Bivalves play a key role in aquatic ecosystems and are a valuable commercial resource. The prosperity of these aquatic organisms depends mainly on the effectiveness of their immune defense, in which the hemolymph plays a central role. Hemolymph may be used as an effective non-lethal criterion of health. However, the predictive value of hemolymph analysis depends on the comparison between the obtained results and reference data from healthy individuals living in natural aquatic environments. We collected hemolymph from 15 commercially important species from wild populations at stations located in non-impacted and impacted water areas of the Sea of Japan. Of the 11 hemolymph parameters we analyzed, the total hemocyte count, percentage of hemocyte types, phagocytic activity, presence of reactive oxygen species, and protein concentration differed significantly between populations from non-impacted and impacted water areas. The most responsive species to pollution were Magallana gigas, Crenomytilus grayanus, Mizuhopecten yessoensis, and Mactra chinensis. This work is the first to examine a large number of commercially important species simultaneously. The results of this study are the basis for establishing the health status criteria of commercial bivalves for veterinary control in aquaculture and biomonitoring.

Hemocyte phagocytosis assay in vitro revealed no significant differences in the average values of phagocytic activity (PA) and phagocytic index (PI) in samples of the molluscs Modiolus kurilensis that inhabit non-impacted water areas (Kievka, Vostok, and Troitsa bays, the Sea of Japan). The only exception was an insignificantly lower value of PI for individuals from Troitsa Bay. The average values of PA for hemocytes of the total population of bivalves from non-impacted water areas were 52.0 ± 1.0%. On the basis of the interannual analysis of PA, the bivalve population from Vostok Bay was chosen as an appropriate reference group that is characterized by relatively stable PA values. In comparison with molluscs from the reference group and other investigated samples, M. kurilensis from the impacted water area (Sportivnaya Gavan, Amursky Bay) had considerably lower values of average PA (36.0 ± 2.0%) and PI (2.1 +- 0.05). The presented cell-mediated immunity assay can serve as an effective method for estimation of the physiological state of the bivalve in natural and aquacultural populations in order to differentiate molluscs from impacted and non-impacted water areas.

Alterations of the cell-mediated and humoral immune parameters were revealed in the bivalves Modiolus kurilensis from the Sea of Japan after injection with heat-inactivated Staphylococcus aureus. Preliminary screening of the in vitro phagocytic activity (PA) and hemagglutination (HA) enabled to obtain homogeneous groups of individuals for all of the parameters to be measured. Changes in hemolymph parameters were studied over a 14-day period. High values for hemolytic activity (HL) and protein concentration (PC) persisted until 36 h, with high levels of HA persisting until 168 h. The in vitro PA level was low from 3 to 6 h postchallenge, which was caused active in vivo hemocyte elimination of the injected antigen in the first 12 h. A significant increase in the in vitro PA up to 36 h was related to a rise in the circulating total hemocyte concentration (from 6 to 12 h) and to in vivo S. aureus clearance (in the first 48 h). For the first time, a complex correlation analysis of the interplay betwee...

Fisheries and Aquatic Sciences

Bivalves, such as Modiolus are used as indicator organisms to monitor the state of the marine environment. Even though hemocytes are known to play a key role in the adaptive and protective mechanisms of bivalves, these cells are poorly studied in horse-mussel Modiolus kurilensis. In this paper, we present classification of horse-mussel hemocytes based on their immune functions, including the production of specific immune-related molecules, as well as their morphological composition after isolation by density gradient centrifugation. An effective fractionation protocol was adapted to separate four hemocyte subpopulations with distinct morphofunctional profiles. First subpopulation consisted of small under-differentiated hemoblasts (2.20 ± 0.85%) with a bromodeoxyuridine positive nucleus, and did not show any immune reactivity. Second was represented by agranulocytes (24.11 ± 2.40%), with evenly filled cytoplasm containing a well-developed protein-synthesizing apparatus, polysomes, smooth endoplasmic reticulum and mitochondria, and positively stained for myeloperoxidase, acidic proteins, glycogen and neutral polysaccharides. Third subpopulation consisted of eosinophilic granulocytes (62.64 ± 9.32%) that contained the largest number of lysosomes, peroxisomes and vesicles with contents of different density, and showed the highest phosphatase, reactive oxygen species (ROS) and phagocytic activities. Lastly, fourth group, basophilic granulocytes (14.21 ± 0.34%), are main producers of lectin-like protein MkC1qDC, recently discovered in M. kurilensis and characterized by pronounced antibacterial and anticancer activity. These cells characterized by intracytoplasmic of the MkC1qDC localization, forming granule-like bodies visualized with specific antibody. Both granulocytes and agranulocytes showed phagocytic activity and ROS production, and these reactions were more pronounced for eosinophilic granulocytes, suggesting that this group is the key element of the cell-mediated immune response of M. kurilensis. Our results support a concept of bivalve's hemocyte specification with distinct phenotypes.