Modulation of endogenous antioxidants by zinc and copper in signal crayfish (Pacifastacus leniusculus)

Abstract

Metal pollution is a long-standing concern and bioindicators are commonly used in ecotoxicological studies to monitor impacted wildlife populations for evidence of sublethal effects. Significant variation in the response of common biomarkers to metals is reported across taxa, thus necessitating careful characterization in model organisms. In this study, we describe the regulation of glutathione S-transferase (GST), glutathione (GSH), and metallothionein (MT) by zinc chloride (0.6, 0.9, 1.2, 2.4, 4.8, 9.6 μg g⁻¹) and copper chloride (0.6, 0.9, 1.2 μg g⁻¹) in signal crayfish (Pacifastacus leniusculus). Zinc chloride did not alter GST activity relative to controls in the hepatopancreas. Crayfish exposed to copper chloride exhibited decreased GST activity at the lowest dose tested (0.6 μg g⁻¹) with no change observed at the higher doses. GSH did not change in response to either metal when sexes were grouped together. MT concentrations increased in response to zinc (2.4, 4.6, and 9.6 μg g⁻¹ doses) and copper (0.6, 0.9, and 1.2 μg g⁻¹ doses) in gill tissue. In tail tissue, MT increased at the 2.4 and 4.8 μg g⁻¹ zinc chloride doses and all the concentrations of copper tested. Sex-specific differences in endogenous antioxidant expression were also analyzed with no clear patterns emerging. We concluded that these endpoints are sensitive to zinc and copper in signal crayfish, although careful interpretation is needed when applying them in field studies given the variation in responses, non-monotonic dose responses, and differences in biotic and abiotic factors that inevitably exist in different aquatic ecosystems.

1. Introduction

Metals entering the environment through anthropogenic activities are a concern for wildlife and human populations and the utilization of biomarkers can serve as a tool to monitor whether organisms exhibit sublethal biological responses to pollution in impacted ecosystems (Breton and Prentiss, 2019; Duan et al., 2017; Hinojosa-Garro et al., 2020; Qian et al., 2017; Shervette and Van Dolah, 2017). Enzymatic antioxidants such as glutathione S-transferase (GST) as well as non-enzymatic antioxidants including glutathione (GSH) and metallothionein (MT) are all known to change in response to oxidative damage caused by exposure to metals (Amiard et al., 2006; Dickinson and Forman, 2002; Park et al., 2020). A thorough understanding of the expression patterns and regulation of these markers in a given model organism and population is required, and careful characterization of responses to different classes of contaminants is essential (Gunderson et al., 2018). This is highlighted by studies reporting species-specific responses of antioxidant and detoxification pathways to pro-oxidants (i.e., metals) (Amiard et al., 2006; Dobritzsch et al., 2020; Perić et al., 2020). Furthermore, studies demonstrate that antioxidant biomarker expression (i.e. GST, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), reduced glutathione (GSH)) is tissue specific, sex-specific, and varies seasonally in crayfish based on studies conducted in 2 species (Astacus leptodactylus and Procambarus clarkii) (Barim-Oz et al., 2017; Elia et al., 2006). Interestingly, sex-specific and seasonal changes in expression of antioxidants have also been reported in crabs and amphipods (Paital and Chainy, 2013; Sroda and Cossu-Leguille, 2011). In this study, we examined the regulation of GST, GSH, and MT by zinc and copper, two metals that commonly pollute aquatic ecosystems, in signal crayfish (Pacifastacus leniusculus) collected from C.J. Strike Reservoir, located on the Snake River in Idaho (USA). We were interested in determining whether these metals modulate the above-mentioned biomarkers in this species, as well as examining sex-specific patterns of expression and response to zinc and copper.

Crayfish are useful ecotoxicology model organisms and studies have examined the expression of biomarkers in crayfish inhabiting polluted systems (Elia et al., 2006; Faria et al., 2010). Signal crayfish populate bodies of water throughout the pacific northwest of the United States and Canada and have been introduced into aquatic systems in Europe and Japan (Holdich, 2002; Larson and Olden, 2011). Crayfish are considered keystone species due to their omnivorous diet (animals, plant, detritus, etc.) making them an important link in the flow of energy and nutrients, as well as the biomagnification of pollutants, since they serve as a food source for both aquatic and terrestrial predators (fish, racoons, otters, birds, etc.) (Bowling et al., 2011; Larson and Olden, 2011). Metals are known to accumulate in crayfish tissues and they can thus serve as bioindicators of heavy metal exposure in impacted ecosystems (Kouba et al., 2010). Studies examining the mobility of signal crayfish report they inhabit relatively narrow home ranges for extended periods of time (Guan and Wiles, 1997), enabling their use by ecotoxicologists to examine localized populations within polluted and reference systems. Furthermore, common biomarkers used in ecotoxicology are expressed in signal crayfish tissues and are sensitive to exposure to contaminants such as organophosphate pesticides and mercury (Gunderson et al., 2018).

Zinc and copper are essential trace elements required for normal growth and development, although toxic responses can result from excess exposure given that they are both common pollutants in aquatic ecosystems located near anthropogenic activities (mining, smelting, agriculture, sewage sludge, use in biocides, etc.) (Eisler, 1993, 1998). Zinc is an essential trace element that is a component of at least 200 metalloenzymes, plays a stabilizing role for biomolecules, and is found in naturally high levels in crayfish (Kouba et al., 2010). Crustaceans can accumulate zinc from both water and food, and while one study found that arthropods were the most zinc sensitive group among invertebrates tested, crayfish (Orconectes virilis) were among the more zinc tolerant crustaceans (Eisler, 1993). Excess zinc exposure in crustaceans can result in symptoms that include gill histopathology, increased tissue total protein, decreased glycogen, decreased acid phosphatase activity, and retardation of limb generation (Eisler, 1993). MT is believed to be involved in zinc metabolism (Bremner and Davies, 1975). Copper is a component of the respiratory metalloprotein-hemocyanin in crustaceans and relatively high concentrations are present in crayfish, particularly in hepatopancreas tissue (Kouba et al., 2010). Copper uptake in aquatic arthropods occurs primarily through dietary intake and uptake in the gut and/or across the gills from the water (Eisler, 1998). High concentrations of copper interfere with respiratory processes in the rusty crayfish (Orconectes rusticus) and the mechanism of action likely relates to its coagulatory action on cellular proteins, while lower concentrations of copper lead to tissue degeneration and changes in glutathione equilibrium (Eisler, 1998; Hubschman, 1967). Studies in crustaceans demonstrate that copper primarily accumulates in the hepatopancreas (Procambarus clarkii) where detoxification presumably occurs (Penaeus monodon) (Dan et al., 2019; Vogt and Quinitio, 1994).

In this study, we examined the regulation of GST, GSH, and MT by zinc and copper in a localized population of signal crayfish inhabiting the Snake River (Idaho, USA) with the goal of adding to the body of comparative literature reporting on the regulation of endogenous antioxidants and detoxification pathways by metals in wildlife. We injected zinc chloride and copper chloride into the hemolymph of the crayfish and measured the response of these biomarkers after 72 h of exposure. Past data collected by our research group demonstrated that mercury chloride decreased GST activity in the hepatopancreas, had no effect on GSH concentrations in the hepatopancreas, and induced MT in the gills, while exhibiting no effect on MT in tail muscle (Gunderson et al., 2018). We predicted that zinc and copper would have similar effects but with changes manifesting at higher doses, given that they are essential metals. Furthermore, we were interested in determining whether sex-specific differences existed in response to these metals in signal crayfish, given the report of sex-specific expression of antioxidants in narrow-clawed (Astacus leptodactylus) and red swamp (Procambarus clarkii) crayfish (Barim-Oz et al., 2017; Elia et al., 2006).

2. Methods

2.1. Animal collection and exposure

Crayfish (body length (rostrum to tail tip) 109 +/− 1.6 mm; body weight 49.7 +/− 1.8 g (mean +/− S.E.)) were collected from C.J. Strike Reservoir (N 42.95148° W 115.97243°) on the Snake River (Idaho, USA) using traps during May-July 2017 under Idaho Department of Fish and Game permit # F-12-04-17. Animals were acclimated in dechlorinated tap water for at least two weeks at room temperature on a light cycle approximating early summer day length in southwestern Idaho (~6:30 − 22:30). Crayfish were then injected with saline vehicle (400 mM NaCl), zinc chloride (0.6, 0.9, 1.2, 2.4, 4.8, 9.6 μg g⁻¹) or copper chloride (0.6, 0.9, 1.2 μg g⁻¹) using previously described protocols (Gunderson et al., 2018). We predicted that zinc and copper would have effects similar to mercury, but at higher concentrations than mercury chloride concentrations utilized in our previous study (0.3–0.9 μg kg⁻¹)(Gunderson et al., 2018). We chose to test concentrations that are close to zinc and copper concentrations reported in crayfish tissues in the literature (Kouba et al., 2010). For example, zinc concentrations range from 18.0 – 127.4 mg kg⁻¹ (dry weight) in abdominal muscle and 25.1 – 506.0 mg kg⁻¹ (dry weight) in hepatopancreas. Likewise, copper concentrations in abdominal muscle and hepatopancreas range from 2.97 – 147 mg kg⁻¹ and 4.93–1510.0 mg kg⁻¹ (dry weight) respectively. A sham group receiving a needle stick with no injection solution was included. Animals were euthanized (chilling on ice and decapitation) after 72 h of exposure. The tissues were collected, flash frozen, and stored at −80°C until being processed for assays.

2.2. Glutathione S-transferase activity (GST)

GST activity (EC 2.5.1.18) was measured in hepatopancreas tissue using a previously published protocol (Gunderson et al., 2018; Gunderson et al., 2004; Gunderson et al., 2016). Briefly, 2.5 μg of cytosolic total protein was run in quadruplicate on 96-well plates and read kinetically @ 340 nm using a BioRad Benchmark Plus © spectrophotometer microplate reader. Activity was then calculated and reported as nmol GSH conjugated/min*mg protein (Gunderson et al., 2004).

2.3. Glutathione concentrations (GSH)

Total GSH concentrations in hepatopancreas tissue were quantified based on a previously published protocol with modification for signal crayfish (Tietze, 1969). One hundred milligrams of hepatopancreas tissue was homogenized in 10 volumes of ice cold 5% sulfosalicylic acid using a Bullet Blender (Next Advance, Inc., BBUC3137) (10–20 zirconium oxide beads, Speed 7 for 4 min @ 4°C). The homogenate was then centrifuged (Eppendorf 5418) for 2 min @ 16,813 g (4°C). A DTNB (5,5’-dithiobis(nitrobenzoic acid)) based colorimetric assay using 96-well plates was used to quantify total GSH in the hepatopancreas homogenate. DTNB (10 mM), NADPH (280 mM) and GSH-reductase (from baker’s yeast, diluted to 50 units per ml) were all prepared in stock buffer (143 mM sodium phosphate, 6.33 mM Na₄EDTA, pH 7.5). To each sample well, 100 μl of NADPH, 32.5 μl of deionized water, 15 μl of GSH reductase, 2.5 μl of tissue homogenate, and 15 μl DTNB were added. The plate was incubated at 30°C for 10 min and then read for 4 min @ 412 nm (BioRad iMark^© microplate reader). A GSH standard curve (0, 5, 9, 18, 36, 55, 73 μM) was run on each plate and contained 100 μl of NADPH, GSH standard and deionized water to total 32.5 μl, 15 μl of GSH reductase, 2.5 μl of 5% sulfosalicylic acid, and 15 μl DTNB. The standard curve was read for 4 min @ 412 nm after a 10 min incubation. GSH concentrations in the samples were calculated based on the standard curve and reported as nmol GSH/gram tissue.

2.4. Metallothionien concentrations (MT)

MT concentrations were measured in gill and tail muscle tissues using a previously published protocol with slight modifications for crayfish (Gunderson et al., 2016). Approximately 0.3 grams of tissue was homogenized using a Bullet Blender (Next Advance, Inc., BBUC3137) (10–20 zirconium oxide beads, Speed 7 for 4 min @ 4°C) and MT isolated using centrifugation. Colorimetric assay utilizing DTNB quantified cysteine residues by measuring absorbance at a wavelength of 412 nm (BioRad iMark^© microplate reader), with a GSH standard curve serving as a cysteine reference (1 cysteine/molecule). MT concentrations (nmol MT/gram tissue) were calculated as previously described with the assumption that signal crayfish have 18 cysteine residues/MT molecule (Faria et al., 2010; Linde and Garcia-Vazquez, 2006).

2.5. Statistical analyses

Datasets were determined to be parametric or non-parametric using tests for normality (Shapiro-Wilk) and equal variance (Brown-Forsythe). A two-tailed t-test (parametric) or Mann-Whitney Test (non-parametric) was used to examine sexually dimorphic patterns of expression within each treatment and to compare the sham treatment to the saline control for each endpoint. Comparisons among the saline control and zinc or copper treatments were conducted using a One-Way ANOVA with Tukey Method for multiple comparisons (parametric) or a Kruskal-Wallis One-Way ANOVA on Ranks with a Mann-Whitney Test for pair-wise comparisons (non-parametic). The software program OriginPro 2020b (OriginLab Corporation) was used for all statistical analyses.

3. Results

3.1. Sham injection

No significant difference between the sham and saline control was observed for GST activity (Figures 1A & 1B; p = 0.53), GSH concentrations (Figures 2A & 2B; p = 0.31), MT concentrations in gill tissue (Figures 3A & 3B; p = 0.14), and MT concentrations in tail muscle (Figures 3A & 3B; p = 0.25) when males and females were grouped. Furthermore, no difference between sham and saline controls were observed in GST activity (Table 1; Male p = 0.83, Female p = 0.8), GSH concentrations (Table 1; Male p = 0.97, Female p = 0.27), MT concentrations in gill tissue (Table 2; Male p = 0.15, Female p = 0.51), and MT concentrations in tail muscle (Table 2; Male p = 0.24, Female p = 0.68) when males and females were considered separately.

Figure 1:

Average GST activity (mean +/− S.E.) in signal crayfish hepatopancreas 72 h after (A) ZnCl₂ or (B) CuCl₂ treatments (injected into hemolymph). *Denotes statistical significance (p ≤ 0.05) compared to the saline control.

Figure 2:

Average GSH concentration (mean +/− S.E.) in signal crayfish hepatopancreas 72 h after (A) ZnCl₂ or (B) CuCl₂ treatments (injected into hemolymph). No significant difference (p > 0.05) was observed between ZnCl₂, CuCl_2, or sham treatments and the saline control group when males and females were grouped.

Figure 3:

Average MT concentration (mean +/− S.E.) in signal crayfish gill (G)and tail (T) tissues 72 h after (A) ZnCl₂ or (B) CuCl₂ treatments (injected into hemolymph). *Denotes statistical significance (p ≤ 0.05) compared to the saline control for the respective tissue. ZnCl₂ (gill n = 9,8,9,10,10,8,7,7; tail n = 9,8,10,9,10,8,7,7), CuCl₂ (gill n = 9,8,8,7,7; tail n = 9,8,8,7,8).

Table 1:

Mean GST activities and GSH concentrations in males (M) and females (F) 72 h after ZnCl₂ and CuCl₂ treatments (mean +/− S.E. (N).

	GST (nmol GSH/min*mg total protein)		GSH (nmol/gram)
	M	F	M	F
Saline	700.7 +/− 95.7 (4)	726.0 +/− 107.7 (5)	931.6 +/− 253.2 (4)	1288.4 +/− 102.5 (5)
Sham	730.8 +/− 92.2 (4)	767.1 +/− 111.7 (4)	943.7 +/− 189.6 (4)	968.1 +/− 265.2 (4)
ZnCl2 0.6 μg g−1	746.9 +/− 82.1 (5)	753.9 +/− 58.8 (5)	1015.6 +/− 120.9 (4)	1063.6 +/− 155.9 (5)
ZnCl2 0.9 μg g−1	736.0 +/− 76.9 (5)	691.3 +/− 110.0 (5)	1098.0 +/− 219.1 (5)	1052.6 +/− 112.7 (4)
ZnCl2 1.2 μg g−1	712.4 +/− 69.8 (5)	735.8 +/− 50.3 (5)	936.4 +/− 294.6 (5)	1110.8 +/− 142.7 (5)
ZnCl2 2.4 μg g−1	510.8 +/− 59.3 (4)*	752.3 +/− 65.8 (4)	1266.6 +/− 258.6 (4)	930.6 +/− 27.2 (4)
ZnCl2 4.8 μg g−1	511.2 +/− 10.3 (3)	589.7 +/− 29.3 (4)	1207.6 +/− 97.0 (3)	1092.6 +/− 129.4 (4)
ZnCl2 9.6 μg g−1	1014.0 +/− 52.6 (3)	885.5 +/− 62.5 (4)	1062.3 +/− 121.7 (3)	840.6 +/− 118.2 (4)
CuCl2 0.6 μg g−1	237.5 +/− 116.1 (4)	261.5 +/− 136.8 (4)	1099.6 +/− 183.6 (4)	1008.6 +/− 91.5 (4)
CuCl2 0.9 μg g−1	515.8 +/− 227.6 (4)	381.5 +/− 287.2 (3)	939.6 +/− 115.2 (4)	603.6 +/− 105.8 (3)ǂ
CuCl2 1.2 μg g−1	662.9 +/− 146.1 (4)	532.7 +/− 159.3 (4)	1043.6 +/− 83.4 (4)	985.6 +/− 202.9 (4)

^*Denotes a significant difference (p < 0.05) between males and females for a given endpoint at the respective concentration.

^ǂDenotes a significant difference relative to the saline control (within a column).

Table 2:

Mean MT concentrations in males (M) and females (F) 72 h after ZnCl₂ and CuCl₂ treatments (mean +/− S.E. (N)).

	MT gill (nmol/gram)		MT tail (nmol/gram)
	M	F	M	F
Saline	5.3 +/− 0.9 (4)	5.0 +/− 0.9 (5)	97.7 +/− 14.0 (4)	89.9 +/− 13.8 (5)
Sham	3.4 +/− 0.7 (4)	3.8 +/− 1.5 (4)	75.0 +/− 10.7 (4)	81.1 +/− 15.3 (4)
ZnCl2 0.6 μg g−1	5.3 +/− 0.9 (5)	4.6 +/− 0.7 (4)	123.1 +/− 15.7 (5)	96.3 +/− 14.3 (5)
ZnCl2 0.9 μg g−1	3.2 +/− 0.5 (5)	4.6 +/− 0.8 (5)	124.9 +/− 8.9 (5)	95.6 +/− 15.6 (4)
ZnCl2 1.2 μg g−1	3.8 +/− 0.7 (5)	3.2 +/− 0.6 (5)	117.8 +/− 26.6 (5)	105.3 +/− 17.8 (5)
ZnCl2 2.4 μg g−1	55.0 +/− 6.7 (4)ǂ	51.4 +/− 9.1 (4)ǂ	139.7 +/− 9.8 (4)	163.4 +/− 23.2 (4)
ZnCl2 4.8 μg g−1	71.4 +/− 21.7 (3)	57.8 +/− 8.2 (4)ǂ	109.9 +/− 15.5 (3)	133.1 +/− 6.7 (4)
ZnCl2 9.6 μg g−1	31.0 +/− 5.2 (3)	28.7 +/− 4.2 (4)ǂ	102.1 +/− 14.9 (3)	88.5 +/− 5.8 (4)
CuCl2 0.6 μg g−1	75.0 +/− 12.1 (4)ǂ	50.5 +/− 7.1 (4)ǂ	146.7 +/− 11.6 (4)	139.2 +/− 13.8 (4)ǂ
CuCl2 0.9 μg g−1	61.8 +/− 10.2 (4)ǂ	53.0 +/− 6.3 (3)ǂ	140.0 +/− 25.1 (4)	138.9 +/− 4.7 (3)
CuCl2 1.2 μg g−1	47.6 +/− 6.5 (3)ǂ	53.8 +/− 4.3 (4)ǂ	148.3 +/− 16.9 (4)	133.3 +/− 8.2 (4)

^*Denotes a significant difference (p < 0.05) between males and females for a given endpoint at the respective treatment concentration.

^ǂDenotes a significant difference relative to the saline control (within a column).

3.2. GST activity

Differences in GST activity between ZnCl₂ the saline control were not observed when males and females grouped together (Figure 1A; p ≥ 0.09) or when the sexes were examined separately (Table 1; p > 0.05). Sexually dimorphic patterns of activity were only observed in animals treated with 2.4 μg/g ZnCl₂, (p = 0.03) with no significant difference between males and females exhibited within the other treatments (p ≥ 0.08) (Table 1). GST activity decreased in animals treated with 0.6 μg/g CuCl₂ (p = 0.002), when males and females were grouped for analysis (Figure 1B). No significant differences in activity between the CuCl₂ treatments and the saline control were observed when males (p = 0.12) or females (p = 0.29) were considered separately (Table 1). Furthermore, sexually dimorphic patterns of GST activity were not observed in the saline control, sham, or CuCl₂ treatments (p ≥ 0.47). (Table 1).

3.3. GSH concentrations

No significant difference was observed in GSH concentrations between ZnCl₂ treatments and the saline control group when males and females were grouped for analyses (Figure 2A; p = 0.94). Sexually dimorphic patterns in GSH concentrations were not observed within any of the ZnCl₂ treatments (Table 1; p ≥ 0.2) nor were differences exhibited when the sexes were analyzed separately (Table 1; males p = 0.62; females p = 0.29). No significant difference in GSH concentrations was observed between CuCl₂ treatments and the saline control group when males and females were grouped (Figure 2B; p = 0.22). Sexually dimorphic patterns of GSH concentrations were not observed within any of the CuCl₂ treatments (Table 1; p ≥ 0.09). CuCl₂ did not alter GSH concentrations in male crayfish (Table 1; p = 0.88). GSH concentrations were significantly lower than the saline control in female crayfish treated with 0.9 μg/g CuCl₂ (p = 0.02) with no significant difference being observed in the other CuCl₂ treatments (p ≥ 0.25) (Table 1).

3.4. MT gill tissue

ZnCl₂ treatment significantly increased MT concentrations in gill tissue at the 2.4, 4.8, and 9.6 μg/g doses when males and females were grouped (Figure 3A; p ≤ 0.001). When males and females were analyzed separately, MT concentrations increased at the 2.4 ZnCl₂ μg/g dose in males (p = 0.03) and the 2.4, 4.8, and 9.6 μg/g ZnCl₂ doses in females (p = 0.02) (Table 2). Sexually dimorphic patterns were not observed within any of the ZnCl₂ treatment groups (Table 2; p ≥ 0.17). CuCl₂ treatment significantly increased MT concentrations in gill tissue at the 0.6, 0.9, and 1.2 μg/g doses when males and females were grouped (Figure 3B; p < 0.0001). MT concentrations increased at all the CuCl₂ doses tested in both males (p ≤ 0.04) and females (p < 0.0001), and sexually dimorphic patterns were not observed within any of the groups (p ≥ 0.13) (Table 2).

3.5. MT tail muscle

ZnCl₂ treatment significantly increased MT concentrations in tail tissue at the 2.4 and 4.8 μg/g doses when males and females were grouped (Figure 3A; p ≤ 0.03). MT concentrations did not change in males (p = 0.68) or females (p = 0.07), when analyzed separately (Table 2). Sexually dimorphic patterns were not observed within any of the groups (Table 2; p ≥ 0.19). CuCl₂ treatment significantly increased MT concentrations in tail tissue at all the doses tested when males and females were grouped (Figure 3B; p ≤ 0.02). MT concentrations were increased in females treated with 0.6 μg/g CuCl₂ (p = 0.04) and exhibited a trend towards increase at the 0.9 μg/g (p = 0.07) and 1.2 μg/g (p = 0.08) doses (Table 2). No change in MT was observed in tail tissue in males (Table 2; p = 0.19). Sexually dimorphic patterns in MT were not observed within any of the treatments in tail tissue (Table 2; p ≥ 0.45).

4. Discussion

In this study, the response of GST activity, GSH concentrations, and MT concentrations to zinc and copper was examined in signal crayfish and our results add to the body of literature reporting variation across taxa in the response of these endpoints to metals. Zinc exposure did not alter GST activity, demonstrating that crayfish are less sensitive to zinc than to mercury, which inhibited activity at the lowest dose tested (0.3 μg kg⁻¹) (Gunderson et al., 2018). GST activity decreased in animals treated with the lowest dose of copper chloride tested in this study (0.6 μg g⁻¹), with no changes being observed at the higher doses (non-monotonic dose response). GSH concentrations did not change in response to zinc or copper. MT exhibited tissue specific responses to zinc and copper. Concentrations increased in response to zinc and copper in gill tissue and copper in tail tissue. A non-monotonic dose response was observed in MT concentrations (tail muscle tissue) with a significant increase being observed in animals treated with 2.4 and 4.6 μg g⁻¹ zinc chloride, but not the highest dose tested in this study. These endpoints are all common biomarkers used in ecotoxicology and careful interpretation of data is needed when applying them in field-based studies given the variability in responses to different doses, classes of compounds, and across taxa.

4.1. Sex- specific differences

Sex-specific differences in the expression and response of GST, GSH, and MT to zinc and copper were examined with no clear patterns emerging. Studies of crayfish, amphipods, and crabs report sex differences, as well as seasonal variation, in antioxidants that included reduced GSH, SOD, GSH-Px, and CAT (Barim-Oz et al., 2017; Elia et al., 2006; Paital and Chainy, 2013; Sroda and Cossu-Leguille, 2011). Sexually dimorphic patterns were not observed within control groups for any of the endpoints examined in this study. Interestingly, non-monotonic patterns were observed with a sexually dimorphic pattern in GST activity being observed at the 2.4 μg g⁻¹ dose of zinc and a sex-specific depletion of GSH in females at the 0.9 μg g⁻¹ dose of copper. At this point, we cannot conclude that sex differences exist in the expression of GST, GSH, or MT in this species, although we feel it is worthwhile to continue to watch for sex-specific patterns in future studies based on the above-mentioned studies that report sex differences in other endogenous antioxidants in invertebrates. The remainder of the discussion primarily focuses on the results from the datasets where males and females are grouped.

4.2. Glutathione S-transferase

GST is a phase II detoxification enzyme and enzymatic antioxidant that attaches GSH to xenobiotics, thus facilitating cellular protection from and clearance of reactive electrophiles and toxins from the body (Park et al., 2020). It is suggested that GSTs are adaptations which enable organisms to manage exposure to oxidative stress inducing chemicals, such as metals and xenobiotics (Park et al., 2020). GST is modulated (induced or inhibited) by different classes of compounds and is therefore used as a biomarker in ecotoxicology studies (Dobritzsch et al., 2020; Park et al., 2020). GST expression is regulated through the transcription factor NRF2 (nuclear factor erythroid-2 related factor) pathway, a master controller of stress response genes, and can also involve antioxidant or electrophile response elements (ARE/EpRE) (Park et al., 2020). GSTs are present in invertebrates and known to be modulated by metals (Dobritzsch et al., 2020; Park et al., 2020). Prior work by our research group on signal crayfish demonstrated that GST is sensitive to mercury chloride, with inhibition taking place at the lowest dose we tested (0.3 μg kg⁻¹) (Gunderson et al., 2018).

The reports on the effects of zinc and copper on GST vary across taxa, dose, sampling window, and can depend on non-pollution related factors (i.e. temperature). In this study, GST activity was not sensitive to zinc exposure but decreased at the lowest dose of copper chloride tested (0.6 μg g⁻¹). The response to zinc is less sensitive than to mercury chloride as previously reported by our research group, where inhibition was observed at the lowest concentrations tested (0.3 μg kg⁻¹) (Gunderson et al., 2018). Furthermore, a pattern suggestive of increased activity was observed at the highest dose of zinc tested (9.6 μg g⁻¹, p = 0.09) which is consistent with reports in other invertebrates. Zinc increased GST in the bivalve (Chlamys farreri) (Zhang et al., 2010), whereas in the abalone (Haliotis discus hannai), lower concentrations of dietary zinc supplementation (33.8 & 710.6 mg/kg) increased GST mRNA with excess zinc (3462.5 mg/kg) leading to decreased in expression (Wu et al., 2011). In field studies, GST activity exhibited positive correlations with zinc in mussels (Mytilus galloprovincialis) and annelid worms (Benali et al., 2017; Ojo et al., 2016).

Copper exposure at the lowest dose tested resulted in inhibition of GST activity (p = 0.002) in signal crayfish, an effect that was not observed at the higher doses tested. Proposed mechanisms for inhibition of GSTs by metals include direct inhibition of the enzyme by metals or a metal induced reduction in GST’s substrate GSH (Dobritzsch et al., 2020). At this point, we cannot explain this low-dose (non-monotonic) effect, although one possible explanation could relate to the upregulation of other protective mechanisms against oxidative stress at the higher doses of copper tested, thus shielding GST from inhibition by copper at the 0.9 and 1.2 μg g⁻¹ treatment doses. For example, we did not measure MT in hepatopancreas tissue, but we did observe modulation by copper in the gill tissue and tail muscle (increased at all concentrations tested). Future studies could examine the regulation of MT (and other endogenous antioxidants) in the hepatopancreas to determine whether it is induced at the concentrations tested and provide a possible protective mechanism against copper induced inhibition of GST.

The low-dose (non-monotonic) response of GST to copper observed in our study could alternatively relate to our exposure range spanning the concentrations that lead to inhibition versus induction of activity in this species. Inhibition and induction of GST by copper have both been reported in the literature. Studies in worms (Tubifex tubifex), scallops (Adamussium colbecki), and the freshwater prawn (Macrobrachium rosenbergii) report decreased GST activity in response to copper (Li et al., 2008; Mosleh et al., 2006; Regoli et al., 1998). A field-based study demonstrated that GST exhibited a strong negative relationship with metal body burden (copper and chromium) in wolf spiders (Pardosa oakleyi) (Butt and Aziz, 2016). Species-specific effects of copper on GST have been reported in marine gastropods, where it inhibited activity in Nucella lapillus but not Monodonta lineata (Cunha et al., 2007). Studies have also reported increases in GST activity in response to copper. Copper increased GST activity in the green mussel (Perna viridis) and in the polychaete worm Laeonereis acuta, where it increased GST in both acute and long-term exposure studies, and the response demonstrated regional specificity within the body (Ferreira-Cravo et al., 2009; Geracitano et al., 2002; Geracitano et al., 2004; Goswami et al., 2014). In our experiment, we could be observing an inhibitory effect of copper through direct interaction with GST at the low dose which is then offset by induction by copper mediated oxidative stress at the higher doses tested.

4.3. Glutathione (GSH)

GSH is the most abundant non-protein thiol in the cell and changes in thiol content are often associated with exposure to ROS generating compounds making it a common biomarker used in ecotoxicology studies (Abarikwu et al., 2017; Dickinson and Forman, 2002; Ulrich and Jakob, 2019). GSH is conjugated enzymatically in association with GST, or non-enzymatically with electrophilic compounds, and is thus involved in xenobiotic metabolism and can also act as an antioxidant. GSH is detectable in gill, muscle, and hepatopancreas of crayfish, and is a valuable tool in assessing the glutathione redox status in this organism (Gunderson et al., 2018; Kovacevic et al., 2008). In a previous study, our group observed no change in GSH in response to dimethoate or mercury chloride at the doses tested (0.3, 0.6, 0.9 μg kg⁻¹) (Gunderson et al., 2018).

GSH is not sensitive to zinc and copper at the concentrations tested in animals from this population when males and females were grouped. Studies have examined the production of ROS and modulation of GSH concentrations in response to copper exposure in other invertebrate species. In the polychaete worm Perinereis nuntia, ROS and GSH increased in response to copper (Won et al., 2012), whereas copper increased ROS with no increase in GSH (decrease after 12 h) in the rotifer Brachionus koreanus (Han et al., 2013). Interestingly, we did not observe changes in GSH concentrations in response to mercury chloride in a previous study on signal crayfish (Gunderson et al., 2018). Future studies could test a broader range of concentrations to determine whether higher doses of these metals modulate GSH concentrations. A likely explanation for a lack of change in GSH concentrations in response to metal exposure could be that the endogenous antioxidant systems in signal crayfish from this population are capable of providing protection against the ROS formation at the concentrations tested, as proposed in a study in polychaete worms (Laeonereis acuta) (Geracitano et al., 2002). This hypothesis is supported by the fact that MT was induced at all the copper concentrations tested in gill and tail tissues and at the higher concentrations of zinc (2.4, 4.6, and 9.6 μg g⁻¹) in gill tissue. As mentioned above, a sex-specific depletion of GSH was observed in females treated with 0.9 μg g⁻¹ of copper, the middle dose tested. At this point, we do not have an explanation to explain the non-monotonic dose response observed in females and future studies examining this are warranted to determine whether it is a pattern that emerges with exposure to metals and other ROS inducing xenobiotics.

The regulation of GSH concentrations is complicated and likely explains the variations reported in its response to zinc and copper in different studies. Perturbations can result from enzymatic or non-enzymatic functions associated with the metabolism of metals and xenobiotics, or alterations in any of the steps involved in biosynthesis (Dickinson and Forman, 2002). In the literature, zinc or copper exposure has been shown to increase or decrease GSH concentrations in different species. For example, in silkworms (Antheraea mylitta), GSH concentrations increased with exposure to zinc whereas GSH decreased in response in zinc in two bivalve species (Ruditapes decussatus and Perna perna) (Sahu et al., 2015; Soliman et al., 2015; Trevisan et al., 2014). Increases in GSH in response to copper have been reported in bivalves (Diplodon chilensis) and polychaete worms (Perinereis nuntia) (Sabatini et al., 2011; Won et al., 2012). Alternatively, decreases in GSH in response to copper and metals have been demonstrated in bivalves (Adamussium colbecki) and spiders (Agelena labyrinthica) (Regoli et al., 1998; Wilczek et al., 2004). Interestingly, GSH increased at low concentrations of copper and decreased at high concentrations in the mud snail (Bellamya purificata) (Zhang et al., 2009), and exhibited tissue specific response patterns to copper in the freshwater pond snail (Lymnaea stagnalis), with increases being observed in hepatopancreas and mantle tissues and decreases observed in foot muscle (Atli and Grosell, 2016).

4.4. Metallothionein (MT)

MTs are non-enzymatic cysteine rich proteins that are inducible through exposure to metals and used as biomarkers in ecotoxicology studies. MTs have several purported biological roles that include sequestration/detoxification of heavy metals within the cell, serving as a reservoir for essential metals such as zinc and copper, protecting against ionizing radiation, and acting as non-enzymatic antioxidants (Amiard et al., 2006; Kouba et al., 2010). MTs (and MT-like proteins) have been described in a broad range of invertebrate taxa including molluscs, annelid worms, and crustaceans(Amiard et al., 2006). Inconsistencies exist in the literature regarding the inducibility of MT by metals across taxa and tissue types, and careful characterization of the response in a given model organism is needed when using it as a biomarker in field-based studies (Amiard et al., 2006). Work published by our group demonstrated that mercury chloride induced MT concentrations in gill but not muscle tissues in signal crayfish (Pacifastacus leniusculus) (Gunderson et al., 2018).

Copper and zinc induce MT, and MT-like proteins, in a broad range of invertebrate taxa that include bivalves, crustaceans, polychaete worms, and gastropods, although variation exists in the response to these metals (Amiard et al., 2006). In our study, the response of MT to zinc and copper was tissue specific and exhibited a non-monotonic dose response to zinc in tail muscle. Tail muscle in this species is sensitive to these metals with zinc exposure inducing MT at two of the middle doses tested (2.4 & 4.8 μg g⁻¹) and copper inducing MT at all the doses tested (0.6, 0.9, 1.2 μg g⁻¹). Non-monotonic dose response curves have been reported when studying endocrine disrupting compounds (Vandenberg et al., 2012) and we observed a similar pattern in the response of MT to mercury, where concentrations increased in gill tissue at the two lower doses tested (0.3 and 0.6 μg kg⁻¹) but not the highest (0.9 μg kg⁻¹) (Gunderson et al., 2018). Interestingly, in the red swamp crayfish (Procambus clarkii), zinc increased MT in hepatopancreas tissue with a more pronounced increase being observed at the lower concentration, which is similar to our results with a response being measured at middle doses (Martin-Diaz et al., 2006). In gill tissue, MT increased at the 2.4, 4.6, and 9.6 μg g⁻¹ zinc chloride doses and all the concentrations of copper tested. As noted earlier, a previous study published by our research group reported that gill tissue, and not tail muscle, was sensitive to mercury chloride exposure at lower concentrations (0.3 and 0.6 μg kg⁻¹) than were tested in this study (Gunderson et al., 2018).

4.5. Conclusions

The endpoints examined in this study are commonly used in ecotoxicology studies and our results add to comparative literature reporting on the modulation of antioxidant and detoxification pathways by metals in aquatic organisms. Careful interpretation of field-based data is needed given the variability across taxa, classes of compounds, tissues, and the fact that non-monotonic dose responses are sometimes observed. The strength of this approach lies in searching for patterns based on the combined use of a broad range of biomarkers along with field contaminant data (Liu and Wang, 2016). The response of markers should be characterized for each model organism, with biotic and abiotic factors (i.e., seasonal variation, temperature, freezing, etc.) studied and accounted for in biomonitoring projects (Acs et al., 2016; Elia et al., 2006; Fisker et al., 2016; Kerambrun et al., 2016; Louiz et al., 2017). The results presented in this study are for a localized population on the Snake River in Idaho. Animals were all collected from the same location to reduce potential variation due differences among populations. It would be interesting to examine the response of other populations of signal crayfish to these metals to determine whether the results can be generalized to the species, or whether they are specific to this population. Finally, the fact that we did not observe clear evidence for sexually dimorphic or sex-specific patterns of expression in the endpoints examined is interesting based on studies conducted in other species of crayfish that report sex differences in endogenous antioxidant pathways (Barim-Oz et al., 2017; Elia et al., 2006). Future studies in this species should continue to examine sex-differences to determine if patterns emerge with further investigation.

Highlight.

• Inhibition of GST by copper exhibited a non-monotonic dose response.

• Copper depleted GSH in sex-specific manner in females.

• MT was induced by both zinc and copper in gill and tail muscle tissues.

• MT in zinc treated crayfish exhibited a non-monotonic dose response in tail tissue.

• Sexually dimorphic patterns were not observed in control animals for any markers.