Journal of biological and health sciences http://biotecnia.unison.mx

Universidad de Sonora

ISSN: 1665-1456

Origial Article

Physiological response of Crassostrea gigas during exposure and depuration period to dinoflagellate Prorocentrum lima

Diana Medina-Felix1 , Luis Rafael Martínez-Cordova2 , Alberto Macías-Duarte1 , José Luis Niebla-Larreta2 , Carmen Isela Ortega-Rosas1 , Norma García-Lagunas3*

1 Universidad Estatal de Sonora, Ley Federal del Trabajo s/n. Col. Apolo CP. 83100, Hermosillo Sonora.

2 Departamento de Investigación y Posgrado en Alimentos. Universidad de Sonora. Avenida Luis Donaldo Colosio s/n Edificio 7G, Col. Centro CP. 83000, Hermosillo Sonora.

3 SECIHTI, Avenida Luis Donaldo Colosio s/n Edificio 7G, Col. Centro CP. 83000, Hermosillo Sonora.

Respuesta fisiológica del ostión Crassostrea gigas durante un período de exposición y depuración al dinoflagelado Prorocentrum lima

ABSTRACT

Dinoflagellate Prorocentrum lima blooms produce toxins causing significant health risks when present in mollusks consumed by humans; thus, short-term exposure and de- puration periods were studied to assess the induced effects on Crassostrea gigas physiological system. Three treatments were evaluated: control diet with 2 × 106 cell/mL Chaetoceros muelleri; T1 oysters fed with P. lima (6 × 103 cell/mL); T2 oys- ters fed with P. lima (6 × 103 cell/mL) and C. muelleri (2 × 106 cell/mL) in day 13 exposure, followed by 15 days depuration. Differentiation and hemocyte count were examined, as well as hemolymph parameters (protein, glucose, lactate, choles- terol, and triglycerides). Accumulation of the okadaic acid in

C. gigas digestive gland was quantified after 13 days of expo- sure. A significant decrease in total hemocyte, granulocyte, and hyalinocyte counts was detected in the treatments with

P. lima after 13 days exposure, followed by a small increase during depuration; C. gigas physiological response against the dinoflagellate was also affected with significant differen- ces in glucose, lactate, cholesterol, and triglyceride compared between exposure and depuration. The experimental results suggest a detriment to C. gigas physiological response by P. lima exposure with recovery in hemocyte count after 8 days with control diet (depuration phase).

Keywords: Harmful algae; Crassostrea gigas; Prorocentrum lima; hemocyte; physiological response; immune system.

RESUMEN

El dinoflagelado Prorocentrum lima produce toxinas dañinas para la salud humana por consumo de moluscos; la exposi- ción y depuración de Crassostrea gigas fue analizada para co- nocer los efectos en el sistema fisiológico. Tres tratamientos fueron evaluaron: dieta control, 2 x 106 células/mL Chaetoce- ros muelleri; T1 alimentados con P. lima (6 x 103 células/mL); T2 alimentados con P. lima (6 x 103 células/mL) y C. muelleri (2 x 106 células/mL) por 13 días, seguido de una fase de 15 días de depuración. Se examinó diferenciación y recuento de hemocitos, así como parámetros en hemolinfa: proteína, glucosa, lactato, colesterol y triglicéridos; cuantificación de la

*Author for correspondence: Norma García-Lagunas e-mail: ngarcial@secihti.mx

Received: October 29, 2024

Accepted: February 12, 2025

Published: March 7, 2025

toxina ácido okadaico en glándula digestiva de C. gigas des- pués de 13 días de exposición. En los tratamientos con P. lima se detectó disminución significativa en hemocitos totales, granulocitos e hialinocitos. El sistema fisiológico de C. gigas también se vio afectado, reportando diferencias en la con- centración de glucosa, lactato, colesterol y triglicéridos entre las fases de exposición y depuración. El sistema fisiológico de

C. gigas se ve afectado como consecuencia de la exposición a P. lima, además se reporta una recuperación en hemocitos después de 8 días con la dieta control (fase de depuración). Palabras clave: Alga nociva, Crassostrea gigas, Prorocentrum lima, hemocitos, respuesta fisiológica, sistema inmune.

INTRODUCTION

Microalgal bloom events, also known as harmful algal blooms (HABs), are cellular proliferation of toxic or noxious algal spe- cies potentially harmful to aquatic animals, consequentially affecting food web interaction (Neves et al., 2021). HABs can occur by several factors, including temperature increase, coastal eutrophication, wind, and human dispersal (Glibert and Burkholder, 2006; Gobler, 2020).

HABs development in aquatic environments results in accumulation of phycotoxins that can be captured by sus- pension feeders. Furthermore, the excessive accumulation of HABs could induce damage to the animal respiratory system, resulting in death, and high biomass bloom can result in oxygen depletion (Karlson et al., 2021). HABs consist of multiple algal species capable of producing a variety of bioactive compounds and toxins, which are responsible for human poisoning syndromes related to crustacean shellfish, mollusk, and fish consumption. Toxins are known as paralytic, neurotoxic, amnesic, azaspiracid, and diarrhetic shellfish poi- soning according to their effects on human health (Anderson et al., 2021; Lad et al., 2022; Lassudrie et al., 2020).

Filter-feeding bivalves represent an important compo- nent of the coastal ecosystem; specifically, Crassostrea gigas is one of the most important shellfish produced worldwide due to adaptability, rapid growth rate, and nutritional value (Bai et al., 2024). However, C. gigas farming is frequently affected by

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Volume XXVII

DOI: 10.18633/biotecnia.v27.1103

toxic species produced by algae of the genus Prorocentrum, Dinophysis, Ostreopsis, and Gambierdiscus (Gaillard et al., 2020; Accoroni et al., 2024; Neves et al., 2019; Economou et al., 2007). The benthic dinoflagellate Prorocentrum lima is widely distributed in tropical and temperate regions (Nishimura et al., 2020) and produces okadaic acid and dinophysistoxins, causing gastrointestinal symptoms (Grigoriyan et al., 2024).

P. lima induces diarrhetic shellfish poisoning (DSP) in humans by the consumption of bivalves contaminated with toxins, due to the accumulation of toxins produced by dinoflagella- te (Ayache et al., 2023; Faustino et al., 2021). DSP toxins are lipophilic polyether compounds, including okadaic acid (OA), dinophysistoxin-1 (DTX1), DTX2, and other derivative forms (Gerssen et al., 2010). Lipophilic toxins are captured and di- gested to accumulate in the digestive gland of filter-feeding mollusks, and even concentrations as low as 5 cells/mL may accumulate toxins to induce DSP in humans (Yasumoto et al., 1984; Huguet et al., 2020).

Mollusk bivalves have an innate immune system, based on nonspecific reactions from cellular and humoral respon- ses. The cellular part is formed by hemocytes, which are a key factor in the immune system together with the barrier tissues, synthesizing humoral factors with antimicrobial acti- vity (Tan et al., 2020; Andreyeva et al., 2022). Hemocytes are involved in phagocytosis, encapsulation, nodule formation, cytotoxicity, and antigenic self/non-self discrimination (Ales- ci et al., 2023). Therefore, the quantification of the circulating hemocytes is a useful determination of the immune system response (Faustino et al., 2021; Andreyeva et al., 2021).

Bivalve exposure to P. lima induces damage to the di- gestive system, negative effects on feeding, respiratory rate, and alteration in the circulating hemocyte concentration (Neves et al., 2019; Faustino et al., 2021). Nonetheless, lack of information still exists of the oyster physiological response to dinoflagellate toxins, thus, hemolymph parameter con- centrations and hemocyte count could serve as a valuable tool for rapid quantification of the physiological response to stress in C. gigas. Therefore, the present study aims to des- cribe C. gigas cellular and immune physiological response to DPS toxin producer dinoflagellate P. lima.

MATERIAL AND METHODS

Oyster acclimatization

Healthy adults of the Japanese oyster Crassostrea gigas were obtained with an initial weight of 40 ± 5 g, and 50 ± 10 mm shell length, from a local oyster farm (Estero la Cruz, Kino Bay, Sonora, Mexico). Oysters were transported to the laboratory to be cleaned of epibionts. The animals were maintained in 50-L tanks with controlled salinity (34 practical salinity units, PSU), and temperature (24 ± 1°C) for a 15-day acclimation period. Oysters were fed with Chaetoceros muelleri daily, according to Helm et al. (2004).

Algal culture

The benthic dinoflagellate Prorocentrum lima (strain PLHV- 4) was obtained from CIBNOR (Centro de Investigaciones

Biológicas del Noroeste, S.C). P. lima was cultivated using mo- dified F/2 medium + Se in Fernbach flasks and glass carboy, maintained at 22 °C ± 1 °C, light/dark= 12:12, and 200 µmol photons m−2 s-1 light intensity, according to Núñez-Vázquez et al. (2003). P. lima strain can produce OA = 2,041 pg/cells, DTX1 = 1.33 pg/cells, and DTX2 = 0.09 pg/cells of toxins.

The dinoflagellate P. lima was harvested at the late exponential growth phase at day 20 (determined before the experimental phase) to feed the oysters, whose cell density was quantified after cell fixation with Lugol’s solution (Gifford and Caron, 2000), using a 1 mL Sedgwick-Rafter counting chamber (Olympus BX41, Tokyo, JP) under a microscope.

The control microalgae used for the present experiment was Chaetoceros muelleri, provided by the Universidad de Sonora (DICTUS), and cultured in Bubble column photobio- reactors (PBRs) using F/2 medium (Guillard and Ryther, 1962), with constant temperature 22 ± 1 °C and illumination 250 µmol photons m-2 s-1.

Experimental exposure and sampling

After the acclimation period, oysters were fed once a day for 13 days of trial in three treatments: Control or non-toxic diet with 2 x 106 cell/mL of C. muelleri; T1 oysters fed with P. lima cells only (6 × 103 cell/mL); and T2 oysters fed with a combina- tion of P. lima (6 × 103 cell/mL) + C. muelleri (2 × 106 cell/mL). Dinoflagellate concentrations were based on field observa- tion data of HAB events in the Gulf of California (Hallegraeff et al., 1995). The exposure effects of the toxic dinoflagellate P. lima on C. gigas were examined in triplicate samples for each treatment, using 20 oysters by replica, placed in 10-L plastic containers with constant aeration.

During the exposure phase, four oysters from each ex- perimental unit were sampled randomly on days 1, 5, 10, and

13. For the depuration phase, four oysters from each experi- ment were collected on days 3, 6, and 15 after the dinoflage- llate exposure. Sampled organisms of each experimental unit were replaced by the same number of organisms (previously marked, and kept under the same experimental conditions) to maintain the number of organisms constant in each unit; additionally, each experimental unit received 100% water exchange every 24 h, before feeding, and a P. lima count was performed to record the consumption percentage. Oyster mortality, feeding behavior, hemocyte count, and cell-free hemolymph metabolite quantification were evaluated.

To determine okadaic acid toxin accumulation in C. gigas after 13 days of feeding with P. lima, the digestive gland of four animals was removed and lyophilized. Okadaic acid extraction was performed according to Lee et al. (1987) with some modifications, while okadaic acid quantification was performed by high-performance liquid chromatography (HPLC) analysis using Agilent 1200 Series Liquid Chroma- tographer, with a diode array detector (DAD) (240 nm), fluorescence detector (FLD) (266 nm excitation and 316 nm emission), and an ultrasep C18 column (250×5 mm, Agilent Bonus); a mobile phase flux of 1.250 mL/min (acetonitrile: water 80:20) and 20 μL of the sample were injected (Pinto- Silva et al., 2005).

Hemocyte count

Hemolymph was obtained from the oyster pericardium using a 1-mL syringe; a hemolymph pool was prepared for each replicate. Immediately after sampling, 100 μL of hemolymph from 3 individual oysters were added to 900 μL of anticoa- gulant (sodium citrate, pH 7.5), and the rest of the samples were centrifuged at 9000g for 5 min (4°C) to separate plasma for analysis. Hemocytes were counted using a Neubauer chamber; the granulocyte and hyalinocyte cell classification was performed according to the morphologic parameters under a light microscope (Olympus BX41) (Faustino et al., 2021). Total hemocyte count was expressed as the number of hemocytes (× 106) mL-1 of hemolymph.

Physiological analysis

The plasmatic parameter concentrations, such as protein, glucose, lactate, cholesterol, and triglycerides were quan- tified using the commercial Home Testing Kits RANDOX® (Great Britain), following the manufacturer’s specifications, with some modifications. Absorbance was read in a micro- plate scanner (Bio Rad, iMark ™), according to Sánchez-Paz et al. (2007).

Statistical analysis

Data were analyzed using a normality test; homogeneity of variances was verified by Leven’s test. A two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to compare metabolite concentration and hemocyte counts among treatments and time. Differences were considered significant when P < 0.05. Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed with JMP® Pro 16.0.0 Software.

RESULTS AND DISCUSSION

Crassostrea gigas feeding behavior

Oysters filtered and ingested both microalgal cells. The control group (fed exclusively with C. muelleri) maintained a normal feeding behavior; no closure of valves or mantle retraction was observed, and the cilia were observed outside the valves during feeding; additionally, a high-water clarifica- tion and rich stool production was recorded during the trial. The oysters ingested 100% of C. muelleri cells, and up to 93% of P. lima (Table 1).

On the other hand, oysters fed with the dinoflagellate

P. lima, produced fecal pellets partially degraded, indicating a partial degradation of the cells. However, a change in C. gigas feeding behavior with P. lima diets was observed at the beginning of the depuration phase with a delay in feed captured (C. muelleri) by the oysters; after two days of depu- ration, the oysters were capable of recovery and capture all feed in the first hours.

Quantification of the toxin Okadaic acid, was determi- ned in the digestive gland of oysters after 13 days of feeding

C. gigas with P. lima (T1 and T2), reporting 250 ng OA.g-1 for the P. lima treatment group (T1), and 200 ng OA.g-1 for the C. muelleri + P. lima treatment (T2).

Table 1. Consumption percentage of Prorocentrum lima after 24 hours feeding of Crassostrea gigas with T1 (6 ×103 cell/mL of P. lima), and T2 (C. muelleri + 6 ×103 cell/mL of P. lima).

Tabla 1. Porcentaje de consumo en Prorocentrum lima despues de alimentar

Crassostrea gigas por 24 horas, con T1 (6 ×103 cel/mL de P. lima), y T2 (C. muelleri + 6 ×103 cel/mL de P. lima).

Effects of Prorocentrum lima exposure in Crassostrea gigas hemocytes

Total hemocyte count (THC) reported significant differences between exposure time and experimental groups (P < 0.05). The control group showed the highest hemocyte concen- tration with no significant variation during the trial (Figure 1A). Nevertheless, groups fed with P. lima (T1) and P. lima + C. muelleri (T2), reported differences compared to the control group and during the time with a decrease in hemocyte con- centration, reaching the lowest concentration on day 13 of the dinoflagellate exposure. Furthermore, a recovery in the hemocytes was detected during the depuration phase (15D) in T1 and T2.

The granulocyte concentration reported significant differences (P < 0.05) between treatments and during the exposure and depuration phases (Figure 1 B). The control group presented the highest granulocyte concentration compared to T1 and T2, and no differences were reported between T1 and T2 during the exposure and depuration phases. Conversely, the hyalinocyte concentration reported significant differences between the control group with the highest concentrations, and the groups exposed to P. lima (P

< 0.05) (Figure 1 C); however, no differences were reported during the time among treatments. Finally, the lowest con- centration of granulocytes and hyalinocytes was reported on day 13 of the exposure phase with an increase on day 15 of the depuration phase.

Physiological analysis in Crassostrea gigas exposed to

Prorocentrum lima

Alterations in C. gigas plasma parameters were detected after P. lima exposure, except for protein concentration, which remained constant during the dinoflagellate expo- sure phase with no significant differences (P < 0.05), and an average of 25.8 ± 0.8 mg/mL (Figure 2A). On the other hand, glucose concentration in C. gigas plasma reported significant differences (P < 0.05) between exposure and depuration

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Figure 1. Total circulating hemocytes (TCH) (A), granulocytes (B) and hyalinocytes (C) counts in the hemolymph of Crassostrea gigas during an exposure phase with the dinoflagellate Prorocentrum lima (13 days) and a depuration phase (15 days). Letters indicates significant differences between exposure and depuration phases (p < 0.05); the mean ± standard deviation (SD) is indicated.

Figura 1. Recuento total de hemocitos circulantes (TCH) (A), Granulocitos (B) hialinocitos (C) en la hemolinfa de Crassostrea gigas, durante una fase de exposición al dinoflagelado Prorocentrum lima (13 días) y una fase de depuración (15 días). Letras indican diferencias significativas entre las fases de exposición y depuración (p < 0.05); se indica la media ± desviación estándar

phases; nonetheless, no differences were reported among treatments. After 5-day exposure to the dinoflagellate, gluco- se concentration increased in T1 and T2; the lowest glucose concentration was reported in the control group at day 15, with a concentration of 1.05 mg/mL (Figure 2B).

Lactate levels on plasma reported significant differences (P < 0.05) with the highest lactate concentrations on day 5 of the exposure phase for T1 (0.34 mg/mL), while, a significant decrease was reported on day 8 of the depuration phase in T2 (0.29 mg/mL) (Figure 3A). Additionally, a negative corre- lation of r = - 0.2 was detected between lactate and glucose parameters during the exposure phase.

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Cholesterol concentration in C. gigas plasma reported significant differences over time of exposure to P. lima, with the lowest concentration on day one in T1; after the exposure

Chaetoceros muelleri control diet

Prorocentrum lima diet T1

phase, a significant increase was reported in all experimental groups (Figure 3B). Similarly, a significant increase (P < 0.05) was reported in triglyceride concentration on day 15 of the depuration phase, while, the lowest one was reported on day 10 of the dinoflagellate exposure phase (0.32 mg/mL) (Figure 4).

DISCUSSION

Oysters filtered and ingested both microalgal cultures used in the experiment, producing fecal pellets containing partia- lly degraded cells of the dinoflagellate P. lima in the form of pseudo-feces. Similar C. gigas feeding behavior exposed to P. lima and reduction of the clearance rate were reported pre- viously (de Romero-Geraldo et al., 2014; de Romero-Geraldo et al., 2016; García-Lagunas et al., 2019). This phenomenon,

Days

Chaetoceros muelleri Prorocentrum lima combined diet T2

Figure 2. Protein (A) and glucose (B) values in Crassostrea gigas plasma, during an exposure phase with the dinoflagellate Prorocentrum lima (13 days) and a depuration phase (15D depuration days). Letters indicates significant differences between exposure and depuration phases (p ˂ 0.05); the mean ± SD is indicated.

Figura 2. Valores de proteína (A) y glucosa (B) en plasma de Crassostrea gigas, durante una fase de

exposición al dinoflagelado Prorocentrum lima (13 días) y una fase de depuración (15D días). Letras indican diferencias significativas entre las fases de exposición y depuración (p < 0.05); se indica la media

± desviación estándar.

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Figure 3. Values of plasma lactate (A) and cholesterol (B) in Crassostrea gigas, during an exposure phase with the dinoflagellate Prorocentrum lima (13 days) and a depuration phase (15D depuration days). Letters indicates significant differences between exposure and depuration phases (p ˂ 0.05); the mean

± SD is indicated.

Figura 3. Valores de lactato (A) y colesterol (B) en plasma de Crassostrea gigas, durante una fase de exposición al dinoflagelado Prorocentrum lima (13 días) y una fase de depuración (15D días). Letras indican diferencias significativas entre las fases de exposición y depuración (p < 0.05); se indica la media

± desviación estándar.

where the harmful algal cell can pass as a viable cell through the digestive system and be introduced again into the envi- ronment, is a real challenge to mollusk aquaculture (Hégaret et al., 2007).

No mortality was observed in oysters by the toxic dinoflagellate exposure. Evidence suggests that bivalves can survive and develop during HABs by the action of the immune system responding to DSP produced by dinoflage- llates, activating protection against okadaic acid (Hégaret et al., 2011; Mello et al., 2013). Similarly, previous research demonstrates that C. gigas exposure to 30 × 103 cell/mL of P.

lima did not result in mortalities (de Jesus Romero-Geraldo et al., 2014; Tan et al., 2023; García-Lagunas et al., 2019).

Diarrhetic shellfish toxin accumulation occurs mostly in the digestive gland; the amount of toxins accumulated by mollusks depends on their ingestion rates, particle capture, and capacity for selective feeding (do Prado Leite et al., 2021; Bricelj and Shumway, 1998). The okadaic acid concentration reported in C. gigas digestive gland after 13-day exposure to

P. lima, are above the regulatory limit for human consump- tion (160 ng OA per gram of meat) (Moreira-González et al., 2022). In the same way, okadaic acid values are similar to

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Figure 4. Values of plasma triglycerides in Crassostrea gigas, during an exposure phase with the dinoflagellate Prorocentrum lima (13 days) and a depuration phase (15D depuration days). Letters indicates significant differences between exposure and depuration phases (p ˂ 0.05); the mean ± SD is indicated.

Figura 4. Valores de triglicéridos en plasma de Crassostrea gigas, durante una fase de

exposición al dinoflagelado Prorocentrum lima (13 días) y una fase de depuración (15D días). Letras indican diferencias significativas entre las fases de exposición y depuración (p < 0.05); se indica la media ± desviación estándar).

those reported by Mafra et al. (2015), 183.3 ng OA.g-1 in C. gigas visceral tissue exposed to the dinoflagellate Dinophysis acuminata (13 750 cell/L) after two weeks.

Hemocytes modulate the invertebrate immune system as a key immune response component, responsible for pha- gocytosis, recognition, and cytotoxic reactions (Weng et al., 2022). Additionally, hemocytes participate in biological pro- cesses, such as shell production, nutrition, endocrine signals, and wound healing (Song et al., 2010). Hemocytes function as a rapid, and efficient response to stress (Weng et al., 2022; Lassudrie et al., 2020), and the effects of harmful algal toxins have been reported in bivalve hemocyte response (Tan et al., 2023).

Exposure to harmful algal toxins could induce an im- munostimulant or an immunosuppressive effect, depending on the dinoflagellate toxicity and health status of the animal (Hégaret et al., 2011). In the present study, a significant decrease of hemocytes was recorded on the first 13-days of the exposure phase to P. lima. The decrease of hemocyte concentration is related to the toxins assimilated by C. gigas, because of the activation of the oyster immune system in res- ponse to the toxins. In the same way, a decrease in hemocyte concentration was reported for the bivalve Perna perna after 48 h exposure to 9 × 105 cell/mL of P. lima (Neves et al., 2019), and a 54% decrease in the mussel hemocyte count after P. perna and Dinophysis acuminate exposure (Simões et al., 2015). Similarly, exposure of the bivalve Argopecten irradians to different concentrations of okadaic acid (50, 100 and 500 nM) produced by the dinoflagellate Alexandrium tamarense induced a decrease in hemocyte concentration after 12 h (Chi et al., 2016).

Granulocytes and hyalinocytes can be distinguished by the presence or absence of cytoplasm granules. Both cells contribute to the immune system response, through diffe- rent mechanisms such as phagocytosis and encapsulation. Moreover, the main distinction between these two hemo- cytes has been suggested to be the different life stages of the same cell type, however, they perform different functions (De la Ballina et al., 2022). In the present study, granulocyte and hyalinocyte were also affected by P. lima with a significant decrease in both cell types. Mello et al. (2010) reported a 12% decrease in bivalve granulocytes during the natural bloom of Dinophysis acuminata; the author also suggests a similar hyalinocyte and granulocyte production, but a granulocyte migration to the affected tissue (gills and intestines) could explain the decrease of this type of cell.

Plasmatic parameters in the invertebrate hemolymph can be a useful indicator of the physiological status (López- Elías et al., 2016). To our knowledge, this is the first report on C. gigas physiological status using commercial quick response kits. It is important to determine the normal rate of each parameter, as a rapid way of determining C. gigas physiological and health status (Medina Félix et al., 2017).

The molluscan hemolymph plays an important role in metabolite and oxygen transport through the open cir- culatory system. The main protein in mollusk hemolymph is hemocyanin; this respiratory pigment is a glycoprotein, responsible for oxygen transportation (Machałowski and Jesionowski, 2021). Additionally, some proteins in mollusk hemolymph have been related to the immune response, such as coagulation and melanization (Pascual et al., 2006). Hemolymph protein concentration in bivalves could be

affected by a stress situation indicating a physiological res- ponse (Simões et al., 2015). Protein concentration in C. gigas hemolymph was not significantly affected by the dinoflage- llate P. lima exposure.

Glucose concentration in C. gigas hemolymph was affected by the dinoflagellate exposure, inducing an in- crease in glucose concentration after 5 days of exposure, and a decrease at day 10 of the exposure phase. Glucose in the mollusk hemolymph is the main source of energy regulating glycogen metabolism and other ATP-dependent physiological mechanisms (Borges et al., 2004; Principe et al., 2019). Glucose concentration in plasma is a powerful tool to estimate the health status of animals, nevertheless, it may be dependent on the diet, temperature, and immune system (Shadenko and Sidorov, 2020). During temperature stress, the concentration of cortisol and glucose increases in C. gigas hepatopancreas, indicating a physiological stress response, where the cortisol hormone played a significant role in glu- cose metabolism (Wang et al., 2022). In the present research, glucose concentration was maintained from 0.7-1.0 mg/mL with a significant increase as a response to the dinoflagellate exposure phase.

Lactate dehydrogenase is an indirect lactate measure- ment, which is the final product of anaerobic metabolism. In mollusks, the lactate dehydrogenase enzyme has been detected in the foot and adductor muscle, and is responsible for the cytosolic redox balance during anaerobiosis cellular metabolism (Lee and Lee, 2011). Therefore, lactate is a con- finable indicator of stress in mollusks (O’omolo et al., 2003). Lactate concentration increases on day 5 after the dinoflage- llate P. lima exposure, and decreases on day 10; in mollusks during hypoxia conditions, large concentrations of lactate are produced, mainly by muscle; in those cases, lactate is elimi- nated through gluconeogenesis, with a positive correlation between glucose and lactate (Medina Félix et al., 2019; Tripp- Valdez et al., 2019). The present study observed an increase in glucose levels on day 10 after C. gigas exposition to P. lima; at the same time, a decrease in the lactate concentration indicated a negative correlation between these parameters.

Lipids are important energy sources in invertebrate he- molymph; they function as an important energy source and are essential for growth, survival, and reproduction. Lipids can be stored as energy in the digestive gland and mantle of mollusks (Medina Félix et al., 2019; Martínez-Pita et al., 2012). Additionally, aquatic mollusks have a lower lipid concentra- tion in hemolymph than terrestrial species (Machałowski and Jesionowski, 2021). For instance, cholesterol and triglyceride contents in Achatina fulica hemolymph were 0.016 and 0.06 mg/mL respectively (Lustrino et al., 2010), compared to an average of 0.24 mg/mL cholesterol and 0.33 mg/mL triglyce- rides in C. gigas hemolymph.

CONCLUSIONS

The present study evaluated C. gigas immune and physio- logical responses after exposition to the dinoflagellate P. lima demonstrating a significant stress situation. These data

represent valuable tools for rapid physiological evaluation of oysters, providing reference parameters for C. gigas. A negative correlation has been reported in the present study between glucose and lactate in C. gigas hemolymph, with a decrease of lactate on day 5 of the dinoflagellate exposure, and an increase on the same day of glucose concentration, indicating the gluconeogenesis pathway activation. Cras- sostrea gigas exposure to the dinoflagellate P. lima induced accumulation in the digestive gland, and alterations in glucose, lactate, cholesterol, and triglyceride; at the same time, hemocyte composition was also affected by P. lima, with higher total hemocytes, granulocytes, and hyalinocytes in the control group. Moreover, C. gigas exhibited signs of recovery during the depuration phase. Therefore, the present study reaffirms the harmful effect that HAB-forming species possess, such as P. lima, on coastal benthic species as C gigas, and their possible recovery if exposure to toxic cells is for a short period.

ACKNOWLEDGMENTS

The authors are grateful to SECIHTI (Secretaría de Ciencias, Humanidades, Tecnología e Innovación) for the project through the researchers for Mexico program. Authors would like to thank the editor M.C. Diana Leticia Dorantes (Perito Oficial Traductor SGA-294/2007) for English edition.

CONFLICTS OF INTEREST

No conflicts of interest to declare.

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