Copper content lower than safety factor 100-fold of acceptable daily intake negatively affects Drosophila melanogaster fruit flies’ nervous function

Figures & data

Table 1. Development of fruit flies at different concentrations of CuSO₄.

	F0	F1 Larvae			F1 Pupae		F1 Adult flies
Concentration (mM)	^1D7d (flies)	^2A1st (days)	^3Number (flies)	^2A1st (days)	^3Ni (flies)	^4NT11 (flies)	^2A1st (days)	^3Ni (flies)	^4SNT15 (flies)
0.0	0.3	6	4.7^ab±2.3	6	18.3^a±5.9	318.3^a±15.9	11	21.3^a±2.9	215.0^a±5.0
0.15	0.3	7	5.0^ab±2.0	7	3.3^b±0.6	157.3^b±3.1	11	2.7^b±0.6	153.3^b±11.5
1.0	0.7	7	10.0^a±5.6	7	7.7^b±2.1	170.3^b±14.1	11	5.3^b±4.6	130.0^b±10.0
2.0	0.3	9	1.7^b±1.2	9	4.7^b±2.5	18.7^c±14.6	13	3.3^b±1.2	16.7^c±6.8
3.0	1.3	13	2.0^b±1.0	13	3.0^b±1.7	–	18	5.3^b±2.9	–
4.0	1.0	–	–	–	–	–	–	–	–
5.0	0.3	–	–	–	–	–	–	–	–
7.5	0.3	–	–	–	–	–	–	–	–
10.0	0.7	–	–	–	–	–	–	–	–
p-Value	0.898	–	0.036	–	0.001	0.000	–	0.000	0.000

Copper ions were added to the food medium with indicated concentrations. Each culturing vial contained 10 male and 10 female fruit flies, and each treatment concentration contained 3 vials. After 7 days of feeding, 1the quantity of died F0 flies was observed (D7d), then 2the first appearing time (A1st) and 3quantity of F1 individual (Ni) at each maturing stage—larvae, pupae, and adult were calculated. For the last two stages, the total number of individuals was calculated after 11 days and 15 days (NT11, NT15), respectively. The values are introduced as the average ± amount of variation of triplicate experiments, and the difference was measured using the t–test. Letters following the same mean in the same column are not significantly different (p > 0.05). The “-” sign indicates the results recorded were 0.

Figure 1. The decline in learning and memory ability of copper-treated larvae. The F1, F2, and F3 flies were independently trained in two sections, which were prepared using the odors n-amyl acetate, and 1-octanol. The control was the flier fed in the negative copper medium. LI was calculated by the relation of preferable AM or OCT indicated in Equations (1)–(3). LI score > 0 indicated appetitive learning; LI score ∼ 0 indicated no evidence of learning and memory; if the LI score < 0, aversive learning had occurred. The values are introduced as the average ± amount of variation of triplicate experiments, and the difference was measured using t–test (p** <0.01; p***<0.001).

Figure 2. Copper caused locomotor impairment in flies. Twenty randomly chosen adult flies were placed in the bottom of 2 × 10 cm vials as a common starting point for each treatment group. The control was described in the legend of Figure 1. The proportion of flies that could reach the 6 cm height column in 10 s was recorded. Data are introduced as the mean ± standard deviation of five independent experiments and difference was measured using the t–test (p*<0.05; p** <0.01; p***<0.001; p^****<0.0001).

Figure 3. Protein concentration of the treatments. The flies were prepared by homogenized protein concentration in the different concentrations of CuSO₄ (1 mM and 2 mM). The control was described in the legend of Figure 1. Bradford’s method using BSA as the standard was applied to estimate the treatments. The values are introduced as the average ± amount of variation of triplicate experiments, and the difference was measured using the t–test and 2-way ANOVA.

Figure 4. DPPH scavenging percentage reduction by copper toxicity. The mixture containing 20 µL of fly homogenized sample and 50 µL methanol (Blank) added 125 µL of 0.004% DPPH solution was kept warm in the dark for 30 min at room condition and measured OD at the wavelength of 517 nm. The mixture using the homogenized flies grown in the negative copper medium was used as a control. The DPPH scavenging percentage was calculated by Eq. (4)(4) $$I\mathrm{\%}=\frac{\left[\right({\mathit{\text{OD}}}_{\mathit{\text{Control}}}-\left({\mathit{\text{OD}}}_{\mathit{\text{Sample}}}-{\mathit{\text{OD}}}_{\mathit{\text{Blank}}}\right)\times 100}{{\mathit{\text{OD}}}_{\mathit{\text{Control}}}}$$(4) , presented in Materials and Methods. The values are introduced as the average ± amount of variation of triplicate experiments, and the difference was measured using the 2-way ANOVA and the t-test. For 1 mM Cu²⁺ p = 0.033 F0, p = 0.009 F1, p = 0.010 F2, p = 0.005 F3 vs. Control, respectively; for 2 mM Cu²⁺ p = 0.023 F0, p = 0.003 F1, p = 0.005 F2, p = 0.000 F3 vs. Control, respectively.

Figure 5. Copper induces catalase inhibition. The effect of 1 mM (A) and 2 mM (B) Cu²⁺ on catalase activity of variant fly generation, respectively. (C) the effect of Cu²⁺ on the specific catalase activity per minute of the flies. The values are introduced as the average ± amount of variation of triplicate experiments, Nonline fit Michaelis-Menten model, and the difference was measured using the 2-way ANOVA and the t-test. The reaction mixture in a cuvette, containing 100 µL of fly homogenized sample and 900 µL of 50 mM sodium phosphate buffer, was measured OD at the wavelength of 240 nm, then adding 500 µL of H₂O₂ solution and measured OD immediately. The OD value was read every 1 min for 3 min. The 100 mL of 30 mM H₂O₂ in 50 mM buffer phosphate was used as blank. The control was showed in the legend of Figure 4. Enzyme activity (p* < 0.05; p** < 0.01; p*** < 0.001; p^**** < 0.0001) was calculated by Eq. (5)(5) $$\mathit{\text{Catalase activity}}=\frac{\Delta A\times V_t\hspace{0.17em}}{{\epsilon }_{240}\times d\times V_s\times C_t}\times 0.01\left(U.{\text{mg}}^{-1}\right)$$(5) as described in Materials and Methods. (C) For 1 mM Cu²⁺: p = 0.030 F1, p = 0.0224 F2, p = 0.0127 F3 vs. Control, respectively; for 2 mM Cu²⁺: p = 0.010 F1, p = 0.004 F2, p = 0.005 F3 vs. Control, respectively.

Figure 6. Copper induces AChE inhibition. The reaction mixture consisted of 10 µL of fly homogenizer, 10 µL of acetylcholine chloride in 2.6 mM methanol, and 200 µL of 0.1 mM, pH 7.0 sodium phosphate buffer. Measure OD at the wavelength of 405 nm to get blank and add 80 µL of 0.3% Fast blue B salt to the mixture and measure OD at the wavelength every 5 min for 20 min. Protein concentration of the samples helped to standardization of the results. The control was described in the legend of Figure 4. Enzyme activity was calculated by Eq. (6)(6) $$\begin{array}{c}{A}_{\mathit{\text{AChE}}}=\frac{\Delta A\times V_t\hspace{0.17em}}{{\epsilon }_{405}\times d\times V_S\times C_t}\\ \times 0.001\left(\mu \text{mol\hspace{0.17em}\hspace{0.33em}}{\text{min}}^{-1}\text{\hspace{0.33em}mg\hspace{0.33em}of\hspace{0.33em}}{\text{protein}}^{-1}\right)\end{array}$$(6) , described in Materials and Methods. The values are introduced as the average ± amount of variation of triplicate experiments, and the difference was measured using the 2-way ANOVA and the t-test. For 1 mM Cu²⁺: p = 0.0022 F0, p = 0.0014 F1, p = 0.0030 F2, p = 0.0006 F3 vs. Control, respectively; for 2 mM Cu²⁺ p = 0.0015 F0, p = 0.0008 F1, p = 0.0008 F2, p = 0.0006 F3 vs. Control, respectively.

Figure 7. The toxicity of copper metal (Cu) on flies through behavioral and biochemical assays.