Express Diagnosis and Prediction of Remote Mass Mortality of Scallop Mizuhopecten yessoensis in Mariculture Farms Using Biomarkers

Abstract

The cage method for the cultivation of the seaside scallop Mizuhopecten yessoensis is the most developed and popular method at sea farms in Primorsky Krai (Sea of Japan). However, this method of mollusk cultivation requires the careful planning of farming activities. Recently, mariculture farms in different countries have often encountered the mass mortality of cultured hydrobionts. The causes of such diseases are not quite clear, and often their identification requires a large amount of time and financial expenditure. Therefore, the use of predictive mechanisms based on biomarkers can help identify hidden threats in cultured scallop organisms that lead to mass mortality. In this study, we propose a rapid diagnostic method for predicting the distant mass mortality of M. yessoensis cultured in cages using biomarkers. The assessment of the pathological state of cultured mollusks at earlier developmental stages using the DNA comet method and oxidative stress markers (malondialdehyde) will allow the diagnosis and prediction of significant losses of marketable individuals in marine farms. In this study, we evaluated different age groups of mollusks cultured in the different water areas of Peter the Great Bay (Sea of Japan). During the study, we found that the death of cultured mollusks increased with increasing DNA damage and the active accumulation of malondialdehyde in tissues. It was observed that in scallops aged 1+ cultured in Severnaya Bay, high levels of DNA molecule damage and malondialdehyde were registered in the digestive glands and gills, which subsequently led to the death of almost all marketable individuals aged 3+. Therefore, the work is of significant value in assisting the aquaculture industry in solving the emerging problems of scallop farming and preserving marketable products. The proposed markers effectively reflect the condition of molluscs under extreme conditions caused by various factors, making them highly suitable for monitoring studies and forecasts on aquaculture farms.

1. Introduction

Aquaculture is an important and promising sector of global fisheries, contributing to the increase in and conservation of biological resources. According to the United Nations Food and Agriculture Organization (FAO), marine and coastal aquaculture production approached almost 30 million tons (USD 67.4 billion) in 2016, of which bivalves accounted for 16.9 million tons (58.8 percent of total production) [1]. In 2018, global aquaculture production rose to a record 114.5 million tons, amounting to USD 263.6 billion. Total aquaculture production consisted of 82.1 million tons of aquatic animals (USD 250.1 billion), 32.4 million tons of algae (USD 13.3 billion), and 26,000 tons of ornamental shells and pearls (USD 179,000). Shellfish, including bivalves, accounted for 17.7 million tons (USD 34.6 billion). The total value of shellfish was USD 34.6 billion [2]. Despite the impact of the COVID-19 pandemic, global aquaculture produced a record 122.6 million tons in 2020, including 87.5 million tons of aquatic animals valued at USD 264.8 billion [3]. In 2022, the world produced a record 130.9 million tons of aquaculture products, up 6.6 percent from 2020 [4]. Meanwhile, experts have estimated that total fishery and aquaculture production could rise to 201 million tons by 2030 [2].

Recently, many mariculture farms using the method of cage cultivation have begun to face the mass mortality of cultivated hydrobionts [5], which is accompanied by significant financial losses. Mass mortality has been noted in different species of cultured bivalves (e.g., oysters, scallops, and mussels), and is often associated with exposure to parasites, rickettsia-like organisms [6], viruses, bacteria [7,8], and others. There are also other prerequisites affecting the survival of hydrobionts, such as environmental factors, excessive planting density, and inbreeding [6]. In addition, we cannot rule out the negative impact on the condition of cultivated mollusks from the use of various plastic devices, such as cages, buoys, etc. It is known that a large amount of plastic waste is generated in the running of mariculture farms due to the degradation of plastic devices in the aggressive marine environment [9]. When plastic products degrade, various toxic substances can be released into the water, which in turn can cause various negative changes in the bodies of cultivated aquatic organisms [10]. In this regard, the further sustainable development of mariculture farms requires a thorough study of the effects of the industrial cultivation of hydrobionts on the environment, as well as the continuous assessment of the physiological state of the cultured hydrobionts, allowing the early detection of pathological changes, which in turn can lead to the mass death of cultured mollusks [7].

The coastal strip of Primorsky Krai is one of the most promising regions of Russia for mariculture development due to its good climatic conditions and availability of considerable water areas, especially in the southern part of the region, which is suitable for hydrobiont cultivation. More than 50 operating mariculture farms are currently engaged in the cultivation of the most valuable species of mollusks, echinoderms, and algae [11]. In recent decades, the seaside scallop Mizuhopecten yessoensis (Jay, 1857) has been one of the traditional cultivated marine bivalves in Primorsky Krai. However, in recent years, some farms have begun to encounter the mass mortality of mollusks during cultivation.

The aim of this study was to use biomarkers as prognostic signals and compare them with the mortality of M. yessoensis from different water areas. To achieve this aim, indicators of the levels of damage to DNA molecules and malondialdehyde in the tissues of the gills and digestive glands of cultured scallops were determined. This approach is a promising, modern, diagnostic and prognostic tool for detecting hidden pathological shifts in any organism [12]. The obtained biomarker indicators were compared with data on the mortality of scallops from the studied aquafarms.

2. Materials and Methods

2.1. Site of Bivalve Collection and Materials

A comparative study was carried out on scallops grown in seafood farms located in different waters of Peter the Great Bay (Sea of Japan) in Severnaya Bay (Slavyansky Bay), and in the waters of Vostochnaya Bay (Rikord Island) (Figure 1). These water areas have different hydrological regimes, for example, Severnaya Bay is relatively shallow and characterized by weak water exchange, while the farm in Vostochnaya Bay on Rikord Island is located in a deeper water area with intense water exchange. The mollusks were selected in the post-spawning period (November) at ages 1+, 2+, and 3+ from the waters of Rikord Island, and 1+ and 2+ from Severnaya Bay, since after the summer period a mass death of mollusks aged 3+ was registered in this water area.

For the study, scallops were removed from their cages and dissected on ice, and the gills and digestive glands were isolated. The determination of the level of DNA molecule damage using the comet assay was carried out immediately after tissue extraction from mollusks in specialized premises located at the coastal base of Severnaya Bay and Vostochnaya Bay. Residues of tissues intended for the determination of malondialdehyde were immediately frozen in liquid nitrogen (Dewar vessel SDS-6M (JSC “NPO Geliymash”, Moscow, Russia)) and transported to the laboratory for further processing.

2.2. Determination of the Damage to DNA Molecules

To determine the extent of the damage in the DNA molecules, we used an alkaline variant of the comet assay [11,13] successfully adapted to marine organisms [14]. For this purpose, individual cells were isolated using an isotonic solution (500 mM NaCl, 12.5 mM KCl, 5 mM EDTA-Na2, and 20 mM Tris-HCl; pH 7.4). The working concentration was 105 cells/mL. Then, 50 μL of cell suspension was added to 100 μL of 1% low-melting-point agarose (MP Biomedicals, Eschwege, Germany) in 0.04 M phosphate buffer (pH 7.4) at 37 °C, mixed thoroughly, applied to a slide previously coated with 1% agarose solution for better adhesion, and covered with a coverslip. The sample was placed in the refrigerator for 3 min for agarose curing. The coverslip was carefully removed, and the slide was submerged in a lysis solution (2.5 M NaCl; 0.1 M EDTA-Na (1% Triton X-100 and 10% DMSO); 0.02 M Tris; pH 10) for 1 h in the dark at 4 °C. After washing with cold distilled water, the slides were placed in electrophoresis buffer (300 mM NaOH and 1 mM EDTA-Na2) and incubated for 40 min. Electrophoresis was performed at 2 V/cm for 15 min. After neutralization (0.4 M Tris-HCl; pH 7.4), the slides were stained with SYBR Green I. The visualization and registration of DNA comets was performed using a scanning fluorescence microscope (Zeiss, Oberkochen, Germany, AxioImager A1) equipped with an AxioCam MRc digital camera. For digital image processing, we used the computer program CASP software v 1.2.2. (CASPLab, Wroclaw, Poland; https://casplab.com, accessed on 24 April 2022), which allows for the calculation of different parameters of comets, indicating the degree of cellular DNA damage. For each comet, the fraction of DNA in the comet tail (DNAt) was calculated. In the scallop groups studied, 15 gel slides (1 slide = 1 individual) containing at least 50 comets in each were analyzed.

2.3. Determination of Malondialdehyde (MDA) Content

The content of MDA, a product of the oxidative degradation of fatty acids, was determined in tissues and subcellular fractions by color reaction with 2-thiobarbituric acid (TBA) [15]. The tissues were homogenized in a homogenizer (SILENTCRUSHER S; Heidolph Instruments GmbH & Co., Schwabach, Germany) in 0.1 M phosphate buffer (pH = 7.5). To prevent lipid peroxidation during the determination of MDA, an alcoholic solution of butylhydroxytoluene (Merck KGaA, Darmstadt, Germany. CAS-no 128-37-0) was added to the samples to a final concentration of 5 mM. The content of malondialdehyde was determined by color reaction with 2-thiobarbituric acid (TBA, Merck KGaA, Darmstadt, Germany. CAS-no 504-17-6).

Then, 30% trichloroacetic acid (AppliChem GmbH, Darmstadt, Germany. CAS-no 76-03-9) and 0.75% TBA solution were added sequentially to the tissue homogenate. The mixture was thoroughly mixed and heated for 20 min in a water bath (Memmert WNB 7, Memmert GmbH + Co., Schwabach, Germany) at a temperature of 95 °C. After cooling, sediments were separated from the samples by centrifugation at 3000 rpm for 20 min. The measurements were carried out at a wavelength of 580 nm and 532 nm; then, the difference in the readings of the optical density was found. To calculate the MDA content, the molar extinction coefficient was used: 1.56 × 105/cm/M. The relative content of MDA was expressed in nmol per g fresh wet weight of the tissue. The measurements were carried out on a Shimadzu UV-2550 spectrophotometer (Scinteck Instruments LLC, Manassas, VA, USA).

2.4. Statistics

Statistical processing of the results was performed using the application software package STATISTICA 6.0 and Microsoft Excel 2016. The results were evaluated for each experiment by comparing group mean values (p < 0.01 using Mann–Whitney test). Correlation coefficients between MDA and % DNA damage were estimated using Pearson regression.

3. Results

The results of our study showed that one-year-old (1+) and two-year-old (2+) scallops sampled from Severnaya Bay had significantly higher DNA molecule degradation in the gills and digestive glands than those from Vostochnaya Bay (Figure 2).

The level of DNA molecule damage in mollusks cultured in the area of Vostochnaya Bay in both the gills and digestive glands in all investigated age groups was practically at the same level and did not exceed 20%. At the same time, no significant differences between different age groups of mollusks from this water area were revealed. At the same time, in M. yessoensis from Severnaya Bay, the levels of gill and digestive gland DNA damage also did not differ significantly among different-aged individuals. Significant differences were observed in one- and two-year-old individuals of generations from different water areas. Thus, in the gills and digestive glands of scallops from Severnaya Bay, the level of DNA molecule damage was almost three times higher than in scallops from Vostochnaya Bay in both age groups (1+, 2+). The content of MDA in scallop tissue (Figure 3) had a similar pattern to the level of DNA damage. In M. yessoensis from Severnaya Bay, this index was significantly higher, almost three times higher in the gills and more than five times higher in the digestive glands than in scallops from the waters of Vostochnaya Bay (Figure 3).

Thus, in the Severnaya Bay generation, the MDA concentration in the gills was within 2–2.5 nmol/g raw weight, and in the digestive gland was within 5–7 nmol/g. As can be seen from the results obtained, there were no significant differences between age groups within one generation in each water area. At the same time, significant differences were observed between the same-age groups of mollusks from different water areas. Also, there was a significant positive correlation between the level of MDA in tissues and % DNA damage in the tissues studied in two different generations of scallops (p < 0.01) (Vostochnaya Bay—DNA/MDA ratio: gills—R² = 0.4943; digestive gland—R² = 0.2504. Severnaya Bay—DNA/MDA ratio: gills—R² = 1; digestive gland—R² = 1) (Figure S1).

4. Discussion

We would like to emphasize in particular that, in scallops cultured in Severnaya Bay, we recorded a mortality rate of scallops aged 2+ of more than 50%, and a 95% rate at the age of 3+ years. At the same time, in the mollusks in the farm located in the waters of Vostochnaya Bay, mass mortality was not observed: mortality was less than 20% in all age groups (Table S1). Based on the results obtained, we can conclude that the damage to the DNA molecules of the gills and digestive gland cells of M. yessoensis at values over 40% (Figure 2), and high levels of MDA (Figure 3) in tissues, are signals of irreversible effects occurring in the body, resulting in the mass death of hydrobionts. It is known that MDA is a widely used indicator of the development of oxidative stress, and the content of high concentrations of MDA in tissues (Figure 3) can itself lead to DNA molecule damage and mutations [16]. Most likely, during periods of mass mortality, mollusks are in a state of oxidative stress, which is confirmed by a significant increase in MDA content in the tissues of mollusks, with DNA molecule damage reaching critical values of almost 50%. Based on the results (Figure 1 and Figure 2), it can be assumed that the level of damage to DNA molecules is 40% or higher in the gills and digestive glands of scallops aged 2+ from Severnaya Bay; moreover, the concentration of MDA in the gills is higher than 6 nmol/g wet weight, and above 25 nmol/g wet weight in the digestive gland, indicating irreversible processes in the bodies of scallops that lead to mass death, as evidenced by the lack of data for this species at the age of 3+.

Various researchers have noted that the peak mortality in scallop farms corresponds to the late stage of spawning [6,8]. It is believed that the stress associated with spawning is the main factor of shellfish mortality, due to the weakening of the main protective systems of the body’s immune, antioxidant, and reparative systems, and it is during this period that the body is prone to various infections and invasions [6]. As another prerequisite for the high mortality of hydrobionts, some authors refer to excessive planting density, which contributes to the development of infectious processes, as well as inbreeding, which weakens the protective physiological potential of cultured mollusks [5,6,7,8]. In addition, plastic hydraulic devices (e.g., cages, ropes, and buoys), which are widely used in aquaculture, can pose a serious risk to cultivated aquatic organisms [9,10]. Plastic is a complex mixture in which chemical additives are loosely bound and can be washed into the environment [9,17,18]. Also, in the aquatic environment, especially the marine environment, which is characterized by highly dynamic physical and hydrochemical factors, the initial processes of degradation of plastic products occur, accelerating the leaching of toxic chemicals into the environment [19,20,21]. In addition to various diseases and chemical pollutants, the physiological parameters (including mortality) of cultured scallops are directly influenced by the environmental variables (temperature, salinity, oxygen, etc.) of a particular water area. Some researchers note that various types of polymers that interact with marine organisms, as a rule, do not have a noticeable effect on survival, but cause various sublethal effects at the molecular biochemical level associated with the generation of reactive oxygen species (ROS) and the development of oxidative stress processes, which in turn, and in combination with other acting factors, can be prerequisites for the mass mortality of aquatic organisms [22,23,24]. Based on the above examples and our results, we can suggest that, in mariculture farms where there is a high mortality rate, cultivated aquatic organisms are in a state of oxidative stress, which can be caused by the combined effects of negative factors. As is known, oxidative stress results in a weakening of the body’s antioxidant, repair, and immune systems, which in turn entails a cascade of biochemical changes that can lead to irreversible pathological consequences.

The most important manifestation of the negative impact of environmental factors on cultivated aquatic organisms at the molecular level is genotoxicity, which manifests as the accumulation of damage to the DNA molecule. According to the generally accepted perspective, accumulating damage in the cell genome can trigger a cascade of biochemical changes leading to cell apoptosis [24,25,26,27].

Our results confirm these conclusions, since it is the failure of the DNA molecule repair system that leads to the accumulation of damage in its structure, and the weakening of antioxidant protection contributes to the accumulation of lipid peroxidation products in tissues.

5. Conclusions

The analysis of the experimental data indicated that mollusks cultured in Severnaya Bay experienced pronounced oxidative stress, leading to increased MDA content in tissues and significant DNA damage. We believe that these biomarkers are early and sensitive indicators of the physiological state of cultured mollusks, as well as signals of the development of pathologies leading to the mass mortality of hydrobionts. Thus, when the level of DNA molecule damage in scallops aged 1+ is around 40–50%, we can foresee upcoming economic losses due to the mass mortality of hydrobionts in the next year. Of course, to make such predictions with confidence, more data on genome damage and MDA levels at different and earlier age stages of shellfish development must be collected. The results obtained in this work can be used to undertake new and promising research on predicting sustainability using biomarkers, not only for mariculture farms, but also for natural ecosystems in marine areas that are used for the commercial reproduction of hydrobionts.