Smirnov L., Sukhovskaya I., Kochneva A. Variability of some antioxidant defense parameters and concentration of protein in the larvae of the three-spined stickleback (GASTEROSTEUS ACULEATUS) in White sea in the summer // Principy èkologii. 2019. № 2. P. 98‒109. DOI: 10.15393/j1.art.2019.8542

Issue № 2

Original research

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Variability of some antioxidant defense parameters and concentration of protein in the larvae of the three-spined stickleback (GASTEROSTEUS ACULEATUS) in White sea in the summer

Introduction

The three-spined stickleback Gasterosteus aculeatus is the avowed model object of evolutionary biology and related fields (Barber, 2013). In recent years, significant information has been collected on the ecology of this species in the White sea. The long-term dynamics of species number (Лайус и др., 2013), the role in trophic chains (Демчук и др., 2018), morphological variability (Доргам и др., 2018) were studied. These investigations are important for the correct interpretation of another data received on the stickleback, including biochemical ones.

The life cycle of G. aculeatus includes a sharp change in habitat conditions, for example, during spawning migrations from the open sea to coastal and freshwater biotopes with a specific complex of abiotic (temperature, salinity, soil composition, flow rate, etc.) and biotic (forage objects, predators, parasites) factors peculiar to them. Stickleback spawning usually occurs in May-June. Juveniles during July – September stay in the area of spawning regions, where they actively feed and reach a mass of about 150-200 mg and a body length of 25 mm.

The organism first of all responds to changes in environmental conditions by activating biochemical systems of antioxidant protection. Processes of occurrence of free radicals, active forms of oxygen (AFO) and responses to them in the normal physiological conditions in the body are balanced. Under the influence of various factors such as exogenous (physical, chemical, biological) as well as endogenous (defects of mitochondrial respiration, specific enzymes) origin, interaction between prooxidants and antioxidant systems comes in unsustainable state, at which the balance may shift towards an increase in the concentration of free radicals and AFO. In these cases, the oxidative stress appears, to protect against which a complex multi-level system of antioxidant protection, consisting of low-molecular antioxidants, in particular glutathione (Zhang, Forman, 1012), and a complex of specific enzymes, which includes glutathione-S-transferase, various peroxidases and catalase. serves.

The study of the variability of antioxidant protection components during active growth, when the physiological state of fish is very labile, as well as under the influence of various environmental factors will contribute to the understanding of the fundamentals of local adaptations of ectothermal organisms.

The task of this work was to determine the concentration of water-soluble protein, glutathione, activity of glutathione-S-transferase, guaiacol-peroxidase and catalase in young three-spined stickleback caught in July-August in three waters of the Kandalaksha Bay of the White sea, which differ in environmental conditions.

Materials

Three fairly typical biotopes with significantly different environmental conditions were selected for the study (table. 1).

 

Table 1. The description of sampling places (by Dorgam et al., 2018)

 

Place of catching	Seldyanaya bay	Kolyushkovaya lagoon	Sukhaya Salma strait
Geographical coordinates	66°33'80.66" N, 33°62'25.16" E	66°31'32.62" N, 33°64'59.53" E	66°31'16.96" N, 33°64'73.70" E
General characteristic	Triangular bay with a wide entrance (depth about 8 m) and a shallow top	Connected with the sea only in full water. Depth up to 4 m	Biotope with a fairly rapid increase in depth to 5 m
The amplitude of the tide, m	2.5	0.3	2.5
Aquatic vegetation	Fucus on the littoral, very dense zoster on most of the water area	Zoster in the area adjacent to the main entrance, in other parts of the lagoon it is not enough. There are almost no fucus	Fucus on stones, rare zoster in the deeper part
Zooplankton	Coastal community with mass species for the White Sea	Since the second half of June, the absolute dominance of Acartia longiremis. By the end of August it disappears	Coastal community with mass species for the White Sea

 

Methods

Collection and primary processing of samples. The larvae of the three-spined stickleback were caught on July 31 and August 18, 2017. Immediately after capture, the fish was frozen in liquid nitrogen and stored in it until analysis began. Before analysis, the fry were weighed, then crushed by scissors, a buffer solution of 50 mm Tris-HCl, pH 7.5, was added in a ratio of 1:4 (weight:volume) and homogenized in the Disruptor Genie homogenizer (USA). The homogenate was centrifuged at 60,000 g for 1 hour in an Allegra 64r refrigerated centrifuge (Beckman, USA). The resulting supernatant was used for experiments.

The concentration of reduced glutathione (GSH) was determined by Hissin and Hilf (1976) with modifications. Soluble homogenate proteins were precipitated using 5 % trichloroacetic acid. The resulting precipitate was separated by centrifugation at 2500 g for 15 minutes. The super-settling liquid was brought to pH 8.5 using 5N NaOH, then 0.4 M Tris-HCl buffer (pH 8.5) containing 5 mm EDTA was added. Then, a 0.01% solution of orthophthalic aldehyde (Sigma-Aldrich) in methanol, prepared immediately before using, was added to the reaction mixture. After mixing, the mixture was incubated at room temperature for 15 minutes and then its fluorescence was measured (Em-420 nm, Ex-350 nm). The glutathione concentration was calculated using a calibration graph based on the results of measurements of GSN (Sigma-Aldrich) solutions with a concentration from 0.5 to 20 µg / ml (0.0016 to 0.065 µm/ml) in a 0.4 M Tris-HCl buffer solution (pH 8.5) containing 5 mM EDTA.

The activity of glutathione-S-transferase (GST) was determined by the binding rate of reduced glutathione (GSH) to the substrate 1-chloro-2,4-dinitrobenzene (CDNB) (Habig et al., 1974). 225 µl of a reaction mixture containing 1 m C DNB and 1 mM GSH in a 0.125 M phosphate buffer (pH 6.5) was added to the tablet well. The reaction was started by adding 25 µl of a homogenate solution. The increase in the optical density of the solution at a wavelength of 340 nm was recorded continuously for 5 minutes at 25 °C using a CLARIOstar Basic Unit tablet reader (BMG Labtech, Germany). The relative activity of the enzyme in fish tissues is represented as the number of µM of the reaction product formed per minute in terms of mg of soluble protein in the tissue (µM / mgprotein * min).

The activity of guaiacol-dependent peroxidase activity (GuPs) was determined by Chance, Maehly (1955) with modifications. A reaction mixture that contained 10 mM of guaiacol, 25 mM of hydrogen peroxide in a 0.05 M phosphate buffer, pH 7.4, was prepared on the day of analysis. The reaction was started by adding a reaction mixture to the supernatant. The measurement was performed at a wavelength of 470 nm for 3 min at 25°C on a CLARIOstar Basic Unit tablet reader. Relative activity was expressed in µM of the reaction product/mg of soluble protein in tissue * min.

Catalase activity (CAT) was determined by Beers, Sizer (1952) with modifications. A reaction mixture that contained 25 mM of hydrogen peroxide in a 0.05 M phosphate buffer, pH 7.4 was prepared in the day of analysis. After the supernatant was added, hydrogen peroxide degeneration was measured by the decrease in the optical density of the solution at a wavelength of 240 nm for 3 minutes at 26°C. Relative activity was expressed as µM H₂O₂/mg protein * min.

The concentration of soluble protein in the supernatant was determined spectrophotometrically by the absorption of the peptide bond at a wavelength of 220 nm at 26°C (Суховская и др., 2010). To construct a calibration graph, solutions of bovine serum albumin of various concentrations (0.02–0.1 mg/ml) in a buffer solution of 50 mM Tris-HCl, pH 7.5, were prepared in the day of analysis.

Mathematical processing of the results was performed using conventional methods (Ивантер, Коросов, 2014). The reliability of the differences was determined using the nonparametric Mann-Whitney test (the Past 3 program), adjusted for multiple Benyamini – Hochberg comparisons (Benjamini, Hochberg, 2000). The differences were considered significant at the significance level p ≤ 0.05.

The research was carried out on the equipment of the Center for collective use of the Federal research center "Karelian scientific center of the Russian Academy of Sciences".

Results

In juveniles caught on August 18 in the Seldyanaya bay, body weight increased by 20 % (p = 0.02) compared to individuals collected on July 31 (table. 2). Initially, this indicator was significantly lower than that of individuals from the Kolyushkovaya lagoon and the Sukhaya Salma strait (p = 0.001 and p = 0.01, respectively). In the other two biotopes, the change in body weight was slightly different. In the lagoon, the mass of fry in July – August did not change much, and in the strait even decreased (p = 0.03) and in August was significantly lower than that of the young from the Seldyanaya bay (p = 0.045) and July fry from the Kolyushkovaya lagoon (p = 0.006).

 

Table 2. The weight of three-spined stickleback juveniles (g) in different months 

	Seldyanaya bay	Kolyushkovaya lagoon	Sukhaya Salma strait
		July
Average	0.14* (n = 7)	0.18 (n =  7)	0.18* (n = 6)
Average error	0.008	0.004	0.008
Mediain	0.14	0.18	0.17
Standard deviation	0.019	0.011	0.019
Minimum	0.11	0.16	0.16
Maximum	0.17	0.19	0.21
		August
Average	0.17* (n = 7)	0.17 (n = 6)	0.14* (n = 7)
Average error	0.009	0.007	0.009
Mediain	0.18	0.17	0.14
Standard deviation	0.024	0.017	0.025
Minimum	0.14	0.14	0.12
Maximum	0.21	0.19	0.18

 

In July, the concentration of protein in the tissues of juveniles did not depend on the place of catching and had similar values (table. 3). In August, the protein content in the tissues of fry caught in the Seldyanaya bay and the Sukhaya Salma strait increased by 13 and 28%, respectively, and in the Kolyushkovaya lagoon it remained unchanged. The Mann – Whitney test score showed no statistically significant differences.

 

Table 3. The content of water-soluble protein (mg/ml) in three-spined stickleback juveniles is different 

	Seldyanaya bay	Kolyushkovaya lagoon	Sukhaya Salma strait
		July
Average	57.75 (n = 7)	56.37 (n = 6)	54.71 (n = 12)
Average error	5.17	3.12	5.88
Mediain	59.04	56.08	49.34
Standard deviation	12.66	8.24	14.40
Minimum	44.02	44.05	39.75
Maximum	74.02	66.13	79.18
		August
Average	65.06 (n = 7)	57.64 (n = 6)	69.99 (n = 12)
Average error	6.11	3.47	8.53
Mediain	60.89	58.04	62.00
Standard deviation	16.18	8.49	29.55
Minimum	43.35	45.17	42.73
Maximum	92.40	67.86	154.01

 

As can be seen from the table 4, the level of GSH in the tissues of caught in July fry from all the studied biotopes was on average slightly higher than in caught in August, but the differences are statistically unreliable. 

 

Table 4. The content of glutathione (μM/mg protein) in three-spined stickleback juveniles in different months

	Seldyanaya bay	Kolyushkovaya lagoon	Sukhaya Salma strait
		July
Average	0.23 (n = 6)	0.24 (n = 7)	0.26 (n = 12)
Average error	0.024	0.009	0.021
Mediain	0.23	0.25	0.27
Standard deviation	0.059	0.026	0.052
Minimum	0.15	0.2	0.18
Maximum	0.32	0.28	0.31
		August
Average	0.24 (n = 7)	0.23 (n = 6)	0.25 (n = 12)
Average error	0.011	0.02	0.008
Mediain	0.23	0.24	0.26
Standard deviation	0.029	0.050	0.027
Minimum	0.20	0.16	0.21
Maximum	0.28	0.3	0.3

 

In July, the highest recorded GST activity (table. 5) in larvae was from Kolyushkovaya lagoon and Sukhaya Salma strait. However, there were no statistically significant differences in this indicator between individuals from all three biotopes. It is worth noting that the lagoon and the strait are connected to each other, and during high tide, water exchange occurs between them. It is possible that there is a partial mixing of larval herds at the sites of material selection.

In August, the activity of the enzyme in Kolyushkovaya lagoon’s fry increased 1.7 times (p = 0.006) compared to July. Larvae from the Seldyanaya bay also showed some, but statistically unreliable, increase in activity in August. It is noteworthy that the level of GST activity in the tissues of fish from the lagoon in August was significantly higher than in July and August individuals from other biotopes (p < 0.016). And fish from the strait have the opposite effect. GST activity in August was 1.8 times lower than in July (p = 0.005).

 

Table 5. The activity of glutathione S-transferase (μМ of reaction product/mg protein*min) in three-spined stickleback juveniles in different months 

	Seldyanaya bay	Kolyushkovaya lagoon	Sukhaya Salma strait
		July
Average	6.44 (n = 6)	7.80 (n = 7)	7.17 (n = 6)
Average error	1.32	0.89	1.07
Mediain	5.38	8.19	8.27
Standard deviation	3.23	2.37	2.63
Minimum	3.22	5.38	3.33
Maximum	11.21	11.31	9.73
		August
Average	7.91 (n = 7)	13.27* (n = 6)	4.32* (n = 12)
Average error	1.26	1.4	0.55
Mediain	9.33	12.43	3.95
Standard deviation	3.34	3.43	1.89
Minimum	2.7	8.89	1.97
Maximum	11.65	17.32	8.42

 

As can be seen from the table. 6, in July, the highest level of GuPx activity was found in fry from the Kolyushkovaya lagoon, which was 3.0–5.8 times higher than in fish from the strait and Seldyanaya bay (p = 0.007 and 0.003, respectively). In August, there was a three-fold significant difference between fry from the lagoon and fish from the strait and the bay (P = 0.005 and 0.007).

 

Table 6. The guajacol peroxidase activity (nМ of reaction product /mg protein*min) in three-spine stickleback juveniles in different months 

	Seldyanaya bay	Kolyushkovaya lagoon	Sukhaya Salma strait
		July
Average	0.11 (n = 3)	0.64* (n = 5)	0.21 (n = 6)
Average error	0.018	0.099	0.042
Mediain	0.12	0.70	0.16
Standard deviation	0.03	0.22	0.10
Minimum	0.08	0.03	0.11
Maximum	0.14	0.83	0.35
		August
Average	0.19 (n = 5)	0.59* (n = 6)	0.40 (n = 8)
Average error	0.038	0.099	0.072
Mediain	0.16	0.58	0.17
Standard deviation	0.09	0.24	0.53
Minimum	0.12	0.29	0.07
Maximum	0.32	0.95	1.67

 

The fry of the Kolyushkovaya lagoon has lower catalase activity in comparison with the fish from other two habitats, but statistically significant differences were not found (table. 7). Apparently, there was no significant difference in temperature between the water areas at the time of collecting the material.

 

Table 7. The activity of catalase (μМ H2O2/mg protein*min) in three-spine stickleback juveniles in different months 

	Seldyanaya bay	Kolyushkovaya lagoon	Sukhaya Salma strait
		July
Average	1.03 (n = 5)	0.71 (n = 4)	0.98 (n = 5)
Average error	0.14	0.21	0.09
Mediain	1.22	0.87	1.05
Standard deviation	0.32	0.48	0.19
Minimum	0.53	0.52	0.71
Maximum	1.28	1.29	1.16
		August
Average	0.19 (n = 5)	0.59* (n = 6)	0.40 (n = 8)
Average error	0.22	0.10	0.13
Mediain	0.88	0.68	1.00
Standard deviation	0.49	0.20	0.43
Minimum	0.62	0.52	0.55
Maximum	1.85	0.99	1.78

 

Discussion

The change in mass indicators for the period from June 31 to August 19 was different for fry from different water areas. If the average mass of fry in the Seldyanaya bay significantly increased (p = 0.02), then the fry from the lagoon did not change much, and the fish from the Sukhaya Salma strait significantly decreased (p = 0.03). It seems likely that the fry herd in Seldyanaya bay is fairly homogeneous, unlike the other two biotopes. The lack of mass growth in fish from the Kolyushkovaya lagoon and its decline in the Sukhaya Salma strait may be due to the fact that larvae that hatched in July prevailed in the catch. It was found (Демчук и др., 2018) that in the first decade of July, the number of adult sticklebacks in the Kolyushkovaya lagoon was very low (spawning ended), while in the strait there was a high density of males per 1 m², indicating active spawning. The lagoon and the strait are connected to each other, and during high tide there is an active water exchange between them. There may have been partial mixing of the herds at the material from the selection sites.

The concentration of water-soluble protein in tissues is usually used as a secondary indicator for recalculations when determining the activity of enzymes. In our opinion, a comparative study of the variability of the level of water-soluble proteins in fish tissues can provide additional information about the influence of various environmental factors on the state of the body in whole. Despite the fact that the Mann – Whitney test score did not show statistically significant differences between fry from the studied waters for this indicator, it can be assumed that the trend for a faster increase in protein levels in August in larvae from the Seldyanaya bay and Sukhaya Salma strait compared to individuals from the Kolyushkovaya lagoon is associated with the feed database of biotopes. Zooplankton biodiversity in the lagoon is extremely low. In addition, to August, the number of food items falls to the minimum values (Демчук и др., 2018), which is probably reflected in the accumulation of protein in sticklebacks from the Kolyushkovaya lagoon.

GSH concentration is used as a non-enzymatic marker of oxidative stress in cells (Grim et al., 2013). It is known that when the ambient temperature fluctuates, the intensity of redox processes and, consequently, the amount of ROS changes, which is reflected in the level of glutathione in tissues (Yuksel et al., 2008; Суховская и др., 2014). It was shown that in notothenia Notothenia rossii and N. coriiceps, the concentration of GSH in tissues increased during acclimation from 0 to 8° C (Machado et al., 2014). Another team of authors (Souza et al., 2018), which conducted similar studies, did not reveal statistically significant changes in the GSH content in these fish species. Possibly, the absence of changes in the GSH content in the tissues of fry is associated with close temperature values (in the range of 21–24 ºС) in all studied biotopes in July and August. Another group of authors (Souza et al., 2018), who conducted similar studies, found no statistically significant changes in GSH content in these fish species. It is possible that the absence of changes in the GSH content in the fry tissues is due to the close temperature values (in the range of 21-24°C) in all the studied biotopes in July and August.

GSH-dependent antioxidant enzymes, in particular GST and GuPx, are used as markers of oxidative and temperature stress (Leggatt et al., 2007; Grim et al., 2013). In August, there was an increase in GST activity in fish from Seldyanaya bay and Kolyushkovaya lagoon. If the changes were statistically unreliable in individuals from the bay, then the fry from the lagoon showed a statistically significant increase in the activity of the enzyme, which may be related to the deterioration of environmental conditions in August. In fry from the Sukhaya Salma strait a significant decrease in GST activity was recorded. The nature of this phenomenon is still unclear. But we can assume that it is not related to the temperature regime of the water area of catches. In particular, it was found that the activity of the enzyme in the shrimp Crangon crangon L. was not affected by temperature or salinity (Menezes et al., 2006). In nototenes N. rossii, a temperature increase of 4° led to an increase in GST activity after a day (Klein et al., 2017). And other researchers have shown a decrease in the activity of the enzyme in this species when the temperature increases (Machado et al., 2014) or lack of response (Souza et al., 2018). A comparative analysis of GST activity in the winter (+12° C) and summer (+24° C) periods in five species of fish living in the Tagus estuary in Portugal showed an increase in activity with increasing temperature in four species, and the fifth (Gobius niger) had a characteristic cyclical fluctuations (Madeira et al., 2013).

GuPx is one of the enzymes that protect cell membranes from excessive amounts of hydrogen peroxide and lipid peroxidation that exceed the physiological level under various types of stress (Grim et al., 2011), one of which is the temperature factor. Despite the fact that the Mann – Whitney test score did not show statistically significant differences between fry from the studied waters for this indicator, it is nevertheless possible we can assume that the tendency for a faster increase in protein levels in August in larvae from the Seldyanaya bay and the Sukhaya Salma strait compared to individuals from the Kolyushkovaya lagoon is related to the feed database of biotopes. Zooplankton biodiversity in the lagoon is extremely low. In addition, by August, the number of food items falls to the minimum values (Демчук и др., 2018), which is probably reflected in the accumulation of protein in sticklebacks from the Kolyushkovaya lagoon.

CAT is an enzyme that neutralizes hydrogen peroxide, which is one of the ROS that occur in the body in response to various types of stress, including temperature. It is shown, for example, that the catalase activity in the muscles of young sea bass Dicentrarchus labrax on the 15th day of the experiment at a water temperature of 24° C was 2 times higher than at 18° C (Vinagre et al., 2012). In the process of thermal acclimation in N. coriiceps and N. rossii, according to some authors, there was a decrease in CAT activity (Forgatti et al., 2017), and according to others, there was no stimulation of CAT activity (Klein et al., 2017; Souza et al., 2018). During the collection period, the water temperature in the studied biotopes did not have significant differences, so this factor had no effect the activity of СAT. In young fish from the lagoon, the average activity of the enzyme was lower than in fish from other waters. It is possible that the environmental situation in the lagoon has a negative impact on the fry. This can be seen in the example of GST and GuPx, which had higher activity indicators for fish from the lagoon compared to those from other biotopes. However, the situation is not critical and does not stimulate the formation of very high concentrations of hydrogen peroxide as a result of oxidative stress, which are successfully neutralized by GuPx and do not require additional catalase activation.

Conclusions

The environmental conditions of the three studied White sea biotopes (Seldyanaya bay, Kolyushkovaya lagoon, and Sukhaya Salma strait) influenced the protein content, glutathione, and GST and GuPx activity in the tissues of three-spined stickleback fry. Changes in biochemical parameters during growth from July to August differed in larvae from Kolyushkovaya lagoon fish from Seldyanaya bay and Sukhaya Salma strait. The ecological situation in the lagoon had a negative impact on the young of the stickleback, which was expressed in the absence of accumulation of average weight, an increase in the level of water-soluble protein, and the activation of two antioxidant defense enzymes (GST and GuPx). In the Sukhaya Salma strait, the main spawning of stickleback occurs later than in other water areas, so in the August catch  the fry hatched in July dominate, which was reflected in the indicators of average weight, glutathione level and GST activity.  

References