Introduction

Carrageenans are a diverse family of high molecular weight polysaccharides of marine origin, extracted from the cell walls of red seaweeds (Rhodophyceae). Traditionally, carrageenans have been used as food additives due to their gelling and stabilizing properties, as well as their ability to modify texture and improve sensory characteristics (BeMiller 2019). These polysaccharides are composed of alternating D-galactose and L-3,6-galactose subunits linked by α-(1, 3) and β-(1, 4) glycosidic bonds, and they can have varying degrees of sulphation, which leads to different physicochemical properties (Necas and Bartosikova 2013). There are three main types of carrageenans, namely κ-, ι-, and λ-carrageenan, depending on the number and position of sulphates per disaccharide unit (Necas and Bartosikova 2013). κ-carrageenan has one sulphate group and can form rigid gels when bound to monovalent metal ions such as K+, Rb+ or Cs+ (Running et al. 2012; Cao et al. 2018). ι-carrageenan has two sulphate groups and forms soft gels when bound to divalent metal ions such as Ca2+. λ-carrageenan has three sulphate groups and can bind to ferric ions to form a weak gel or may not gel at all in solution (Running et al. 2012; Cao et al. 2018).

Other studies suggest that carrageenans can trigger inflammation in mammalian cells, which has raised concerns about their safety as food additives. Therefore, further research is needed to fully understand the potential health effects of carrageenans. The inflammatory in vivo models of carrageenan, as well known as carrageenan-induced paw edema, consist in the injection of carrageenan in the paw of rodents (rats, mice and guinea pig) with the characteristic production of hyperalgesia, erythema and edema formation (Winter et al. 1962; Morris 2003). In this context, the presence of sulphate groups appears to be of high relevance in the triggering of inflammation, being λ-carrageenan, the main form used for this purpose (Winter et al. 1962; Fujiki et al. 1997; Morris 2003; Bhattacharyya et al. 2010; Sokolova et al. 2014). Some in vitro assays have greatly helped to elucidate the inflammatory signaling pathways as well as elucidate the mechanism of action of carrageenan. For example, two mouse macrophage cell lines (RAW 264.7 and 23ScCr), the human colonic epithelial cell line NCM 460 and primary cultures of human colonic cells have been used to identify several receptors such as Toll-like receptors (TLRs) and B-cell lymphoma/leukemia B-cell protein 10 (BCL10) capable of binding carrageenan (Bhattacharyya et al. 2008, 2010). Both receptors are able to stimulate the NF-κB transduction pathway with subsequent activation of proinflammatory genes and cytokines (Bhattacharyya et al. 2008, 2010). Other in vivo studies indicated that carrageenan induces inflammation in the human gastrointestinal tract when used as a food additive, causing some controversy (Tobacman 2001; Bixler 2017). However, the results of recent in vivo studies in humans with carrageenan in food demonstrate that it does not produce inflammation in the gastrointestinal tract or induce the innate immune system, due to its low absorption and interaction with cells (Necas and Bartosikova 2013; Younes et al. 2018). Likewise, and supporting these data, studies performed with two human intestinal cell lines (HT-29 and HCT-8) did not find permeability to carrageenan through intestinal epithelial cells, cytotoxicity, or induction of interleukin-8 (IL-8), interleukin-6 (IL-6) or monocyte chemoattractant protein-1 (MCP-1) (McKim et al. 2016). In addition, it has also been shown that carrageenan was able to bind strongly to serum proteins, in particular to fetal serum proteins used in cell cultures, which could be involved in its mode of action (McKim et al. 2015). Moreover, this characteristic of binding and inhibiting certain proteins by preventing their interaction with other proteins or receptors, together with its stabilizing properties (gelling, thickening, and binding) suggest that it could be used to bind drugs with different effects such as bactericidal or cytotoxic (Das et al. 2021). However, despite all the progress made in understanding the inflammatory mechanisms of carrageenan in mammals, as well as many of its properties, so little is known to date about its possible effects in fish. In this context, some in vivo studies in fish [zebrafish (Danio rerio), Nile tilapia (Oreochromis niloticus), pacu (Piaractus mesopotamicus) or gilthead seabream (Sparus aurata)] have shown the potential of carrageenan to induce inflammation (Timur et al. 1977; Matushima and Mariano 1996; Fujiki et al. 1997; Martins et al. 2006; Huang et al. 2014; Ribas et al. 2016; Campos-Sánchez et al. 2021a, b, c, 2022a, b). However, there are no data on the many other beneficial properties that carrageenan might have when used in fish. Thus, the aim of the present study was to evaluate the hemagglutinating and hemolytic activity of λ-carrageenan in gilthead seabream erythrocytes, as well as its cytotoxicity on fish cells, and its potential as a bactericidal and bacteriostatic agent against various fish pathogens. To our knowledge, this is the first study that attempts to elucidate these properties on fish cells, which could broaden the use of carrageenan, in addition to its effect as an inflammation inducer, and shed some light on its mechanism of action in fish of commercial interest.

Material and methods

λ-carrageenan

Λ-carrageenan (Sigma, CAS Number: 9064-57-7) was acquired as solid powder and stored at room temperature according to manufacturer recommendations. Carrageenan was diluted in sterile phosphate-salt buffer (PBS; 11.9 mM phosphate, 137 mM NaCl and 2.7 mM KCl, pH 7.4) (Fisher Bioreagents) and used at concentrations of 0.1-1000 µg mL-1. Subsequently, λ-carrageenan was resuspended in the appropriate medium depending on the technique used. Thus, λ-carrageenan was resuspended and adjusted to each concentration in PBS with sodium chloride and glucose for incubation with erythrocytes, Eagle's minimal essential medium (EMEM, Thermo Fisher) for PLHC-1 cell line, L-15 Leibowitz medium (Gibco) for SAF-1 cell line, tryptic soy broth (TSB, Difco Laboratories) for Vibrio harveyi, Vibrio anguillarum and Photobacterium damselae, or Flexibacter maritimus medium (FMM, Conda) for Tenacibaculum maritimum, and dilutions were prepared for each concentration. Erythrocytes, cell lines or bacteria were exposed to a final solution of 0 (PBS diluted in the same volume of culture medium; control), 0.1, 1, 1, 10, 100 and 1000 μg mL-1. Before performing the assays, the osmolarity of these solutions was measured in an osmometer (Wescor) to avoid effects due to this parameter.

Hemagglutinating and hemolytic activity and morphological changes in gilthead seabream erythrocytes

Specimens of the sea water gilthead seabream (Sparus aurata L.) (mean weight: 403.6 ± 16.5 g; mean length 27.3 ± 0.3 cm), obtained from a local farm (Murcia, Spain), were kept in recirculating seawater aquaria (450 L) at the Marine Fish Facility of the University of Murcia (Spain) during a quarantine period of one month. The conditions were as follows: water temperature of 20 ± 2ºC, flow rate of 900 L h-1, salinity of 28 ‰, photoperiod of 12 h light to 12 h dark and continuous aeration. Ammonium and nitrite levels in the tank water were maintained below the limits for the species (0.1 mg L-1 and 0.2 mg L-1, respectively). Fish were fed a commercial diet (Skretting, Spain) at a rate of 2% body weight day-1 and were maintained 24 h without feeding prior to the trial. All experimental protocols were approved by the Ethical Committee of the University of Murcia (protocol code A13150104).

For erythrocyte isolation, six fish were randomly selected, anesthetized with clove oil (20 mg L-1, Guinama®). Immediately, 1 mL samples of blood were drawn with a heparinized syringe by tail vein injection and placed in 7 mL of PBS, containing 0.35 % sodium chloride, to adjust the osmolarity of the medium, and 10 mM glucose (hereafter, PBS-glu), and the fish were returned to the aqua (Morcillo et al. 2016). The blood was placed on a 51 % Percoll density gradient (Pharmacia) and centrifuged (400 × g, 30 min, 4 °C) to separate leucocytes from erythrocytes. The latter cells, which were in the pellets, were collected, washed twice with PBS-glu, counted, and adjusted to 5 x 108 cells mL-1.

The hemagglutination properties of λ-carrageenan were carried out by incubating erythrocytes in a 96-well U-shaped plate (Nunc). Briefly, 50 µL of varying concentrations of λ-carrageenan in PBS-glu were placed in each well in sextuplicate and 25 µL of the erythrocyte suspension was added. Erythrocyte hemagglutination was visualized after 1.5 h of incubation with λ-carrageenan at room temperature, when the erythrocytes in the wells considered as blank had completely sedimented (Li et al. 2008). Positive or negative controls consisted of 25 µL of the erythrocyte suspension and 50 µL of Concanavalin A (lectin that strongly binds erythrocytes) or PBS-glu, respectively. Macroscopic images of the plate were taken from above and below with a Canon 7D camera with a 22 mm 4.5 wide-angle lens (Canon EF) attached to a ring flash with tripod and with a scanner (CanoScan-4400F), respectively.

Erythrocyte lysis causes the release of their contents, hemoglobin, either in the form of oxyhemoglobin (HbO2) or deoxyhemoglobin, being the most abundant protein. Thus, erythrocyte viability was determined by hemolysis and subsequent release of oxyhemoglobin to the medium (Morcillo et al. 2016). After 3, 6, 12 and 24 h of exposure of erythrocytes with varying concentrations of λ-carrageenan in PBS-glu, samples were centrifuged (10000 × g, 1 min) and 100 µL of the supernatant was transferred to 96-well flat-bottom plates (Nunc) and absorbance was measured at 414 nm (the maximum absorbance for oxyhemoglobin) with a plate reader (BMG, Fluoro Star Galaxy). Positive (maximum hemolysis and absorbance) or negative (minimum hemolysis and absorbance) controls consisted of 100 µL of erythrocytes in 1 mL of sterile distilled water or 1 mL of PBS-glu, respectively.

Cell morphology was studied by observing the cells with a phase contrast microscope (Zeiss). For this purpose, 500 µL aliquots containing 200,000 cells per well were seeded in 24-well plates with 500 µL per well of 0, 100, and 1000 μg mL-1 of λ-carrageenan in duplicate, and the cells were incubated for 3 and 6 h at 25°C.

Cytotoxic activity and morphological changes in cells lines

The established cell line PLHC-1 (ATCC CRL2406), a hepatocellular carcinoma from Poeciliopsis lucida, was seeded in 25 cm2 plastic tissue culture flasks in EMEM (Thermo Fisher) supplemented with 5% fetal bovine serum (FBS, Life Technologies), 2 mM mL-1 glutamine, 100 i.u. mL-1, penicillin/streptomycin, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, 95% (Life Technologies). Cells were grown at 30°C in a humidified atmosphere (85 % humidity) and 5% CO2. Exponentially growing cells were detached from the culture flasks by brief exposure to of trypsin (0.05% in PBS, pH 7.2-7.4), according to the standard trypsinization methods.

The established SAF-1 cell line (ECACC nº 00122301), fibroblasts from gilthead seabream fin, was seeded in 25 cm2 plastic tissue culture flasks (Nunc) in L-15 Leibowitz medium (Gibco), supplemented with 10% FBS (Life Technologies), 2 mM mL-1 glutamine, 100 i.u. mL−1 penicillin and 100 mg mL−1 streptomycin (Life Technologies). Cells were grown at 25°C in a humidified atmosphere (85 % humidity). Exponentially growing cells were detached from culture flasks by brief exposure to trypsin (0.25 % in PBS, pH 7.2-7.4), according to the standard trypsinization methods.

The detached cells from the three cell lines were collected by centrifugation (200 × g, 5 min, 37°C) and the cell viability determined as previously described. Aliquots of 100 µL were dispensed into 96-well tissue culture plates (containing 30,000 cells well-1) and incubated (24 h, at the optimum temperature for each cell line). This cell concentration was previously determined to obtain satisfactory absorbance values in the cytotoxic assay and to avoid cell overgrowth. The culture medium was then replaced with 200 µL per well of the λ-carrageenan concentrations to be tested at the appropriate dilution. Cells were incubated for 3, 6, 12 and 24 h and then viability was determined by the MTT assay, which is based on the reduction of the soluble yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT, Sigma) to an insoluble blue formazan product by mitochondrial succinate dehydrogenase (Denizot and Lang 1986; Berridge and Tan 1993). After incubation with λ-carrageenan dilutions, the medium was removed and 200 µL per well of MTT (1 mg mL-1 in culture medium for each cell line) was added. After 4 h of incubation (at the corresponding temperature for each cell line), the formazan crystals were solubilized with 100 µL per well of dimethyl sulfoxide (DMSO, Sigma). Plates were shaken (5 min, 100 rpm) under dark conditions and absorbance was determined at 570 nm and 690 nm in a microplate reader.

Cell morphology was directly observed and photographed from the culture plates using a phase contrast microscope (Zeiss). Each cytotoxicity assay with each cell type was performed in three replicates for each concentration of λ-carrageenan.

Bactericidal and bacteriostatic activities

Four opportunist marine pathogenic bacteria (Vibrio harveyi strain Lg 16/00, V. anguillarum strain CECT4344, Photobacterium damselae subsp. piscicida strain PP3 and Tenacibaculum maritimum strain CECT4276) were used in the bactericidal and bacteriostatic assays. All bacterial strains were grown from 1 mL of stock culture that had been previously frozen at −80 °C. Before use, an aliquot of stock culture from V. harveyi, V. anguillarum and P. damselae was incubated with TSB (Difco Laboratories) medium supplemented with 1.5% NaCl, whilst an aliquot of stock culture from T. maritimum was incubated with FMM (Conda) in flasks which 90% empty. The flasks were incubated at 25ºC with continuous shaking (100 rpm) overnight. Exponentially growing bacteria were resuspended in the sterile corresponding culture medium and adjusted to 108 colony-forming units (c.f.u.) mL-1, according to the McFarland standard curve.

Samples of 20 µL of λ-carrageenan dilutions previously adjusted in PBS were added (in triplicates) to the wells of a 96-well U-shaped plate (Nunc). Aliquots of 20 µL of the previously cultured bacteria were added to obtain 40 µL final volumes and the plates were incubated for 5 h at 25°C for the bactericidal assay (Guardiola et al. 2017). PBS solution was added to some wells instead of the λ-carrageenan and served as positive control, while culture medium without bacteria was added in other wells to ensure the sterility of the tests. Control samples were incubated under the same experimental conditions as described above. Then, 25 µL of MTT (1 mg mL−1) were added to each well and the plates were newly incubated (10 min, 25ºC) to allow the formation of formazan. Plates were then centrifuged (2000 × g, 10 min), and the precipitates dissolved in 200 µL of DMSO, of which 100 µL of were transferred to a flat-bottom 96-well plate (Nunc). The absorbance of the dissolved formazan was measured in a spectrophotometer (BMG, SpectroStarnano) at 570 nm and 690 nm in a microplate reader. Bactericidal activity was expressed as percentage of non-viable bacteria, calculated as the difference between absorbance of surviving bacteria compared to the absorbance of bacteria from positive controls (100 %).

Aliquots of 100 µL of λ-carrageenan dilutions previously adjusted in PBS were added (in triplicates) to the wells of a flat-bottom 96-well plate (Nunc). Samples of 100 µL of the previously cultured bacteria were added to obtain 200 µL final volumes and the plates were incubated at 25°C. The turbidity of the samples was measured in a spectrophotometer at 620 nm every 120 min for 24 h (Sunyer and Tort 1995). PBS solution was added to some wells instead of the λ-carrageenan and served as positive control to evaluate the growth of bacteria without treatment, while culture medium alone was added in other wells to ensure the sterility of the tests. Control samples were incubated under the same experimental conditions as described above. Antimicrobial activity was also analysed by disk diffusion susceptibility (antibiograms) according to Kirby–Bauer’ method (Bauer 1966) with some modifications. Exponentially growing bacteria were propagated on agar plates (in duplicates) containing their corresponding culture medium; Tryptic Soy Agar (TSA; AppliChem Panreac), for V. harveyi, V. anguillarum and P. damselae) supplemented with 1.5% NaCl or Bacteriological European Agar (AppliChem Panreac) with FMM (Conda) for T. maritimum). λ-carrageenan (0, 100 and 1000 µg disc-1) was set on sterile antimicrobial discs (0.5 mm diameter) and these were placed on the inoculated agar surface. Plates were then incubated at 25°C for 24 h. After incubation, the zone of inhibition diameter was calculated and interpreted as per the recommendation of CLSI (CLSI 2018).

Statistical analysis

The results were expressed as mean ± standard error of the mean (SEM). Data were analysed by One-way ANOVA (followed by Tukey tests) to determine differences between the doses of λ-carrageenan tested. Normality of the data was previously assessed using a Shapiro-Wilk test and homogeneity of variance was also verified using the Levene test. All statistical analyses were conducted using the computer package SPSS (25.0 version; SPSS Inc., USA) for Windows. The level of significance used was p < 0.05 for all statistical tests.

Results

Hemagglutinating and hemolytic activity

The hemagglutinating activity of λ-carrageenan on gilthead seabream erythrocytes was only detected after incubation with the highest dose of λ-carrageenan (1000 μg mL-1) compared to negative control (Fig. 1). As for hemolytic activity, values were significantly increased (p < 0.05) in erythrocytes incubated for 3 h with 100 or 1000 μg mL-1 of λ-carrageenan, compared with those incubated without or with 1 and 10 μg mL-1 of λ-carrageenan for the same time (Fig. 2A). At the same time, the increase observed in erythrocytes incubated with 1000 μg mL-1 of λ-carrageenan was also statistically significant compared with those incubated with 0 (control) and 0.1 μg mL-1 of λ-carrageenan. After 6 h of incubation, a dose-dependent increase in hemolytic activity was evident in erythrocytes incubated with the three highest doses of λ-carrageenan, although the increases were only statistically significant (p < 0.05) in erythrocytes incubated with the highest dose of λ-carrageenan, relative to control samples (Fig. 2B). However, no significant effects on the haemolytic activity of erythrocytes were observed after incubation for 12 or 24 h with either dose of λ-carrageenan (Fig. 2C, 2D).

Fig. 1
figure 1

Hemagglutination activity of gilthead seabream (Sparus aurata) erythrocytes incubated with λ-carrageenan (0, 0.1, 1, 1, 10, 10, 100 and 1000 µg mL-1) for 1.5 hours. Representative macroscopic photographs of the plate: top view (A) and bottom view (B). Phosphate buffered saline (PBS, 0.35% sodium chloride, 10 mM glucose) and Concanavalin A were used as negative and positive controls, respectively

Full size image
Fig. 2
figure 2

Hemolytic activity (expressed as percent oxyhemoglobin release) of gilthead seabream (Sparus aurata) erythrocytes incubated with λ-carrageenan (0, 0.1, 1, 1, 10, 100 and 1000 µg mL-1) for 3 (A), 6 (B), 12 (C) and 24 (D) hours. Data represent the mean ± standard error of the mean (n = 6). Different letters denote significant differences between experimental concentrations (ANOVA; p < 0.05)

Full size image

The hemagglutinating and hemolytic activities of λ-carrageenan were readily confirmed by phase contrast microscopy and optical images evidence that the processes were dose and time dependent during the first 6 h of incubation with λ-carrageenan (Fig. 3).

Fig. 3
figure 3

Representative phase contrast microscopy micrographs of gilthead seabream (Sparus aurata) erythrocytes incubated with λ-carrageenan (0, 100 and 1000 µg mL-1) for 3 (A, B, C) and 6 (D, E, F) hours. Scale bar = 50 µm

Full size image

Cytotoxic activity and morphological changes in cells lines

No significant variations (p > 0.05) in cytotoxic activity were observed after incubation for 3 h of the PLHC-1 cell line with λ-carrageenan at any of the concentrations tested (Fig. 4A). However, statistically significant increases in the cytotoxic activity of λ-carrageenan were evident in these cells after 6 or 12 h of incubation compared with control cells (Fig. 4, B, C). Similar results were obtained when cells were incubated for 12 h with the highest dose (1000 μg mL-1) of λ-carrageenan compared with all other doses. However, the variations in cytotoxic activity observed in PLHC-1 cells incubated for 12 h with 100 μg mL-1 were not significant (p > 0.05) compared with the values recorded for cells incubated with the highest dose for the same time (Fig. 4C). At 24 h of incubation the two highest doses of λ-carrageenan caused cytotoxicity (about 40 %) in PLHC-1 cells compared with the cytotoxicity observed in cells incubated with the other concentrations tested (Fig. 4D).

Fig. 4
figure 4

Cytotoxic activity (expressed as percentage of viable cells) of PLHC-1 cell line (Sparus aurata) incubated with λ-carrageenan (0, 0.1, 1, 10, 100, and 1000 µg mL-1) for 3 (A), 6 (B), 12(C) and 24 (D) hours. Data represent the mean ± standard error of the mean (n = 6). Different letters denote significant differences between experimental concentrations (ANOVA; p < 0.05)

Full size image

As for cell morphology, PLHC-1 cells incubated with the highest doses of λ-carrageenan (100 or 1000 μg mL-1) for 12 or 24 h showed obvious changes compared with control cells (Fig. 5). Control cells appeared as large adherent epithelioid cells, whereas after incubation with 100 μg mL-1 of λ-carrageenan, the cells appeared smaller, rounded, and darker than control cells, with a clear tendency to detach from the culture flask, and some cell debris was observed in the culture medium. These changes were most evident in cells incubated for 24 h with the highest concentration of λ-carrageenan assayed (1000 μg mL-1).

Fig. 5
figure 5

Representative phase contrast microscopy micrographs of PLHC-1 cell line incubated with λ-carrageenan (0, 100, and 1000 µg mL-1) for 12 (A, B, C) and 24 (D, E, F) hours. Scale bar = 50 µm

Full size image

Interestingly, no significant variations in cytotoxic activity were observed in SAF-1 cell line after incubation with λ-carrageenan at any of the concentrations and times assayed. Concomitantly, no morphological variations were observed in SAF-1 cells after incubation with λ-carrageenan at any experimental dose or time (data not shown).

Bactericidal and bacteriostatic activity

No significant variations (p > 0.05) were observed in the bactericidal activity of V. harveyi, V. anguillarum and T. maritimum after incubation with λ-carrageenan at any concentration tested (Fig. 6A, B, C). However, a statistically significant dose-dependent increase in bactericidal activity (p < 0.05) was observed after incubation of P. damselae with 100 and 1000 μg mL-1 of λ-carrageenan compared with the values obtained with the other doses tested (Fig. 6C). However, no significant effects (p > 0.05) were observed after incubation of bacteria with λ-carrageenan on bacteriostatic activity at any dose at any experimental time point (data not shown). Likewise, not inhibition halo was observed in the antibiogram of any bacteria after incubation with 100 or 1000 μg mL-1 λ-carrageenan (data not shown).

Fig. 6.
figure 6

Bactericidal activity (expressed as percentage of viable bacteria) of Vibrio harveyi (A), Vibrio anguillarum (B), Photobacterium damselae (C) and Tenacibaculum maritimum (D) (Sparus aurata) incubated with λ-carrageenan (0, 0.1, 1, 10, 100, and 1000 µg mL-1) for 5 hours. Results are expressed as mean ± standard error of the mean (n = 6). Different letters denote significant differences between experimental concentrations (ANOVA; p < 0.05)

Full size image

Discussion

In the present study, taking into account the scarce literature available on the effects of carrageenans in fish, we evaluated the direct effect of λ-carrageenan, using the same doses and incubation times as in our previous work (Campos-Sánchez et al. 2022c), on gilthead seabream erythrocytes, on several fish cell lines and against four opportunistic marine pathogenic bacteria.

Erythrocytes are a perfect cell to study the direct effect of a stimulus (such as λ-carrageenan) due to the special properties and shape of their characteristic cell membrane, which allows them to deform, aggregate or even modify their metabolism as a consequence or any change in the internal environment (Benedik and Hamlin 2014). Moreover, the normal morphology of erythrocytes is essential for survival and the correct development of their functions (Benedik and Hamlin 2014). In this regard, high concentrations of λ-carrageenan have been related to the ability to form macroaggregates of even higher molecular weight in serum-containing medium, due to the generation of negative charges by its sulphate groups that are able to bind proteins with high affinity (Silva et al. 2010; McKim 2014). This ability of carrageenan allows it to produce changes in proteins and cells, and even induce cell recruitment from hematopoietic tissues in both mammals and fish (Silva et al 2010). For example, intravenous administration of carrageenan in mice revealed its ability to reach the peripheral blood and distribute to many organs, where it caused potent suppression of immune cells (Magaña et al 2015). Therefore, after its in vivo administration in fish, it could be thought that carrageenan could also reach the blood circulation, where it could interact with erythrocytes. However, in vivo studies would be necessary to corroborate this hypothesis. We hypothesize that the positive charges of the erythrocyte membrane could be attracted by these negative charges of λ-carrageenan and increase the ability of erythrocytes to adhere and form aggregates (Wang et al. 2021). This fact could explain why in our study, only high doses of the polysaccharide were associated with the hemagglutinating activity detected in erythrocytes. In fact, the hemagglutination observed at the microscopic level in erythrocytes incubated with doses lower than 1000 μg mL-1 of λ-carrageenan would not be sufficient to observe this process at the macroscopic level. Likewise, the same mechanism just described but determined after 3 or 6 h of incubation of erythrocytes with λ-carrageenan could alter the normal morphology of erythrocytes, and therefore, their viability, with consequent hemolysis and release of hemoglobin to the medium. Supporting our data, a study in shrimp showed that λ-carrageenan was able to induce hemocyte degranulation and death of the oldest hemocytes within a maximum time of 2 h. However, the special dynamic shape of the cell, that confers to erythrocytes a great plasticity and deformability could allow them to adapt to the new environment and cease their hemolysis (Wang et al. 2021). This hypothesis would explain the increased hemolytic activity and altered shape of some erythrocytes incubated with high doses of λ-carrageenan at 3 and 6 h, but not at longer exposure times. However, further studies are needed to understand the molecular interaction between λ-carrageenan and the fish erythrocyte membrane.

The cytotoxic activity of λ-carrageenan has been extensively researched in various cell lines, including human tumoral cell lines such as ileocaecal adenocarcinoma (HCT-8), colorectal adenocarcinoma (HT-29), embryonic kidney293 (HEK293), and non-cancer cells such as human umbilical vein cells (HUVEC) (Pacheco-Quito et al. 2020), interestingly, showing only significant effects on tumour cells. Furthermore, in studies carried out with the human HeLa cell line, the mechanism of action of λ-carrageenan was related to its ability to arrest the cell cycle after 3 days of incubation in both G1 and G2/M phase, suppressing the ability of these cells to divide and indicating a strong antiproliferative effect (Prasedya et al. 2016). In mice, s studies have also reported that carrageenans have antiproliferative activity in tumour cell lines in vitro, inhibitory activity of tumour growth, and anti-metastasis activity in mice (Zhou et al. 2004; Yuan et al. 2006). These activities may be attributed to the ability of carrageenans to block interactions between cancer cells and the basement membrane, inhibiting tumour cell adhesion to various substrates, and thereby suppressing its proliferation (Zhou et al. 2004; Yuan et al. 2006). These studies have certain parallels with our results since no cytotoxic activity was observed in SAF-1 cells, which are non-cancerous cells (fibroblasts obtained from seabream fin). In contrast, we observed that λ-carrageenan was cytotoxic for the tumour PLHC-1 cell line after being incubated with high doses of λ-carrageenan for 6 and 12 h, suggesting that it might have a specific cytotoxic effect against tumour cells in. However, more studies are needed to understand the molecular mechanisms by which carrageenan can activate or inhibit tumour cells, enabling the development of new anticancer products that can be used in fish, other animals, or even humans.

To our knowledge, this is the first study that directly investigates the effect of λ-carrageenan on fish bacteria. This is important given the large number of bacteria that have been reported to hydrolyse or degrade carrageenans and use them as a source of galactose in their metabolism in both in vitro and in vivo studies (Tobacman 2001). However, the bactericidal effect of the polysaccharide has received relatively little attention in comparison to its antimicrobial action against biofilms and diets based on carrageenans, with κ- and ι-carrageenan being the types most commonly studied (Pacheco-Quito et al. 2020). In contrast, carrageenan has shown in vitro antiviral activity against some human viruses, such as Herpes Simplex virus (HSV), human cytomegalovirus, human rhinoviruses, and papillomavirus, by preventing virions from binding or entering cells (Necas and Bartosikova 2013). However, the study of its effect on bacteria has been limited. The differences in the bactericidal activity among carrageenans are likely dependent on their sulphate content, as well as the disposition of these sulphate groups in their global structures. These sulphate groups may interact with the negative charges of peptidoglycan and other structures belonging to the bacterial membrane, resulting in varying degrees of alteration (Pacheco-Quito et al. 2020). The limited number of studies on carrageenan's bactericidal effects may also be related to the fact that these effects are often accompanied by modifications due to oxidation or carboxymethylation processes (Yanishlieva et al. 1999). For instance, polymeric carrageenans have been reported to cause intestinal inflammation and colitis in C57BL/6J mice by reducing the population of the anti-inflammatory bacterium Akkermansia muciniphila in gut microbiota (Shang et al. 2017). In contrast, λ-carrageenan has shown immunostimulatory effects in shrimp and increased resistance against V. alginolyticus through dietary administration.

Previously, our research group only investigated the bactericidal activity of carrageenan in the skin mucus of gilthead seabream after intramuscular injection of κ/λ-carrageenan (Campos-Sánchez et al. 2021a). In that study, significantly increased bactericidal activity was observed against V. anguillarum and P. damselae 3 h after carrageenan injection. In this present study, we found that λ-carrageenan had direct bactericidal activity only against P. damselae. This suggests that P. damselae may be more sensitive to the effects of λ-carrageenan than the other bacteria tested (V. harveyi, V. anguillarum, and T. maritimum). Alternatively, the greater effect observed in the previous in vivo assay (Campos-Sánchez et al. 2021a) may have been produced by the κ-carrageenan fraction of the mixture of both carrageenans. It has also been demonstrated that λ-carrageenan has lower antimicrobial effects than other carrageenans (Pacheco-Quito et al. 2020). Further studies are necessary to clarify the differences in bactericidal activity among different types of carrageenans against fish pathogens.

In conclusion, this study provides valuable insights into the effects of λ-carrageenan on fish cells and marine pathogens. The results demonstrate that high doses of λ-carrageenan exhibit hemagglutinating and hemolytic activity on erythrocytes from gilthead seabream. Interestingly, the cytotoxicity assays indicate that fish tumour cells may be more susceptible to λ-carrageenan compared to non-tumoral cells. Furthermore, the bactericidal activity of λ-carrageenan was found to be dependent on the specific bacterial cells used in the assays, with no observed bacteriostatic effects on the tested fish pathogenic bacteria. These findings shed light on the diverse effects of this marine seaweed polysaccharide on fish cells and marine pathogens, offering a novel perspective on its potential applications. Further research is warranted to explore the underlying mechanisms of λ-carrageenan's activities and to elucidate its specific interactions with different fish species and bacterial strains. This knowledge can contribute to the development of new strategies for utilizing λ-carrageenan in various fields, including aquaculture, therapeutics, and antimicrobial interventions in the marine environment.