Heavy metals are known to have an effects on insect growth rates and mortality. Moreover, they have a direct effect on some innate insect resistance mechanisms such as the immune and detoxification systems. Antioxidant and detoxification enzymes are good indicators of oxidative damage caused by metals. Some parameters are used as biomarkers in toxicity measurements such as biotransformation enzymes (CytP450) and their products (protein, RNA, DNA), oxidative stress (CAT, SOD, GPx), hematological (transaminases), neuromuscular (AChE) and genotoxic (DNA damage) parameters. Biomarkers provide information about the potential xenobiotic mechanisms. In this study, the effects of G. mellonella larvae fed with different concentrations of Cu (10 mg /L), Zn (30 mg /L) and thier mixture on the antioxidant enzyme activities and accumulation levels in the midgut and fat body were investigated.
Similar studies have shown that the accumulation of heavy metals in tissues cause toxic effects when they reach critical values (Li et al. 2005; Tkachenko and Kurhaluk 2012). In insects, large amounts of accumulated metals are taken up by metal-binding proteins and stored in intracellular spheres to prevent them from interacting with biomolecules and reactions in midgut tissues. When midgut cells reach the metal storage capacity, cells break down and the accumulated metals are released into the lumen and excreted with feces. This process damages the epithelial cells and disrupts the midgut processes (Peric-Mataruga et al. 2018). Moreover, the gut is the first organ to be exposed to metals and other environmental xenobiotics taken up by insects and also defend against them, in addition, it also include many events such as an antioxidant effects, detoxfication, reformed of digestive enzyme activities, expression of different enzyme isoforms. (Wang et al. 2020).
Fat body of insects is analog with liver in vertebrates and it was known that xenobiotics mostly overaccumulate in this tissue. According to the results of this study, the implementation of metals as a mixture showed a synergistic effect and it has increased the accumulation in both tissues. When using metals singly, Zn levels increased in midgut and fat body. Moreover, Cu level increased in midgut whereas decreased in fat body. Long-term exposure to metals causes metals to accumulate in organisms. The accumulation of metals increases the formation of high superoxide radicals (O2−) or derivatives such as hydrogen peroxide (H2O2) (Gopi et al. 2019). SOD, CAT and GPx are the first defense antioxidant to neutralize any molecule that has the potential to become free radicals or any free radicals that can trigger the production of other radicals (Ighodaro and Akinloye 2018). SOD catalyzes the transformation of O2− to H2O2 and O2 (McCord and Fridovich 1969). H2O2 transforms into OH˙ (hydroxide) radical by Fenton and Haber Weiss reaction in the presence of metal ions (Gomes 2012). Catalase transforme toxic hydrogen peroxide generated as a result of superoxide dismutase activity into water and oxygen (Duthie 1989). GPx plays a important role in the process of preventing lipid peroxidation (Gill and Tuteja 2010). In this study, SOD activity was increased in the Cu and Zn groups in the midgut, while it decreased in the fat body of G. mellonella larvae compared to the control. CAT activity increased in both tissues in Cu applied groups compared to the control, while it decreased in the midgut in Zn applied group. It is thought that due to the high toxic effect of copper, the SOD produces excessive H2O2 and the CAT activity may have increased to eliminate the excessively produced H2O2. In M. posthuma, it was reported that different concentrations of CuSO4 and Cu NP caused a decrease in SOD activity and the highest decrease was occurred at the highest concentration (Gautam et al. 2018). Additionally, it was demonstrated that there was a significant increase in the CAT activity of S. littoralis fed with the extract of castor leaves treated with ZnO NPs and AgNPs. (Ibrahim and Ali 2018). Another study with G. mellonella demonstrated that larvae fed with increasing concentrations of Cr and Pb caused an increase in CAT and SOD enzyme activity compared to control (Wu and Yi 2015). In this study, a significant decrease in GPx activity was observed in all experimental groups compared to control. Moreover, it was observed that GPx activity in the group treated with Cu was lower than CAT and SOD enzymes. In addition GPx activity showed that an increase in Zn and mixture applications compared to SOD and CAT activity. Its known that activity of CAT is more effective in high concentrations of H2O2, while GPx activity is more effective in lower H2O2 concentrations (Duthie et al. 1989). Li et al. (2005) observed that in Oxya chinensis, GPx activity decreased due to increase in Cd concentration. Zinc is a cofactor of thousands of metalloenzyme and proteins (Weiss and Carver 2018) and also a crucial element, forming active sites in a wide range of enzymes and the zinc fingers of many DNA-binding proteins (Dow 2017). Additionally, although copper is an integral part of many vital enzymes, it can caused oxidative damage (Nikolic et al. 2016) and accumulation in tissues at increasing concentrations. In addition, it has been observed that antioxidant enzyme activities change according to the species, developmental stage and tissues.
In this study, CytP450 activity increased in the midgut when Cu and Zn applied singly, while GST activity increased only in the Cu applied group. In fat body, GST activity was increased in Cu and Zn applied groups, while CytP450 only increased in the Zn applied group compared to the control.
P450 enzymes are mainly involved in the first stage of xenobiotic metabolism, while GST enzymes are mainly involved in the second stage and responsible for the modification and conjugation of polar compounds. GST and Cyt P450 enzymes are found in the tissues of many organisms, generally in the liver. GST plays a role in protecting cellular integrity, preventing oxidative stress responses and DNA damage by catalyzing endogenous and exogenous xenobiotics, conjugation of glutathione with various electrophilic substrates (Casalino et al. 2004; Mao et al. 2019). In studies with different insect species, P450 enzyme activities were found to be at the highest levels, especially in the midgut and malpigi tubes (Tuncsoy 2017). In a study with Helicoverpa armigera, as a result of treatment of pentomethylbenzene and naphthalene, it was observed that the amount of increase in CytP450 activity was higher in the midgut compared to the fat body (Qiu et al. 2003). In this study, AChE enzyme activity in midgut has increased in both treatment groups compared with control, while in fat body increased in Zn treatment and reduced in Cu. In a study, it is thought that as a result of the toxicity of Pyriproxyfen and B. Thuringiensis, the increase in free radical formation and the decrease in AChE activity may result from the inactivation of this enzyme by free radicals. In addition, the authors commented that increased free radicals might have reduced the activity of enzymes by interacting with metal ions, which are cofactors of these enzymes, or with active amino acids in the enzyme structure (Tuncsoy 2018). In this study, it was observed that there was a significant decrease in the enzyme activities analyzed both in midgut and fat body of the larvae fed with the mixture applied diet. Also, when mixture grpup is compared with the other two metals, a decrease in enzyme activities in both tissues. Excessive production of ROS with the effect of increased metal accumulation in tissues may have caused deficiency of defense systems or inhibition of enzymes. Also, it is thought that these differences may be due to applied time, different size and concentration of metals (Benavides et al. 2016; İbrahim and Ali 2018; Gautam et al. 2018).
Innate immune parameters and antioxidant enzymes are used as indicators to determine the stress tolerance of invertebrates and environmental pollution. G. mellonella larvae and studies with other species showed that heavy metals accumulate in tissues and when reached the accumulation concentration critical levels, may cause toxic effects and also changes in antioxidant and detoxification enzymes, which are oxidative stress biomarker. Changes in such enzymes are used as biomarkers in determining the toxic effects of xenobiotics.