A comparative study of the oxidative system in Chironomidae larvae with contrasting feeding strategies

Abstract

The aim of the present work was to compare the oxidative system in the fourth-instar larvae of four species of Diptera belonging to the family Chironomidae: Chironomus riparius, Paratanytarsus sp., Prodiamesa olivacea and Conchapelopia melanops. These species were selected based on their ecological and biological differences and were assessed in relation to their ecological and biological characteristics. The following parameters of oxidative stress were determined: the levels of total antioxidant capacity (TAC) and reduced glutathione; the activity of the antioxidant enzyme catalase (CAT); the level of malondialdehyde (MDA), a product of lipid peroxidation; and glutathione S-transferase (GST) activity. Results demonstrated significant differences among taxa in the specific activity of most investigated parameters. Prodiamesa olivacea showed the greatest antioxidant protection (activity of CAT, level of reduced glutathione (GSH)) and the highest detoxification activities (activity of GST and level of GSH). Predatory C. melanops was shown to be the most promiscuous biomarker for assessing the total antioxidant activity. We demonstrate that the four chironomid taxa, occupying the same ecosystem, share an important antioxidant enzymatic and non-enzymatic pool, but with oxidative stress parameters differing among the four species.

Keywords:

• Catalase
• glutathione
• lipid peroxidation
• total antioxidative capacity
• macroinvertebrate

1. Introduction

Numerous environmental pollutants induce oxidative stress. A balance between intracellular pro-oxidants (including extracellular stimuli like pollutants) and antioxidant systems may be used to assess environmental stress. For this reason, antioxidant enzymes have been considered sensitive biomarkers of oxidative stress, particularly in aquatic organisms (Valavanidis et al. 2006). Consequently, the persistence of a population exposed to a range of stressors depends on the protection provided by detoxifying and antioxidant mechanisms, whose function is the restoration of internal cellular homeostasis (Bonnefoy et al. 2002; Vertuani et al. 2004). Methods for studying oxidative stress in living organisms include evaluation of the activity of antioxidant enzymes, the level of low-molecular antioxidants, analysis of lipid peroxidation products and evaluation of the total systemic antioxidant potential. Oxidative stress resulting from exposure to chemicals is associated with changes in glutathione S-transferase activity, which catalyzes the conjugation of glutathione with xenobiotics in the second phase of their metabolism (Bukowska 2005). Levels of glutathione are also informative because this antioxidant plays an important role both in neutralizing reactive oxygen species (ROS) and in decreasing xenobiotic effects.

Figure 1. Study area.

The use of bioindicators for the assessment of risks posed by pollution has grown significantly over the past few years. Bioindicators are useful markers of pollution because they are particularly sensitive and are the first organisms to express detectable changes that demonstrate a stress response to toxins (Huggett et al. 1992). The freshwater aquatic organisms used as bioindicators include some fish species, as well as invertebrates, including representatives of Crustacea, Annelida, Plecoptera, Trichoptera, Ephemeroptera, Diptera, Odonata and Mollusca (de Lafontaine et al. 2000; Berra et al. 2004; Bonnail et al. 2016; Mohanty & Luna 2016). The Chironomidae (Diptera) in particular are considered to be effective indicators of water quality (Saether 1979; Mousavi et al. 2003). They show a broad range of ecological characteristics and can tolerate a wide range of environmental conditions (Battarbee 2000; Cañedo-Argüelles et al. 2016). In addition, they comprise a large number of species and occur in high abundance in all freshwater aquatic ecosystems, and have been successfully used in a variety of biomonitoring-based approaches (Rosenberg 1992). Chironomids are good bioindicators of the aquatic environment because they spend most of their life in bottom sediments, where they are exposed to toxic substances such as heavy metals, radioactive substances, pesticides and other xenobiotics, originating from sources such as industrial waste and agricultural runoff. Chironomids have been shown to be valuable models for research on morphological deformity, trophic classification of lakes and paleolimnology, and in toxicity studies (Meregalli et al. 2000; De Bisthoven et al. 2005; Lee & Choi 2009; Weltje et al. 2010; Rebechi & Navarro-Silva 2012). Studies of their behavior and life history, as well as biomonitoring studies, have been carried out at population and ecosystem levels. In recent years, they have been increasingly used as biochemical and physiological indicators of environmental stress.

The aim of the present study was to investigate parameters of oxidative stress, including total antioxidant capacity (TAC), glutathione, lipid peroxidation and catalase (CAT), in chironomid larvae. We also investigated the ability of chironomid species to metabolize xenobiotics by determining the activity of glutathione S-transferase (GST) and reduced glutathione levels. Four species of Chironomidae – Chironomus riparius (Meigen, 1804) (subfamily Chiromoninae), Paratanytarsus sp. (subfamily Chiromoninae), Prodiamesa olivacea (Meigen, 1818) (subfamily Prodiamesinae) and Conchapelopia melanops (Meigen, 1818) (subfamily Tanypodinae) – were selected for investigation, based on their contrasting ecological and biological characteristics, such as mode of feeding and diet.

The most widely used chironomid species used as bioindicators belong to the genus Chironomus: C. riparius (in Europe and North America), C. dilutus (in North America) and C. yoshimatsui (in Japan). However, laboratory-bred individuals often demonstrate genetic impoverishment (Nowak et al. 2007), limiting their similarity to the wild specimens which they are intended to represent (Brown et al. 2011). In addition, these taxa are not cosmopolitan species, which creates difficulties in comparing results obtained in different geographical regions. Particular attention has been focused on C. riparius, which is becoming increasingly popular as a model organism for aquatic bioassays. This species is easy to culture in the laboratory, is widely distributed in the northern hemisphere in temperate latitudes and is found in both lentic and lotic waters, often at high densities and usually in waters enriched with sediment (Armitage et al. 1995; Péry & Garric 2006). Its life cycle is typically completed within a period of 3 to 4 weeks at 20°C. This species possesses hemoglobin (Hb) with a high affinity for oxygen, which results in a high tolerance of the larvae to a range of contaminants (Osmulski & Leyko 1986; Choi et al. 1999; Choi & Ha 2009; Al-Shami et al. 2010). Therefore, the species prefers eutrophic and organically enriched waters (Armitage et al. 1995). It is an opportunistic feeder on detritus (collector-gatherer feeding on sediment-deposited detritus), building tubes in the substrate and with the larvae growing to a large size (Rasmussen 1985; De Haas et al. 2006).

Paratanytarsus spp. are eurytopic forms with medium-sized larvae, which are adapted to a wide range of ecological conditions. Larvae are filter feeders, of smaller body size than C. riparius, but also build tubes that protect the larvae from the negative effects of chemical contaminants (Winner et al. 1980; Halpern et al. 2002). Notably, P. grimmii, a cosmopolitan chironomid belonging to this family, is considered a pest because of its capacity to breed in water distribution systems. Paratanytarsus grimmii is able to reproduce asexually through aphonic parthenogenesis, reducing the risk of crossbreeding with related individuals and inbreeding depression (Porter 1971). A case has been made for the utilization of this species in routine ecotoxicological studies (Gagliardi et al. 2015).

Prodiamesa olivacea was selected for the present study because it possesses a large larva and occurs in both lotic and lentic systems. It is associated with packets of leaves, i.e. large-grained allochthonic organic matter (De Bisthoven et al. 1992). This species is classified as a picker (collector-gatherer, feeding on leaves), although it shows mobility and contributes to the mechanical fragmentation of the foreign matter. This species, together with C. riparius, is used in biomonitoring studies based on the appearance of deformities (Servia et al. 1998).

The final taxon used in the study was Conchapelopia sp., which is ubiquitous in Europe in both lenitic and lotic ecosystems (Vallenduuk & Moller Pillot 2007). Conchapelopia melanops, the species used here, is a ubiquitous species living in large stagnant waters and diverse running waters and can comprise from 10 to 20% of the total benthos density along a longitudinal river profile (Vallenduuk & Moller Pillot 2007). It belongs to the subfamily Tanypodinae, consisting mainly of predators. The Tanypodinae exhibit well-defined food preferences, feeding mainly on the larval forms of other Chironomidae species (Baker & McLachlan 1979).

2. Material and methods

Fourth-instar chironomid larvae were collected using a net in the upstream part of the River Bzura in autumn 2017 (three sampling occasions). The organisms were stored in polypropylene bottles containing river water and quickly transferred to the laboratory where they were identified under a stereoscopic microscope, using the keys of Wiederholm (1983, 1986, 1989), and frozen. Depth and velocity of the river flow and the area of the river bottom (where the samples were collected) were measured. The physico-chemical parameters analyzed were conductivity, dissolved oxygen, temperature and acidity.

At each site, three samples (0.1 × 0.1 m) of river bottom were collected using a tubular sampler 0.01 m² in cross-sectional area. The size classification of inorganic particles and inorganic substrate index, SI (Cummins 1962; Quinn & Hickey 1990), were calculated. In addition, the amount of benthic particulate organic matter (BPOM) was evaluated. Using sieves and filters, the organic matter was divided into two fractions – coarse (BCPOM > 1 mm) and fine (BFPOM < 1 mm) particulate organic matter – according to Petersen et al. (1989). Periphyton was measured as chlorophyll a concentration using the method of Goltermann et al. (1978).

2.1. Study area

The study was conducted in a low-order stretch of the River Bzura in central Poland. The River Bzura is a tributary of the River Vistula, debouching into the Vistula 587 km from its source. It is 166 km long and its drainage is ca. 7788 km². The Bzura flows close to the city of Łódź. The study sites are located in a first-order stream section (Figure 1). This habitat is characterized by a sandy-bottom substrate with large amounts of allochthonous organic matter, especially tree leaves (Alnus glutinosa (L.)), covering the stream bed over the whole year.

2.2. Cell-free extract preparation and enzyme activity measurements

Chironomidae of a very similar size were selected from each sample for biochemical analyses. Samples (wet mass 0.046–0.083 g) were placed in 100 mM sodium phosphate buffer (pH 7.4, 100 mM KCl and 1 mM EDTA) and maintained on ice until homogenization. Homogenization using a CAT X-120 knife homogenizer was performed on ice at 2000 rpm for 2 min, and the homogenates were then centrifuged at 10,000 rpm for 10 min (4 °C) (Sigma 3–16 kL separator). The supernatants were immediately used for estimations. For each sampling date, three replicates were performed to determine individual parameters.

The protein concentration was determined using a spectrophotometric method with Folin reagent (Lowry et al. 1951).

2.3. Activity of catalase

Determination of catalase activity was performed according to the method of Aebi (1984). The method is based on the decomposition of hydrogen peroxide, which is indicated by a decrease in absorbance at 240 nm. The assay mixture (volume 3 mL) consisted of 2 µL or 3 µL of homogenate (depending on the kind of homogenate), 50 mM potassium phosphate buffer (pH 7.0) and 54 mM hydrogen peroxide. One unit of CAT activity was defined as the enzyme activity that degraded 1 μmol of H₂O₂ in 1 min. Activity of catalase was expressed as (μmol × min^-1) × mg^-1 protein in homogenate.

2.4. Activity of glutathione S-transferase

GST activity was assayed spectrophotometrically by monitoring the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH at λ_max = 340 nm at 37 °C. We used a volume of 15 µL of homogenate (50 µL of 20 mM GSH, 100 µL of 20 mM CDNB and 835 µL of 0.1 M potassium phosphate buffer (pH 6.5)). The rate of increase of product concentration was monitored by measuring absorbance at 340 nm at 25 °C for 3 min in a SPECORD 250 plus spectrophotometer (Analytik Jena). Within this period, the rate of reaction was linear with time. Activity of GST was calculated using an extinction coefficient of 9.6 mM^–1 × cm^–1 and was expressed as nmol/min/mg protein in homogenate (Habig et al. 1974).

2.5. The level of reduced glutathione

The level of reduced GSH in homogenate was determined by a modified method of Ellman (1959). Homogenate was deproteinated by addition of 20% trichloroacetic acid (TCA) to a final concentration of 2%. Than a volume of 5 µL of 10 mM 5,5ʹ-dithiobis(2-nitrobenzoic acid) (DTNB) was added to 50 µL of supernatants cleared by centrifugation (10 min, 15,000 rpm/min). The formation of yellow anion 5-thio-2-nitrobenzoate, which is proportional to the total glutathione concentration, was monitored at 412 nm and 25 °C against reagent controls. The concentration of GSH in the samples was calculated from a millimolar absorption coefficient for DTNB (ε = 13.6 mM⁻¹ × cm⁻¹) and was expressed as µmol/mg protein in homogenate.

2.6. Determination of lipid peroxidation (malondialdehyde level)

Lipid peroxidation (LPO) was measured using thiobarbituric acid (TBA) according to the method of Stocks and Dormandy (1971). The concentration of the end product of LPO, malondialdehyde (MDA; a thiobarbituric acid-reactive substance), is an index of lipid peroxidation and oxidative stress. The level of MDA was monitored at 532 nm and calculated using the MDA absorption coefficient (156 mM⁻¹ × cm⁻¹). The level of MDA was expressed as µmol/mg protein in homogenate.

2.7. Total antioxidant capacity

The total antioxidant capacity in the homogenate was determined by means of reduction of 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Absorbance measurement was performed at 414 nm. Considering that one Trolox molecule reacts with two ABTS⁺ molecules, the calculated values were multiplied by two (unit per µmol of Trolox equivalent L^–1, which is used as a standard in antioxidant capacity measurements) (Bartosz 2003).

2.8. Statistical analysis

All statistical analyses were carried out using Statistica 10.0 (StatSoft Inc.). One-way analysis of variance (ANOVA) was used to examine differences among investigated parameters.

3. Results

River characteristics and water physico-chemical parameters measured in the River Bzura during the study are shown in Table I.

Table I. Physico-chemical parameters for the sampling site. SI - inorganic substrate index, pH – acidity. SD- standard deviation.

River parameters	mean	± SD
width (m)	0.90	± 0.100
depth (m)	0.067	± 0.003
currel velocity (m s^−1)	0.27	± 0.075
SI (mm)	2.37	± 0.741
temperature (°C)	2.97	± 0.567
Dissolved oxygen (mg dm^−3)	6.19	± 0.384
BFPOM (g m^−2)	6164	± 271
BCPOM (g m^−2)	2171	± 77
pH	6.43	± 0.457
Conductivity (μS cm^−^1)	323	± 6.658

Data for oxidative stress parameters of chironomid species are shown in Figure 2. C. riparius shows a low activity of CAT, and the lowest levels of GSH, LPO and TAC. In contrast, the highest enzyme activity for CAT and highest levels of GSH and LPO were observed for P. olivacea. Conchapelopia melanops shows the lowest activity of CAT (Figure 2). Prodiamesa olivacea shows the highest detoxycation activity (GST, Figure 3). Larvae of Paratanytarsus sp. showed average parameter values (Figures 2 and 3).

Figure 2. Catalase (CAT) activity and level of reduced glutathione (GSH), lipid peroxidation (LPO) and total antioxidant capacity (TAC) in the four studied chironomid species.

Figure 3. Activity of glutathione S-transferase (GST) in the four studied chironomid species.

There were significant differences (Table II, ANOVA) in whole-body CAT and GST activities as well as in levels of GSH, MDA and TAC. Differences were observed among the chironomid taxa in GSH content and CAT activity. Prodiamesa olivacea was characterized by significantly (p < 0.05) higher CAT activity and GSH level, with respect to C. riparius and C. melanops. GST levels for P. olivacea (p < 0.001) differed from those of all other investigated taxa. For LPO levels, significant differences were found between P. olivacea and C. riparius. Total antioxidant capacity was different between C. melanops and the other chironomid taxa (post-hoc Tukey test).

Table II. Differences (ANOVA) between taxa studied for parameters examined; df (3,32) Post hoc differences: 1 – Chironomus riparius, 2 – Prodiamesa olivacea, 3 – Paratanytarsus sp. 4 – Conchapelopia melanops df – number of degrees of freedom CAT – activity of catalase GSH – the level of reduced glutathione GST – activity of glutathione S-transferase MDA – level of malondialdehyde TAC – total antioxidant capacity.

ANOVA	df	F	p	Differences between taxa (post hoc)
CAT	(3,32)	5.644	0.0032	2–1, 4
GSH	(3,32)	4.866	0.0067	2–1, 4
GST	(3,32)	35.677	0.0001	2–1, 3, 4
MDA	(3,32)	3.176	0.0373	1–2
TAC	(3,32)	38.381	0.0001	4–1, 2, 3

Post hoc differences: 1 – Chironomus riparius, 2 – Prodiamesa olivacea, 3 – Paratanytarsus sp. 4 – Conchapelopia melanops.df – number of degrees of freedom.

For GSH levels, a statistically significant negative correlation was recorded for size of inorganic substrate (r = −0.359, p = 0.032), and a positive correlation was found for BCPOM (r = 0.372, p = 0.025). Among the enzymes studied, CAT activity showed a positive correlation with dissolved oxygen (r = 0.338, p = 0.044) and a negative correlation with temperature (r = 0.384, p = 0.021) and conductivity (r = −0.361, p = 0.031).

In C. riparius, total antioxidant activity was positively correlated with CAT activity, while in P. olivacea this parameter was positively correlated with GSH level. For TAC only negative correlations, with CAT activity (C. melanops) and GSH level (P. olivacea and Paratanytarsus sp.), were found. LPO was positively correlated with GST activity in C. riparius and negatively correlated with GST activity in Paratanytarsus sp. (Table III).

Table III. Pearson correlaton coefficients between selected chironomidae taxa and investigated parameters. C. rip. – Chironomus riparius, P. oli. – Prodiamesa olivacea, Para. – Paratanytarsus sp., C. mel. – Conchapelopia melanops CAT – activity of catalase GSH – the level of reduced glutathione GST – activity of glutathione S-transferase MDA – level of malondialdehyde TAC – total antioxidant capacity.

Parameters		GSH		GST		MDA		TAC
		r	p	r	p	r	p	r	p
CAT	C. rip.	0.591	0.094	−0.171	0.660	−0.489	0.181	−0.279	0.467
	P. oli.	−0.237	0.540	0.260	0.499	−0.455	0.219	0.511	0.160
	Para.	−0.004	0.992	0.428	0.250	−0.415	0.267	0.522	0.149
	C. mel.	0.437	0.239	0.265	0.490	0.287	0.454	−0.737	0.024
		GSH	C. rip.	−0.454	0.220	−0.557	0.119	0.214	0.580
			P. oli.	0.612	0.080	0.262	0.495	−0.897	0.001
			Para.	−0.131	0.737	0.400	0.286	−0.707	0.033
			C. mel.	−0.274	0.476	0.541	0.133	−0.461	0.212
				GST	C. rip.	0.840	0.005	−0.052	0.895
					P. oli.	−0.050	0.899	−0.379	0.315
					Para.	−0.749	0.020	0.264	0.493
					C. mel.	−0.345	0.363	−0.565	0.113
						TBARS	C. rip.	0.212	0.585
							P. oli.	−0.460	0.213
							Para.	−0.544	0.130
							C. mel.	0.051	0.896

C. rip. – Chironomus riparius; P. oli. – Prodiamesa olivacea; Para. – Paratanytarsus sp.; C. mel. – Conchapelopia melanops.

4. Discussion

Many hydrophobic pollutants that exist in the aquatic environment accumulate in sediment. As a consequence, benthic organisms risk exposure to their effects. Exposure to xenobiotics in sediments may cause adverse effects at lower trophic levels, potentially leading to biomagnification of these toxicants, and thus more serious toxic effects, at higher trophic levels (Newman 1998). Regardless of feeding strategy and quality of ingested food, chironomids are unable to avoid toxic substances contained in water and sediments (De Haas et al. 2002) and, consequently, are considered to be good biomarkers of the condition of the environment. We detected significant differences between taxa with reference to the specific activity of most investigated parameters. Our results emphasize that four chironomid taxa, living in the same ecosystem, share an important antioxidant (enzymatic and nonenzymatic) pool, as well as detoxifying properties, and the values of the investigated parameters are different in each of the taxa.

LPO plays a significant role as a marker of oxidative stress, associated with accelerated aging of the cells (Guéraud et al. 2010; Negre-Salvayre et al. 2010). It involves oxidation of lipids, which is associated with formation of peroxide that is neutralized by utilization of glutathione. LPO is most frequently initiated by the hydroxyl radical. It is the most reactive form of oxygen species, formed as a result of reduction of oxygen. It is created by the interaction of chemical substances (electron donors) and oxygen present in the environment (Bartosz 2003). The primary factor here is oxygen, which transforms into a hydroxyl radical that initiates the peroxidation process. LPO was found to be at a low level in the red larvae of C. riparius and Paratanytarsus sp., while it achieved the highest value in the white larvae of P. olivacea. It is conceivable that the red forms of Hb demonstrate higher peroxidase activity than other types of Hb in the Chironomidae. In the peroxidation reaction, red forms of Hb degrade hydrogen peroxide to water and oxygen, thus preventing the formation of hydroxyl radicals, the initiator of LPO. In studies of chironomids, LPO within 0.05 nmol mg⁻¹ was observed (Campos et al. 2016). Our results are approximately an order of magnitude higher (from 0.12 nmol mg⁻¹ in C. riparius to 0.18 nmol mg⁻¹ in P. olivacea). Comparable results were obtained in studies of a contaminated eutrophic reservoir, where the sensitivity of daphnids (Wojtal-Frankiewicz et al. 2013) and the response of the antioxidant system of zebra mussels, dependent on the environmental characteristics of the ecosystem, were investigated (Wojtal-Frankiewicz et al. 2017). In this study, despite the LPO values differing significantly within the same species among seasons, temperature was strongly, negatively correlated with LPO, with a high level of oxidative stress generated at lower temperatures. In our study, as in the studies of zebra mussels, the temperature in the River Bzura was relatively low, and that may have been the reason for inhibition of the growth of Chironomidae taxa, while LPO values were observed to be significantly higher in P. olivacea, and this taxon has been described as a cold stenothermal species in the profundal zone of lakes (Marziali & Rossaro 2013). Other field and experimental studies on molluscs have demonstrated that a decrease in antioxidant enzyme activity and peroxidase pool causes an increase in LPO (Doyotte et al. 1997; Choi et al. 2001). Oxidative stress may cause partial damage to the internal mitochondrial membranes by LPO, which interferes with the function of respiratory electron transport system (ETS) units and reduces their activity (Cossu et al. 2000).

Superoxide dismutase (decomposing the superoxide anion radical), as well as catalase and glutathione peroxidase, which consume hydrogen peroxide, play an important role in protection against LPO. These enzymes, therefore, prevent the formation of the hydroxyl radical in Fenton’s reaction, which initiates LPO. In our study, no correlation between LPO and CAT activity was observed in any of the studied taxa, similarly to the case in molluscs (Bielen et al. 2016) where prooxidative conditions upon exposure to ZnCl₂ were not evidenced by an increase in CAT activity. CAT is a haem-based protein, which catalyzes decomposition of hydrogen peroxide to water and oxygen. The effectiveness of antioxidant catalase varies depending on the organism, the type of stressor and the time of exposure, and is used as a biomarker of oxidative stress (Halliwell & Gutteridge 2007; Osman et al. 2007). Our results indicate differences in the activity of catalase in the studied taxa. It is noteworthy that CAT exhibits a typical bell-shaped response to toxic chemicals (Viarengo et al. 2007); the initial increase in enzyme activity (reflecting the induction due to pro-oxidant conditions) leads to a decrease, which may be the consequence of a variety of factors (increased catabolic rate, direct inhibition of CAT by toxic chemicals, problems with compensation of oxidative stress). This bell-shaped response is further changed with the concentration of a chemical substance, and with exposure time (Viarengo et al. 2007); both increases and decreases in CAT activity are reported. Our results indicate that P. olivacea and Paratanytarsus sp. exhibit wide ranges of CAT activity. Additionally, P. olivacea, which has the highest CAT activity, shows significant differences in this parameter from all other taxa studied with the exception of Paratanytarsus sp. High CAT activity is associated with high energy expenditure during the transformation of chironomids, which causes a periodic increase in the content of hydrogen peroxide in the mitochondria and the cytosol, due to active adenosine-5-triphosphoric acid (ATP) synthesis (Ahmad 1995). Another reason is the need to compensate for the deficit of peroxidase activity (Ahmad 1992) in the process of hydrogen peroxide decomposition.

The overall CAT activity in bivalves collected from Oxbow Lake in the lower course of the River Drawa was between 9 and 18 μmol min⁻¹ mg⁻¹ protein (Bielen et al. 2016). The studies conducted in two rivers in Italy estimated the activity of this enzyme in winter at 9.4–11.5 μmol min⁻¹ mg⁻¹ protein, whereas in summer its activity reached 13.6 μmol min⁻¹ mg⁻¹ protein for the whole chironomid family. There were seasonal variations, which apparently were independent of the environmental parameters. Interestingly, it was found that the Plecoptera, which includes species requiring well-oxygenated water, shows the highest activity of CAT, suggesting a protective role of this enzyme against oxidative stress. This finding explains the low CAT values in chironomids which have Hb (C. riparius and Paratanytarsus sp.); they are the taxa with optimum development in warm ecosystems that are rich in nutrients, and possess a strong ability to adjust their oxy-regulatory capacity; i.e. the ability to maintain high oxygen exchange (respiration rate) with low oxygen availability (oxy-regulators). It should be emphasized that these larvae have 13 types of Hb (Osmulski & Leyko 1986). Chironomus riparius, which inhabits bottom sediments, is exposed to anaerobic conditions, and thus is adapted to low oxygen concentrations in the environment by production of a high content of Hb. Hb metabolizes xenobiotics (Ha & Choi 2008), which enables this species to survive polluted environments. This capability is associated with oxygenous metabolism, but auto-oxidation of heme in respiratory fluids of this taxon leads to the formation of high amounts of ROS. Consequently, antioxidative enzymes, which neutralize free radicals, play a crucial role in this species (Choi et al. 1999). Other taxa, such as P. olivacea, which sometimes occupy the same sites as C. riparius, have no Hb. High CAT activity was observed in P. olivacea, which has a low temperature optimum and is classified as an oxy-conformer from poor nutrient conditions (Brodersen et al. 2004, 2008; Marziali et al. 2006; Marziali & Rossaro 2013).

During the lipid peroxidation process, ROS and fatty acid peroxides increase and GSH is involved in the degradation of peroxides with glutathione peroxidase. GSH is a tripeptide, which has various functions in living organisms. It belongs to the most important antioxidants, which react with ROS, thus protecting thiol groups of proteins before irreversible inactivation (Bartosz 2003). It plays a crucial role in cellular protection against reactive species and electrophiles, and it is involved in regulation and maintenance of the cellular redox status and activity of specific enzymes (Lushchak 2012). In aquatic ecosystems, GSH enables effective detoxification of microcystins (Wiegand & Plussmacher 2005). A decrease in GSH content is usually associated with intensive detoxification and/or with the presence of oxidative stress, and also limits LPO (Doyotte et al. 1997; Choi et al. 2001). Increased oxidation reactions contribute to increased LPO and decrease in GSH, which stimulates an increase in the activity of the arachidonic acid cascade enzymes: cyclooxygenase and lipoxygenase, and causes an influx of calcium ions into the cells leading to damage to the cellular membrane and its receptors. A consequence of oxidative stress is cytoplasmic membrane permeability, causing depolarization. In our study, a correlation between GSH level and amount of coarse particulate organic matter was noted. The lowest GSH level was determined in the tissues of the predatory species C. melanops. Because of its diet, it is particularly vulnerable to toxic chemicals, which must be detoxified in the presence of GSH. This is associated with the fact that predators are susceptible to biomagnification of pollutants and toxins accumulated in their prey.

GST is the primary enzyme responsible for detoxification of xenobiotics (Bukowska 2005). GST activity has been well recognized, in many biomonitoring programs, as a biomarker of contamination of aquatic environments (Livingstone 1993; Van der Oost et al. 1997; Cajaraville et al. 2000). The activity of GST is an indicator of exposure to organic pollutants and metals (McLoughlin et al. 2000). This indicator has been used in studies of environmental pollution, including exposure to sediment-related carcinogens in fish (Gallagher et al. 1998; Melgar Riol et al. 2001), as well as in studies using molluscs (Sheehan & Power 1999). The application of GST activity as an indicator associated with pollution or potential resistance to pesticides has been assessed in the case of many species of insects (Hodge et al. 2000). The Paratanytarsus sp. chironomids are, similarly to molluscs, filtering invertebrates able to accumulate pollutants at analytical levels in their soft tissues (Goldberg & Bertine 2000). However, in our study the activity of GST was high in P. olivacea, inhabiting leaf packets, and was even higher than the maximum standard value reported for non-exposed C. riparius (Domingues et al. 2007). Nevertheless, these values were not as high as those obtained by Palacio-Cortes et al. (2017) in a study of C. sancticaroli; the detoxification of xenobiotics is presumably efficiently performed in this genus (Palacio-Cortes et al. 2017). In contrast, red chironomid larvae, both of filtering Paratanytarsus sp. and of pickers C. riparius, showed average GST values comparable to those of previous studies (Choi et al. 2000; Domingues et al. 2007).

We also examined TAC as an essential biomarker of oxidative stress (Słowińska et al. 2016). High TAC values were obtained for predatory C. melanops, while this species generally showed relatively low enzymatic activity. Probably a high level of TAC; e.g. the presence of a large number of low-molecular antioxidants (vitamins A, C, E and other compounds), compensates for enzymatic activity, which is at a moderate level.

On the basis of our research, it can be concluded that chironomids are a diverse and flexible group of insects, reflecting the morphological, physiological and environmental characteristics of these species as well as their resilience to anthropogenic changes to the environment. The efficient defense mechanisms of these insects appear to have contributed significantly to their evolutionary success (Cytryńska et al. 2016). The results obtained in the present study indicate a range of defense mechanisms, including antioxidant defense and detoxification.

The usefulness of species, or groups of species, whose behavior can serve as biological indicators that reflect changes in pollution is increasingly recognized (Dziock et al. 2006). Depending on the sensitivity of the organism, various responses are observed, from changes in behavior and physiology, through changes in morphology and biochemistry, and ultimately reflected in changes in mortality rates. Pander and Geist (2013) showed the role of bioindication in the analysis of the status of water pools and the methods to rehabilitate water resources. The organisms most commonly used are crustaceans, molluscs, fish, protozoa and algae (Schneider & Lindstrøm 2009; Sasikumar & Krishnakumar 2011; Negishi et al. 2013). Among chironomids, the most commonly used species is C. riparius. Our studies indicate, however, that each of the taxa examined reacted to the quality of the environment in a different way. The predator C. melanops appeared to have the greatest utility for bioindication. Therefore, it is emphasized that owing to the effects of biomagnification, ecotoxicological studies should be verified under natural conditions, through biological and chemical monitoring of aquatic ecosystems, in order to assess the exposure of ecosystems to toxic substances and their effects on the organisms in the food chain, as well their ultimate bioaccumulation and toxicity.

5. Conclusion

The organisms studied expressed a variety of feeding strategies, which was reflected in their metabolism and capacity for antioxidant defense, detoxification and neurological response.

We have observed that, in spite of a low activity of the biomarkers studied, which are characteristic for predators, C. melanops has the highest TAC activity (sum of the various antioxidants) which shows an excellent adjustment of metabolism of this taxon, due to food pull from which the valuable components may be used to neutralize xenobiotics and alleviate oxidative stress. The way of nutrients achievement makes it possible to tight oxidative stress; the low activity of the studied biomarkers is compensated by substances having the ability to scavenge free radicals.

Another strategy is characteristic of P. olivacea, which inhabits leaf packets and is exposed to chlorophyll a metabolites and an elevated level of ROS, and is thus characterized by high CAT and GST activity and a high GSH level.

Acknowledgements

The authors wish to thank Professor Bożena Bukowska for her expert support and assistance.

Disclosure statement

No potential conflict of interest was reported by the authors.

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