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Dynamics of the immune response of the horse mussel Modiolus kurilensis (Bernard, 1983) following challenge with heat-inactivated bacteria.

ABSTRACT 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 between the cellular and humoral parameters against bacterial challenge in a bivalve revealed significant inverse correlations of PA with HL and HA during the period of these active immune responses from 3 to 48 h. The investigated immune parameters can serve as effective methods for estimation of the physiological state of the bivalve in natural and aquacultural populations under normal environmental conditions and significantly alterations under conditions of stress.

KEY WORDS: innate immune response, phagocytosis, protein concentration, hemagglutination, hemolytic activity, Modiolus kurilensis, horse mussel

INTRODUCTION

The manifestation of physiological adaptations and defense reactions to environmental factors in organisms is associated with the development of the immune system. Despite the absence of adaptive immunity bivalves have simple and effective mechanisms of immune protection that can resist persistent attacks from various pathogens present in the environment (Olafsen et al. 1993, Paillard et al. 2004, Song et al. 2010, Husmann et al. 2011). The innate immune defense is comprised primarily by the circulating hemocytes. Cellular responses include hemocytosis, phagocytosis, encapsulation, nodule formation, and intracellular degradation of foreign material (Vasta 1986). Humoral protection is provided by a set of hemolymph nonspecific soluble factors. Humoral factors are able to recognize foreign antigens, acting as surface phagocyte receptors and regulating the migration and concentration of hemocyte opsonins (Anderson & Beaven 2001, Yang et al. 2011). Other soluble factors with lytic and cytotoxic activity are responsible for the direct destruction of pathogens that are circulating or have avoided capture by phagocytes (Cheng et al. 1975), serving as an additional mechanism of cellular protection. The best proof of the unity of humoral and cellular defense mechanisms in invertebrates is the production of hemolymph phagocyte cells, coelomocytes, or special lymphoid formations of soluble factors (Macey et al. 2008). The formation of cellular and humoral factors of different types is in part associated with certain stages of the organism's development and is also regulated by a complex array of external factors that affect the organism, such as changes in weather conditions (Mydlarz et al. 2006), damage, bacterial infection or infestation (da Silva et al. 2008), and interactions with other organisms (La Peyre et al. 1995, Goedken et al. 2005). The isolation of foreign particles by hemocyte phagocytosis or encapsulation can be performed within a few minutes after the appearance of pathogen, whereas the humoral response time is usually measured in hours (Mydlarz et al. 2006). The response speed of different cell types also varies (Parisi et al. 2008). It is supposed that there are different mechanisms for their activation and cascades of reactions, leading to the involvement of different types of hemocytes in this process. The dynamics of the immune response depends on the individual characteristics of the molluscs (Oubella et al. 1993) and the immunogenicity of the injected bacteria (Allam et al. 2006). Despite the level of attention now given to the mechanisms of pathogenic organism reception and the modern methods for analyzing the structure of cells and molecules responsible for the immune response, data on the time dynamics of this process that shows the immune status of an organism under various conditions are contradictory and fragmentary.

The horse mussel Modiolus kurilensis is one of the dominant macrozoobenthos species in communities of the Far Eastern Seas and traditional object of ecological biomonitoring of physiological state and aquatic area conditions (Podgurskaya & Kavun 2005, Sokolnikova et al. 2015). Unfortunately, the data on the immune defense mechanisms for the widespread genus Modiolus are lacking. Despite the good knowledge of the morphological and functional parameters of hemolymph in mussels such us Mytilus edulis (Wootton et al. 2003, Gauthier-Clerc et al. 2013), Mytilus galloprovincialis (Parisi et al. 2008, Ghersi et al. 2011), and Mytilus trossulus (Luengen et al. 2004, Karetin 2010), only very limited data about hemocytes (Anisimova 2012, Sokolnikova et al. 2015) and humoral factors (Tunkijjanukij & Olafsen 1998) are available in the literature for the Modiolus. The great majority of the protozoa that occur in the mussels are commensals. There are parasitic forms of the genus Nemcitopsis, Pseudoklossia, Haplosporidium, Minchinia, Marteilia, and Perkinsus capable of causing diseases and epizootic among mussels (Gaevskaya 2007). To understand the mechanism of innate immunity by which bivalves defends themselves against pathogens, the heat-inactivated Staphylococcus aureus were injected and several immunity-related parameters at different exposure times were compared.

MATERIALS AND METHODS

Experimental Animals and Conditions

Sexually mature Modiolus kurilensis bivalves with a shell length of 75-95 mm were collected from wild populations in the intertidal zone in the Vostok Bay in the Sea of Japan (42[degrees] 52' 52.3" N 132[degrees] 44' 41.6" E). Animals were acclimated in an open-circuit aquaria system with aeration at 13-15[degrees]C for 1 wk before processing. All animals were maintained under the same conditions for all experimental periods. All assays were performed on individual specimens.

To exclude individuals with possible physiological abnormalities before the experiments, a primary screening of phagocytic activity (PA) in the hemocytes and hemagglutination (HA) in the plasma hemolymph was performed. Animals with PA and HA that varied in the range of the median [+ or -] 30%, [13,000-22,000 relative fluorescence units (RFU)] and 3-6 -[log.sub.2] of titer, respectively, were used as test specimens. After this screening, 320 of the 1,016 collected bivalves (31.5%) were chosen for the following experiments.

Bacterial Suspension

Bacteria Staphylococcus aureus are naturally present in the Sea of Japan (Beleneva 2011). A strain of S. aureus, 636, which was previously isolated from marine aquatic organisms and stored in the collection of bacterial cultures at the Zhirmunsky Institute of Marine Biology of the Far Eastern Branch of the Russian Academy of Sciences (1MB FEB RAS) at -85[degrees]C, was used as the source of biotic particles for initiating the immune process. Bacteria were grown on Chapman solid medium (pH 7.0 [+ or -] 0.2) at room temperature to form well-defined colonies of a rounded shape; the cell mass was then washed by artificial seawater (ASW) with a 1,090 mOsm osmolality containing 460 mM NaCl, 9.4 mM KC1. 48.3 mM Mg[Cl.sub.2] x 6[H.sub.2]O, 6 mM NaHC[O.sub.3], 10.8 mM Ca[Cl.sub.2] x 2[H.sub.2]O, and 10 mM (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid. A portion of the heat-inactivated (1 h at 72[degrees]C) bacterial suspension was stained with fluorescein-5-isothiocyanate (FITC, MP Biomedicals), and another portion was stained with rhodamine-B-isothiocyanate (RITC, MP Biomedicals). Bacterial suspensions used in the inoculation experiments were made in sterile seawater (SSW) with stained bacteria [3 x [10.sup.7] colony-forming units (CFU) [mL.sup.-1]].

Challenge Experiment

All molluscs (320 individuals) were divided into experimental (Exp) and control (Cont) groups. The experimental group (160 bivalves) was injected with 500 of Staphylococcus aureus stained with RITC in the posterior adductor muscle. The control group (160 bivalves) was injected with 500 [micro]l of SSW by the same method. Prior to injection of the bacterial suspension (Exp1) or SSW (Cont1), 500 of hemolymph from each individual was collected and analyzed to provide parameters regarding the initial state of the animals. Thereafter, the individuals of the Exp and Cont groups were labeled and placed in separate aquaria. Hemolymph samples (500 [micro]l) were collected from 16 individuals per group at 3, 6, 12, 24, 36, 48, 72, 120, 168, and 336 h postinjection with the bacterial suspension (Exp2) or SSW (Cont2).

Hemolymph Sampling

Hemolymph was collected from the posterior adductor muscle sinus using a 1-ml sterile syringe into a precooled microtube to avoid hemocyte aggregation and centrifuged at 800 x g for 12 min at 15[degrees]C. The supernatant was then transferred to a cryotube, frozen in liquid nitrogen at -196[degrees]C, and stored in a freezer at -85[degrees]C.

Hemocyte Phagocytosis and Concentration

The hemocyte pellet was washed with a calcium- and magnesium-free salt solution [436 mM NaCl, 10 mM KCl, 22 mM [Na.sub.2]HP[O.sub.4] x 7[H.sub.2]O, 16 mM glucose, 12 mM N-(2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid and 45 mM ethylenediaminetetraacetic acid (EDTA)], centrifuged at 800 x g for 12 min at 15[degrees]C and resuspended in ASW. The hemocyte suspensions from each individual were plated in three replicates in a 96-well black plate (Cellstar. Greiner Bio-One) and onto glass slides. The samples were incubated in a moist chamber at 15[degrees]C for 20 min for maximum cell adhesion to the substrate.

Hemocyte counts were carried out at that time microscopically using a Goryaev's hemocytometer.

To assess the in vivo hemocyte PA, the glass slides with hemocytes were fixed for 1 h with a 4% paraformaldehyde solution prepared with ASW and embedded in the water-soluble medium Mowiol 4-88.

To initiate the in vitro phagocytic reaction in the plate, a bacterial suspension stained with FITC was added to the hemocytes at 30-35 cells per hemocyte (at 15[degrees]C). Fluorescence quenching of the noninternalized bacteria was carried out by incubating the preparations with a 0.1 % trypan blue solution in ASW for 12 min. The preparations were washed three times with ASW. The phagocytosis reactions were stopped by fixation in 4% paraformaldehyde solution for 80 min after the addition of bacteria to the cells. The fluorescence intensities of the samples in the 96-well plates were analyzed using the DTX 880 Multimode plate reader (Beckman Coulter) with 485 nm-excitation and 535 nm-emission filters. The data were expressed in RFU. The resulting preparations were photographed with a Zeiss Axio Imager A1 fluorescence microscope (Germany) with an AxioCam MRc5 camera using a fluorescent filter (Zeiss BP 546/12, FT 560, BP 575-640). The images were analyzed with the AxioVs40 and V4.6.3.0 package of computer programs.

Hemagglutination Reaction

To assess HA, a human erythrocyte (Er) suspension (blood group O) was centrifuged at 300 x g for 15 min at 4[degrees]C, washed with a phosphate buffered saline solution (PBS; 0.8% NaCl, 0.02% KCl, 10 mM [Na.sub.2]HP[O.sub.4]-K[H.sub.2]P[O.sub.4]. pH 7.4) and fixed with a 0.25% glutaraldehyde solution prepared in PBS for 30 min, followed by neutralization of the aldehyde groups with glycine amino groups (1% glycine solution in PBS) for 8 h at 4[degrees]C. The suspension of Er was then transferred into a Tris-buffered saline (TBS) solution.

Hemagglutination assessment was carried out in round-bottom 96-well immunological plates using mollusc plasma and human fixed Er in a TBS, solution (10 mM Tris-HCl, 150 mM NaCl, 15 mM Ca[Cl.sub.2], pH 7.5). One hundred microliters of [TBS.sub.1] was placed in each well of the plate, and 100 [micro]l of plasma was placed into the bottom row; then, a series of double dilutions (from [2.sup.-1] to [2048.sup.-1]) were performed. Thereafter, 50 [micro]l of Er (6 x [10.sup.7] cells [ml.sup.-1] in [TBS.sub.1]) was added to each well. The upper row of the plate without plasma was used as a negative control. After incubation (2 h at 23[degrees]C) the results of HA were examined visually and expressed as -[log.sub.2] of titer. The titer of HA is the highest dilution giving unequivocal agglutination of Er.

Degree of Hemolysis

To assay hemolytic activity (HL), the native Er were incubated with plasma to estimate the degree of hemoglobin release in the solution. Fifty microliters of plasma, 50 [micro]l of [TBS.sub.2] solution (10 mM Tris-HCl, 150 mm NaCl, 2 mM Ca[Cl.sub.2], pH 7.5) and 400 [micro]l of the Er (1.25 x [10.sup.8] cells [ml.sup.-1] in [TBS.sub.2]) were added into the microtubes. The reaction mixture was incubated for 1 h with periodic shaking every 15 min. The reaction was stopped by adding 1 ml of [TBS.sub.3] solution (10 mM Tris-HCl, 150 mM NaCl, 30 mM [Na.sub.2]EDTA, pH 7.5). The microtubes were centrifuged for 15 min at 300 x g at 4[degrees]C. The supernatant was then transferred into a 3 ml spectrophotometer-fused silica cell with a path length of 10 mm (Shimadzu, Cat. No. 200-34442) and diluted 6-fold with a [TBS.sub.4] solution (10 mM Tris-HCl, 150 mM NaCl, pH 7.5). The optical density (OD) was detected by a Shimadzu BioSpec-mini spectrophotometer at 514 nm. For each series of reactions, the negative control (spontaneous hemolysis) was carried out where the plasma was replaced by [TBS.sub.2], and the positive control (complete hemolysis) was carried out where the plasma was replaced by a 0.1 % solution of Triton X-100 in [TBS.sub.2]. The degree of the HL was calculated according to the following equation:

Percent hemolysis = [(X - [X.sub.0]) / ([X.sub.1] - [X.sub.0])] x 100%,

where:

X is the OD solution after incubating with plasma:

[X.sub.0] is the OD solution in the negative control;

[X.sub.1] is the OD solution in the positive control.

Protein Assay

Determination of the protein concentration (PC) was performed by the ultraviolet (UV) absorption (280 nm) method (UV-method), which depends on the presence of aromatic amino acids in proteins. For this purpose, 100 [micro]l of plasma was placed into the spectrophotometer black micro cell for protein quantitation (Shimadzu, Cat. No. 046-25302-11), and the absorbance was detected by the Shimadzu BioSpec-mini spectrophotometer using the default settings of the UV-method (optical wavelength = 280 nm, absorption coefficient = 0.667).

Data Analysis

A comparison of the immune parameters of the molluscs samples of the Expl and Cont1 groups from all time points was performed using Kruskal-Wallis analysis of variance and multivariate analysis of variance. Compliance of the studied parameters to the normal distribution was assessed by the Kolmogorov-Smirnov test. Comparison of the immune status of the animals from each time point before the injection of SSW (Cont1), bacteria (Expl), or postchallenge (Cont2, Exp2) were performed using the Wilcoxon test and t-test for dependent samples. To identify possible correlations between HA and other parameters, the method of [gamma]-correlation for rank scales with frequent repeats in the data was used. To identify correlations of other parameters, a Pearson R correlation was performed. A multifactorial regression analysis was used for the estimation of this relationship. All data in this work are presented as the mean [+ or -] confidence interval (CI) 95%.

RESULTS

Screening and Primary Analysis of the Data

A Kolmogorov-Smirnov test revealed a significant (P < 0.05) deviation from the normal distribution for all of the investigated immune parameters, both in the preliminary screening of individuals (n = 1,016) and in the Cont1 and Expl samples for the whole community (n = 320) and each particular individual (n = 16). The initial screening likely influenced the HL and PC distributions, which had not been evaluated previously, and thus the methods of parametric and nonparametric statistics were used for more reliability in all cases of comparisons. These preliminary assessments of the PA and HA (n = 1,016) revealed their high variability: PA ranged from 8,197 to 30,517 RFU, and HA ranged from 0 to 11 -[log.sub.2] of titer. Some specimens in the Cont1 and Exp1 groups (n = 320) showed negligible deviations from the initially established values (median [+ or -] 30%) for the PA (4.7%) and the HA (2.8%). High ranges of variation (n = 320) were also observed for the HL (0-98.3%) and the PC (0.59-2.67 mg [ml.sup.-1]).

The results of the comparisons of the different time point samples of the Cont1 and Expl by Kruskal-Wallis analysis of variance and multivariate analysis of variance showed homogeneity (P > 0.05) for all of the studied parameters and a lack of the initial heterogeneity observed for all of the specimens. Pairwise comparisons of the immune parameters of the dependent samples (Cont1 and Cont2) by Wilcoxon test and r-test revealed no significant differences (P > 0.05) before and after injection with SSW (Fig. 1).

In Vivo Detection of the Staphylococcus aureus in Hemolymph

The injection of the heat-inactivated bacteria into the posterior adductor muscle of the Modiolus kurilensis caused an immediate activation of the immune defense cascade (Exp2). Injected bacteria stained with RITC were found in the hemolymph for the first 2 days (Fig. 2). Some bacteria were adhered to the surface of the hemocytes, whereas some had already been engulfed by phagocytes at 3 h postchallenge (Fig. 2A). Some hemocytes contained up to 20 bacteria. Adherent bacteria were not easily detected after 6-12 h. Only a small number of cells containing bacteria were observed during the next few days (Fig. 2B). Molluscs hemolymph did not contain bacteria at the experimental stages from 72 to 336 h.

The Dynamics of Changes in Hemolymph Parameters after Injection with SSW or Bacterial Suspension

Hemocyte Concentration Changes in Bivalves following Challenge with Heat-Inactivated Staphylococcus aureus

Quantification of the total hemocyte concentration (THC) in modiolus hemolymph revealed a significant gradual increase in the abundance of the circulating hemocytes at 12 h postchallenge with Staphylococcus aureus from 1,245 x [10.sup.3] [+ or -] 329 x [10.sup.3] cells/mL to 3,405 x [10.sup.3] [+ or -] [10.sup.8] x [10.sup.3] cells/ml (mean [+ or -] CI 95%). The level of THC of the Exp2 specimens returned to their initial values by 36 h (1,209 x [10.sup.3] [+ or -] 314 x [10.sup.3] cells/mL). Injection of SSW did not statistically significantly modify the THC (Fig. 1A).

In Vitro Hemocyte Phagocytosis

Analysis of the in vitro phagocytosis showed that after 3 h postchallenge with Staphylococcus aureus, there was a significant (P < 0.05 according to Wilcoxon criterion and t-test for dependent samples) decline in the hemocyte PA (up until 6 h). A significant PA level increase was observed starting at 12 h. reaching a peak in value at 36 h. The level of PA then gradually decreased, returning to its initial values without significant differences (P < 0.05) in the animals of the Expl and Exp2 groups at 72 h (Fig. 1A).

Humoral Factors in Plasma

The dynamics of the activity of Modiolus kurilensis hemolymph humoral factors was very different from those for the PA (Fig. 1B-D). A significant increase in the levels of HA, HL, and PC (P < 0.05 for Wilcoxon and a t-test for dependent samples) were observed starting at 3 h, which remained high for HA without pronounced peaks up until 168 h and returned to initial values at 336 h (Fig. 1B). Significantly high (P < 0.05) values of HL and PC were revealed only up until 24 h (Fig. 1C, D), with a maximum difference between the samples Exp1 and Exp2 at 3 h (HL) and 12 h (PC).

Correlations of Immune Parameters

The correlation analysis of the immune parameters of the Cont1 and Exp1 (n = 320) specimen groups showed a significant (P < 0.05) correlation only between HA and HL ([gamma] = 0.28) and HL and PC (R = 0.16), whereas PA and THC had no correlations with the humoral parameters. Multiple regression analysis also showed no significant correlation between the HA and HL ([R.sup.2] = 0.11, [beta] = 0.33, partial correlation = 0.30, P < 0.01). The estimation of the dependences of the dynamics of the immune parameters (Exp2) was performed by the difference between Exp2 and Exp1 (Table 1) for all studied individuals. The most pronounced direct correlation was revealed between HA and HL (but only at 48 and 168 h). The level of PA had an inverse correlation with levels of HL and HA at 3-48 h, which corresponded to the phase of the active immune responses. The last result was also confirmed by the multiple regression analysis, which showed a coefficient of partial correlation (-0.48) between the PA and HL in the period from 3 to 48 h of [R.sup.2] = 0.24, [beta] = -0.48, P < 0.001.

DISCUSSION

Despite the intensive study of issues related to the immune resistance of molluscs to various microorganisms, such as Perkinsus (La Peyre et al. 1995, Ordas et al. 2000, Goedken et al. 2005, da Silva et al. 2008), Vibrio (Oubella et al. 1993, Oubella et al. 1994, Lopez-Cortesa et al. 1999, Allam et al. 2000, Allam et al. 2002, Allam et al. 2006, Labreuche et al. 2006, De Decker & Saulnier 2011), Shigella flexneri, and Salmonella typhimurium (Hartland & Timoney 1979), information on the key immune defense mechanisms and reactions still remains largely inconsistent and does not give a complete picture of the innate immune response. Lopez-Cortesa et al. (1999) revealed, that Ruditapes phUippinarum and Ruditapes decussatus showed greater activity in response to the live bacteria of the Vibrio and Escherichia coli species than to the inactivated bacteria. A somewhat different picture was shown for R. philippinarum in the work from Allam et al. (2006), where both heat-inactivated and live Vibrio tapetis (5 x [10.sup.7] CFU in 0.1 ml) were used, which demonstrated an increase in the concentration of circulating hemocytes at 2 h postchallenge, peaking at 6 h in all experimental groups. R. philippinarum showed longer dynamics in THC changes (up to 168 h) in comparison with R. decussatus (up to 72 h). The study by Oubella et al. (1993) did not describe analogous dynamics for the same mollusc species and demonstrated that a significant difference with control specimens remained even at the end of the experiment (more than 168 h). Similar results to Allam et al. (2006) were obtained in this research. An increase in the number of circulating cells in the experimental group of animals in comparison with the control group was detected already at 3 h after injection of the heat-inactivated Staphylococcus aureus, but a significant increase occurred at 6 h, reaching its maximum after 12 h, followed by a gradual decrease and returning to initial values at 36 h. A similar dynamic in the changes in cell concentrations in the initial period post-challenge of the bacterial particles can be observed in other published works (Oubella et al. 1993, Oubella et al. 1994, Allam et al. 2000, Labreuche et al. 2006).

Unfortunately, the studies on foreign particle clearance in organisms are rare. This is likely due to the long duration of the immune response or to the initiation of diseases in molluscs, as in the case of Brown ring disease. Unlike live bacteria injected into molluscs, heat-inactivated bacteria allow for observing the consistency and mechanism of activation of the various cascade reactions, the bacterial particles elimination kinetics, which excludes the unpredictable course of the immune response depending primarily from the reproduction ability of the bacteria. One study showed the dynamics of Mercenaria mercenciria and Crassostrea virginica clearance of different strains of live bacteria ([10.sup.3] CFU in 0.02 ml) injected by an intracardial method (Hartland & Timoney 1979). The most rapid elimination was observed with Escherichia coli (at 12-16 h after postchallenge), and the longest clearance was displayed for Shigellaftexneri (at 36-48 h) at 20[degrees]C. The in vivo PA in the Ruditapes phUippinarum increased 30% with respect to the control group in 30 min after injection into the adductor muscle with the live bacteria Vibrio tapetis (5 x [10.sup.7] CFU in 0.1 ml) stained with the vital dye cyanoditolyl tetrazolium chloride (CTC; 1 mM), which was followed by the gradual return to control values after 24 h (Allam et al. 2002). The highest number of free bacteria was found up to 24 h, but bacteria were not found in hemolymph after 72 h. The three bacteria species were found inside hemocytes of Mytilus galloprovincialis from the first hour to 48 h postinjection (Parisi et al. 2008). In this work, the injection of bacteria stimulated the active in vivo hemocyte phagocytic reaction in the first hours of the experiment, the intensity of which gradually decreased as the bacteria were eliminated. The complete clearance of hemolymph was observed after 48 h. At the same time, the in vitro phagocytic reaction had the opposite dynamics, i.e., PA showed significantly lower values at 3 and 6 h, and the cell response was extended after 6 h with a pronounced reaction at 36 h after bacterial suspension injection. Hemocytes returned to PA initial levels at 72 h. The low in vitro PA of hemocytes in the early hours was likely due to their decrease in phagocytic capacity, as the active in vivo phagocytic reaction took place in the first few hours; the subsequent rise in in vitro phagocytosis levels was associated with an increase in the number of circulating hemocytes, which was consistent with the works cited earlier (Hartland & Timoney 1979, Allam et al. 2002). Differences in the immune responses of two species of oysters, Crassostrea gigas and C. virginica, were revealed by Goedken and colleagues (Goedken et al. 2005). At 72 h postchallenge, Perkinsus marinus and C. virginica showed increases in in vitro PA in response to fluorescence beads, whereas the hemocytes of C. gigas did not. Completely different results were obtained for the same species of molluscs after injection of zymosan (La Peyre et al. 1995), where C. gigas hemocytes displayed significantly higher in vitro PA than C. virginica did. Increase in the in vitro PA was observed in hemocytes of Scapharca subcrenata (Zhi-Hong et al. 2002) from 4 h to 72 h after stimulation with Vibrio. In the first hours of the experiment, Chlamys farreri PA increased and returned to initial values at 144 h after injection of Listonella anguillarum (Cong et al. 2008).

In the past years, much attention has been given to the expression and activity of hemolymph factors of bivalves in response to a variety of particles. For instance, the increased expression of humoral factors in the first hours of experiments was observed in the hemocytes of Solen grandis (Wei et al. 2012), Chlamys farreri (Zhang et al. 2008, Zhang et al. 2010, Yanget al. 2011), Pinctadafucata (Anjuet al. 2013), Crassostrea ariakensis (Xu et al. 2012), Mytilus galloprovincialis (Romero et al. 2011), and Tegillarca granosa (Bao et al. 2013) postchallenge with a variety of pathogen associated molecular patterns, including peptidoglycan from Staphylococcus aureus (Chellaram et al. 2004, Romanenko et al. 2008). Similar processes occurred after the injection of Vibrio, Micrococcus, Staphylococcus and Saccharomyces bacterial species into the adductor muscle of M. galloprovincialis, T. granosa, Ruditapes phUippinarum, Argopecten irradians, Cristaria plicata, Crassostrea hongkongensis, Mytilus coruscus, P. fucata, and Pinctada martensii (Zhang et al. 2009, Zhu et al. 2009, Gestal et al. 2010, Song et al. 2010, Zhao et al. 2010, Chen et al. 2011, Estevez-Calvar et al. 2011, He et al. 2011, Romero et al. 2011, Wang et al. 2011, Bao et al. 2013, Liu et al. 2014, Qu et al. 2014, Xiang et al. 2014). Increases in PC, HL, and HA in the first 3 h of the experiment were observed, but HL and PC rapidly recovered to initial values by 36 h, which was consistent with an quick increase in the transcriptional activity, a characteristic feature of many of the factors described in a number of studies. The levels of PC and HL constancy at 72 h was in accord with earlier works of the influence of Vibrio on R. phUippinarum (Oubella et al. 1994, Paillard et al. 2004, Allam et al. 2006). Long-term and constantly high HA can be associated either with a longer activity of the corresponding genes or with a Modiolus kurilensis hemagglutinins being more resistant to depletion or degradation. The observed dynamics of M. kurilensis HA compared favorably to data in the paper by Olafsen et al. (1992), where HA in Crassostrea gigas after injection with Vibrio increased at the only investigated time point (24 h) and was not changed after 336 h in another study (Olafsen et al. 1993).

Studies on the immediate display of protective physiological activity of immune factors are becoming increasingly rare, which is likely due to their significant variability and, consequently, the complexity of their analysis and interpretation. As seen from the literature, a high variability of cellular and humoral immune parameters, including PA, HA, PC and lytic factors, is specific to native (control) samples of different molluscs, including but not limited to: Mytilus galloprovincialis (Santarem et al. 1994), Crcissostrea virginica, Crassostrea gigas (La Peyre et al. 1995), Ruditapes decussates (Ordas et al. 2000), Ruditapes philippinarum (Paillard et al. 2004), and Mactra veneriformis (Yu et al. 2010). This is likely because the immune factors have integrative and homeostatic functions, subtlety responding to many exogenous and endogenous factors. Therefore, a preliminary screening of individuals for the parameters to be investigated is a prerequisite to finding good control samples that have the values corresponding to normal levels of these parameters.

In this study, the preliminary screening of the collected animals was able to ensure the homogeneity of the specimens in Cont1 and Expl in the aggregate. This enhanced the significance of the results obtained for the relatively small samples (n = 16); it also allowed confidence that the identified dynamics were typical and that the results were not distorted by data from individuals with initial levels not within the determined normal ranges. It was also important to correctly estimate and interpret the possible correlations between the studied parameters. Data analysis of all of the initial (Cont1 and Exp1, n = 320) values revealed a weak correlation between the HA and HL, expressed by the coefficients of y-correlation (0.28) and a partial correlation (0.30) for the linear multifactorial regression analysis ([R.sup.2] = 0.11, [beta] = 0.33, P < 0.01). A similar relationship has been previously shown for Mytilus galloprovincialis (Santarem et al. 1994). Likely, these two most important factors of humoral immunity can work together, but not always. There was a very weak correlation between the HL and PC (R = 0.16) and a lack of correlation between HA and PC, probably due to the presence of other soluble factors, as well as to their asynchronous expression and release into the hemolymph. Obtained results displayed no correlation between PA and any of the studied humoral parameters that were consistent with the data from a study carried out on Mercenaria mercenaria (Tripp 1992) and Elliptio complanata (Bouchard et al. 2009), which showed that the ratio of cellular and humoral activity is highly individual and has a more complex nonlinear relationship. The correlation of the individual's changes in the immune parameters (Exp2 to Exp1) was of special interest, as these relationships were expressed in the dynamics of the immune response. A marked correlation between HA and HL and between HL and PC slightly increased only at the individual time points and were likely random and associated with the small sample size. In addition, at two time points of the active cellular responses period (3 h - 48 h), there was an inverse relationship between PA and HA changes, which was observed for absolute values of the Ruditapes decussatus from the Perotrochus atlanticus enzootic zone (Ordas et al. 2000). The most rational explanation of the observed process was the opsonizing effects of the many agglutinating factors, i.e., the agglutinins depleted their activity by binding to the bacterial particles but increased the PA at the same time. Another observed inverse correlation between PA and HL was also expressed in the active cellular response period (3-48 h, n = 96). A multifactorial linear regression analysis confirmed this relationship and indicated that ([R.sup.2] = 0.24) PA had a linear dependence on HL with [beta] = - 0.48 (P < 0.001) in a quarter of the cases. This could mean that PA and HA worked by compensatory mechanisms in some organisms, i.e., an insignificant increase or decrease of the PA compensated the activity of the lytic factors and vice versa.

Thus, several stages in the dynamics of the Modiolus kurilensis immune response to the challenge of heat-inactivated bacteria Staphylococcus aureus were identified. The induction of the immune response, with the initiation of active bacterial particles engulfment by hemocytes and humoral factors release, was a process that took place in the first 3 h. Immediate implementation of the immune response, the effector stage, involved the active destruction of the bacteria, which took place from 3 to 6 h. Due to the PA increase observed from 12 to 36 h, which was most likely related to the exit of excess hemocytes from their storage depot, humoral component activity decreased at 36 h. As the organism was subsequently free from bacterial particles (clearance) the next stage included a reduction or ceasing of the activity of both humoral (from 36 h) and cellular (with 72 h) immune factors followed by the return to the initial state of the immune system (36-72 h).

ACKNOWLEDGMENTS

We thank their coworkers at the Department of Cell Biology and Genetics of the Far Eastern Federal University, the collective of the Zhirmunsky Institute of Marine Biology of the Far Eastern Branch of the Russian Academy of Sciences Laboratory of Pharmacology and the staff of the Vostok Marine Biological Station for their help in this research. This work was supported by the Russian Science Foundation (RSCF) (number 15-15-20026).

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DOI: 10.2983/035.034.0321

ANDREI GRINCHENKO, (1) YULIA SOKOLNIKOVA, (1) * DENIS KORNEIKO (1) AND VADIM KUMEIKO (1,2)

(1) Far Eastern Federal University, 8 Sukhanova Str., Vladivostok, 690950 Russia; (2) Zhirmunsky Institute of Marine Biology, Far East Branch, Russian Academy of Sciences, 17 Palchevskogo Str., Vladivostok, 690041 Russia

* Corresponding author. E-mail: yulia.sn@mail.ru

TABLE 1.
The correlation between changes in the investigated
immune parameters.

Time                Compared     Correlation
points (h)    n    parameters    coefficient

    3         16      --
    6         16    PA-HA       [gamma] = -0.48
   12         16      --
   24         16      --
   36         16    PA-HA       [gamma] = -0.60
   48         16    HA-HL       [gamma] = 0.48
 3-48         96    PA-HL       R = -0.54
                    HL-HA       [gamma] = 0.19
                    HL-PC       R = 0.25
   72         16    HA-PC       [gamma] = 0.45
  120         16      --
  168         16    HA-HL       [gamma] = 0.57
  336         16      --
3-336        160    PA-HL       R = -0.31
                    HL-PC       R = 0.24
                    HA-HL       [gamma] = 0.13

Exp2 to Expl, the individual parameter values before (Expl)
minus the values after injection of Staphylococcus aureus
(Exp2); [gamma], gamma correlation coefficient; R, Pearson's
correlation coefficient (only values with P < 0.05 shown).
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Author:Grinchenko, Andrei; Sokolnikova, Yulia; Korneiko, Denis; Kumeiko, Vadim
Publication:Journal of Shellfish Research
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Date:Dec 1, 2015
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