Immune condition reflects animal’s state of health in many ways as one of the most important physiological parameters. Necessity for simple and quick methods of ecological monitoring and health evaluation of commercial aquatic organisms increases with each year. Development of such methods must include a series of steps; vital ones are finding of chosen parameter’s variability and its relative norm taking into account an effect from environment natural changes as well as organism response to possible adverse factors.

Lectins are one of the most important factors of invertebrates’ humoral immunity having a main function of agglutination and opsonization of antigens. Modiolus modiolus is a widely spread bivalve mussel, which makes it highly appropriate for ecological monitoring being also a subject of fishery and sea farming.

M. modiolus from multiple aquatic areas of the Japanese Sea (illustration 1) with shell length of 75-95 mm were used in the given piece of work. Hemolymph was taken by disposable sterile syringes from the back adductor muscle, and then centrifuged during 15 min at 800g. Supernatant fluid (plasma) was immediately frozen in liquid nitrogen (-196°C), long-term storage was performed at 85°С.

Hemagglutination reaction (HR) was executed for evaluation of activity level of plasm’s agglutinating factors as one of the main indicators of the humoral immunity. HR was performed with donor human erythrocytes (ER) of several blood types (0, A, B, AB) that were cleaned from preservative solution by phosphate isotonic buffer medium (PBM: 0,9% NaCl, 10mM К₂HPO₄-NaH₂PO₄, pH=7.4). ER suspensions  based on tris-buffered saline medium were used directly in the reaction (TBS: 10 mM Tris-HCl, 150 mM NaCl, 15 mM CaCl₂, pH=7.5), that were prepared by two methods: volumetric and hemocytometric.

Volumetric method was used in numeral works (Olafsen et al., 1992; Tunkijjanukij et al., 1997) and consists of breeding a dense deposit of ER in certain volume ratio (1.5% concentration was used in this work). Hemocytometric method lies in calculation of ER concentration in hemocytometer (Goryaev chamber) with resulting amount of ER 6×10⁴ cells in microliter. HR titer indicator was specified as  –log₂(titer), which gives each plasm breeding its own rang: 1/2 – “1”, 1/4 –“2”, …, 1\2048 – “11”. Methods of nonparametric statistics were used when analyzing data due to the specific rang scale.

HR inhibition reaction with 30 mM solutions of 24 carbohydrates of different structure was performed for defining carbohydrate specificity of M. modiolus plasm.

Originally 30 species were examined from background (water clear) aquatic areas: Kiyevka bay, Troitsi bay and Vostok gulf. In this case, HR was executed with use of one ER suspensions of II (A) blood type prepared by volumetric method. General bounds of indicator variation were from 3 to 10 (illustration 2A), whereat figures 3 and 10 were found in single species. 77% (69 out of 90) fit into the joint bounds of quartile ranges (4-7). Used criteria of Kolomogorov-Smirnov and Mann-Whitney paired comparison, as well as ANOVA Kraskell-Wallace shows absence of authentic differences between all three points with significance level p>0.05.

Unicellular green algae were found in some of the described M. modiolus. However invasion of such type on mussel physiology is poorly studied. Connection between HR results and presence of microalgae in the tissues has not been established. Part of the species from Troitsi bay and Vostok gulf were examined on subject of histpathology of kidneys and digestive glands (DG). (…)

Also, 30 species each time were obtained from Amurskiy Zaliv (near the Skrebtsov island) on 20^th of March, 4^th of May, 26^th of May and 22^nd of November. Positive dynamics of agglutinating activity of M.modiolus’ hemolymph can be described during the spring season with its maintaining until late autumn, up to period of ice cover (illustration 3). Paired comparisons of Kolmogorov-Smirnov and Mann-Whitney show reliable differences (p>0.05) between selections of 20-Mar and 26-May, as well as 20-Mar and 22-Nov.

In next work, HR values were compared of animals from aquatic area with different anthropogenic load: Vostok gulf (background) and Sportivnaya bay of Amurskiy Zaliv (impact). HR was performed with use of ER suspensions of four blood types (0, A, B, AB) prepared by both volumetric and hemacytometric methods. All types of applied criteria for comparison of independent selections by aquatic areas indicate absence of significant differences (p>0.05). Analyze of values from HR setup with different ER showed absence of dominating affinity of M. Modiolus plasm’s agglutinins to the human blood type groups of AB0 system, also greater accuracy HR in case of hemacytometric method of ER suspension preparation compared to the volumetric one.

When examining M. modiolus in changes of HR values in response to injection of thermally inactivated Staphylococcus aureus, animals’ immunity status was checked for the sake of excluding species significantly deriving from the general variability bounds. Checkpoints were determined for specifying dynamics of plasm’s agglutinin activity after antigen injection: 3 hrs, 6 hrs, 12 hrs, 1 day, 1.5 day, 2 days, 3 days, 7 days, 14 days. As a result, 80 species included into experiment were divided into groups of 8 animals for each time checkpoint. Each animal has been sampled for 300 mcL of hemolymph before antigen injection, after which S. aureus suspension was immediately injected in same amount with concentration 7,5×10⁸. Used paired comparison of Wilkinson for dependent selections shows that in points of 3hrs.-3 days after antigen injection, HR is definitely increasing (p<0.05). No differences were established in points 7 and 14 days.

For examined samples of hemolymph, activity was found, established for selected and previously described lectines (modiolines E and H) of M. modiolus from North Atlantic. For the first time, species of given systematic group showed hemolymph’s agglutinin activity in relation to D-glucuronic and D-galacturonic acids, which receded in range of homological sugars of somewhat other structure (N-acetylated amines of neutral sugars and neutral mono-sugars  correspondingly). Attained data gives evidence of finding of new lectine or group of lectines in the contents of M. kurilensis plasm’s hemolymph.