The induction of cellular stress response systems, heat shock protein hsp70/Hsp70 and multixenobiotic transporter abcb1, by cadmium chloride (CdCl 2 ) was explored in amphipod species with different stress adaptation strategies from the Lake Baikal area. Based on the lethal concentrations (LC) of CdCl 2 , the sensitivities of the different species to CdCl 2 were ranked [24 hr LC50 in mg/L CdCl 2 (mean / 95% confidence interval)]: Gammarus lacustris (1.7/1.3-2.4) < Eulimnogammarus cyaneus

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... 1-4.0) < Eulimnogammarus verrucosus (8.3/3.8-8.7) < Eulimnogammarus vittatus (18.2/12.4-26.6). Conjugated dienes, indicating lipid peroxidation, were significantly increased after 24 hr exposures to 5 mg/L CdCl 2 only in the more CdCl 2 -sensitive species G. lacustris and E. cyaneus. Upon treatment with 0.54 to 5.8 mg/L CdCl 2 for 1, 6 and 24 hrs, hsp70 transcript levels were generally more increased after the longer exposure times and in the more CdCl 2 -sensitive species. Relating the CdCl 2 exposure concentrations to LCx values revealed that across the species the increases of hsp70 transcript levels were comparatively low (0.2-to 2.6-fold) at CdCl 2 concentrations ≤ LC50. Relative hsp70 transcript levels were maximally increased in E. cyaneus by 5 mg/L CdCl 2 (≙ LC70) at 24 hrs (9.1-fold increase above the respective control). When G. lacustris was exposed to 5 mg/L CdCl 2 (≙ LC90) for 24 hrs, the increase in hsp70 was in comparison to E. cyaneus considerably less pronounced (3.0-fold increase in hsp70 levels relative to control). Upon exposure of amphipods to 5 mg/L CdCl 2 , increases in Hsp70 protein levels compared to untreated controls were highest in E. cyaneus at 1 and 6 hrs (5 mg/L CdCl 2 ≙ LC70) and in E. verrucosus at 24 hrs (5 mg/L CdCl 2 ≙ LC45). Thus, when the fold increases in Hsp70 protein levels in the different amphipod species PeerJ reviewing PDF | (Manuscript to be reviewed were related to the respective species-specific LCx values a similar bell-shaped trend as for hsp70 transcript levels was seen across the species. Transcript levels of abcb1 in CdCl 2 exposed individuals of the different amphipod species varied between 0.6-and 4.7-fold in relation to the respective controls. In contrast to hsp70/Hsp70, abcb1 transcripts in CdCl 2 exposed individuals of the different amphipod species did not indicate similar levels of induction of abcb1 at equal LCx levels across the species. Induction of hsp70 and abcb1 genes and Hsp70 proteins by CdCl 2 in the lethal concentration range shows that these cellular responses are rather insensitive to CdCl 2 stress in the examined amphipod species. Furthermore, the increase of expression of these cellular defense systems at such high stress levels suggests that induction of these genes is not related to the maintenance of normal metabolism but to mitigation of the effects of severe toxic stress. PeerJ reviewing PDF | (Manuscript to be reviewed 1 Title: Changes of cellular stress response related hsp70 and abcb1 transcript and Hsp70 protein 2 levels in Siberian freshwater amphipods upon exposure to cadmium chloride in the lethal 3 concentration range 4 5 6 7 Authors: Manuscript to be reviewed 21 Abstract: The induction of cellular stress response systems, heat shock protein hsp70/Hsp70 and 22 multixenobiotic transporter abcb1, by cadmium chloride (CdCl 2 ) was explored in amphipod 23 species with different stress adaptation strategies from the Lake Baikal area. Based on the lethal 24 concentrations (LC) of CdCl 2 , the sensitivities of the different species to CdCl 2 were ranked [24 25 hr LC50 in mg/L CdCl 2 (mean / 95% confidence interval)]: Gammarus lacustris (1.7/1.3-2.4) < 26 Eulimnogammarus cyaneus (2.9/2.1-4.0) < Eulimnogammarus verrucosus (8.3/3.8-8.7) < 27 Eulimnogammarus vittatus (18.2/12.4-26.6). Conjugated dienes, indicating lipid peroxidation, 28 were significantly increased after 24 hr exposures to 5 mg/L CdCl 2 only in the more CdCl 2 -29 sensitive species G. lacustris and E. cyaneus. Upon treatment with 0.54 to 5.8 mg/L CdCl 2 for 1, 30 6 and 24 hrs, hsp70 transcript levels were generally more increased after the longer exposure 31 times and in the more CdCl 2 -sensitive species. Relating the CdCl 2 exposure concentrations to 32 LCx values revealed that across the species the increases of hsp70 transcript levels were 33 comparatively low (0.2-to 2.6-fold) at CdCl 2 concentrations ≤ LC50. Relative hsp70 transcript 34 levels were maximally increased in E. cyaneus at 5 mg/L CdCl 2 (≙ LC70) at 24 hrs (9.1-fold 35 increase in hsp70 levels in the CdCl 2 treatment above the respective control). The increase in 36 hsp70 was considerably less pronounced in G. lacustris [3.0-fold increase in hsp70 levels 37 relative to control by 5 mg/L CdCl 2 (≙LC90), 24 hr exposure]. Upon exposure of amphipods to 38 5 mg/L CdCl 2 , increases in Hsp70 protein levels compared to untreated controls were highest in 39 E. cyaneus at 1 and 6 hrs (5 mg/L CdCl 2 ≙ LC70) and in E. verrucosus at 24 hrs (5 mg/L CdCl 2 40 ≙ LC45). Thus, when the fold increases in Hsp70 protein levels in the different amphipod 41 species were related to the respective species-specific LCx values a similar bell-shaped trend as 42 for hsp70 transcript levels was seen. Transcript levels of abcb1 in CdCl 2 exposed individuals of 43 the different amphipod species varied between 0.6-and 4.7-fold in relation to the respective PeerJ reviewing PDF | ( Manuscript to be reviewed 44 controls. In contrast to hsp70/Hsp70, abcb1 transcripts in CdCl 2 exposed individuals of the 45 different amphipod species did not indicate similar levels of induction of abcb1 at equal LCx 46 levels across the species. Induction of hsp70 and abcb1 gene and Hsp70 protein by CdCl 2 in the 47 lethal concentration range shows that these cellular responses are rather insensitive to CdCl 2 48 stress in the examined amphipod species. Furthermore, the increase of expression of these 49 cellular defense systems at such high stress levels suggests that induction of these genes is not 50 related to the maintenance of normal metabolism but to mitigation of the effects of severe toxic 51 stress. 52