Drosophila melanogaster as a Bioindicator in Comparative Copper and Lead Toxicology: Exploring the Health Implications of Heavy Metal Exposure

1. Introduction

Drosophila melanogaster is a standard model for the study of various diseases, being an important bioindicator for testing the influence of chemical factors [1,2], for the development of treatments [3,4], diagnosis and understanding of toxicity phenomena in the body [5,6,7,8,9], as well as in the study of some neurodegenerative diseases [10].

Drosophila melanogaster (2n=8), exhibits a wide array of mutant genotypes alongside with a short life cycle, providing a significant advantage in the comparative study of toxicology [5,11]. Studies under the influence of heavy metals in D. melanogaster have stimulated increased numbers of reactive oxygen species (ROS) [6,12,13,14], leading to vacuolation of cells, a phenomenon associated with programmed cell death [15,16,17,18], damage to genetic material [19] and eventually apoptosis [18,20]. Numerous studies have highlighted the possibility of using antioxidants to reduce the toxic effects of heavy metals in Drosophila melanogaster [16,21,22]. Being thus an excellent bioindicator, the assay provides valuable information for the development of therapeutic strategies and personalized treatments for various diseases such as Parkinson’s [10,23,24,25,26], Alzheimer [27,28,29], cancer [30,31,32], kidney disease [33] and diabetes [34,35,36], being a model for investigating insulin action [37]. In terms of the action of metals, D. melanogaster may be an essential indicator for determining and understanding how they act on organisms beyond normal limits [6,38,39,40]. D. melanogaster is not only used for understanding the physiological aspects of essential metals [41,42,43], but also for assessing the toxic impact of heavy metals [44,45] having numerous genes and detoxification mechanisms in its composition similar to those found in humans [46,47,48,49].

Metals with metabolic implications, such as copper, although indispensable for the growth and development of organisms by participating in enzymatic reactions and maintaining homeostasis in the body [50,51], become toxic if they accumulate in high concentrations [52,53].

Copper intake in the body is provided by copper-rich foods such as vegetables, fruit [54,55,56], in meat products [57] or in cereals [1,2]. Drinking water also contributes about 0.12-0.26 mg (≈20%) [1] of copper to the average daily intake. The maximum concentration of copper contamination in drinking water is about 1.3 mg. The natural concentration of copper in soil is about 50 mg/kg. The atmospheric copper content also varies between 5 and 20 ng Cu/m³ [61].

Copper is found in nature in combinations, mainly as sulphides [62], participating in numerous biochemical processes based on oxidation-reduction reactions [63], via the various enzymes in whose composition it enters. In normal concentrations it has a positive impact on the functioning of the nervous and immune systems. In the nervous system, it facilitates the transmission of signals and the maintenance of neuronal integrity and in the immune system it contributes to the activation of defence cells and the protection of the body against pathogens.

The link between copper and these systems highlights its importance in maintaining health, with recommended doses of 1.5-3.0 mg in adults and 1 mg in children [52]. High concentrations of copper [64] act as an enzyme inhibitor and limit the activity of alkaline phosphatase [65], catalase [22], xanthine oxidase [66] and ribonuclease [67,68].

Heavy metals, such as lead, are toxic to the body [69,70,71], affecting a number of physiological and biochemical processes, such as metabolism, functioning of internal organs, and can contribute to diseases [5] and in plants affect [6,19,72], nutrition, photosynthesis [73], respiration [74], growth and development their [77,78].

Accumulation of heavy metals [78] leads to altered nutrient absorption capacity, deregulation of metabolic functions and eventually even death of organisms in case of intoxication [72,79,80]. In the context of heavy metal, pollution and climate change have a negative impact on the quality of life [81].

The maximum concentration limits for lead according to the EU Food Regulation are 0.1 mg/kg in meat products, 0.2 mg/kg in cereals and vegetables, 0.1 mg/kg and 0.02 mg/kg in milk. The normal limits for total lead in the human body in a person weighing 70 kg is on average 120 mg lead, with 0.2 mg/L in blood and between 0.2 and 3 in tissues. The U.S. Centers for Disease Control and Prevention has established standard elevated blood lead levels for adults and children (10 μg/dL and 5 μg/dL) [82].

Lead poisoning in concentrations between 0.2 mM-5 mM can manifest acute or chronic forms, and clinical symptoms usually become evident after 2-3 days [83].

Looking at the impact of lead on organisms, we see that exposure to lead can have significant health consequences. Studies show that lead can affect various organs and systems, including the nervous, cardiovascular and reproductive systems [16,84].

In the present study we aimed to monitor the effect of heavy metals on the life cycle of several mutant genotypes and the standard genotype, with a view to using them as bioindicators of comparative toxicity between copper and lead, as well as identifying toxicity limits. Drosophila melanogaster offers the possibility of establishing toxicity limits between heavy metals, their combinations, and interactions between them, which recommends it as a toxicological bioindicator for assessing impacts on ecosystems and organisms.

2. Materials and Methods

2.1. Description of biological Material

In the toxic effects research, we opted to use four genotypes of Drosophila melanogaster, including both wild and mutant variants.

The wild type genotype was used as a control variant, having a light grey body, scarlet (dull red) eyes and wings longer than the body (Figure 1a) [85].

The white mutant genotype is different from the wild type due to the presence of the recessive white gene which determines the white colour of the eyes. This gene is located on chromosome I (heterozygous X) at a distance of 1.5 centimorgans from the 0 end and is denoted by ,,w” (Figure 1b) [86].

The brown mutant genotype has as a defining feature the presence of the recessive bw gene, located on chromosome II at 104 cMo from the 0 end. This gene blocks the formation of the normal red pigment, resulting in a brown eye colour (Figure 1c) [87].

The white-vestigial mutant genotype is characterized by the white colour of the eyes, generated by the presence of the recessive gene w, located on chromosome I and the presence of reduced and rudimentary wings determined by the presence of the recessive gene ,,vg”, located on chromosome II (Figure 1d).

2.2. Preparation of culture medium and inoculation of D. melanogaster

The preparation of the culture medium consists of 200 ml of water to which 2 g of gelatine, 3.6 g of brewer’s yeast, 16.4 g of sugar and 20 g of grey are added.

The mixture obtained is boiled for 20 minutes and then 1 ml of propionic acid is added to the hot medium. The resulting composition is poured (30 ml) into culture dishes and covered with sterile stoppers until the culture medium solidifies. Subsequently, the medium is treated with a solution of yeast and distilled water, using a brush over which fine particles of dried yeast are sprinkled. Varying concentrations of lead acetate and copper sulphate were added to the prepared medium where adults of Drosophila melanogaster were inoculated. After etherization and examination, selected individuals were inoculated onto the culture media.

2.3. Growing environment and experimental metal varinates Cu²⁺ (CuSO₄) and Pb²⁺ (Pb(C₂H₃O₂)₂)

Four concentrations of Cu²⁺ and Pb²⁺ were used in this study to determine the effect on lifecycle proliferation. Due to the varying toxicity limits between Cu²⁺ and Pb²⁺ according to the literature we used intermediate epidermal variants to determine the proliferation inhibition concentration (>IC₅₀) in the four genotypes of D. melanogaster studied (wild, white, brown and white-vestigial) and to compare the toxicity levels between the two metals.

The experimental variants used were:

-

normal environment with grey (control) [81];

-

medium with grey, supplemented with copper sulphate solution CuSO₄ -5H₂O (0.50 mM, 1.00 mM, 2.00 mM and 4.00 mM);

-

medium with grey, supplemented with Pb(C₂H₃O₂)_2.·3H₂O lead acetate solution (0.50 mM, 1.00 mM, 2.00 mM and 4.00 mM);

Determination of the inhibition index (CI₅₀) of proliferation following exposure to Cu²⁺ (CuSO₄) and Pb²⁺ (Pb(C₂H₃O₂)₂)

After pregrafting the experimental variants, Drosophila melanogaster adults were inoculated in a 1:1 ratio (♀ : ♂) for the wild-type and mutant genotypes: white, brown and white-vestigial.

The experiment was carried out in three replicates during the life cycle (egg-adult, Figure 2) ,during which the proliferation of different genotypes of Drosophila melanogaster was monitored under normal conditions and exposed to Cu²⁺ and Pb²⁺ concentrations of 0.50 mM, 1.00 mM, 2.00 mM and 4.00 mM.

In the research, the impact of Cu²⁺ and Pb²⁺ was monitored at all developmental stages of Drosophila melanogaster (egg, larvae I, II, III, pupa and adult) (Figure 1) in order to determine the prolificacy as well as their inhibition concentrations.

After the establishment of the proliferation indices under normal conditions, they were compared with the experimental variants with different concentrations of Cu²⁺ and Pb²⁺ to determine the proliferation inhibition index (IC) according to formula (1):

$$\mathit{I}\mathit{C}=100-\frac{\mathit{N}\mathit{u}\mathit{m}\mathit{b}\mathit{e}\mathit{r} \mathit{o}\mathit{f} \mathit{i}\mathit{n}\mathit{d}\mathit{i}\mathit{v}\mathit{i}\mathit{d}\mathit{u}\mathit{a}\mathit{l}\mathit{s} \mathit{i}\mathit{n} \mathit{e}\mathit{x}\mathit{p}\mathit{e}\mathit{r}\mathit{i}\mathit{m}\mathit{e}\mathit{n}\mathit{t}\mathit{a}\mathit{l} \mathit{g}\mathit{r}\mathit{o}\mathit{u}\mathit{p}}{\mathit{N}\mathit{u}\mathit{m}\mathit{b}\mathit{e}\mathit{r} \mathit{o}\mathit{f} \mathit{i}\mathit{n}\mathit{d}\mathit{i}\mathit{v}\mathit{i}\mathit{d}\mathit{u}\mathit{a}\mathit{l}\mathit{s} \mathit{i}\mathit{n} \mathit{c}\mathit{o}\mathit{n}\mathit{t}\mathit{r}\mathit{o}\mathit{l} \mathit{g}\mathit{r}\mathit{o}\mathit{u}\mathit{p}}\ast 100$$

(1)

The proliferation inhibition index >IC₅₀ was considered the maximum toxicity level of the metal taken in the study [89,90].

Figure 2. Developmental stages of Drosophila melanogaster larvae, (a.1)- larva stage I, (a.2)- larva stage II, (a.3)- larva stage III, (a.4)- larva pupation; Evolution of pupal to adult formation: (b.1)- pupal, (b.2)- hatching, (b.3)- hatched pupal, (b.4)- adult;.

Figure 2. Developmental stages of Drosophila melanogaster larvae, (a.1)- larva stage I, (a.2)- larva stage II, (a.3)- larva stage III, (a.4)- larva pupation; Evolution of pupal to adult formation: (b.1)- pupal, (b.2)- hatching, (b.3)- hatched pupal, (b.4)- adult;.

2.4. Statistical calculation methods used to determine proliferation and toxicity levels between Cu²⁺ and Pb²⁺

In this study, we investigated the behavior of the genotypes concerning adaptability and prolificacy under exposure to various concentrations of heavy metals. We conducted statistical analysis using a multivariate ANOVA test, which uncovered the effects of genotype, metal type (Cu²⁺ or Pb²⁺), toxicity levels, and concentration on the developmental period from egg to adult. Significance was determined at a confidence level of p <0.05. The obtained results underwent comparative analysis using the Tukey test. The statistical interpretation was carried out using Rstudio software.

3. Results

This study determined the prolificacy of D. melanogaster genotypes following the application of different concentrations of copper and lead at different life cycle stages.

The results showed differences in the evolution of the number of larvae (I, II and III), as well as in the formation of pupae and adults, depending on the genotype, the type of metal used (Cu²⁺ or Pb²⁺) and the concentration used. Using the D. melanogaster genetic model, research has identified the influence of genotype and metal concentrations used in determining toxicity levels.

3.1. Influence of genotype, concentration and type of metal Cu²⁺ and Pb²⁺ on proliferation in Drosophila melanogaster

In the study of the influence of different wild-type genotypes (control) and variations between brown, white and white-vestigial mutant genotypes, a significant difference in the number of larvae formed in stages I, II and III under different experimental conditions was observed.

The results show a directly proportional correlation between the concentrations of heavy metals used and their type (Cu²⁺ and Pb²⁺), as well as the nature of the genotype, in terms of the number of larvae formed (p < 0.001).

There was also a significant influence of genotype on the number of individuals resulting from exposure to these metals.

Thus, from the results obtained, it can be deduced that the type of genotype studied can exert a significantly different influence on the number of individuals resulting from exposure to Cu²⁺ and Pb²⁺, thus confirming the significant role of genetic predisposition to toxicity (Table 1).

As in the larval stage, the influence of genotype and metal concentrations used was found to show significant variation in prolificacy levels throughout the life cycle, including in pupation and adult formation (p < 0.001) (Table 2).

Analysis of the interaction between the genetic factor and metal concentration levels (Cu²⁺ and Pb²⁺ ) significantly influenced proliferation in D. melanogaster throughout the life cycle, both in the larval stages (Table 1) and in the pupal and adult formation process (Table 2) (p < 0.001). Thus, as concentration increases, proliferation capacity specifically affects certain genotypes, with some being more tolerant (showing a lower inhibition concentration) and others more sensitive.

Looking at the interaction between concentration and metal type (Cu²⁺ or Pb²⁺), it had a significant impact on the number of individuals during the developmental cycle (p < 0.001). This reflects differences in the response of proliferation rates depending on the type of metal to which D. melanogaster genotypes are exposed.

The results obtained show the same impact of decreased prolificacy for both Cu²⁺, and Pb²⁺, obtaining the same effect of decreased number of inidivids in larval stages of pupal formation (Genotype*Metal, Concentration*Genotype: Metal, p >0.05). Toxicity levels (degree of inhibition of proliferation, >IC₅₀) are influenced by genotype, concentrations and type of metal (Cu²⁺ or Pb²⁺) used in the medium (Concentration, Genotype, Metal, Concentration*Genotype, Concentration*Metal, p <0.001) (Table 1 and Table 2).

Table 2. Analysis of factors involved in the process of inhibition of proliferation at different concentrations of copper (CuSO₄) and lead (Pb(C₂H₃O₂)₂ in pupal and adult formation.

	Analysis factor	df	SS	S2	F	Pr(>F)
Pupal stage	Concentration	4	24437	6109	203.090	< 2e-16 ***
	Genotype	3	5526	1842	61.234	< 2e-16 ***
	Metal	1	1687	1687	56.094	1.45e-13 ***
	Concentration: Genotype	12	1132	94	3.136	0.000211 ***
	Concentration:Metal	4	585	146	4.861	0.000691 ***
	Genotype:Metal	3	80	27	0.884	0.448785 Ns
	Concentration: Genotype: Metal	12	179	15	0.496	0.918151 Ns
	Residuals	1060	31886	30
Adult stage	Concentration	4	12209	3052.3	135.964	< 2e-16 ***
	Genotype	3	4569	1522.9	67.837	< 2e-16 ***
	Metal	1	893	893.5	39.799	5.55e-10 ***
	Concentration: Genotype	12	551	45.9	2.044	0.0189 *
	Concentration:Metal	4	274	68.5	3.051	0.0166 *
	Genotype:Metal	3	65	21.7	0.968	0.4075 Ns
	Concentration: Genotype: Metal	12	89	7.4	0.331	0.9836 Ns
	Residuals	586	13155	22.4

Note: SS- sum of square, dF-degrees of freedom; S² -mean square. “Ns”-not significant, “*”- p < 0.05, “**”- p < 0.01, ***significant at p < 0.001.

Table 2. Analysis of factors involved in the process of inhibition of proliferation at different concentrations of copper (CuSO₄) and lead (Pb(C₂H₃O₂)₂ in pupal and adult formation.

	Analysis factor	df	SS	S2	F	Pr(>F)
Pupal stage	Concentration	4	24437	6109	203.090	< 2e-16 ***
	Genotype	3	5526	1842	61.234	< 2e-16 ***
	Metal	1	1687	1687	56.094	1.45e-13 ***
	Concentration: Genotype	12	1132	94	3.136	0.000211 ***
	Concentration:Metal	4	585	146	4.861	0.000691 ***
	Genotype:Metal	3	80	27	0.884	0.448785 Ns
	Concentration: Genotype: Metal	12	179	15	0.496	0.918151 Ns
	Residuals	1060	31886	30
Adult stage	Concentration	4	12209	3052.3	135.964	< 2e-16 ***
	Genotype	3	4569	1522.9	67.837	< 2e-16 ***
	Metal	1	893	893.5	39.799	5.55e-10 ***
	Concentration: Genotype	12	551	45.9	2.044	0.0189 *
	Concentration:Metal	4	274	68.5	3.051	0.0166 *
	Genotype:Metal	3	65	21.7	0.968	0.4075 Ns
	Concentration: Genotype: Metal	12	89	7.4	0.331	0.9836 Ns
	Residuals	586	13155	22.4

Note: SS- sum of square, dF-degrees of freedom; S² -mean square. “Ns”-not significant, “*”- p < 0.05, “**”- p < 0.01, ***significant at p < 0.001.

3.2. Influence of copper (CuSO₄) on proliferation in Drosophila melanogaster during the life cycle (egg-adult)

Investigations of the prolificacy rate of Drosophila melanogaster genotypes at various copper concentrations show a decrease directly proportional to the increase in copper concentration, both in the three larval developmental stages (stage I, II and III) and in the pupal and adult formation stages (Table 3).

Significant differences were observed in the response to copper concentrations between the genotypes used (p < 0.05). Brown and white-vestigial genotypes showed higher sensitivity to the highest Cu⁺² concentrations compared to wild and white genotypes.

The wild genotype showed prolificacy rate values below 50% (>IC₅₀ ) at copper concentrations of 4.00 mM in larval stages I, II and III. Close values of proliferation inhibition >IC₅₀ were observed at 2.00 mM concentration of Cu⁺². The same toxicity effect was observed in pupal and adult formation with >IC₅₀ values being present at 4.00 mM concentration (Table 3).

Table 3. Results on the proliferation of D. melanogaster genotypes following exposure to various concentrations of copper.

Genotype	Concentration (mM)	Copper (CuSO4)
		StageI	IC	StageII	IC	StageIII	IC	Pupal	IC	Adult	IC
		Mean		Mean		Mean		Mean		Mean
wild	Control	35.97a	0.00	28.33a	0.00	26.39a	0.00	25.41a	0.00	24.87a	0.00
	0.50	29.85b	17.03	24.58abc	13.24	22.24ab	15.73	20.67ab	18.66	20.27abc	18.50
	1.00	25.41bcd	29.37	19.58cdef	30.88	17.85bcde	32.38	15.67cde	38.34	16.40cdef	34.05
	2.00	21.56de	40.06	16.58defgh	41.47	15.03defg	43.05	13.33def	47.52	14.20def	42.90
	4.00	13.23fgh	63.22	10.14ijk	64.22	9.24hijk	64.98	7.90ghi	68.92	8.39gh	66.26
brown	Control	27.08bc	0.00	20.83cde	0.00	19.52bcd	0.00	18.74bc	0.00	17.73bcd	0.00
	0.50	22.28cde	17.71	17.83defgh	14.40	16.39cdef	15.99	14.96cdef	20.16	14.47def	18.42
	1.00	19.97de	26.23	16.03efgh	23.07	14.88efg	23.76	13.04def	30.43	12.73defg	28.20
	2.00	16.85efg	37.78	12.56hij	39.73	11.67ghij	40.22	10.22fgh	45.45	10.80fgh	39.10
	4.00	10.82hi	60.04	7.03kl	66.27	6.48kl	66.77	6.00hi	67.98	6.94hi	60.84
white	Control	30.54b	0.00	26.64ab	0.00	26.18a	0.00	24.48a	0.00	22.31ab	0.00
	0.50	28.00bc	8.31	21.47bcd	19.40	19.82bc	24.31	17.26bcd	29.50	18.33bcd	17.83
	1.00	22.33cde	26.87	17.14defgh	35.66	16.30cdef	37.73	14.07cdef	42.51	14.27def	36.06
	2.00	17.85ef	41.56	13.53fghi	49.22	12.30fghi	53.01	10.85efg	55.67	11.67efgh	47.71
	4.00	10.46hi	65.74	8.33ghij	68.72	7.18jkl	72.57	7.30ghi	70.18	6.56hi	70.62
white-vestigial	Control	23.10cd	0.00	21.85bcd	0.00	19.70bc	0.00	18.56bc	0.00	16.93bcde	0.00
	0.50	19.79de	14.32	18.42defg	15.72	15.94cdefg	19.08	14.63cdef	21.16	13.87defg	18.11
	1.00	17.05efg	26.19	15.19fghi	30.47	13.27efgh	32.62	12.74def	31.34	11.33efgh	33.07
	2.00	11.51ghi	50.17	9.03jkl	58.69	8.45ijkl	57.08	7.22ghi	61.08	6.27hi	62.99
	4.00	5.97i	74.14	4.53l	79.27	4.58l	76.77	3.64i	80.37	2.94i	82.63

*Superscript letters are assigned based on the results of the Tukey pairwise comparison test, indicating the significance levels of each column value obtained relative to the copper concentration and exposure time. The highest-ranked values are denoted, beginning with the first letter “a”. Similar superscript letters indicate statistically non-significant differences between the obtained values (p >0.05).**IC denotes the prolificity inhibitaion concentration of copper.

Table 3. Results on the proliferation of D. melanogaster genotypes following exposure to various concentrations of copper.

Genotype	Concentration (mM)	Copper (CuSO4)
		StageI	IC	StageII	IC	StageIII	IC	Pupal	IC	Adult	IC
		Mean		Mean		Mean		Mean		Mean
wild	Control	35.97a	0.00	28.33a	0.00	26.39a	0.00	25.41a	0.00	24.87a	0.00
	0.50	29.85b	17.03	24.58abc	13.24	22.24ab	15.73	20.67ab	18.66	20.27abc	18.50
	1.00	25.41bcd	29.37	19.58cdef	30.88	17.85bcde	32.38	15.67cde	38.34	16.40cdef	34.05
	2.00	21.56de	40.06	16.58defgh	41.47	15.03defg	43.05	13.33def	47.52	14.20def	42.90
	4.00	13.23fgh	63.22	10.14ijk	64.22	9.24hijk	64.98	7.90ghi	68.92	8.39gh	66.26
brown	Control	27.08bc	0.00	20.83cde	0.00	19.52bcd	0.00	18.74bc	0.00	17.73bcd	0.00
	0.50	22.28cde	17.71	17.83defgh	14.40	16.39cdef	15.99	14.96cdef	20.16	14.47def	18.42
	1.00	19.97de	26.23	16.03efgh	23.07	14.88efg	23.76	13.04def	30.43	12.73defg	28.20
	2.00	16.85efg	37.78	12.56hij	39.73	11.67ghij	40.22	10.22fgh	45.45	10.80fgh	39.10
	4.00	10.82hi	60.04	7.03kl	66.27	6.48kl	66.77	6.00hi	67.98	6.94hi	60.84
white	Control	30.54b	0.00	26.64ab	0.00	26.18a	0.00	24.48a	0.00	22.31ab	0.00
	0.50	28.00bc	8.31	21.47bcd	19.40	19.82bc	24.31	17.26bcd	29.50	18.33bcd	17.83
	1.00	22.33cde	26.87	17.14defgh	35.66	16.30cdef	37.73	14.07cdef	42.51	14.27def	36.06
	2.00	17.85ef	41.56	13.53fghi	49.22	12.30fghi	53.01	10.85efg	55.67	11.67efgh	47.71
	4.00	10.46hi	65.74	8.33ghij	68.72	7.18jkl	72.57	7.30ghi	70.18	6.56hi	70.62
white-vestigial	Control	23.10cd	0.00	21.85bcd	0.00	19.70bc	0.00	18.56bc	0.00	16.93bcde	0.00
	0.50	19.79de	14.32	18.42defg	15.72	15.94cdefg	19.08	14.63cdef	21.16	13.87defg	18.11
	1.00	17.05efg	26.19	15.19fghi	30.47	13.27efgh	32.62	12.74def	31.34	11.33efgh	33.07
	2.00	11.51ghi	50.17	9.03jkl	58.69	8.45ijkl	57.08	7.22ghi	61.08	6.27hi	62.99
	4.00	5.97i	74.14	4.53l	79.27	4.58l	76.77	3.64i	80.37	2.94i	82.63

*Superscript letters are assigned based on the results of the Tukey pairwise comparison test, indicating the significance levels of each column value obtained relative to the copper concentration and exposure time. The highest-ranked values are denoted, beginning with the first letter “a”. Similar superscript letters indicate statistically non-significant differences between the obtained values (p >0.05).**IC denotes the prolificity inhibitaion concentration of copper.

For the brown genotype, at the concentration of 4.00 mM Cu⁺², the results indicate a decrease in proliferation for larval stages I, II and III (>IC₅₀), highlighting a manifestation of increased toxicity in this genotype (p <0.05). A significant decrease in the number of pupae and adults compared to the control variant is also noted, indicating the negative impact of copper on their formation (p <0.05).

In the case of the white genotype, the concentration of 4.00 mM (>IC₅₀) of copper shows the same toxic effect, causing a reduction in the proliferation of this genotype during the life cycle.

The highest sensitivity was recorded in the white-vestigial genotype in which the 2.00 mM Cu⁺² concentration (>IC₅₀) showed a decrease in proliferation of more than 50% compared to the control group for all developmental stages (p <0.05).

According to the results obtained (Table 3 and Figure 3), the proliferation rate of the genotypes was inhibited by the presence of copper in all larval stages, showing the significant reduction of the proliferation rate starting with the dose of 0.50 mM Cu⁺²(p <0.05).

In the early stages of the developmental cycle, concentrations of 0.50-1.00 mM Cu⁺² resulted in decreases in the proliferation rate, which increased as the concentration of Cu⁺² increased.

Pup and adult formation of the genotypes studied was influenced directly proportional to increasing concentrations for all stages followed (Table 3 and Figure 4).

Thus, from the presented results it was evident that the value of >IC₅₀ at the concentration of 4.00 mM is present in all studied genotypes (4/4). At 2.00 mM concentration of Cu⁺² the white-vestigial genotype showed the >IC₅₀ value throughout the development cycle (1/4).

The results obtained in this study highlight the impact of copper concentrations (CuSO₄) on the proliferation of Drosophila melanogaster during their life cycle, showing an inhibition with increasing copper concentration and genotype under study.

3.3. Influence of lead (Pb(C₂H₃O₂)₂ on proliferation in Drosophila melanogaster during the life cycle (egg-adult)

Analysis of the number of stage I, II and III larvae of Drosophila melanogaster genotypes exposed to varying concentrations of Pb²⁺ revealed significant differences in the rate of proliferation as an effect of toxicity. The results showed significant variation in the number of individuals developed under different experimental conditions.

Investigations found that genotype had a significant influence on development in D. melanogaster following exposure, confirming a higher genetic predisposition to toxicity for certain genotypes. The highest values of prolificacy were recorded for the wild and white genotypes, the average values for the brown genotype and the lowest values for the white-western genotype (Table 4). The >IC₅₀ value at all developmental stages is present only in the white-vestigial genotype at a concentration of 4.00 mM (1/4, of genotypes).

In the early stages of the developmental cycle, larval stages exposed to concentrations ranging from 0.50 to 1.00 mM of lead exhibited a decrease in prolificacy. However, this decrease was not statistically significant concerning genotype prolificacy (p >0.05).

The wild genotype showed a significant reduction in the number of larvae in stages I. II and III at higher concentrations, especially at 4.00 mM. The same trend was observed for the white and brown genotypes, suggesting a common sensitivity to lead.

As for the white-vestigial genotype, a more pronounced sensitivity to lead was observed. with a significant reduction in larval numbers in stages I, II and III starting at 1.00 mM concentration. in contrast to the other genotypes which showed a higher tolerance at this concentration. The results suggest that genotypes show different responses to lead concentrations. influencing development in D. melanogaster to varying degrees.

Figure 5. Effect of lead on different genotypes of Drosophila melanogaster in adult formation: (a)- wild genotype. (b)- brown genotype, (c)- white mutant genotype, (d)- white-vestigial mutant genotype.

Figure 5. Effect of lead on different genotypes of Drosophila melanogaster in adult formation: (a)- wild genotype. (b)- brown genotype, (c)- white mutant genotype, (d)- white-vestigial mutant genotype.

Figure 6. Effect of lead on different genotypes of Drosophila melanogaster in adult formation: (a)- wild wild genotype (b)- brown genotype (c)- white mutant genotype (d)- white-vestigial mutant genotype.

Figure 6. Effect of lead on different genotypes of Drosophila melanogaster in adult formation: (a)- wild wild genotype (b)- brown genotype (c)- white mutant genotype (d)- white-vestigial mutant genotype.

Analysing the influence of Drosophila melanogaster genotypes on the number of pupae and adults obtained we found that the diversity of genotypes had a significant impact on the results. while also influencing the number of pupae in the control samples (p <0.001). In the case of lead exposure, the results obtained indicate a significant link between lead concentration and the effects generated.

Studies on mutant genotypes revealed that the white-vestigial genotype showed significantly fewer pupae formed in most of the variants studied, compared to the wild wild genotype which showed the highest tolerance at the pupal and adult stages.

Analysis of the effect of lead in Drosophila melanogaster reveals the significant impact of this metal on the development and health of organisms highlighting the importance of further research in this field and the development of effective strategies for environmental and public health protection.

Comparative study of toxicity effects on drosophila melanogaster at various concentrations of lead and copper

The comparative study of copper and lead toxicity revealed significant variations in the formation of pupae and the emergence of adult flies. The analysis of toxicity differences demonstrated that copper exhibited higher toxicity at both low and high concentrations compared to lead. In the context of adult development, it was observed that at a concentration of 0.50 mM, no significant differences between the two metals were identified (p < 0.05).

Figure 7. Effect of copper on various genotypes of Drosophila melanogaster in adult development.

Figure 7. Effect of copper on various genotypes of Drosophila melanogaster in adult development.

These differences persisted across other concentrations, indicating a continuous dominant effect of copper toxicity over that of lead. Consequently, the level of significance increased with rising concentrations. Therefore, at a concentration of 1.00 mM, the toxicity degree was significantly more pronounced for copper with p < 0.05 compared to lead. At concentrations of 2.00 mM and 4.00 mM, the degree of significance reached p < 0.001. The intervals were significantly different from each other (Figure 8) and also compared to the control group. These findings demonstrate a more acute toxic effect of copper compared to lead. This outcome might be inherently understandable considering that copper (CuSO₄) is often used as an antimicrobial and antibacterial agent, playing a crucial regulatory role in homeostasis, whereas the accumulation of lead exhibits a significantly different toxicological profile.

The study on D. melanogaster can provide valuable insights into both types of toxicity, considering its rapid life cycle and the ease with which multiple generations can be generated and observed.

4. Discussion

Following literature reviews the results obtained complete the explanation of the Cu²⁺ toxicity phenomenon. (i) The same toxic effect has been observed by other authors using wild type genotype at concentrations of 0.50 mM [61,62,63], the most damaging concentration of 3.00 mM of Cu²⁺, values that justify the toxicity values obtained by us (2 mM-4 mM, Table 3). (ii) In addition to the results we can say that there is a significant influence of the genotype used, as the authors also indicate an inhibition of proliferation on Cu²⁺ depending on the sex [93,94] or the developmental stage [2], the number of individuals starting to decrease significantly depending on the dose applied 0.40 mM for females and 0.75 mM for males [93]. (iii) Research shows the possibility of development of genotypes with increased tolerance to Cu²⁺ concentrations up to 50 mM of more than 75% as opposed to other genotypes with survival rates below 18%. As a result. genotype influence plays a determined role in the assessment of metalor toxicity above normal doses.

Copper is an essential element for the proper functioning of all organisms having an essential role in the composition of enzymes [95,96], antimicrobial and antiviral against Escherichia coli [97,98], Staphylococcus aureus (MRSA) [99,100,101], SARS-coronavirus [102,103,104], influencing virus A [104,105,106] and fungi [107]. However, even though the effects of copper are beneficial at high concentrations the results obtained for D. melanogaster indicate a negative effect of Cu²⁺, manifested by reduced numbers of larvae. pupae and adults at concentrations starting at 0.50 mM Cu²⁺.

Research has shown that the toxicity causes a reduction in the number of individuals and the impact is significant in all genotypes at the highest concentrations used in the experiment of 2.00-4.00 mM. Our results indicate different degrees of toxicity depending on the genotype of D. melanogaster used, which was observed for both Cu²⁺, and Pb²⁺ as well as results obtained by other authors [20].

As regards lead accumulation research has shown the appearance of oxidative stress [109] at concentrations of 0.20 mM Pb ²⁺ [16] from the larval stage and even the appearance of spots on the wings. showing a mutagenic effect of Pb²⁺ from concentrations of 0.40 mM [110].

Cumulative cytotoxic effects of Pb²⁺ and Cu²⁺, have also been shown in the organisms Ceriodaphnia dubia and Daphnia carinata [111].

Studies by other researchers on the impact of heavy metals on Emiliania huxleyi indicate a stimulation of copper uptake in the presence of lead at low concentrations [111]. However, at higher lead levelsm a reverse effect was observed. characterized by a significant reduction in copper uptake [112].

Influence on the reduction of absorption has also been identified for iron (Fe) indicating a decrease in Fe absorption under the influence of Pb²⁺, at a concentration of 342 µg Pb/mL [109].

These results were also obtained in mouse experiments, which indicated that DMT1, responsible for iron (Fe) uptake, facilitates lead transport across the blood-brain barrier to the brain [113] (2.00 mM Pb²⁺ ). This explains the toxic effect of lead on the nervous system with long-term exposure, with an even more pronounced impact on children [84].

Due to the considerable number of generations undergone by Drosophila melanogaster, this species offers a precise opportunity for localizing Quantitative Trait Loci (QTL), not only concerning genetic effects and biological impacts in heavy metal resistance, but also for analyzing the toxic effects of various chemicals and treatments. Consequently, by furnishing substantial and efficient biological support, Drosophila melanogaster emerges as a pivotal model and valuable indicator in health analysis and maintenance. This contributes significantly to the identification, monitoring, and development of solutions for various diseases, including cases of heavy metal poisoning [26].

5. Conclusions

This study unveiled the influence of genetic factors on the proliferation capacity of Drosophila melanogaster. The most sensitive genotype, white-vestigial, exhibited lower prolificacy compared to wild genotypes or other mutant variations. Prolificity varied across both control and experimental copper and lead variants throughout the life cycle.

Metals toxicity was elucidated through the proliferation inhibition index, which fluctuated according to genotype and concentration, with proliferation inhibition directly correlating with increasing concentration.

Copper displayed toxicity across all genotypes within the 0.50 mM to 2.00 mM concentration range (>IC₅₀ valid for all genotypes), emphasizing the necessity of regulating and monitoring copper levels in the environment. Lead exhibited toxicity within the concentration range of 1.00 mM to 4.00 mM, with an inhibitory concentration exceeding IC₅₀ solely for the white-vestigial genotype.

The comparative analysis between copper and lead highlighted that the adverse effects of copper are more enduring and acute than those of lead, suggesting the requisite for appropriate management of heavy metal exposure in natural environments. Differences in proliferation toxicity in D. melanogaster between copper and lead are substantial, escalating with increasing concentrations.

These findings underscore the significance of further research in ecotoxicology and the formulation of effective strategies for environmental protection and public health concerning heavy metal pollution, utilizing the D. melanogaster genetic model.

In conclusion, we aim to replicate the experiments with other metals and develop genotypes resistant to the most influential health-affecting factors. These research directions hold the potential to offer significant insights and practical solutions in addressing complex health issues, drawing upon the contributions of Drosophila melanogaster in the experiments.