Effects of the Toxic Dinoflagellate Protoceratium reticulatum on Physiological Rates of Juvenile Scallops Argopecten purpuratus

Abstract

Protoceratium reticulatum, a dinoflagellate species known for producing yessotoxins (YTX), can form harmful algal blooms (HABs) impacting marine life. This study examined how P. reticulatum influenced the physiological rates and affected the tissue health of juvenile scallops, Argopecten purpuratus. The scallops were exposed to non-toxic algae Isochrysis galbana (diet A) and mixtures where 30 and 70% of the non-toxic algae were replaced by the toxic algae P. reticulatum (diet B and C, respectively) for 15 days, followed by a 15-day recovery period (I. galbana). Results showed that the clearance rate (CR), inorganic ingestion rate (IIR), organic ingestion rate (OIR), and absorption rate (AR) were significantly reduced within the first seven days of exposure to toxic diets, with reductions of approximately 25% and 50% in diets B and C, respectively, compared to the non-toxic diet, and an increase in these parameters during the recovery stage. Histological observations revealed greater tissue damage in the digestive gland than in the gills, with a higher frequency of scallops with severe damage correlating with higher toxic diet content. Despite no direct impact on survival, the compromised physiological health of A. purpuratus juveniles suggests increased vulnerability to other stressors. These findings provide new insights into the filter-feeding behavior and selective filtration capabilities of scallops in the presence of toxic dinoflagellates and how scallops health can be compromised, contributing to the understanding of how HABs and associated toxins affect A. purpuratus.

1. Introduction

Protoceratium reticulatum (Claparède and Lachmann) Bütschli 1885 is a marine dinoflagellate species that is distributed worldwide (reviewed by Paz et al. [1], Paz et al. [2], Wang et al. [3]). These species can produce toxic harmful algal blooms associated with their ability to synthesize lipophilic toxins, including yessotoxin (YTX) and its analogs [3,4]. In addition to P. reticulatum, other dinoflagellates such as Lingulaulax polyedra comb. nov. (Lingulodinium polyedra) [2,5], Gonyaulax spinifera [6,7,8], and Gonyaulax taylorii [9] are also known to produce YTXs.

YTX is a marine polyether compound that was initially isolated from the marine scallop Patinopecten yessoensis in Japan in 1986 [10]. Since its discovery, numerous analogs and derivatives have been described in P. reticulatum [11] and several shellfish species. These toxins were originally classified under the diarrhetic shellfish poisoning (DSP) group due to their presence in shellfish causing gastroenteritis in humans [12,13]. However, toxicological studies in mice have shown that these toxins, when administered intraperitoneally or orally, do not induce diarrhea or affect protein phosphatase activity [14,15]. Furthermore, the intraperitoneal administration of YTXs, and to a lesser extent the oral administration, has been linked to cardiotoxic effects in mice [12,13], as well as other effects at the cellular and molecular levels [15,16,17,18,19]. Although there have been no reported cases of human intoxication [20], the toxic effects led European authorities to set a regulatory limit of 3.75 mg/kg of YTX equivalents to ensure shellfish safety for human consumption [21]. This regulation may vary according to the regulation of each country, as shown in New Zealand, where it was deregulated.

The toxic dinoflagellate P. reticulatum can produce harmful algal blooms (HABs) in diverse regions globally. It has been connected to mass die-offs of marine species in South Africa, such as clams (Donax serra) and mussels (Choromytilus meridionalis and Aulacomya atra) [22,23]. Pitcher et al. [24] reported the death of millions of farmed abalone in South Africa due to a severe summer bloom of the toxic dinoflagellates L. polyedra (max. 1.6 × 10⁵ cells L⁻¹) and G. spinifera (max. 6.11 × 10⁵ cells L⁻¹). Rogers-Bennett et al. [25] reported a massive mortality associated with YTX of the abalone (Haliotis rufescens) in the natural environment of the California Coast, USA. Jurgens et al. [26] reported a severe mortality event, likely caused by a toxic bloom of Gonyaulax spp., which impacted the sea urchin Strongylocentrotus purpuratus and the starfish Leptasterias sp., resulting in nearly 99% mortality across approximately 100 km of the California coastline.

The specific mechanism by which YTX toxicity affects bivalves remains unclear [27]. In Mytilus galloprovincialis, YTX has been found primarily in circulating immunocytes and the digestive gland, particularly within the lumen of tubules and ducts, suggesting its presence in the gills as well [28,29]. Similarly, King et al. [27] observed significant damage to the digestive gland of clams (Venerupis philippinarum) during a mass stranding linked to a P. reticulatum bloom, with toxicity levels measured between 0.09 and 0.27 mg Eq. YTX kg⁻¹. Studies assessing histological analysis in marine mollusks to understand the effects of YTX are scarce, but some have reported tissue damage in the gill and digestive gland [24,27]. In the clam V. philippinarum, abnormalities in the digestive gland tubules, such as a reduction in tubule height and the sloughing of epithelial cells, have been observed following exposure to the toxic dinoflagellate P. reticulatum, which produces YTX [27]. Additionally, in the abalone Haliotis rufescens, the presence of YTX has been reported to impact the gill epithelium, leading to the degeneration and necrosis of epithelial cells, along with a mild inflammatory response [24]. Tissue damage may be caused by YTX’s negative impact on the cytoskeleton, particularly on actin filaments and cadherin transmembrane proteins [28,30]. Additionally, it is possible that the damage is related to cell death through the apoptotic process [31,32].

In Chile, a country with a coastline extending over 4000 km, P. reticulatum and YTX have been reported in widely separated locations [33]. YTX was first detected in southern Chile in the mussel Mytilus chilensis from the Chonos Archipelago (43 °S) [34], where P. reticulatum was identified as the main producer of YTX in the area. Likewise, [35] associated the presence of P. reticulatum with a high content of YTXs (9.4 to 52.2 pg cell⁻¹) in Puyuhuapi Fjord, NW Patagonia. In northern Chile, YTX was initially detected in phytoplankton samples from Bahía Arica (18 °S), where it was proposed that P. reticulatum was the source [36]. Subsequently, Alvarez et al. [37] confirmed YTX presence in phytoplankton samples from a dense bloom of P. reticulatum (approximately 350 cells mL−1) in Bahía Mejillones (23 °S) and identified this species as the primary YTX producer in the region. During this event, the high mortality of juvenile scallop Argopecten purpuratus (12 ± 2 mm) from aquaculture facilities was reported for the first time.

Since then, there have been no other reports of mass mortalities in marine invertebrates until January 2019, when a massive beaching of clams (Ameghinomya antiqua) and echinoderms, including the red urchin (Loxechinus albus) and the star (Stichaster striatus), occurred on the coast of Pabellón de Pica in the Tarapacá Region. This event was associated with low concentrations of YTX (0.1–0.4 mg YTX kg⁻¹) [38]. Two weeks later, a mass stranding of approximately 12 tons of Humboldt squid (Dosidicus gigas) was observed in Bahia Inglesa, Atacama Region, with toxin concentrations of 0.42 mg YTX kg⁻¹ recorded in their visceral tissues. A similar event occurred at the end of March 2019, when 130 tons of Humboldt squid were stranded in Puerto Aldea, Coquimbo Region, with a YTX concentration of 0.12 mg YTX kg⁻¹ [38].

The scallop Argopecten purpuratus is an endemic species on the Pacific coast of South America, where it is found from Paita (5 °S) in northern Peru to Valparaíso (33 °S) in Chile [39,40]. It is a key commercial species in international markets, including Spain, Belgium, and Brazil, prized for its high nutritional value [41], large size, rapid growth, and high price [42,43,44,45].

Although several toxic outbreaks of the dinoflagellate P. reticulatum and/or the presence of YTX have been detected in different locations in Northern Chile, causing massive mortality in marine invertebrates and probably in juveniles of A. purpuratus, no information is available on the effects of the toxic dinoflagellate in this important aquaculture species from the Pacific coast of South America. The aim of this study was to determine the effects of toxic P. reticulatum on the physiological rates of A. purpuratus juveniles.

2. Materials and Methods

2.1. Experimental Animals

Juvenile Argopecten purpuratus (height: 29.8 ± 4.0 mm) were obtained from the Laboratorio Central de Cultivos Marinos at Universidad Católica del Norte, Chile. Prior to the experiment, the scallops were acclimated for one week in 14 L aquariums containing filtered seawater, with constant aeration, a temperature of 15 °C, and a salinity of 34. They were fed Isochrysis galbana, with the daily ration set at 3% of the scallop dry weight. This amount, calculated based on the average weight of 25 mm juveniles, was equivalent to 3.06 mg per individual per day.

2.2. Culture of Protoceratium reticulatum and Isochrysis galbana

The diet comprised two species: the haptophyte Isochrysis galbana and the toxic dinoflagellate Protoceratium reticulatum. I. galbana was cultured in 250 mL Erlenmeyer flasks with 200 mL of f/2 medium [46], maintained under controlled conditions at 20 ± 1 °C, a salinity of 34 ± 1, an irradiance of 50 μmol m⁻² s⁻¹, and a 12:12 h light/dark cycle. For the toxic diet, monoclonal P. reticulatum (PRIEM strain) was cultured in batch conditions in 250 mL Erlenmeyer flasks with 150 mL of L1 medium [47], under similar controlled conditions at 20 ± 1 °C, a salinity of 28 ± 1, an irradiance of 50 μmol m⁻² s⁻¹, and a 12:12 h light/dark cycle. Each culture was maintained until the mid-exponential phase, and the cell density (cells mL⁻¹) was estimated using a CKX41 inverted microscope (Olympus, Tokyo, Japan) and Neubauer counting chamber (Paul Marienfeld GmBH & Co, Lauda-Königshofen, Germany) for I. galbana and a Sedgewick-Rafter counting chamber for P. reticulatum.

2.3. Experimental Design

To estimate the toxic effect of P. reticulatum on the physiological rates of scallop juveniles, three treatments based on toxic and non-toxic diets were established. The amount of diet was estimated to supplement a daily amount of food equivalent to 3% of its dry weight (

Table 1. Composition of the experimental diets used to feed A. purpuratus juveniles.

Diet	Protoceratium reticulatum	Isochrysis galbana
A	0%	100%
B	30%	70%
C	70%	30%

). Each treatment was carried out in triplicate in 14 L aquariums containing 54 individuals maintained in filtered seawater with constant aeration, at 16 °C, and at a salinity of 34. The diets were delivered continuously using a peristaltic pump (Lead Fluid BT100S-1) for 15 d (intoxication period). After this period, all treatments were fed a non-toxic diet containing 100% I. galbana for 15 days.

Physiological rates were measured in triplicate, taking random individuals from each experimental aquarium on days 0 (first day of the intoxication period), 2, 7, 11, and 15 (intoxication period) and on days 16, 22, 25, and 30 (detoxification period). For the experiments, one individual was placed in a 250 mL beaker (experimental chamber) with filtered seawater (0.45 µm) (closed system), with a final volume of 200 mL, and their corresponding diet. After completing the physiological determinations, the individuals were dissected, and the total weight (including the shell) and weight of the soft tissues were recorded using an electronic balance Scout^TM Pro (0.1 g) (Ohaus Corporation, Parsippany, NJ, USA). From the dissected tissues, one portion (2 mm cross section) of the digestive gland and gill from each organism were placed in histological cassettes Leica Jet II (Leica, Wetzlar, Germany) and fixed in Davidson’s solution for 48 h for histological analysis. Additionally, the shell length was measured using a Vernier (0.01 mm).

2.4. Clearance Rate

For the individual experiments to determine the physiological rates, the particle concentration constituting the diets was estimated considering the amount of food (0.06 mg) that one individual required for feeding each 30 min. Cells (particles) corresponding to the different diets were controlled every 30 min for 3 h. Each 30 min period, a 15 mL sample was taken, fixed with Lugol iodine solution, and stored in a dark place. Subsequently, these samples were quantified using Neubauer counting chambers (for counting I. galbana) and a Sedgwick-Rafter chamber (for counting P. reticulatum) under an optical microscope. The clearance rate was estimated using the following equation [48]:

$$CR \left({\displaystyle \frac{L}{h}}\right)={\displaystyle \frac{\ln Cb-\ln Ce}{t}}\ast V$$

(1)

where CR is the clearance rate (L h⁻¹), Cb is the initial concentration of microalgae (cells mL⁻¹), Ce is the final concentration of microalgae (cells mL⁻¹), V is the total volume of seawater (L), and t is the time of the experiment.

2.5. Ingestion Rates

The ingestion rate was estimated by taking three samples of 100 mL for each diet. Each sample was concentrated on GF/C glass fiber filters (47 mm; 1.2 µm) (Microclar, Buenos Aires, Argentina) that had been previously washed, ashed, and weighed. The blank filters and filter-retained diet were rinsed with an isotonic solution of ammonium formate (3.4%) to eliminate salts and avoid cell lysis. To estimate the inorganic and organic content of the diets (mg L⁻¹), the filters were dried at 50 °C for one week in an oven (VMTECH 101-3AB, Wincom Company Ltd, Hunan, China) and then weighed using an analytical balance (BOECO BAS 31 plus, 0.0001 g) (BOECO, Germany). They were then ashed at 450 °C for 5 h in a muffle furnace (VMTECH TC-5-12, Wincom Company Ltd, Hunan, China) and reweighed after cooling in a desiccator. The inorganic ingestion rate (IRR) and the organic ingestion rate (OIR) were calculated according to the equations described by Bayne et al. [49]:

$$IIR\left({\displaystyle \frac{mg}{h}}\right)=CR\left({\displaystyle \frac{L}{h}}\right)\times inorganic material \left({\displaystyle \frac{mg}{L}}\right)$$

(2)

$$OIR\left({\displaystyle \frac{mg}{h}}\right)=CR\left({\displaystyle \frac{L}{h}}\right)\times organic material \left({\displaystyle \frac{mg}{L}}\right)$$

(3)

where IIR is the inorganic ingestion rate (mg h⁻¹), OIR is the organic ingestion rate (mg h⁻¹), and CR is the clearance rate (L h⁻¹).

2.6. Absorption Efficiency

Absorption efficiency was determined by taking three samples of 50 mL of each diet and recollecting all feces accumulated during the clearance rate experiments. Both samples were concentrated on GF/C glass fiber filters (47 mm; 1.2 µm), and the weights of inorganic and organic matter were estimated following the same methodology used to determine the ingestion rates. Calculations to determine the absorption efficiency (EA) were carried out according to the equations described by Conover [50]:

$$AE={\displaystyle \frac{A-F}{\left(1-F\right)\times A}}$$

(4)

where AE is the absorption efficiency, A is the proportion of the weight of organic matter to the total weight of the diet, and F is the ratio of the weight of organic matter to the total weight of feces.

2.7. Absorption Rate

The absorption rate (AR) was estimated using the following equation from Bayne, et al. [49]:

$$AR\left({\displaystyle \frac{mg}{h}}\right)=AE\times OIR\left({\displaystyle \frac{mg}{h}}\right)$$

(5)

where AR is the absorption rate (mg h⁻¹), AE is the absorption efficiency, and OIR is the organic ingestion rate (mg h⁻¹).

2.8. Excretion Rate

The excretion rate was determined after 3 h using a 100 mL sample from each experimental chamber and were filtered using GF/C glass fiber filters (47 mm; 1.2 µm), then a 10 mL subsample was analyzed following the method of [51]. For this determination, a calibration curve was prepared with known concentrations of the ammonium sulfate stock solution. The samples were read using a UV/VIS spectrophotometer (Rayleigh, Beifen-Ruili, China) at a wavelength of 640 nm. The equation described by [52] was used to calculate the excretion rate:

$$ER={\displaystyle \frac{28\times X\times V}{t}}$$

(6)

where ER is the excretion rate (μg NH₄-N·h⁻¹), X is the ammonium concentration, V is the total volume of seawater (L), t is the duration of the experiment, and 28 is the conversion factor of μM to μg NH₄-N.

2.9. Toxin Analyses

The YTX content in juvenile scallop samples (n = 3) was determined during the intoxication period on days 0, 2, 5, 7, 11, and 15. Individuals were separately packed in sealed bags and frozen at −20 °C until analysis. YTXs were extracted from whole shellfish tissues following the methodology described by the European Union Reference Laboratory for Marine Biotoxins [53].

The presence of YTX and homo-yessotoxin (homo-YTX) in the extract was determined using a method adapted from Regueiro et al. [54]. Instrumental analysis was conducted with a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Sunnyvale, CA, USA), utilizing a reversed-phase HPLC column Gemini NX-C18 (50 mm × 2 mm; 3 μm) with an Ultra Guard column C18 (Phenomenex, Torrance, CA, USA). The flow rate was 0.35 mL min⁻¹, and the injection volume was 10 μL. The mobile phase was run in gradient mode as follows: 81% eluent A (100% water with 6.7 mM NH₄OH) and 19% eluent B (90% acetonitrile: 10% water with 6.7 mM NH₄OH) for 1 min, followed by a linear increase to 95% B over 5 min, maintained for 2 min, and then returned to initial conditions of 19% B. The column was then re-equilibrated for 5 min. YTX and homo-YTX were detected with a Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Sunnyvale, CA, USA) equipped with a HESI II electrospray interface. The HESI was operated in negative ionization mode with a spray voltage of 3 kV and in positive ionization mode with a spray voltage of 3.5 kV. The ion transfer tube and HESI vaporizer temperatures were set to 200 °C and 350 °C, respectively. Nitrogen (>99.98%) was used as the sheath and auxiliary gas at pressures of 30 and 4 arbitrary units, respectively. Data were acquired in a selected ion monitoring (SIM) mode for quantification and a data-dependent (ddMS²) mode for confirmation. The analysis was conducted using a mass inclusion list with precursor ion masses, expected retention times, and collision energies (CE) for each toxin. In the SIM mode, the masses were set to 570.2330 m/z for YTX and to 577.2396 m/z for homo-YTX, with a scan range of m/z 100–1000, a mass resolution of 35,000, automatic gain control (AGC) at 2 × 10⁵, and a maximum injection time (IT) of 3000 ms. In the ddMS² mode, the mass resolution was 70,000, AGC was 2 × 10⁵, and IT was 3000 ms, with an isolation window of 2 m/z for both modes. The toxin concentrations in the extracts were quantified by comparing the peak areas with those of certified reference materials from NCR, Canada. The method’s quantification limits were 6 ng mL⁻¹ for YTX and 5 ng mL⁻¹ for homo-YTX.

2.10. Histological Analysis

Histological analyses were performed in triplicate, taking random individuals from each experimental aquarium on days 0 (first day of the intoxication period), 15 (end of intoxication period), and 30 (end of detoxification period). Fixed tissues were dehydrated using an ascending ethanol gradient (70%, 80%, 95%, and 100%), cleared in xylene, embedded in paraffin (Paraplast X-Tra, McCormick, CA, USA), and sectioned into 4 µm slices. These sections were mounted on glass slides and stained with hematoxylin–eosin, following the procedure outlined by Kim et al. [55].

From each stained slide, the whole tissue was inspected using a Nikon eclipse light microscope (Nikon Instruments Inc., Japan). To record the tissue damage associated with YTX, three digital microphotographs of each tissue (digestive gland and gill) were captured at 20× using a Nikon DS-Fi3 camera (Nikon Instruments Inc., Tokyo, Japan). This information was used to determine the tissue damage grade in the digestive gland and gills. We established a five-level classification system to assess tissue damage across the entire slide as follows: unaffected (no damage), minimal (<25% damage), low (25–50% damage), moderate (50–75% damage), and severe (75% damage).

2.11. Statistical Analyses

The physiological rates, such as the clearance, ingestion, absorption efficiency, absorption rate, and excretion, of A. purpuratus juveniles exposed to different toxic diets were evaluated using the two-factor Permutation Test. This statistical approach was used because after applying conventional linear models (ANOVA), the residuals did not meet the basic assumptions of normality and homoscedasticity required for this type of model. The Permutation Test, developed by Fisher [56] and Pitman [57], assesses differences between groups when they do not meet previously mentioned assumptions. Additionally, an a posteriori test (PostHocTest) was applied using Scheffe’s method, which identifies significant differences between the groups. All analyses and graphic representations were performed using R 3.5.1 statistical and programming software (R Development Core Team, 2018) ‘Survival’ Therneau [58] and “lmPerm” packages Wheeler and Torchiano [59], available through the CRAN repository (www.r-project.org/, accessed on 12 May 2023).

3. Results

3.1. Clearance Rate

During the intoxication period, significant differences were observed in the clearance rate (CR) among the three diets (p < 0.05) (Figure 1). The highest values of CR correspond to the control group (diet A; 100% I. galbana) with a maximum of 0.65 ± 0.30 L h⁻¹ on day 15 and an average of 0.56 ± 0.07 L h⁻¹. Regarding scallops fed with diet B (30% P. reticulatum), a CR decrease by 17.85% was observed as compared with the control group with a minimum value of 0.40 ± 0.22 L h⁻¹ on day 2 and an average of 0.46 ± 0.06 L h⁻¹. For individuals fed diet C (70% P. reticulatum), the filtration rates were the lowest during this period, decreasing by 48.21% compared to the control group. The maximum value recorded corresponded to 0.41 ± 0.34 L h⁻¹, whereas the average was 0.29 ± 0.08 L h⁻¹.

In the detoxification stage, no significant differences were observed between the toxic diets (B and C) and control diet (p < 0.05) (Figure 1). The CR of the control group (A) remained relatively constant at an average of 0.62 ± 0.13 L h⁻¹. In the case of individuals consuming diet B (30% P. reticulatum), the CR increased by 24.49% to 0.61 ± 0.3 L h⁻¹ on the first day of detoxification; subsequently, it remains stable with an average of 0.56 ± 0.09 L h⁻¹. With respect to diet C (70% P. reticulatum), the change in food to I. galbana notably increases the CR by 130.77%, reaching 0.60 ± 0.31 L h⁻¹ during the first day of detoxification, and the rate then increases with a maximum of 0.84 ± 0.29 L h⁻¹ on the 25th day.

3.2. Ingestion Rates

During the intoxication period, significant differences were reported in the inorganic ingestion rates (IIR) of the toxic diets (B and C) compared to the control diet (A) (p < 0.05) (Figure 2). The highest IIR values were observed in the control group (diet A), with a maximum of 1.76 ± 0.81 mg h⁻¹ on day 15 and an average of 1.61 ± 0.18 mg h⁻¹. In diet B, the IIR was reduced by 22.36% compared to the control diet, with a maximum of 1.48 ± 0.97 mg h⁻¹ on the first day and an average of 1.25 ±0.18 mg h⁻¹. For diet C, the IIR was the lowest during the intoxication period, decreasing by 52.79% compared with diet A. The maximum IIR value corresponded to 1.03 ± 0.84 mg h⁻¹ on day 11, with an average of 0.76 ± 0.16 mg h⁻¹.

During the detoxification period, significant differences were observed between the different diets (p < 0.05) (Figure 2). The average IIR of juveniles fed diet A was 2.34 ± 0.82 mg h⁻¹. For diet B, the IRR increased from 125.19% to 2.95 ± 1.43 mg h⁻¹, while the IRR in individuals fed with on diet C increase considerably by 318.84%, reaching an average of 2.42 ± 0,63 mg h⁻¹.

During the intoxication period, significant differences were reported in the organic ingestion rates (OIR) of the toxic diets (B and C) compared to the control diet (A) (p < 0.05) (Figure 3). The OIR followed the trend of IIR, with the highest rate value for diet A corresponding to 1.62 ± 0.88 mg h⁻¹. In individuals fed diet B, the OIR was reduced by 16.43% compared to the control diet (A), reaching an average value of 1.22 ± 0.26 mg h⁻¹. Regarding diet C, the OIR was the lowest during this period, decreasing by 44.52% compared to that of the control, reaching an average value of 0.81 ± 0.23 mg h⁻¹.

During the detoxification period, significant differences were observed between the different toxic diets (p < 0.05) (Figure 3). Juveniles fed diet A reached an average OIR value of 1.91 ± 0.47 mg h⁻¹. The OIR of juveniles fed diet B increased by 105.55%, reaching an average of 1.77 ± 0.62 mg h⁻¹. Finally, in diet C, the OIR increased by 106.84% to an average value of 2.05 ± 0.70 mg h⁻¹.

3.3. Absorption Efficiency

No significant differences in absorption efficiency (AE) were observed between the toxic and control diets (p = 0.02537) (Figure 4). Similarly, in both periods, the AE values were similar between the diets. For the control diet (A), the minimum observed was 68.21 ± 3.56% and the maximum was 76.15 1± 1.77%, with an average of 72.24 + 2.36%. Regarding diet B, the minimum was 66.84 ± 6.70% and the maximum was 76.12 + 1.71%, with an average of 72.03 ± 3.02%. For diet C, the minimum value observed in absorption efficiency was 60.55 ± 7.82% and the maximum was 75.65 ± 1.75%, with an average of 69.03 ± 4.38%.

3.4. Absorption Rate

Significant differences were observed in the absorption rate (AR) between the control diet (A) and toxic diets (B and C) (p < 0.05) (Figure 5). The highest AR was reported in juveniles of the control group (diet A), with a maximum of 1.25 ± 0.19 mg h⁻¹ on day 15 and an average of 1.10 ± 0.12 mg h⁻¹. In the case of diet B, the AR decreased by 16.7% as compared to the control, with a maximum of 1.10 ± 0.21 mg h⁻¹ on day 11 and an average of 0.92 ± 0.21 mg h⁻¹. During the intoxication period, the AR was 52.06% lower than that of the control diet. For this treatment, the maximum AR corresponded to 0.84 ± 0.30 mg h⁻¹, while the average was 0.54 ± 0.19 mg h⁻¹.

During the detoxification period, there were no significant differences between the toxic diets (B and C) and control diet (A) (p = 0.05179) (Figure 5). The AR of the control diet (A) increased slightly, reaching an average value of 1.45 ± 0.31 mg h⁻¹. In the case of individuals on diet B, the absorption rate increased by 45.65% to 1.34 ± 0.48 mg h⁻¹, with an average of 0.92 ± 0.12 mg h⁻¹. Regarding diet C, the change in non-toxic food notably increased the AR by 178.57%, reaching an average value of 1.42 ± 0.38 mg h⁻¹.

3.5. Excretion Rate

During the intoxication period, no significant differences in excretion rates (ER) were observed between juveniles fed the control diet and those exposed to toxic diets (p = 0.0507) (Figure 6). During this period, the highest value obtained in the control group (diet A) was 53.85 ± 17.61 µg NH₄ h⁻¹ on day 11, with an average of 41.81 ± 9.82 µg NH₄ h⁻¹. Regarding juveniles fed with diet B, the maximum value observed was 69.35 + 20.81 µg NH₄ h⁻¹ for day 15, with an average of 50.73 ± 16.95 µg NH₄ h⁻¹. For diet C, the maximum value recorded corresponded to 60.63 ± 19.74 µg NH₄ h⁻¹, while the average was 49.45 ± 15.76 µg NH₄ h⁻¹.

In the detoxification period, no significant differences were observed between the toxic diets (p = 0.2227) (B and C), and the control diet (A) reached an average of 42.66 ± 6.53 µg NH₄ h⁻¹, while diet B has an average of 46.42 ± 8.08 µg NH₄ h⁻¹. Finally, diet C resulted in an average of 47.77 ± 7.07 µg NH₄ h⁻¹.

3.6. Toxin Analyses

No YTX was detected in individuals fed the control diet (Figure 7). Regarding diet B, individuals accumulated low concentrations of YTX during the toxicity period, reaching 2.47 ng g⁻¹ on the second day of experimentation. Subsequently, no toxins were recorded on day 7, which increased quickly to 72 ng g⁻¹ on day 11, and then decreased to values near 50 ng g⁻¹. In the case of diet C, the concentration did not exceed 25 ng g⁻¹ at the beginning of the intoxication period. Then, the concentration stabilized at day 5, increasing abruptly to 100 ng g⁻¹ by day 7. After that, the concentration decreased to approximately 50 ng g⁻¹ on day 15.

3.7. Histopathological Responses

Tissue damage was observed in the digestive glands and gills when the organisms were exposed to the toxic dinoflagellate P. reticulatum (Figure 8). Specifically, the digestive glands of the affected organisms showed tissue damage characterized by significant hemocyte infiltration, irregular and damaged digestive tubules with sloughing and disintegrating digestive cells, and a reduction in epithelial cell height in the digestive tubules. In the gills, damage is primarily associated with hemocyte migration, epithelial cell melanization, and the disintegration of epithelial cells. The percentage of affected tissue in the samples was evaluated to determine the extent of damage and record the level of affectation (unaffected, minimal, low, moderate, and severe damage) by organism for each experimental condition.

Significant variability in tissue damage levels was observed among individuals, affecting both the digestive gland and gills. A higher frequency of scallops with severe tissue damage in the digestive gland was registered in scallops fed diet B during T0 (40%) and those fed diet C at T15 and T30 (20 and 40%, respectively). Organisms with low and minimal tissue damage in the digestive gland were present in all treatments, and a higher frequency of organisms with minimal damage was observed in scallops fed diet A at T0 (100%). The tissue damage level in the gills was less evident than that in the digestive gland. At T0, scallops from diets B and C did not present tissular affectations (both 100%), and only those fed by control diet A presented a minimal percentage of organisms with minimal affectation (20%). During T15, only scallops fed diet C presented organisms with tissue damage in the gill, where all affected organisms presented moderate damage levels (40%). At the end of the recovery stage (T30), higher tissue damage in the gills was observed in organisms fed C (moderate: 40%; severe: 40%), followed by those fed diet B (moderate: 40%).

4. Discussion

The present study provides evidence that the clearance rate (CR), inorganic ingestion rate (IIR), organic ingestion rate (OIR), and absorption rate (AR) of A. purpuratus juveniles were significantly reduced when fed diets that included the toxic dinoflagellate P. reticulatum during the first seven days of exposure. In both diets B (30% P. reticulatum) and C (70% P. reticulatum), the CR, OIR, IIR, and AR were reduced by nearly 25 and 50% compared with the non-toxic diet (Isochrysis galbana). This reduction in CR or microalgal filtration has been highlighted previously in other scallop species exposed to toxic dinoflagellates [60,61,62]. In the scallop Pecten maximus, the filtration and respiration rates are reduced when organisms are exposed to the microalgae Alexandrium minutum for two days [60]; in contrast, juveniles of the scallop Nodipecten subnodosus have shown reduced feeding and increased pseudofeces production when exposed to the toxic dinoflagellate Gymnodinium catenatum, which produces paralytic shellfish toxins (PST) [63]. The same behavior has been reported in the scallops Pecten novaezelandiae in adults exposed to low concentrations of the toxic PST-producing dinoflagellate A. tamarense with lower CRs during the first three days of exposure [61]. The behavior modulating, filtering, and ingestion of microalgae caused by the addition of a toxic dinoflagellate suggests the ability of the scallop A. purpuratus to reject specific microalgae cells by selective feeding. This assumption is validated by the fact that scallops exposed to the toxic dinoflagellate accumulate lower amounts of YTX during the first five days, and the fact that scallops fed a toxic diet showing an increased CR, OIR, and IIR during the recovery stage (T30) were organisms from all diets fed by non-toxic microalgae (I. galbana). Low toxin accumulation has been observed in clams that close their shells when they are exposed to toxic algae [64]. This finding supports the idea that A. purpuratus can detect and reject P. reticulatum.

Selective feeding has previously been suggested for A. purpuratus. In the larval stage, some authors have suggested that organisms might selectively retain bacteria [65], and, in the adult stage, the pre-ingestive selection of food has been reported [66]. Perhaps selective feeding in marine bivalves is poorly understood. At least three mechanisms of selecting suspended particles have been reported: Preferential clearance on the ctenidia (e.g., selective removal of specific microalgae), (2) pre-ingestive selection by the labial palps (rejection through pseudofaeces), and (3) differential absorption in the gut [67,68]. Because the CR decreased and no pseudofeces were observed, we suggest that A. purpuratus preferentially filtrates non-toxic microalgae, rejecting P. reticulatum cells. The species-specific selection and avoidance of particular types of microalgae have been observed in clams Mercenaria mercenaria, Meretrix meretrix, and Ruditapes philippinarum [69]. In the scallop Argopecten irradians, the authors suggested that the organisms are able to recognize algal cells as non-self and non-food, avoiding the filtration of the toxic dinoflagellate Prorocentrum minimum [70]. Additionally, it has been reported that Mytilus edulis can distinguish algal food particles even when they are of similar size [71]. Similar observations have been reported in the tropical bivalve Lithophaga simplex, which selectively filtrates particles (bacteria and algae) despite their numerical dominance in plankton and their size [72]. Researchers have linked the selectivity of microalgae to the ctenidial architecture [67] and the interactions between the physicochemical properties of the particles and the mucus covering the pallial organs [67,72,73,74,75].

The filtration selectivity of A. purpuratus was supported by the fact that CR decreased and the ammonium excretion rate (ER) remained stable for scallops fed different diets. The ammonium ER can be related to the food consumption of the organisms. In marine bivalves, when organisms stop feeding, the rate of ammonia excretion is reduced [76]. In contrast, Navarro and Contreras [77] reported that in the mussel Mytilus chilensis, ammonia excretion is increased when organisms are exposed to the toxic dinoflagellate A. catenella for eight days due to the degradation of PSTs. However, the ER of A. purpuratus exposed to toxic algae was the same as that of the control. In this context, our findings suggest that the ammonium rate did not differ among organisms, as they maintained feeding on the non-toxic algae (I. galbana) provided. This highlights the species’ ability to adapt its metabolism to the different experimental diets tested in this study. The mechanism behind this detection still unknown and requires further validation and exploration [78]. It is important to note that there is a lack of understanding of the mechanisms related to avoiding the filtration of toxic or harmful microalgae. However, we hypothesize that the ctenidum or labial palps may be important organs containing receptors that may control filtration behavior.

Even lower CRs, OIRs, and IIRs were reported in scallops fed diets B and C during the first seven days of exposure to toxic algae, after which the organisms slightly increased these parameters. The same scenario has been reported for the scallop P. novaezelandiae. The authors reported that scallops reduced clearance rates when exposed to toxic PSTs produced by the dinoflagellate A. tamarense during the first three days and increased clearance rates before day six [61]. In our study, the increase in the CR, OIR, and IIR after day seven in scallops fed with toxic diets B and C was attributed to the loss of functionality of organs related to toxic algae detection caused by tissular damage that may have affected microalgae selectivity if recognition mechanisms are attributed to gill and pallial organs reported by other authors [72,73,74,75]. Organisms exposed to the toxic dinoflagellate in this study presented tissue damage in the digestive gland and gill, attributed to the presence of YTX. In the digestive gland, tissue damage is associated with significant hemocyte infiltration, irregular and damaged digestive tubules with sloughing and disintegrating digestive cells, and a reduction in epithelial cell height in the digestive tubules. In the gills, damage is primarily associated with hemocyte migration, epithelial cell melanization, and the disintegration of epithelial cells. Similar tissue damage has been observed in different shellfish species exposed to toxic dinoflagellates, associated with toxins produced by different toxic algae [24,70]. In A. irradians, the toxic dinoflagellate P. minimum has been associated with the sloughing of digestive cells from the digestive tubule epithelia and the migration of hemocytes to the stomach and intestine [70].

In the abalone Haliotis midae, exposure to YTX led to severe damage to the gill and external epithelia, characterized by a significant loss of cell adhesion [24]. The loss of functionality in the digestive gland caused by YTX has been reported in the abalone H. discus hannai, which damages the metabolic and digestive physiological activities [79]. These results suggest that the loss of function of these organs may be related to the observed physiological changes in scallops exposed to P. reticulatum in this study.

Cellular lysis and tissue damage in the gill and digestive gland caused by toxins in mollusks, particularly YTX, have been associated with the induction of oxidative stress (increases in reactive oxygen species and lipid peroxidation), the disruption of the antioxidant and defense systems, reduction in digestive physiological activity, and apoptosis mediated by caspase proteins [27,32,79,80]. Both the digestive glands and gills are tissues vulnerable to oxidative stress and apoptosis when exposed to toxic substances [81]. This susceptibility explains the observed tissue damage in A. purpuratus when exposed to P. reticulatum. Additionally, the loss of tissue function may indicate that the scallops’ recovery of CR is hindered while still exposed to the toxic dinoflagellate. The suggested loss of the ability to detect toxic algae cells caused by YTX was also supported by the increase in YTX content in A. purpuratus juveniles after day seven when the CR increased. Interestingly, during the recovery stage, the number of scallops with severe tissue damage increased, reflecting the presence of the toxin. In scallops, the digestible gland is an important energetic reserve tissue that stores lipids [82], and YTXs is a lipid-soluble toxin [80]; the tissular damage seen in the recovery stage of the scallops exposed to P. reticulatum was associated with remnant toxins contained in the digestible gland trapped in lipidic storage reserves that remain affecting tissues of the organ.

In this study, the massive infiltration of hemocytes was observed in the digestible gland and gill of scallops exposed to P. reticulatum. The migration of hemocytes through the epithelia into the alimentary tract, including the stomach and intestines, has been observed in A. irradians irradians exposed to the toxic dinoflagellate Prorocentrum minimum [70]. These authors reported the encapsulation of toxic algae by hemocytes, suggesting the recognition of the toxic dinoflagellate. Hégaret et al. [83] also reported hemocyte migration to the alimentary tract in Manila clams exposed to P. minimum. Similarly, in Mytilus edulis, hemocytes were observed migrating into the stomach and intestines following exposure to P. minimum [84]. The migration of hemocytes has been attributed to the elimination of harmful algal cells and toxin removal. It has been suggested that hemocyte cells in M. edulis can detect P. minimum cells as parasites [84]. Additionally, the presence of YTX has been observed in the hemocytes of the mussel Mytilus galloprovincialis [29]. Hemocytes are versatile cells that carry out several crucial physiological functions, such as nutrient digestion, transportation and distribution, wound healing, detoxification, shell mineralization, and excretion [85]. Among these functions, the recognition of non-self molecules is one of the most important [86,87], including the detection of toxic dinoflagellates and toxins [29,84,86,87,88]. In this context, the detection of dinoflagellates and toxins is feasible, and whether this recognition can be performed by tissue cells to change the physiological responses of the scallop and avoid the filtration of toxic dinoflagellates remains unknown.

The gill tissue melanization observed in scallops affected by P. reticulatum was attributed to modulation of immune response mechanisms that may be related to the migration of hepatocytes to sites where toxic algae or toxins were detected. Large foreign organisms are typically neutralized by hemocyte encapsulation, a process often accompanied by melanization [89]. Cellular melanotic encapsulation is a complex mechanism involving the coordination of hemocytes and various plasma proteins, including components of the phenoloxidase (PO) activation system and certain pattern recognition receptors [90]. Melanization reactions have been reported to be toxic to a wide range of potential pathogens (bacteria, fungi, and viruses) [90]. When organisms encounter potential pathogens, pattern recognition receptors trigger downstream serine protease cascades, ultimately leading to the activation of prophenoloxidase (PPO) [90]. It is suggested that this defense mechanism can be activated by the presence of toxic dinoflagellates, as the melanization of tissues occurs when different shellfish species are exposed to these organisms. Increased melanization in A. purpuratus gills has been reported in scallops exposed to A. catenella and its toxins [91]. In Nodipecten subnodosus, gill melanization has been observed in scallops exposed to PST-producing G. catenatum and is related to hemocyte aggregation and partial shell closure [62,63]. Additionally, in agreement with the melanization mechanism triggered by hemocyte encapsulation, Wikfors and Smolowitz [92] reported hemocyte aggregations containing melanized algal cells in different tissues when juvenile scallops were exposed to P. minimum. The fact that hemocytes activate melanization reactions highlights the idea that toxic algae are detected by the biological system (scallop), supporting previous ideas related to the presence of unknown recognition mechanisms in A. purpuratus that allow scallops to detect P. reticulatum.

In this study, scallops with a higher YTX content reached values below the regulatory limit established by the European Union for humans (3.75 mg Eq. YTX kg⁻¹). However, this toxin has been associated with shellfish mortality (Crassotrea gigas, Venerupis philippinarum, Panopea generosa, Mytilus galloprovincialis, and Mytilus trossulus) when P. reticulatum blooms are registered [27]. YTX has been associated with shellfish mortality in various parts of the world, including Japan [93], Norway [94], Canada [95], Chile [37,38], South Africa [22,23], and New Zealand [96]. These mortalities have been associated with damages in the digestive gland even when toxin is present in organisms tissues at low concentrations (0.28 mg kg⁻¹ equivalent to 2200 ng g⁻¹) [27]. In addition, a recent laboratory study described a reduction in the survival of Argopecten purpuratus veliger larvae exposed to a high cell density of P. reticulatum [97]. In this sense, we recorded a very low concentration of the toxin (below 100 ng g⁻¹), even when feeding organisms with a higher percentage of P. reticulatum (70%). However, digestive gland tissue was affected by YTX. Perhaps survival did not affect the results of this research, providing insight into how P. reticulatum can affect the healthy physiological state of the scallop A. purpuratus, making the organisms more susceptible to other stressful agents such as upwelling, acidification, hypoxia, temperature increase, opportunistic pathogens, and anthropogenic pollution, among others. Attention must be paid to the future scenarios of this dinoflagellate as a potential trigger that compromises A. purpuratus health.

It is worth noting that A. purpuratus exhibited considerable inter-individual variability in its response to P. reticulatum across different response variables in this study. This level of variability has also been documented in other marine bivalves exposed to toxic dinoflagellates, including C. gigas and P. maximus [98]. Previous research in oysters has suggested that this high variability in toxin accumulation is related to differences in feeding behavior [60,98,99,100,101]. Despite the inter-individuality variation results of the present research, particular consideration must be paid if researchers include more sensitive tools to understand the mechanisms underlying the interaction between the scallop A. purpuratus and the toxic dinoflagellate P. reticulatum.

Based on the parameters discussed above, we propose a hypothetical model for the interaction between A. purpuratus juveniles and the toxic dinoflagellate P. reticulatum. In this model, the exposed scallops initially recognize the presence of the dinoflagellate (the recognition of the dinoflagellate per se or the produced toxin) and respond by reducing filtration rates, assimilation rates, and toxin accumulation, possibly through selective filtration. The closure of valves or the cessation of filtration due to the presence of P. reticulatum may be a possible explanation. However, we discarded it as the ammonium excretion rate remained stable in scallops fed different diets and as the filtration rate was only reduced not stopped. On the other hand, prolonged exposure (7 days) leads to tissue damage, which may impair the scallops’ ability to detect the dinoflagellate, resulting in increased toxin accumulation, higher filtration rates, and further damage to the digestive gland and gill tissues (Figure 9). This idea needs further validation by different research groups in order to clarify these interactions. We encourage the application of novel technologies to validate the proposed hypothesis. Furthermore, this study significantly enhances our understanding of the non-food safety impacts that HABs may have on A. purpuratus, as previous studies in the field have reported mortalities in marine invertebrates at lower concentrations of P. reticulatum than those used in our study. Our findings suggest that the observed damage in exposed organisms could make scallops more susceptible to other stressors, such as pathogenic agents and environmental changes. This increased vulnerability may become particularly significant in future scenarios of pathogen expansion and global warming.

5. Conclusions

The present study demonstrates that the physiological responses of A. purpuratus juveniles are significantly impacted by exposure to the toxic dinoflagellate P. reticulatum. Specifically, feeding on diets containing 70% of P. reticulatum resulted in a marked reduction in the clearance rate (CR), organic ingestion rate (OIR), inorganic ingestion rate (IIR), and absorption rate (AR), particularly during the initial seven days of exposure. The tissue damage observed in the digestive gland and gills of scallops exposed to P. reticulatum, including hemocyte infiltration, melanization, and cellular disintegration, underscores the potential for YTX toxins to compromise the health of A. purpuratus. These findings highlight the vulnerability of A. purpuratus to P. reticulatum exposure, with implications for its overall physiological state and resilience to environmental stressors. Further research is necessary to elucidate the underlying mechanisms of toxin detection and the long-term effects of such exposure on scallop health and survival.