Abstract
Induction of stress response genes hsp70 and abcb1 and Hsp70 protein by cadmium chloride (CdCl2) was explored in amphipod species with different stress adaptation strategies from the Lake Baikal area. Based on lethal concentrations (LC) the sensitivities to CdCl2 were ranked (24 hr LC50 – mg/L CdCl2): Gammarus lacustris (1.7) < Eulimnogammarus cyaneus (2.9) < E. verrucosus (8.3) < E. vittatus (18.2). Conjugated dienes indicating lipid peroxidation were significantly increased by 5 mg/L CdCl2 (24 hr exposure) only in G. lacustris and E. cyaneus. Upon treatment with 0.54 – 5.8 mg/L CdCl2 hsp70 transcript levels were more increased in the toxicologically more sensitive species. Relating the exposure concentrations to LCx values revealed that across the species the increases of hsp70 transcript levels were comparatively low (up to 2.6-fold) up to LC50; at higher LCx values hsp70 induction was more pronounced (up to a 9.1-fold by 5 mg/L CdCl2 (≙LC70) in E. cyaneus). In contrast, abcb1 inductions did not correspond with CdCl2 LCx values across species; abcb1 induction was highest (4.7-fold) in E. verrucosus by 5.0 mg/L CdCl2 (≙LC45, 24 hr exposure). Induction of stress gene responses by lethal CdCl2 concentrations indicates that in the amphipods they are rather insensitive.
Introduction
Cadmium is a non-essential heavy-metal that reaches the environment via various anthropogenic and natural sources and at low concentrations causes poisoning in humans and wildlife [1]. Toxic cadmium effects have often been attributed with increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) causing damages of biological macromolecules such as proteins [2]. Exposure to cadmium was found to lead to the induction of cellular stress response genes hsp70 [3–12] and abcb1 [13, 14] in a range of aquatic organisms. This induction of hsp70 and abcb1 by cadmium can be related to the increased abundance of damaged cellular macromolecules such as cellular membrane fragments or misfolded proteins [15, 16].
Lake Baikal in Eastern Siberia, the oldest, deepest and by volume largest lake in the world, is a biodiversity hotspot with high degrees of endemicity of species [17, 18]. Baikal’s water is generally highly pristine, however, the risk of water contamination by heavy metals is increasing especially through the Selenga river, which is the largest tributary of Lake Baikal and comprises almost half of the riverine inflow into the lake [19, 20]. Amphipods are a dominant taxon of the benthic communities of Lake Baikal and the more than 350 endemic species and subspecies represent 45.3% of all freshwater amphipod species of the world [21, 22]. The numerous phylogenetically closely related species featuring a range of adaptation strategies are interesting models for comparative studies [23]. In the here studied amphipod species constitutive expression levels of cellular stress response genes vary within an order of magnitude which could be related with differences across species regarding stress tolerance. Thus, constitutive hsp70 levels relate to the species-specific differences in thermotolerance [24–26]. Furthermore, species-specific degrees of gene responses of hsp70 and abcb1 to organic compounds as humic substances [25] and phenanthrene [27] were found.
It is stated in many studies on the effects of cadmium on hsp70/Hsp70 levels that the test organisms were exposed to sublethal cadmium levels but the published molecular effect data are generally not related to the general level of stress caused by the cadmium concentration applied, such as lethal cadmium concentrations. It is thus unclear how sensitive the observed gene response to cadmium is.
We therefore here addressed the question at which stress levels cellular stress response genes hsp70 and abcb1 are induced upon exposure to CdCl2 in amphipod species with different cellular stress response capacities. The lethal concentrations (LCx 24h) of CdCl2 served as a measure for the cadmium-related stress levels. Transcript levels of hsp70 and abcb1 were quantified via quantitative polymerase chain reaction (qPCR) in RNA extracted from tissue of amphipods upon exposures of up to 24 hrs to different CdCl2 concentrations. The study was performed with four amphipod species that differ with regard to their ecological preferences and habitats and accordingly with their physiological and cellular adaptations to environmental conditions. Three of the species are littoral amphipods from Lake Baikal belonging to the Eulinogammarus genus; the Holarctic species Gammarus lacustris that occurs in waters connected to Lake Baikal but not in areas of the lake inhabited by endemic fauna was also examined.
Materials and Methods
Studied species and animal sampling
The experimental species were Eulimnogammarus cyaneus (Dyb., 1874), Eulimnogammarus verrucosus (Gerstf., 1858) and Eulimnogammarus vittatus (Dyb., 1874), endemic to Lake Baikal, and Gammarus lacustris (Sars, 1863) common in surface waters across the Holarctic. Eulimnogammarus cyaneus is most abundant along the shallow shoreline (mainly up to 1 m water depth), few individuals occur at water depths up to 20 m [28] at temperatures between 5-13°C [22]. Habitats of E. vittatus are at water depths of up to 30 m, the abundance peak is at 2–3 m depth [29]; the water temperatures in the habitat range between 9-13°C [22]. Eulimnogammarus verrucosus commonly occurs close to shore across Lake Baikal at water depths of less than 1 m to up to 10–15 m [29] at temperatures of 5-13°C [22]. Gammarus lacustris is common in shallow, eutrophic lakes with seasonal temperature fluctuations in the Lake Baikal region at water depths of 0-7m but the species is not found in the open Baikal [30]. Constitutive transcript/protein levels of heat shock protein 70 (hsp70/Hsp70) and transcript levels of ATP binding cassette (ABC) transporter 1 (abcb1) indicate the cellular stress response capacities of the species in the order: Eulimnogammarus vittatus ≈ Eulimnogammarus verrucosus < < Eulimnogammarus cyaneus < Gammarus lacustris. Indeed, the species-specific constitutive/induced hsp70/Hsp70 levels correlate with higher degrees of thermotolerance of E. cyaneus and G. lacustris compared to E. verrucosus [24–26, 31].
Eulimnogammarus specimens for the experiments were sampled at the Baikal shoreline close to Irkutsk State University’s biological station at the Bolshie Koty settlement (Southern Baikal) and G. lacustris was collected from a small shallow artificial lake close to Bolshie Koty (“Lake 14”, for specifications of the sampling points refer to [25]). Body lengths and weights of the animals used in the experiments were: E. verrucosus – 20-25 mm, 403 ± 95 mg; E. vittatus – 18-20 mm, 121 ± 16 mg; E. cyaneus – 9-14 mm, 17 ± 3 mg; G. lacustris – 14-18 mm, 80 ± 16 mg. Upon sampling the animals were brought to the lab in a cold box with water from the sampling sites. Animals were acclimated to lab conditions in aerated water in 2 L tanks at 6.5-7°C for 1-3 days prior to the experiments. The water used for maintaining the animals in the tanks and for the experiments was withdrawn from Lake Baikal with buckets from the pier next to the Biological Station in Bolshie Koty. Lake Baikal water instead of water from the pond where G. lacustris was collected was also used for G. lacustris exposures to avoid that differences in water contents (minerals, organic matter) affected the toxic effects of cadmium.
3.2. Acute toxicities of CdCl2
Acute toxicity of CdCl2 to amphipods was determined in 24 hr exposure experiments. Aqueous CdCl2 solutions for the experiments were set up in glass tanks with 2 L of well-aerated water along a control with clean water. The water temperature in the tanks was maintained at 6.5-7.0°C during the exposures. Twenty amphipods of each species were placed in separate tanks with eight different CdCl2 concentrations and a control. The CdCl2 concentration ranges were 0.5 – 8 mg/L for G. lacustris and E. cyaneus and 0.625-30 mg/L for E. verrucosus and E. vittatus. During the experiments, tanks were regularly checked for dead animals that were removed and recorded. After 24 hrs alive and dead animals in all tanks were counted. Exposure experiments were repeated two to three times with different CdCl2 concentrations that were chosen depending on the mortality rates determined for the already tested concentrations. Cadmium concentrations in water from a control and a CdCl2 exposure were determined with atomic absorption spectroscopy (AAS) at the Analytical Chemistry Department at the UFZ. The actual deviated from the nominal Cd concentration by 15 % (nominal: 10 CdCl2 mg/L; actual: 8.5 CdCl2 mg/L) and all nominal CdCl2 concentration values were adapted accordingly.
3.3. Measurements of conjugated diene (CD) levels
Levels of conjugated dienes (CD) were measured in amphipods upon exposure to 5 mg/L CdCl2 for 1, 6 and 24 hrs according to [32] with some modifications. Several entire animals were pooled to weights of 150 to 800 mg depending on the species and homogenized in a heptane/isopropyl alcohol mixture (1:1, v/v) using a Potter-Elvehjem tissue homogenizer, the extract was then filled in a glass tube and brought up to a volume of 4.5 ml with a heptane/isopropyl alcohol mixture. One ml of distilled water was added, the mix was vigorously shaken and incubated at 25°C for 30 min for phase separation. 0.5 ml of the heptane phase were mixed with ethanol in a 1:3 ratio (v/v) and the absorbance at 233 nm was measured on a SmartSpec Plus spectrophotometer (Bio-Rad) with extraction blanks used as references. An extinction coefficient of 2.52×104 M−1cm−1 [33] was used to determine the amount of CD present. The data are reported as nmol g−1 wet weight.
3.4. CdCl2 exposures for investigating cellular stress responses
Two sets of exposure experiments were performed for examining cellular responses of the different amphipod species to CdCl2 exposure: 1) CdCl2 exposure concentrations were scaled to species-specific LC10 and LC50 values enabling to compare responses at equal stress levels. Respective CdCl2 exposure concentrations (LC10/LC50) were: E. cyaneus – 0.68/2.89 mg/L, E. verrucosus – 0.54/5.75 mg/L, G. lacustris – 0.59/1.7 mg/L; E. vittatus was only exposed to 5 mg/L corresponding to LC10. 2) Exposures were with all species at 1.7 (corresponding to the LC50 for G. lacustris) and 5.0 mg/L CdCl2 (corresponding to the LC90 for G. lacustris) enabling to determine species-specific responses to equal concentrations. CdCl2 exposures were performed in aerated 2 L tanks (n=5/concentration) with Baikal water at 7°C. Specimens were sampled after 1, 6, and 24 hr exposures, frozen and stored in liquid nitrogen for extraction of total protein and diene conjugates or placed in QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and frozen in liquid nitrogen for RNA isolation. Control animals were kept in CdCl2 uncontaminated water but otherwise at equal temperature and aeration conditions as CdCl2 exposed animals. One whole specimen of E. verrucosus or a pool of several whole animals of the other species equalling 200 to 500 mg of tissue were used for the further analyses. All exposure experiments were repeated at least five times and all samples were assessed in triplicate.
3.5. Isolation of total RNA and cDNA synthesis
Isolation of total RNA and cDNA synthesis were performed as described in [27]. Tissue preserved in QIAzol Lysis Reagent was homogenized with a MM400 homogenizer (Retsch, Haan, Germany) followed by a phenol-guanidine-chloroform based extraction of total RNA. To improve the separation of the organic and aqueous phases MaXtract gel (Qiagen) was used according to the manufacturer. Total RNA was purified from the obtained aqueous phase with the miRNeasy kit with DNase treatment using a QIAcube instrument (Qiagen). One μg of total RNA was reverse-transcribed to single-stranded cDNA using Oligo(dT)18 primer (Fermentas, USA, #SO131) and H Minus Reverse Transcriptase system (Fermentas, USA, #EP0452) following the manufacturer’s instructions.
3.6. Quantitative real-time PCR analysis
Gene expression levels were analysed with quantitative polymerase chain reaction (qPCR) using a StepOnePlus Real-Time PCR System (Applied Biosystems) as described previously [25]. The amplification was performed in a final volume of 12.5 μl using the SensiMix SYBR Low-ROX Kit (Bioline) with each primer at 200 nM. Primer sequences for qPCR of hsp70 and abcb1 and housekeeping genes β-actin, gapdh and ef1-a were from [25]; qPCR conditions were according to [25, 27]: 94 °C for 4 min; 35 (for hsp70 and references genes) and 45 cycles (for abcb1) with 95 °C for 15 sec, 60-62 °C for 15 sec and 72 °C for 15 sec followed to fluorescent measurement step with 78-79°C (close to the melting temperature of the PCR products) for 10 sec. The extra step for fluorescence measurement was added to avoid detection of unspecific amplification products and primer dimers. Relative expression levels of hsp70 and abcb1 genes were calculated with the comparative ΔΔCt method [34] using efficiency corrected calculation models and Best Keeper levels based on the geometric mean of the Ct values of the housekeeping genes [35–37].
3.7. Western-blotting
Isolation of total protein from amphipod tissue and Western-blotting were performed as described in [25]. Tissues were homogenized in 0.1 M Tris buffer (pH=7.6) containing 1.5 mg phenylmethylsulfonyl fluoride (PMSF) and 1% protease inhibitor cocktail (Amresco, USA); proteins were then subjected to denaturation in Tris-HCl buffer (0.25 M Tris-HCl (pH = 6.8), 5% sodium dodecyl sulphate (SDS), 0.5 mM ethylenediamine tetraacetic acid (EDTA), 10% glycerol, 5% ß-mercaptoethanol and 0.03% Bromophenol Blue) at 95 °C for 5 min. Total protein concentrations were determined following [38]. Equal amounts of total protein per sample were separated by SDS gel electrophoresis on a 12.5% polyacrylamide gel [39] using a Mini-PROTEAN II Electrophoretic Cell (Bio-Rad, USA). Wet-transfer of the proteins from the gel to a polyvinylidene difluoride transfer membrane (GE Healthcare, UK) was according to [40] with minor modifications. Hsp70 protein was labelled with anti-HSP70 (Sigma-Aldrich, # H9776) at 1:3000, which detects both the inducible and constitutive forms of HSP70, and with secondary antibodies conjugated with alkaline phosphatase (Anti-Mouse IgG:AP Conj., Stressgen # SAB-101) at 1:1000. Anti-actin antibody (Sigma-Aldrich, # A2668) dissolved 1:400 and secondary antibodies (Anti-Rabbit IgG, Sigma, # A9919) dissolved 1:1000 were used for actin labelling. Sixty ng of bovine Hsp70 (Sigma-Aldrich, # H9776) and 60 ng of bovine actin (Sigma-Aldrich, A3653) were used as positive controls. Bands were visualized with 0.4 mM BCIP (5-bromo-4-chloro-3-indolyl phosphate) and 0.4 mM nBT (nitroblue tetrazolium). The Gel Explorer software package (DNAtechnology, Russia) was used for semi-quantitative measurements of Hsp70 and actin levels on the membranes and Hsp70 levels were normalized relative to actin for each sample.
3.8. Data analysis and statistics
Regressions of concentration-mortality relationships were calculated by fitting mortalities at the respective concentrations to the non-linear HILL model: where
- Y
- is the mortality at the CdCl2 concentration c
- X
- is the logarithm of the CdCl2 concentration
- min
- is the minimum percentage mortality (control, constrained to 0)
- max
- is the maximal percentage mortality (constrained to 100)
- P
- is the shape parameter
- LC50
- is the constant describing the CdCl2 concentration causing 50% mortality
Regressions of hsp70 transcript/Hsp70 protein levels at respective CdCl2 concentrations expressed as LCx values were calculated by fitting the observations at the respective concentrations to the Gaussian distribution model: where
- Y
- is the gene expression level at LCx (lethal concentration for x% of individuals)
- X
- is LCx
- ampl
- (amplitude) is the height of the center of the distribution in Y units
- SD
- is a measure of the width of the distribution (in LCx)
Data for qPCR, Western-blot and lipid peroxidation analyses were found to satisfy the assumptions of equal variance and normality and parametric statistics were used. For pairwise comparisons of abcb1 and hsp70/Hsp70 levels in treatments and respective controls at each time point the t-test was applied. Multiple comparisons of several treatments with one respective control were done with two-way ANOVA and Dunnett’s test. To analyse the significance of observed changes in each time-point, pair-wise comparisons with the respective control were done. The results of multiple pair-wise comparisons were done using Steel-Dwass method for all pairs’ comparison. Differences were regarded as significant if P < 0.05. Regressions were calculated with Graphpad Prism version 7. Statistical analyses were performed with JMP version 10.0 (SAS Institute, Cary, NC).
Results
4.1. Lethal CdCl2 concentrations
Acutely toxic CdCl2 concentrations were across the different amphipod species from < 1 to < 100 mg/L (Fig. 1, Table 1). The range of LCx values calculated from CdCl2 concentration – lethality relationships were over an order of magnitude across the species. LCx values were highest for E. vittatus indicating the lowest sensitivity of this species to CdCl2. The order of the species with regard to their sensitivities to CdCl2 was: G. lacustris < E. cyaneus < E. verrucosus < E. vittatus (Fig. 1, Table 1).
4.2. Conjugated diene (CD) levels
After 1 and 6 hr exposures to 5 mg/L CdCl2 no significant changes in CD levels were seen in any of the examined species. Significant increases of CD levels by about 58% and 47% occurred only in E. cyaneus and G. lacustris, respectively, after 24 hr exposures (Fig. 2). The CdCl2 concentration of 5 mg/L corresponds to LC70 and LC90 for E. cyaneus and G. lacustris, respectively. For E. vittatus and E. verrucosus this CdCl2 concentration is equivalent to LC10 and LC45, respectively (Fig. 1, Table 1).
4.3. Heat shock protein 70 transcript/protein levels
Hsp70 transcript levels across the amphipod species were upon exposures to CdCl2 significantly (p < 0.05) increased by between 1.4 - (E. cyaneus at LC30 for 24 hrs) to up to 9.1-fold (E. cyaneus at LC70 for 24 hrs) (Fig. 3a, b). With longer exposure times to CdCl2 changes in hsp70 transcript levels were in all amphipod species generally more pronounced: After 1 hr exposures hsp70 levels were significantly (p < 0.05) increased in two cases (E. cyaneus at LC30, G. lacustris at LC90), after 6 hr exposures in four cases (E. cyaneus at LC30, E. verrucosus and G. lacustris at LC50, G. lacustris at LC90) and after 24 hr exposures in nine cases (E. vittatus at LC10, G. lacustris at LC10, LC50 and LC90, E. verrucosus at LC25, LC45 and LC50, E. cyaneus at LC30 and LC70; Fig. 3a). Although significant changes in hsp70 transcript levels were also seen with CdCl2 at lower LCx, changes were more pronounced at higher LCx levels and could be described with a regression curve over all data following a Gaussian shape with an overall maximum hsp70 transcript level at LC70 followed by a decrease (Fig. 3b).
Hsp70 protein levels across amphipod species from CdCl2 treatments also followed a Gaussian function when expression data were related to the respective LCx values (Fig. 4). Significantly (p < 0.05) increased Hsp70 protein levels were seen in E. cyaneus at 1.8- and 1.7-fold, respectively, after 1 and 6 hr exposures to 5 mg/l CdCl2 corresponding to the species-specific LC70; and in E. verrucosus after 24 hrs exposure with a 2.6-fold increased protein level above the control. For this species 5 mg/l CdCl2 corresponded to the LC45. In E. vittatus and G. lacustris exposed to the species-specific LC10 and LC90, respectively, Hsp70 protein levels were not significantly different from the respective controls (Fig. 4).
4.4. Abcb1 transcript levels
Upon exposures of amphipods to CdCl2 abcb1 transcript levels were significantly (p < 0.05) increased by between 2.2- (E. cyaneus at LC50 for 24 hrs) to up to 4.7-fold (E. verrucosus at LC45 for 24 hrs). Over the different exposure times the number of cases of abcb1 increases only slightly changed. Thus, abcb1 transcript levels were increased in three cases at 1 hr exposures (E. verrucosus at LC25, E. cyaneus at LC70, G. lacustris at LC90), in two cases at 6 hr exposures (E. cyaneus at LC50, G. lacustris at LC90) and in four cases at 24 hr exposures (E. verrucosus at LC45, E. cyaneus at LC50, G. lacustris at LC90, E. vittatus at LC10). Significant abcb1 transcript increases at all three time points occurred only in G. lacustris at LC90; in E. cyaneus at LC50 abcb1 transcripts were increased at two time points. In the other cases, significant abcb1 transcript increases were seen only at one time point. Changes in abcb1 transcript levels did not seem to depend on CdCl2 concentration / LCx value; the significant changes were seen across different LCx values and were not generally induced at higher or lower LCx values (Fig. 5).
Discussion
The four examined amphipod species showed clear differences regarding toxic sensitivities to CdCl2 that became evident by LC50 values ranging over one order of magnitude (Table 1). Along those lines the levels of conjugated dienes, indicating cadmium effects on the molecular level, were at equal CdCl2 exposure concentrations only increased in the toxicologically more sensitive species E. cyaneus and G. lacustris (Fig. 2). Conjugated dienes are primary products of lipid peroxidation resulting from oxidative stress [41, 42], a main cause of the toxic action of cadmium [43, 44]. This effect of CdCl2 on gammarids was indicated in a previous study where it was found that CdCl2 exposure leads to the inhibition of common antioxidant enzymes in gammarids [45]. Differences in internal cadmium levels as a consequence of different cadmium uptake rates can, to a certain extent, be seen as a reason for the differences in toxicological sensitivities of the different species. Body size has previously been shown to be an important determinant of cadmium uptake by aquatic insects [46]. Along those lines, cadmium uptake rates during CdCl2 exposures were higher in the concurrently toxicologically more sensitive E. cyaneus than in E. verrucosus that differed by 10- to 50-fold in freshweight [47]. In the present study, G. lacustris individuals that were similar in size to E. cyaneus individuals showed toxic sensitivities that were accordingly in the same range as for E. cyaneus (Table 1). Eulimnogammarus vittatus, however, was from the examined species least sensitive to CdCl2 although the body sizes of the individuals were clearly below those of E. verrucosus and it can be assumed that the cadmium uptake rate was accordingly higher. It was previously found that the occurrence of toxic effects by CdCl2 in E. verrucosus was in addition to a in comparison to E. cyaneus lower cadmium uptake rate further depressed by a reaction to CdCl2 exposure with metabolic depression [47]. This was not examined here but it seems conceivable that the in comparison low toxic sensitivity of E. vittatus to CdCl2 may be due to particularly pronounced metabolic depression as a reaction to CdCl2 exposure.
Although metabolic depression by cadmium may have decreased activity on a physiological level it appeared to have no effect on the transcription activity. Thus, hsp70 was significantly induced in E. verrucosus that previously showed a metabolic depression response to CdCl2 exposure but not in E. cyaneus that does not respond to CdCl2 with metabolic depression [47] in the 6 and 24 hr CdCl2 treatments at LC50 (Fig. 3). Furthermore, a significant abcb1 increase was seen in E. verrucosus at LC25 but not in E. cyaneus at LC30 after 1 hr exposure to CdCl2 and in E. vittatus but no other species at LC10 after 24 hrs exposure to CdCl2 (Fig 5).
The CdCl2-dependent hsp70 transcript and Hsp70 protein response levels could across the species be related to the respective CdCl2–related stress levels. When plotting all hsp70 transcript or Hsp70 protein levels across species against the respective species-specific LCx values the values showed an increase to a maximum followed by a decrease at higher LCx values (Figs. 3, 4). When assuming that the increasing LCx values represent increasing degrees of stress this course represents stress-level dependent expression degrees that for Hsp70 has been described before: The Hsp70 titre increase in the “compensation phase” is followed by a Hsp70 titre decrease in the “non-compensation phase” at higher toxicity levels when the ability of the cells to react to the toxicant is increasingly degraded [48]. The applied CdCl2 concentration represented different degrees of stress for the different amphipod species; at this stage it is assumed but it cannot be definitely asserted that the hsp70 transcript/Hsp70 protein responses are equal in the different amphipod species at corresponding stress levels.
Provided that across the gammarid species equal internal, toxicologically relevant cadmium concentrations cause equal levels of toxic effects, as was indicated earlier [47], the LCx values can be regarded as approximate measures of the respective internal cadmium concentrations. Hence, across the species the degrees of hsp70 transcript and Hsp70 protein induction appear to be approximately equal at corresponding internal cadmium levels.
Induction of abcb1 transcripts tends to depend on LCx levels and time of exposure, i.e., internal cadmium levels. Increased abcb1 levels were more seen at higher CdCl2 LCx values and occurred at lower LCx levels after longer times of exposure (Fig. 5). However, across species abcb1 induction levels are not uniform at equal LCx levels. Thus, after 24 hrs exposure to CdCl2 at LC10 abcb1 levels were only significantly increased in E. vittatus but not in E. verrucosus, E. cyaneus and G. lacustris; after 6 and 24 hrs exposure to CdCl2 at LC50 significant abcb1 increases were only seen in E. cyaneus but not in E. verrucosus and G. lacustris (Fig. 5).
The differences in sensitivity of the hsp70 and abcb1 transcript responses among the gammarid species may confirm that induction of the two genes is via different pathways. Misfolded proteins that are damaged e.g. by the impact of cadmium-induced ROS [15, 16] trigger the induction of the hsp70 gene as, according to the chaperone titration model [49], heat shock factor (Hst1) is released when Hsps bind to the misfolded proteins thus initiating hsp transcription. Induction of abcb1 appears to be triggered via another pathway involving the transcription factor NF-κB [15, 16].
Both Hsp70 and Abcb1 proteins act as protective proteins against cadmium toxicity: Hsp70 as chaperone preserves the structure of intact proteins and re-folds damaged proteins; Abcb1, a cellular efflux transporter, appears to efflux cell membrane components, such as ceramides, that result from membrane damage by ROS and can induce apoptosis [15, 16]. It was found earlier that E. cyaneus that shows higher constitutive and induced levels of Hsp70 than E. verrucosus [24, 25] detoxifies a higher proportion of cadmium by binding it to proteins of the biologically detoxified heat stable protein fraction (BDF) [47]. The in comparison high constitutive Hsp70 titres in Gammarus lacustris [25] may indicate that also in this species a comparatively high level of the internal cadmium is bound to the BDF. However, when considering that the toxicologically more sensitive gammarid species have higher titers of cadmium detoxifying proteins and that the transcripts of both here examined proteins are induced by cadmium only at comparatively high stress levels, it seems obvious that overall the detoxifying effect by those cellular stress response proteins on cadmium is only minor. Major determinants of the cadmium sensitivity of the species appear to be body size and in relation to that the cadmium uptake rate and one additional parameter that could be the degree of metabolic depression caused by cadmium.
Summary and Conclusions
In this study cadmium induced hsp70/Hsp70 and abcb1 levels in gammarids exposed to CdCl2 were for the first time phenotypically anchored, i.e., exposure concentrations of treatments for transcript measurements were equated with the respective lethal concentrations (LCx). When gene/protein responses were related to LCx values the four gammarid species examined showed a relatively uniform hsp70/Hsp70 response with pronounced responses in CdCl2 treatments at higher LCx and in lower LCx treatments after longer times of exposure to CdCl2 (Figs. 3, 4). For abcb1, responses to CdCl2 exposure when related to LCx levels were not uniform across the species; however, abcb1 was overall also induced by CdCl2 in the lethal concentration range (Fig. 5). Induction of hsp70/Hsp70 and abcb1 responses by CdCl2 in the lethal concentration range in the gammarids indicates that changes in transcript levels of those genes are rather insensitive markers for cadmium stress.
Data accessibility statement
All raw data can be obtained from the authors upon request.
Acknowledgements
We would like to thank Rolf Altenburger, Andreas Schüttler, Daria Bedulina and Maxim Timofeyev for their input and discussions related to the study; Sibylle Mothes for Cd analyses of water samples; and Stephan Schreiber for his help with the QIAcube at the Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig. This study was financially supported by scholarships from the Deutscher Akademischer Austauschdienst (DAAD) and the Russian Ministry of Education and Science (“Mikhail Lomonosov” Programme) (M.P., V.P.), by an international DAAD scholarship (M.P., V.P.), by an Erasmus Mundus (MULTIC II) scholarship (V.P.), the project of Goszadaniye of Siberian Institute of Plant Physiology and Biochemistry (No 0343-2017-0006; M.P., V.P.) and by the bilateral funding programs “Helmholtz-Russia Joint Research Groups” (HRJRG) from the Helmholtz Association and the Russian Foundation for Basic Research (RFBR) (LaBeglo project HRJRG-221; M.P., V.P., TL) and „Helmholtz-RSF Joint Research Groups” from the Helmholtz Association and the Russian Science Foundation (RSF) (LaBeglo2/RSF 18-44-06201; T.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.