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Труды ИБВВ РАН, вып. 83(86), 2018
Transactions of IBIW, issue 83(86), 2018
COMPARATIVE STUDY OF IMMUNE-BIOCHEMICAL INDICES OF FAMILY DREISSENIDAE MUSSELS INHABITING THE VOLGA RIVER WATER RESERVOIRS
A. S.Sokolova, D. V. Mikryakov, S. V. Kuzmicheva
Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, 152742 Borok, Russia
e-mail: [email protected]
The results of the study of some immuno-biochemical parameters of the mollusks Dreissena polymorpha and D. bugensis inhabiting the Volga River reservoirs are presented. The content of non-specific immune complexes, malondialdehyde and antioxidant activity of tissues was studied. Interspecific and intraspecific differences of studied parameters in samples from different waterbodies were recorded. Both species showed higher levels of immune complexes in the Rybinsk and Gorky reservoirs, higher intensities of lipid peroxidation in the Gorky, Cheboksary and Kuibyshev reservoirs and a low content of antioxidants in D. polymorpha from the Saratov reservoir. Significant differences in the content of antioxidants between D. polymorpha and D. bugensis from the Gorky, Saratov and Volgograd reservoirs were probably associated with species characteristics of the organism.
Keywords: Volga River reservoirs, bivalve mollusks, immune complexes, lipid peroxidation, antioxidant activity
DOI: 10.24411/0320-3557-2018-10031
INTRODUCTION
The Volga River is one the greatest rivers of Russia and the longest and largest river in Europe in terms of discharge. It flows across the European part of Russia, accepting around 150000 tributaries and discharges into the Caspian Sea. Nine hydroelectric stations were built on the Volga since 1930's. This lead to the formation of the following water reservoirs: Verkhnevolzhskoye, Ivankovo, Uglich, Rybinsk, Gorky, Cheboksary, Kuibyshev, Saratov, Volgograd [Gidrologija, gidrohimija ..., 2018].
Mollusks are an important component of benthic communities. Freshwater species including bivalve mussels from family Dreissenidae: D. polymorpha (Pallas, 1771) and D. bugensis (Andrusov, 1897) strongly affect ecosystems of waterbodies. Dreissenids are aggressive invasive species actively colonizing different regions of the world due to high rates of dispersal and the ability to replace other mussel species [Pollux et al., 2010]. The first description of D. polymorpha from the Volga was made by P.S. Pallas [Pallas, 1773]. Its congener, D. bugensis was first observed in the waterbodies of Middle and Lower
Volga in 1980's [Antonov, 1993] and has later dispersed across the Volga's reservoirs, appearing in the samples from the Upper Volga waterbodies in 1996 [Orlova, Shcherbina, 2001].
There is little information on physiological, biochemical and immunological indices of family Dreissenidae mussels inhabiting the aforementioned water reservoirs. Studies of oxidative processes intensity and level of antioxidant defense in D. polymorpha and D. bugensis from the Rybinsk reservoir have shown both seasonal and interspecific differences as well as dependence on the anthropogenic pollution level [Klimova, Chuiko, 2017; Klimova et al, 2017]. However, there are no comparative studies on indices of dreissenids inhabiting the Volga River water reservoirs with different hydrological regimes, hydrochemical characteristics, ecological state etc.
Aim of the present study - to perform a comparative assessment of immune-biochemical indices content in mollusks from family Dreissenidae inhabiting the Rybinsk, Gorky, Cheboksary, Kuibyshev, Saratov and Volgograd water reservoirs.
MATERIALS AND METHODS
Materials for the study have been collected during a complex hydrobiological expedition (August of 2017) on RV "Akademik Topchiev" of IBIW RAS. Mussels were sampled using a dredge and modified Ekman-Birge (DAK-250) bottom sampler with 1/40 m2 area, one lift per sampling site. After capturing the mussels, their soft tissues were separated from the shell and immediately frozen in a freezer. Total homogenates of one mollusk species were prepared from tissues using 0.6% physiological solution with 1:6 ratio (mass to volume) in laboratory conditions. These ho-mogenates were used to study the content of immune complexes (IC), products of lipids' peroxi-
dation (LP) and the level of antioxidant defense (AD).
The content of IC was measured on spec-trophotometer with 450 ^m wave length using the method of selective precipitation with 4% polyethylene glycol of 6000 molecular mass [Grinevich, Alferova, 1981].
The intensity of PLP was evaluated by the accumulation of malondialdehyde (MDA), one the final products of peroxidation. MDA concentration was determined according to the quantity of LP products reacting with thiobarbituric acid and forming a colored complex with it. The intensity of coloration was measured using a spectropho-
tometer by the change of absorption maximum at 535 pm wave length. The content of MDA was calculated taking the coefficient of molar extinction into account (1.56 * 105 M-1 cm-1) and expressed in nanomoles per 1 g of tissue [Andreeva et al, 1988].
The level of AD was estimated according to the kinetics of substrate oxidation of the reduced form of 2.6 dichlorphenolindophenol by atmospheric oxygen using standard method adapted for mollusks. The method is based on the fact that the higher is the substrate's oxidation rate in the presence of biological material the lower is the antioxidants' content in tissues. Substrate oxidation in-
RESULTS
hibition constant (SOC) which is the index of tissue's antioxidative activity was determined relative of the control using the formula: Ki = Kcon -Kexp/C, where Kcon and Kexp are the constants of substrate oxidation in the control and in the experiment, correspondingly; C - concentration of ho-mogenate in the cuvette [Semenov, Yarosh, 1985].
Graphic and statistical treatment of data was performed using Microsoft Office Excel and Statistica 6.0 software. Significance of differences was calculated using Student's t-test. Differences were significant at p < 0.05.
Analysis of obtained data has shown that there are both interspecific differences of studied indices as well as differences between samples of the same species from different waterbodies. High
values of IC have been observed for both species from the Rybinsk and Gorky reservoirs and for D. bugensis from the Saratov reservoir.
Fig. 1. Level of immune complexes content. Note: Hereinafter: D.B. - Dreissena bugensis, D.P. - Dreissena polymorpha. A - Rybinsk, B - Gorky, C - Cheboksary, D - Kuibyshev, E - Saratov, F - Volgograd; "*" - significant differences between species, a, b, c , d, e - significant intraspecific differences in samples from different reservoirs at p < 0.05.
Dreissena bugensis from the Gorky, Cheboksary and Kuibyshev reservoirs were found to demonstrate more intensive processes of LP. In D. polymorpha processes of LP were on the same level throughout allof the reservoirs except for Saratov (Fig. 2). High MDA content may be the result of activation of lipid peroxidation processes.
SOC index in both mussel species in all water reservoirs fluctuated within 3.5-4.8, except for D. polymorpha from the Saratov reservoir (Fig. 3). A significantly high level of SOC indicates lower antioxidants' content in mussels' tissues.
DISCUSSION
It is known, that IC are complexes antigen-antibody and complement's components linked with it formed during the interaction with low-molecular alien compounds (haptens, soluble antigens and autoantigens). They play an important role in the processes of immune reactions' regulation, elimination of xenobiotics from the organism and maintenance of immunological homeostasis.
When the body is saturated with foreign bodies, excessive IC formation occurs due to suppression of the clearing function of the phagocytic system cells, which leads to accumulation of IC in the tissues and disruption of organs [Freidlin, Kuznetsova, 1999; Roit et al, 2000; Koyko et al, 2008].
Fig. 2. Malondialdehyde level.
Fig. 3. The substrate oxidation inhibition constant.
Excessive accumulation of IC in dreissenids from the Rybinsk, Gorky and Saratov reservoirs indicates a violation of the mechanisms of elimination of these complexes, suppression of the mechanisms of humoral immunity and the presence of uncontrolled pathological processes. The revealed differences in the IC content in mollusks from different reservoirs may be due to the influence of several factors: the hydrological regime of the reservoir, the level of anthropogenic pollution and the concentration of microorganisms.
The high content of MDA recorded in D. bugensis and D. polymorpha may be the result of the activation of LP processes associated with the accumulation of reactive oxygen species (ROS) and a decrease in the activity of enzymatic and non-enzymatic antioxidants. Excessive ROS
(superoxide and hydroxyl radicals, singlet oxygen, peroxides, and many other compounds) causes activation of the LP of cell membranes, destruction of nucleic acids, proteins, damage to DNA, mitochondria, destruction of polyunsaturated fatty acids of cell membranes, lipid peroxidation and inactivation of antioxidant protection structures [ Baraboy et al. 1992; Zenkov et al. 1999; Menshikova et al., 2008; Winston, 1991; Fiho, 1996; Grubinko, Leus, 2002]. The accumulation of toxic lipid peroxidation products in the body tissues can cause secondary damage to cell membranes, a decrease in the content of antioxidants, suppression of immunological and regenerative functions, as well as premature aging, decreases of growth and development rates and overall loss of viability. Uncontrolled build-up of lipid perox-
idation products is known to be hindered by a multilevel AD system consisting of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione-s-transferase) and low-molecular antioxidant compounds (reconstituted with glutathione P-tocopherol), phenol form of Qi0 coenzyme, P-carotine, ascorbic acid, etc.) [Zenkov et al., 1999; Menshikova et al., 2006; Soldatov et al, 2007; Winston, 1991]. The AD system plays an important role in the neutralization of molecular mechanisms that initiate the activation of peroxidation processes and the implementation of adaptive compensatory reactions in the body, since its components are involved in the regulation of metabolic functions. Intensification of free-radical processes accompanied by the accumulation of LP products and a deficiency of antioxidants is known to occur in the organism of hydrobionts during adverse effects [Nemova, Vysotskaya, 2004; Rudneva et al., 2005; Winston, 1991; Rudneva, Kuzminova, 2011; Silkina et al, 2012]. With a lack of antioxidants in the body,
CONCLUSION
processes of oxidative stress develop, accompanied by an imbalance in the system of prooxidants - antioxidants. The balance in the PL - AD system shifts in the direction of PL amplification. Similar violations were found in mollusks living in areas with increased anthropogenic load [Panasyuk, Lebedeva, 2008; Belcheva et al., 2014; Dovzhenko et al., 2014; Viarengo et al., 1988; Romeo, Gnassia-Barelli, 1997].
Interspecific differences of the studied parameters are conditioned by the following physiological and ecological features of D. bugensis compared to D. polymorpha: higher filtration rate and tolerance to silty bottom, resistance to low temperature and oxygen deficiency, lower selectivity in food, lower oxygen demand and metabolic rate during long-term anaerobic conditions [Lyakhnovich et al., 1994; Shkorbatov et al., 1994; Pryanichnikova, 2015; Mills et al., 1993; Karatayev, 1998; Jones, Ricciardi, 2005; Farr, Payne, 2010].
Thus, conducted studies have shown interspecific and intraspecific differences in the content of non-specific immune complexes, malondialdehyde and antioxidant activity of tissues in D. polymorpha and D. bugensis from the reservoirs of the Volga River. The hydrological regime of waterbodies, the level of anthropogenic pollution, the concentration of microorganisms
and the saturation of water with oxygen, as well as species characteristics are probably causing these differences. Analysis of the data showed the advisability of further studies of immuno-biochemical parameters of family Dreissenidae mollusks, since the results can be used for ecological status monitoring of the Volga River and other water objects.
The work was performed as part of the state assignment of the FANO of Russia (subject No. AAAA-A18-118012690123-4).
Authors are grateful to R.Z. Sabitova, D.P. Karabanov and D.D. Pavlov for their help during sampling.
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