RU2571817C1 - Method of assessment of health of marine bivalved molluscs and condition of their habitat - Google Patents

Abstract

FIELD: ecology.

SUBSTANCE: in bivalved molluscs haemolymph is taken, divided into plasma and hemocytes, determining their phagocytic activity. Additionally, haemolytic activity of plasma and concentration of total protein in it are determined as biomarkers.

EFFECT: method enables to evaluate the state of health of marine bivalved molluscs and their habitat based on biological indication of cellular and humoral factors of haemolymph of molluscs while simplifying and reducing the labour intensity of the method.

3 cl, 3 dwg, 5 ex

Description

The invention relates to the field of ecology, environmental protection, immunology and physiology, in particular to methods for assessing the health of marine bivalves.

Shellfish play an important role in the structure and functioning of marine benthic communities and are valuable food items. The effectiveness of the cultivation and extraction of fishing facilities is associated with the need for timely assessment of their health and constant monitoring of the state of marine ecosystems. The lack of clear criteria for assessing the physiological state of mollusks, the low level of development of veterinary control methods and the lack of information about the relationship of morphophysiological characters with the limits of norm and pathology in assessing the health status of these animals significantly limit the economic efficiency of marine farms. In this regard, the development of criteria and the optimization of diagnostic methods for monitoring the physiological state of commercial mollusk populations is an important practical task for solving a wide range of problems related to both the development of marine economies and the conservation of marine biodiversity.

The most popular method for assessing the physiological state of bivalves is the method for assessing their motor activity, which manifests itself in registering the position of the valves. Under normal conditions, the valves are open most of the time to provide continuous nutrition and absorption of oxygen from the water. In response to water pollution or other deterioration of its quality, mollusks initially close the sink, then the nature of their behavior, the rhythm of alternating periods of closed and open leaves change.

One of the latest in this area is a method of biological monitoring of the aquatic environment based on the registration of the valve leaflets of bivalve shell mollusks and a system for its implementation (RU 2361207, IPC G01N33 / 18, 07/10/2009). The method includes securing the valve of the position of the valves on the shell flaps of the shell, placing the clams with the sensors in controlled water, generating and transmitting optical radiation through the optical fibers of the transmission line, one of the sections of each of which is made in the form of a loop and mounted on one of the flaps of the fixed base of the sensor with focusing one side of the loop into the bottom of the groove made at the base of the sensor, with the possibility of mechanical interaction of the other side of the loop with the element of influence on the sensitive electric cop mounted on other casement shell, and with the possibility of deformation of the loop shape in its plane as a result of mechanical interaction with a loop member influence on the sensor element and the bottom of the sensor base of the groove. Further, the method provides for the conversion of optical radiation transmitted through the optical fibers into electrical signals by optical radiation receivers. In the complex, the system contains position sensors for the mollusk shell valves, amplifiers, analog-to-digital converters, and a computer.

The technical drawbacks of the system are that these sensors are difficult to manufacture, fasteners are bulky and unreliable, difficult for normal operation of mollusks, which leads to a restriction of the animal’s natural movements, the system is vulnerable to real environmental conditions (currents and waves) due to protruding parts; wires and sensors are easily susceptible to rapid fouling (algae, hydroids, mollusk juveniles, etc.), affecting the operation of the system, which leads to the need for periodic maintenance and calibration; when the animal is killed (replaced), equipment is lost or dismantled. In addition, for the implementation of the claimed method, it is necessary to place the mollusks in a separate container, for example a flowing aquarium; there is no possibility to trace the effect of chronic pollution on the state of mollusks. This method, based on the description of the proposed system, includes a complex instrument base, requiring significant economic costs. A limitation of this method is also the use of only bivalve mollusks leading an attached lifestyle.

The use of biochemical indicators seems to be promising, since any physiological, and even more morphological deviation from the norm is preceded by certain biochemical processes, which are the root cause of a change in the physiological state of the body, deterioration in the quality of its offspring or death. Biochemical markers of toxic water pollution include, first of all, changes in the concentration and activity of molecules such as enzymes - universal catalysts and regulators of metabolic processes, as well as accumulated harmful substances from the environment.

The method for determining toxic pollution of wastewater and natural fresh water (RU 2308719, IPC G01N33 / 18, 10.20.2007) involves placing mollusks in separate aquariums with running water, subsequent killing, removal of the liver, obtaining an extract of liver proteins, determining the activity of hydrolytic enzymes in it - acid phosphatase or deoxyribonuclease (DNase). A significant change in the total activity of the test enzyme or at least one qualitative change in the composition of its multiple forms in the experimental version as compared with the control is taken as an indicator of the toxicity of the tested water.

Technical disadvantages are the use of a complex instrument base, the need for a separate content and the death of the mollusk under study.

A known method of monitoring water pollution (US 20130118236 A1, IPC G01N33 / 18, 05.16.2013), which involves the use of sensors for oxygen consumption, particle clearance and apical growth of mollusks placed in a special chamber.

A known method for determining the lysozyme activity of biological objects (RU 2294373, IPC C12Q001 / 02, G01N033 / 48, 07.20.2006), which involves the cultivation of a test culture of Micrococcus lysodeikticus strain No. 2665 GISK them. Tarasevich on a liquid nutrient medium at a temperature of 27 ° C to an optical density of 3.2-3.4, exposure of the medium to freeze drying, preparation of a suspension from a test culture, which is introduced into the biological object under study and lysozyme activity of the biological object is evaluated according to the known method.

The disadvantage of this method is the difficulty of working with a bacterial culture that requires sterile conditions and constant monitoring of its condition.

The closest in technical essence to the claimed technical solution is a method for assessing the state of an ecosystem based on determining the concentration of a set of stress biomarker molecules in animal cells of various systematic groups (WO 2002022859A2, IPC C12Q1 / 00, G01N33 / 50, 03.21.2002). In this method, among the many methods for assessing the concentration of biomaker molecules, the authors recommend using methods based on interaction on the principle of "antigen-antibody" (various types of enzyme-linked immunosorbent assay (ELISA), Western blotting, etc.). Antigen is a biomarker of interest in a cell, antibodies are isolated and purified molecules after immunization of laboratory animals. The authors recommend the use of high performance liquid chromatography with protein A specific for Fc-legs of antibodies for the isolation and purification of antibodies. The obtained purified antibodies, in turn, are used to create affinity columns specific for the biomarker of interest. With their help, one can already repeatedly select target molecules from the material of organisms subjected to stress. If the concentration of the obtained protein allows, then it is possible to study its amino acid composition using one of the standard methods, for example, Edman sequencing. The authors also recommend supplementing the analysis of the studied molecules with one-dimensional polyacrylamide gel electrophoresis according to Lemmley (ID SDS-PAGE) followed by Western blotting. The boundaries of the normal biomarker content are determined by Western blotting and / or ELISA using samples obtained from animals subjected to and not subject to stress. As an alternative, the known method proposes to determine the "normal" and "abnormal" concentration values from the published data. As a result, the studied samples (liquid homogenates of cells or tissues of organisms subjected to stress) are divided by the number of biomarkers used in the analysis and for each of them an analysis is carried out according to a well-developed standardized method suitable for laboratory analysis and for working in the field. The results are compared with previously defined normal limits.

The disadvantages of this method include:

- complexity, multi-stage procedure for sample preparation and analysis;

- the use of a large set of studied biomarker molecules;

- high complexity and a significant lag in obtaining results in time that limit the possibilities of using this method for conducting express monitoring studies;

- death of the test object, since the homogenate of all animal tissues is used.

This method also has significant economic costs because it involves the maintenance of test objects in controlled conditions requiring separate aquariums, as well as the use of expensive reagents and equipment (based on the list in the description of this method).

The objective of the present invention is to develop a method of biomonitoring based on the bioindication of cellular and humoral factors of mollusk hemolymph, which is sufficiently sensitive to environmental changes, simplifying and reducing the complexity of the method of biomonitoring.

The problem is solved in that in the known method for assessing the health of marine aquatic organisms and the state of their habitat, including the selection of marine aquatic organisms, preparing tissue samples, determining the biomarker and comparing the biomarker values of animals subject to stress with animals not subject to stress, according to the present invention in the role of marine hydrobionts take bivalve mollusks. As the test tissue, hemolymph is used, which is then divided into plasma and hemocytes. The phagocytic activity of hemocytes is determined as a biomarker for assessing the health of marine aquatic organisms and the state of their environment.

In the proposed technical solution, attached and non-attached bivalve mollusks, in particular, scallop (Mizuhopecten yessoensis), mya arenaria (Mya arenaria), Kuril / Long-stemmed modiolus, and Pacific igreas (Pacific) various types of mussels. Bivalve mollusks, which are test objects, are used directly on the day of their catch, i.e. they do not require special conditions for their maintenance, as a result of which the laboriousness of biomonitoring is greatly simplified and the time spent on it is reduced.

The use of hemolymph as the test tissue significantly increases the accuracy and sensitivity of assessing the health status of the mollusk and its habitat. Due to the fact that hemolymph is a protective and transport type of tissue of the internal environment, responsible for maintaining homeostasis, providing immune defense and the formation of physiological adaptations to changes in the environment. Taking hemolymph does not require the destruction of hydrobionts, simplifies sample preparation and eliminates the use of sophisticated expensive equipment.

The separation of hemocytes from hemolymph plasma allows us to evaluate the health of mollusks based on the diagnosis of a biomarker, which uses the phagocytic activity of hemocytes. Phagocyte cells absorb and neutralize invading foreign agents by digesting them with hydrolytic enzymes of lysosomes. Phagocytosis as a fundamental component of the protective function is a highly sensitive indicator of the health status of living organisms. As a result, the sensitivity of the claimed method is significantly increased.

To increase the sensitivity and reliability of the method for assessing the health of mollusks and the state of their environment, it is proposed to additionally use hemolytic plasma activity as a biomarker. This operation does not require additional taking of material, since plasma is used, separated from hemolymph cells. Lytic substances (lysine proteins) carry out the lysis (destruction) of foreign agents (cells, bacteria, etc.).

Additionally, it is proposed to use the concentration of total plasma protein as a biomarker. The plasma is constantly contained in various concentrations of substances: lectins (participate in all cell-cell interactions, which is manifested in the mitogenetic, signaling, cytotoxic and other properties of these molecules, provide agglutination (gluing) of cells and molecules), lytic and antimicrobial (have bacteriolytic or inhibiting bacterial growth, exhibiting bacteriostatic properties). The effectiveness of the implementation and interaction of protective reactions depends on the concentration of a set of plasma proteins. Unlike the prototype, the content of a certain protein is not determined, but the total protein content in the plasma. As a result of which there is no need to carry out complex manipulations on their separation, cleaning on columns, etc. This not only simplifies the process of biomonitoring, reduces the time for its implementation. In the end result, this increases the sensitivity of the method and the accuracy of assessing the health status of bivalves and their habitat.

The use of all three biomarkers in a complex, their comparison in animals prone and not prone to stress, allows to achieve the claimed technical result, namely, sufficient sensitivity to environmental changes, simplification and reduction of the complexity of the method of biomonitoring.

The biomonitoring method is carried out in several stages:

Stage 1 - selection of marine aquatic organisms - they capture the tested sexually mature individuals of bivalves of the same age category, which is determined by measuring the size of the shell in the impact water area and conditionally background (clean) water area;

Stage 2 - preparation of tissue samples, i.e. taking hemolymph - from each animal, 2700 μl of hemolymph is taken with a syringe from the hemal sinus of the posterior adductor muscle. For this, the scalpel tip is placed along the valves and, pressing on it, slightly open the mollusk shell;

Stage 3 - separation of hemolymph into hemocytes and plasma - the taken hemolymph is transferred to test tubes in an "ice bath" until the cells are separated from the plasma by centrifugation. The tubes are centrifuged for 12 min at 800g in a refrigerated centrifuge at 15 ° C. After centrifugation, the supernatant representing the plasma is removed and transferred to cryovials, which are frozen in liquid nitrogen at −195.8 ° C; the resulting precipitate containing hemocytes is used to determine phagocytic activity (FA).

Stage 4 determination of the biomarker - FA. This stage is carried out according to generally accepted methods and includes three operations:

a) preparation of a cell suspension of hemocytes - the remaining precipitate after plasma separation is washed by adding (with subsequent resuspension) 2700 μl of physiological calcium-free salt medium (PBS) of the following composition: 436 mm NaCl, 10 mm KCl, 22 mm Na ₂ HPO ₄ · 7H ₂ O, 16 mM glucose, 12 mM N- (2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES) containing 0.045 M ethylenediaminetetraacetic acid (EDTA). An aliquot (10 μl) of the resulting suspension is used to count the number of hemocytes in the Goryaev’s chamber. The calculation of the concentration is carried out according to the well-known formula: the number of cells / ml = the number of cells over 1 large square * 250 * 1000. To obtain a primary cell culture, the cells are again centrifuged under the same conditions and resuspended in 2700 μl of artificial salt medium (ASC) with an osmosis of 1090 mOsM containing 460 mM NaCl; 9.4 mM KCl; 48.3 mM MgCl ₂ · 6H ₂ O; 6 mM NaHCO ₃ ; 10.8 mM CaCl ₂ · 2H ₂ O; 10 mM HEPES;

b) the in vitro reaction of phagocytosis - to set the phagocytosis reaction, 50 μl of the cell suspension from each animal is placed in the wells of a black 96-well plate. Samples are incubated in a humid chamber at 17 ° C for 40 min for maximum cell adhesion to the substrate. As in vitro biotic particles, the phagocytosis reaction is performed using heat-inactivated and stained with a solution of fluorescein-5-isothiocyanate (FITC, MP Biomedicals) bacteria at the rate of 15-20 cells per 1 hemocyte. Every 10 minutes after adding bacteria to the cells in the wells for 3 hours, the fluorescence of non-internalized bacteria is quenched by selecting the supernatant from the wells, adding 50 μl of a 0.1% trypan blue solution prepared on ASC and incubating the preparations for 12 minutes. The drugs are washed three times alternating the selection and addition of ASC. The phagocytosis reaction is stopped after 1 h 20 min from the moment of adding bacteria to the cells by fixing 50 μl of a solution of 4% paraformaldehyde prepared on ASC for 1 h. The phagocytosis reaction is set in three parallel repeats.

c) assessment of FA - the activity of phagocytes is analyzed by the fluorescence intensity of the samples in the tablets using a flatbed fluorimeter with an excitation light filter of 485 nm and an absorbing 535 nm. The result of the assessment is the optimal time for the in vitro reaction of phagocytosis, which includes the maximum value of phagocytic activity. Data are expressed in arbitrary units (conventional units).

Stage 5 - determination of a biomarker - hemolytic activity (GA) of plasma. This stage is carried out according to generally accepted methods and includes two operations:

a) determination of HA - a hemolytic reaction is carried out, for which donor erythrocytes (Er) are pre-washed from the preservation solution with an isotonic phosphate buffer mixture (0.8% NaCl, 0.02% KCl, 10mM Na₂HPO_four-KH₂PO_four, pH 7.4) by centrifugation for 12 min at 300g and 15 ° C until the supernatant is completely clarified. The amount of Er in the resulting suspension is determined by counting them in the Goryaev chamber with its further dilution to a concentration of 1.25 × 10⁵ cells / μl Tris-HCl buffer with pH 7.5 (TBS) (10 mm Tris-HCl, 150 mm NaCl, 2 mm CaCl₂) 50 μl of previously frozen plasma, 50 μl of TBS and 400 μl of Er suspension are added to 1.5 ml tubes. The resulting resuspended suspension is incubated for 1 hour, with periodic shaking on a shaker every 15 minutes. The reaction is stopped by adding 1 ml of TBS_one(10 mM Tris HCl, 150 mM NaCl, 30 mM Na₂EDTA), which includes a chelating agent that binds Ca ions²⁺necessary for the activity of most invertebrate lysines. Centrifuged for 12 min at 800g and 15 ° C. The supernatant is diluted with TBS₂(10 mM Tris-HCl, 150 mM NaCl) 6 times. The resulting suspension is transferred to a spectrophotometric cell and the absorbance is measured at a wavelength of 514 nm in a spectrophotometer. In each series of reactions, a negative control is carried out (spontaneous hemolysis of Er). For this purpose, at the very beginning, instead of plasma, 50 μl TBS are added to the tubes; positive control (complete lysis of Er) - instead of plasma, add 50 μl of 1% Triton X-100 solution on TBS.

b) GA score - GA score is expressed as a percentage according to the formula:

GA index = ((XX ₀ ) / (X ₁ -X ₀ )) * 100%, where

X is the optical density of the solution after incubation with plasma,

X ₀ is the optical density of the solution in the negative control,

X ₁ is the optical density of the solution in the positive control.

Stage 6 - determination of the biomarker - the concentration of total plasma protein - is carried out using the UV method, based on the ability of aromatic radicals, present mainly in amino acids of proteins, to absorb light with a wavelength of 280 nm. For this purpose, 100 μl of plasma is added to a quartz microcuvette (cat. No. 046-25302-11) with an optical path length of 10 mm and the optical density is measured on a spectrophotometer at a light wavelength of 280 nm and an absorption coefficient of 0.667.

Stage 7 - statistical processing of the obtained data is carried out using the software package Microsoft Excel 2007 and Statistica 6.0. The Kolmogorov-Smirnov test shows that the obtained values obey the law of normal distribution with significance levels p> 0.05 for all samples; based on this, further statistical analysis is carried out using parametric criteria. One-way analysis of variance (ANOVA) and paired t-test are used to test the significance of the hypothesis of the absence or presence of differences between the studied animal samples. Data are presented as the average ± confidence interval (FA in conventional units, GA in%, total protein concentration in g / ml).

Stage 8 - analysis of hemocyte FA, GA, and total plasma protein concentration compares biomarkers in stressed animals caught in the studied (impact) water area and animals not exposed to stress from the background water area.

Example 1

30 specimens of sexually mature bivalve mollusks of the same age category of the Kuril / Long-boiled Modiolus (Modiolus kurilensis) are caught from conditionally background water (an area with a concentration of a chemical that is characteristic of areas not directly affected by human activity and caused by local natural sources) - . East of Peter the Great Bay of the Sea of Japan. The Gulf of Vostok State Natural Complex Marine Sanctuary is located in this region, human economic activity is insignificant, geochemical conditions and pollutant content in water and soil correspond to a conditionally background level ([5], [1], [2]). At the same time, animals are collected from the impact water area (the water area located near a point source of pollutant emission and exposed to local toxic load from this source) - Amursky Bay (30 specimens). According to physical and chemical monitoring of the Far East, the Amur Bay, located near the agglomeration of the city of Vladivostok, does not meet sanitary and epidemiological standards and natural environmental indicators for phenols, petroleum products, the number of pathogenic bacteria, in particular enterococci, oxygen, phosphorus, heavy metals, and a number of other parameters that vary significantly from year to year ([5], [6], [3], [4]). Animals used in the experiment on the day of their capture.

From each animal, 2700 μl of hemolymph is taken with a syringe from the hemal sinus of the posterior adductor muscle.

The taken hemolymph is separated into hemocytes and plasma by centrifugation for 12 min at 800 g in a refrigerator centrifuge at 15 ° C. After centrifugation, the plasma is removed and transferred to cryovials, which are frozen in liquid nitrogen at −195.8 ° C.

The phagocytic activity of the remaining pellet from the cells is determined, for which a cell suspension prepared after washing the hemocytes is placed in the wells of a black 96-well plate. Samples are incubated in a humid chamber at 17 ° C for 40 min for maximum cell adhesion to the substrate. An in vitro phagocytosis reaction is performed. The activity of phagocytes is evaluated by the fluorescence intensity of the samples in the tablets using a plate fluorimeter.

FA modiolus of the Kuril from the impact area of 9000 ± 2000 conv. units

FA of the Kurilsky modiolus from the conditionally background water area of 17,400 ± 3,700 conv. units

Example 2

Carried out analogously to example 1. Only conduct an additional determination of GA biomarkers and the concentration of total plasma protein.

To determine the HA, 50 μl of previously frozen plasma are taken, mixed with 50 μl of TBS and 400 μl of Er suspension. The resulting resuspended suspension is incubated for 1 hour with periodic shaking on a shaker every 15 minutes. The reaction is stopped by adding 1 ml of TBS_one. Centrifuged for 12 min at 800g and 15 ° C. The supernatant is diluted with TBS₂6 times. The resulting suspension is transferred to a spectrophotometric cell and the absorbance is measured at a wavelength of 514 nm in a spectrophotometer.

GA of the Kuril modiolus from the impact water area 20.7 ± 10.3%.

GA of the Kuril modiolus from the conditionally background water area is 31.1 ± 10.6%.

The concentration of total plasma protein is determined using the UV method, for which 100 μl of plasma is added to a quartz microcuvette (cat. No. 046-25302-11) with an optical path length of 10 mm and the optical density is measured on a spectrophotometer at a light wavelength of 280 nm and the absorption coefficient of 0.667.

The concentration of total plasma protein of the Kuril modiolus from the impact water area is 0.87 ± 0.09 g / ml.

The concentration of total plasma protein of the Kuril modiolus from the conditionally background water area is 1.03 ± 0.07 g / ml.

Example 3

Carried out analogously to examples 1 and 2, only as a bivalve mollusk take the scallop (Mizuhopecten yessoensis).

FA of the scallop from the impact area 9000 ± 2000 conv. units

FA of the scallop from the conditionally background water area of 13400 ± 4000 conv. units

GA of the coastal scallop from the impact water area 12.8 ± 7.5%.

GA of the scallop from the conditionally background water area is 19.7 ± 10.9%.

The concentration of total plasma protein of the coastal scallop from the impact water area is 0.6 ± 0.1 g / ml.

The concentration of total plasma protein of the coastal scallop from the conditionally background water area is 0.9 ± 0.10 g / ml.

Example 4

Carried out analogously to examples 1 and 2, only as a bivalve mollusk they take mya arenaria (Mya arenaria).

FA missions of the arenaria from the impact water area 13000 ± 3000 conv. units

FA missions of the arenaria from the conditionally background water area 19500 ± 4000 conv. units

GA of the arenaria from the impact water area 32.7 ± 6.8%.

GA missions of arenaria from the conditionally background water area 46.2 ± 9.6%.

The concentration of the total plasma protein of the arenaria mission from the impact water area is 1.26 ± 0.17 g / ml.

The concentration of the total plasma protein of the arenaria mission from the conditionally background water area is 1.78 ± 0.07 g / ml.

Example 5

Carried out analogously to examples 1 and 2, only as a bivalve mollusk take a giant mussel / Gray (Crenomytilus graynus).

FA of mussel giant from the impact water area 9300 ± 4100 conv. units

FA of mussel giant from the conditionally background water area 18600 ± 3300 srvc. units

The giant mussels from the impact water area are 62.9 ± 9.4%.

GA of mussel giant from conditionally background water area 96.7 ± 27.3%.

The concentration of the total plasma protein of the giant mussel from the impact water area is 1.9 ± 0.1 g / ml.

The concentration of the total plasma protein of the giant mussel from the conditionally background water area is 3.45 ± 0.57 g / ml.

An analysis of the phagocytic activity of modioli showed that animals from the impact water area have significantly lower values of the average phagocytic activity (9000 ± 2000 conventional units) compared with those from the conventionally background water area (17400 ± 3700 conventional units). The applied t-test confirmed the significance of differences (p> 0.05) (Example 1). A comparative analysis of the hemolytic activity and total plasma protein concentration of M. kurilensis from conditionally background and impact water areas showed a significant decrease in those in animals from the impact - Amursky Bay (p <0.05). The hemolytic activity of plasma of modioluses from the impact water area reached 20.7 ± 10.3%, the concentration of the total protein was 0.87 ± 0.09 g / ml, while the values of the same parameters in animals of the conditionally background water area were 31.1 ± 10.6 % and 1.03 ± 0.07 g / ml (Example 2).

A comparative analysis of the phagocytic activity of hemocytes in the scallop revealed a significant decrease in such in animals from the impact water area (8500 ± 3200 conventional units). The average phagocytic activity of mollusks from the hall. East was 13400 ± 4000 conv. units The concentration of total protein was 0.9 ± 0.10 g / ml, and hemolytic activity of 19.7 ± 10.9%; from the Amur Bay - 0.6 ± 0.1 g / ml and 12.8 ± 7.5%. Applied t-test confirmed the significance of differences (p> 0.05) (Example 3).

A comparative analysis of the phagocytic activity of hemocytes of the myria arenaria revealed a difference in animals from impact (13000 ± 3000 conventional units) and conditionally background (19500 ± 4000 conventional units) waters. Also, mollusks from the impact water area showed a decrease in the concentration of total protein 1.26 ± 0.17 g / ml and hemolytic activity - 32.7 ± 6.8%, whereas from the hall. East, the same parameters were 1.78 ± 0.07 g / ml and 46.2 ± 9.6%, respectively. Applied t-test confirmed the significance of differences (p> 0.05) (Example 4).

The average phagocytic activity of the giant mussel from the conditionally background water area was 18600 ± 3300 conv. units A comparative analysis of the phagocytic activity of hemocytes revealed a decrease in such in mollusks from the impact water area (9300 ± 4100 conventional units). The concentration of total protein in mussels from the hall. East was 3.45 ± 0.57 g / ml, and hemolytic activity was 96.7 ± 27.3%, from the Amur Bay - 1.9 ± 0.1 g / ml and 62.9 ± 9.4%. Applied t-test confirmed the significance of differences (p> 0.05) (Example 5).

As can be seen from examples 1-4, the average values of hemocyte FA, GA and total plasma protein concentrations were significantly reduced in bivalves from the impact water area compared to a sample from the conditionally background water area, which can be clearly seen from FIG. 1, 2, 3.

In FIG. Figure 1 shows the comparative average values of the phagocytic activity (FA) of hemocytes ± the confidence interval for bivalves from conditionally background water (light gray columns) and impact water (dark gray columns), where 1 is the Kuril modiolus, 2 is the scallop, 3 - mia arenaria, 4 - giant mussel.

In FIG. 2 shows comparative average values of hemolytic activity (GA) of the plasma ± confidence interval for bivalves from conventionally background water (light gray columns) and impact water (dark gray columns), where 1 is the Kuril modiolus, 2 is the scallop, 3 - mia arenaria, 4 - giant mussel.

In FIG. Figure 3 presents comparative average values of the concentration of total plasma protein ± confidence interval for bivalves from conventionally background water (light gray columns) and impact water (dark gray columns), where 1 is Kuril modiolus, 2 is the scallop, 3 is mia arenaria, 4 - giant mussel.

The data obtained indicate that the developed method for assessing the health of marine bivalves and the state of their habitat is highly sensitive and can be implemented on the basis of existing domestic or foreign equipment using a simple set of tools and reagents, which simplifies and reduces the complexity of the method of biomonitoring. The proposed method allows you to track the influence of not only anthropogenic, but also the natural current changes of natural factors on marine organisms, recording the cumulative effect of their action in real field conditions. At the same time, changes at various levels of organization of living organisms are evaluated: cellular and molecular. The method works, even if only FA is determined, since phagocytosis is an innate mechanism of the body's defensive reaction during the invasion of foreign agents.

Information sources

1. Galysheva Yu.A., Khristoforova N.K., Chernova E.N. et al. Some ecological parameters of the aquatic environment and bottom sediments of Kievka Bay, Sea of Japan, Izv. TINRO. 2008.V. 154.P. 114-124.

2. Zhuravel EV, Khristoforova NK, Drozdovskaya OA et al. Assessment of the state of the waters of Vostok Bay (Peter the Great Bay, Sea of Japan) by hydrochemical and microbiological indicators // Bulletin of the Samara Scientific Center of the Russian Academy of Sciences. 2012.V. 14, No. 1. S. 2325-2329.

3. Korshenko A.N., Matveychuk I.G., Plotnikova T.I. et al. Yearbook of seawater quality by hydrochemical indicators (Sea of Japan) for 2009. Vladivostok: PUGMS. 2010.28 p.

4. Markina Zh.V., Zhuravel E.V., Aizdaycher N.A. Assessment of the state of the waters of Peter the Great Bay (Sea of Japan) based on hydrochemical indicators and analysis of the growth and content of chlorophyll A of the microalgae Phaeodactylum tricornutum // Izv. Samar. scientific center of Ros. Acad. sciences. 2011.Vol. 13, No. 1. S. 1460-1463.

5. Naumov Yu.A. Anthropogenesis and ecological state of the geosystem of the coastal-shelf zone of Peter the Great Bay of the Sea of Japan. Vladivostok: Dalnauka. 2006.300 s.

6. Nigmatulin L.V. Estimation of the anthropogenic load of coastal sources on the Amur Bay (Sea of Japan) // Tomsk State University Journal. FEB RAS. 2007. No. 1. S. 73-76.

Claims (3)

1. A method for assessing the health of marine aquatic organisms and the state of their habitat, including the selection of marine aquatic organisms, preparing tissue samples, determining the biomarker and comparing the biomarker of animals subject to stress with animals not subject to stress, characterized in that bivalve mollusks are used as marine aquatic organisms, as the test tissue, their hemolymph is taken, which is then divided into plasma and hemocytes, and the phagocytic activity of hemocytes is determined as a biomarker. 2. The method according to claim 1, characterized in that as an additional biomarker, hemolytic activity of the plasma is determined. 3. The method according to claim 1, characterized in that as an additional biomarker, the concentration of total protein in plasma is determined.

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