays require a lot of time and large economic and physical resources. Here, we describe an in vitro procedure to evaluate the toxicity of functional additives, used in the production of shrimp Penaeus vannamei. This method adapted the cell viability assay based on the reduction of tetrazolium salts (MTT) to primary cell cultures of shrimp hemocytes.•A simple and reliable tool that requires few physical and economic resources to evaluate in short time (6?h) the cytotoxic effect of therapeutic products and additives to be included in shrimp culture•This inexpensive method requires only a modified Hank's balanced salt solution (HBSS) containing Ca2+ and Mg2+ to keep hemocytes metabolically active to successfully carry out the cytotoxicity assay•This toxicity in vitro assay does not require exposure of the shrimp to compounds at toxic concentrations.

Primary culture of Zhikong scallop Chlamys farreri hemocytes as an in vitro model for studying host-pathogen interactions

Primary cultured cells can be a useful tool in studies on physiology, virology, and toxicology. Hemocytes play an important role in animal rapid response to pathogen invasion. In this study, an appropriate medium for primary culture of hemocytes of the bivalve Chlamys farreri was developed by adding 5% fetal bovine serum and 1% C. farreri serum to Leibovitz L-15 medium. These primary cultured hemocytes were maintained for more than 40 d in vitro and were classified into 3 types: (1) granulocytes containing numerous granules in the cytoplasm, (2) hyalinocytes with no or few granules, (3) a small percentage of macrophage-like cells. Furthermore, the primary cultured hemocytes were observed to be sensitive to bacterial and viral challenges. These hemocytes could phagocytose the bacterium Vibrio anguillarum, and presented cytopathic effects on the extracellular products (ECPs) of V. anguillarum; the mRNA level of QM, which plays an important role in immune response, also significantly increased 12 h after infection. When these hemocytes were challenged with ostreid herpesvirus 1 (OsHV-1), virus particles and empty capsids in the cells infected for 48 h were observed by transmission electron microscopy, and the QM mRNA level increased significantly at 12 h and 24 h following OsHV-1 challenge. This primary culture system is available for C. farreri hemocytes which can be used in the future to study host-pathogen interactions.

Antiviral Activity of Ctn[15-34], A Cathelicidin-Derived Eicosapeptide, Against Infectious Myonecrosis Virus in Litopenaeus vannamei Primary Hemocyte Cultures

The shrimp farming has been converted into a mature aquaculture industry dealing with over millions of metric tonnes of processed commodities. Nevertheless, the global shrimp productions are constantly threatened by disease outbreaks, mainly triggered by rapidly disseminating viruses. Infectious myonecrosis virus (IMNV) is one of these epizootic agents affecting shrimp production in Brazil, of which no treatment exists. Herein, the antiviral activity against IMNV of an eicosapeptide, named Ctn[15-34], derived from a member of the cathelicidin family of antimicrobial peptides, was demonstrated. Cultures of hemocytes from Litopenaeus vannamei were established that support IMNV replication and infectivity titration. The cytotoxic effect of IMNV in culture and the in vitro anti-IMNV activity of Ctn[15-34] were assessed using a high-sensitive fluorescent-based method in combination with quantitative PCR. The Ctn[15-34] (<12.5µM) neutralized the toxic effects of IMNV at loads sufficient to kill 50% of shrimp hemocytes. This study reported for the first time the replication of IMNV in vitro and the employment of a straightforward methodology to assess cell viability and viral/antiviral activities. In addition, it provided the basis for the development of the anti-infective multi-effector Ctn[15-34] eicosapeptide and analogs as components of antiviral formulations against shrimp viral diseases.

Impact of Common Cytostatics on DNA Damage in Freshwater Mussels Uniopictorum and Unio tumidus

The impact of etoposide (ETO), vincristine sulfate (VIN), and cisplatin (CP) on the DNA damage level was studied in vivo and in vitro on hemocytes of freshwater mussels Unio sp. (Unio pictorum/Unio tumidus) using the alkaline comet assay. For in vivo experiments, the mussels were exposed in static system for 72 h. For in vitro experiments, treatment was performed in primary cultures of hemocytes for 22 h. The DNA damage level was analyzed by Comet Assay IV software. Increase of damage was detected for ETO (40 and 100 μM) in vivo and in vitro and for VIN (0.04 and 0.1 μM) in vivo. CP did not induce an increase of DNA damage, but post‐treatment with hydrogen peroxide indicated existence of DNA crosslinks in specimens treated with 4 μM CP. Effective concentrations of selected cytostatics are at least one (CP) or several orders of magnitude (ETO, VIN) higher comparing to the concentrations in hospital wastewater.

Reactive oxygen intermediates promotes the expression of an antimicrobial peptide in the northern scallop Argopecten purpuratus hemocytes in vitro

Reactive oxygen intermediates are metabolites normally produced on aerobic metabolisms and has been linked to processes like oxidative stress and aging. Despite this negative paradigm about ROI, another set of evidence in mammals suggest that these reactive intermediates can act as important second messengers in a variety of cellular signaling processes such as those involved on the immune response. Primary cultures of hemocytes from the Chilean scallop Argopecten purpuratus were developed to study a possible link between the production of reactive oxygen intermediates and the expression of an antimicrobial peptide, big defensin, under a context of immune response. A modified Leibovitz L-15 medium supplemented with 50% of sterile plasma from scallop was essential for hemocyte adhesion an survival. The ability of these primary cultures to develop an immune response was confirmed by phagocytic activity and ROI production essays. The antimicrobial peptide big defensin was up-regulated in response to the β-glucan zymosan and this up regulation could be diminished near to constitutive levels by the treatment of cultured hemocytes with the antioxidant trolox for 3 hours before the immune induction. This results constitutes the first evidence on invertebrates that supports a rol of intracellular oxygen intermediates as promoters of the expression of an antimicrobial effector during an immune response.

A novel Eriocheir sinensis primary hemocyte culture technique and its immunoreactivity after pathogen stimulation

Abstract In order to advance immunological research, a modified primary cell culture method for hemocytes from Eriocheir sinensis (Chinese mitten crab) was established using L-15 medium lacking FBS and CO 2 at 28 °C. Under these conditions, cultured cells were stably propagated for 30 days. To evaluate cellular activity, expression levels of certain critical immune-related genes, melanization of the hemocytes, and the phosphorylation status of key molecules in cell signaling pathways were analyzed. mRNA expression level of EsDWD1, EsLecG and EsToll2 was upregulated after LPS, PGN and GLU stimulation. Meanwhile, stimulated by Vibrio parahemolyticus led to the melanization of the hemocytes, and the melanization was more obviously with the increasing concentration of bacteria. Moreover, exposure to V. parahemolyticus significantly induced JNK, ERK and p38 phosphorylation, with the greatest extent of phosphorylation observed 30 min post-induction. Collectively, our results establish a technique for E. sinensis hemocyte primary culture and demonstrate appropriate immunological response, regulation and activity. Statement of relevance We declare that we have no conflict of interest statement.

Assessment of cytotoxic and immunomodulatory properties of four antidepressants on primary cultures of abalone hemocytes (Haliotis tuberculata)

Pharmaceutical compounds like antidepressants found in surface waters raise concerns due to their potential toxicity on non-target aquatic organisms. This study aimed at investigating the in vitro cytotoxicity and immunomodulatory properties of four common antidepressants, namely Amitriptyline, Clomipramine, Citalopram and Paroxetine, on primary cultures of abalone hemocytes (Haliotis tuberculata), after 48h-exposure. Effects on immunocompetence (phagocytosis, levels of reactive oxygen species, esterase activity and lysosomal membrane destabilization) were assessed. Results obtained by MTT assays revealed that acute toxicity is unlikely to occur in the environment since the LC50s of the four antidepressants are at the mg/L level. The different immunological endpoints displayed a biphasic response, with an increase at the lowest concentration (i.e. 1μg/L) followed by a decrease at higher concentrations. Overall, Amitriptyline and Clomipramine, the two tricyclic antidepressants, had higher immunomodulatory capacities than the two selective serotonin reuptake inhibitors Citalopram and Paroxetine. Amitriptyline was the most potent and Citalopram the least potent drug in altering immune function in H. tuberculata.