Investigation of the Effects of Silicon Dioxide Nanoparticles and Environmental Contaminants on Immunocytotoxic and Antioxidant Defence Systems in Model Organism Galleria mellonella L

Investigation of the Effects of Silicon Dioxide Nanoparticles and Environmental Contaminants on Immunocytotoxic and Antioxidant Defence Systems in Model Organism Galleria mellonella L | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of the Effects of Silicon Dioxide Nanoparticles and Environmental Contaminants on Immunocytotoxic and Antioxidant Defence Systems in Model Organism Galleria mellonella L Benay Tuncsoy, Murat Idikut, Mustafa Tuncsoy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6653945/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jun, 2025 Read the published version in Biological Trace Element Research → Version 1 posted 15 You are reading this latest preprint version Abstract Silicon dioxide (SiO₂) nanoparticles are chemically stable, biocompatible, abundant and inexpensive. Moreover, they have highly surface-reactive. These properties make them suitable for environmental applications but also raise questions about their reactivity with environmental contaminants. In the study, enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), cytochrome P450 (Cyt P450), glutathione-s-transferase (GST), acetylcholinesterase (AChE) and total, differential haemocyte counts and apoptotic index were investigated in haemolymph, midgut and fat body of Galleria mellonella exposed to LD 50 value of SiO 2 NP, environmental concentration of abamectin, cadmium sulphate (CdSO 4 ) and copper sulphate (CuSO 4 ) singly and in mixture. The total hemocyte count decreased in the SiO 2 and CdSO 4 singly applied groups however an increased were observed in the SiO 2 NPs + CdSO 4 mixture and a decreased were observed in the SiO 2 NPs + abamectin group compared with the control. As for differential hemocyte counts, prohemocytes, plasmatocytes, spherulocytes, granulocytes, and oenocytoids was altered following treatment with SiO 2 NPs, CdSO 4 , and abamectin singly and in mixture. As a result of this study, it was determined that SiO 2 NPs, CdSO 4 and Abamectin lead to toxic effects in G. mellonella larvae as a result of single and mixture applications and it was also observed that SiO 2 NPs may increase the toxic effects of environmental pollutants on antioxidant defence and immune system depending on tissue differences as a result of mixture applications. G. mellonella Silicon dioxide nanoparticle (SiO2 NPs) cadmium sulfate (CdSO4) and Abamectin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Nowadays, nanotechnology has led to significant advances in many fields such as materials, electronic devices, medical applications and energy production. The physicochemical features of NPs and nanomaterials, such as shape, physicochemical stability, chemical composition, size, surface energy surface area, crystal structure and surface, all have an effect on their toxicity indicators ( 1 ). Furthermore, although people are becoming more aware of the potential effects of nanoparticles on human health and the environment, their commercial use is increasing and concerns about their potential harmful effects are growing. Recently, several researches have focused on the toxicity impacts of diverse nanoparticles in order to discover different elements of nanotoxicity. Humans are continually exposed to a various of pollutants, including NPs, which are becoming more prevalent as a result of the rapid advancement of nanotechnologies and their unintentional or intentional discharge into the environment. Although these nanotechnological advances have enabled progress, their long-term effects on the environment and non-target organisms remain unclear. Moreover, their excessive use can lead to uncontrolled and excessive transfers to upper trophic levels ( 2 ). Due to their special properties at the nanoscale, nanoparticles can interact physicochemical with organic chemicals or metals in the environment, changing their bioavailability and also causing other different reactions, including synergistic, antagonistic and potentiating effects ( 3 ). The ability of nanoparticles to adsorb an environmental pollutant is critical to the toxicity of the NP or pollutant, as it facilitates the entry of the pollutant into the body, particularly through the absorption of nanoparticle-pollutant complexes. Complexed pollutants can be released into the body after entering the cell, raising their concentration and, as a result, bioavailability and toxicity. This process is known as the Trojan Horse effect ( 3 – 4 ). This idea encourages the entrance of hazardous substances adsorbed on NPs, causing an increase in intracellular pollutant concentration. In other cases, the complexes are effectively swallowed, and toxicity can be reduced if pollutants desorb from nanoparticle pollutant complexes in limited or partial amounts ( 4 ). Silica nanoparticles (SiO 2 NPs) are considered an appropriate material for biomedical applications and are utilized in biosensors, biomarkers, cancer therapy, and DNA/drug delivery ( 5 – 9 ) and is widely researched as enzyme immobilization ( 10 ). Although SiO 2 NPs are often regarded as biocompatible materials for biomedical and biotechnological applications, they can aggregate and remain in the kidney, heart, spleen, liver and brain following ingestion, inhalation, or skin application ( 11 ). It is thought that it may also have insecticidal effects by blocking the digestive system of the insect and causing malformation in its external morphology ( 12 ). The presence of organic and inorganic pollutants in the environment causes deterioration, a severe issue that endangers the global ecosystem ( 13 – 14 ). Environmental pollutants can be defined as substances that harm the environment as a result of human activities and natural consequences. These pollutants include heavy metals and pesticides. Cadmium has become a pollutant in the environment because of industrial expansion and modern advances in technology ( 15 ). The other material utilized in this study is abamectin. It is a natural substance derived from the soil bacterium Streptomyces avermitilis that is a widely used insecticide and anthelmintic. Abamectin affects the nervous system of insects, causing fatal consequences. It specifically targets glutamate-gated chloride channels and gamma-aminobutyric acid (GABA) gated chloride channels ( 16 ). Galleria mellonella larvae have an immune system that is similar to mammals' innate immune response, and in recent years, they have been employed as model organisms to study the virulence mechanisms of human pathogens ( 17 – 19 ). G. mellonella larvae have a wide surface area and are easy to isolate hemolymph, making them ideal for immunological research ( 20 ). They may be cultivated in great quantities in the laboratory without the need for specialized equipment ( 21 ). Furthermore, G. mellonella can live at temperatures ranging from 25 to 37°C, making it useful for infection investigations and allowing experiments to simulate mammalian systems (22; 23). Pollutant-nanoparticle interactions, as well as synergistic toxicity, are often overlooked. The goal of this research is to give a review of the combined toxicity of NPs and co-pollutants in order to emphasize the lack of evidence in the current literature and suggest that this issue deserves immediate attention. However, NPs released indiscriminately into the environment may interact with or absorb other pollutants on their surfaces, allowing them to enter the body ( 17 ). This may cause a significant alteration in the toxicological profile. As a result, while these interactions are difficult to characterize, they need serious consideration. 2. Materials and Methods 2.1. Materials The following materials were purchased from Sigma Aldrich (St. Louis, MO, USA): SiO 2 NPs (nanopowder (spherical, porous), 5–20 nm particle size (TEM), 99.5% trace metals basis), CdSO 4 (≥ 99.99% trace metals basis), 4-nitroanisole (PNOD), 3,4-dihydroxy L-phenylalanine (L-DOPA), Brillant Blue R Concentrate, entellan, ethidium bromide, Triton and CDNB. Giemsa's Azure Eosine and Methylene Blue Solution was purchased from MERCK and abamectin was purchased from Syngenta (Agrimec®EC, Syngenta®; analytical standard). Sephadex® G-25 (PD10, Pharmacia) 2.2. Determining of LD 50 value G. mellonella larvae were reared at 30 ± 1°C, 65 ± 5% RH on a diet composed of bran, honey, glycerol, honeycomb and distilled water ( 23 ). Newly hatched larvae were reared through the last instar at the artificial diet. The last instar G. mellonella larvae were removed from the diet medium and separated into control and treatment groups. The larvae were injected with various concentrations of 50, 100, 250, 400, and 500 µg/ml SiO 2 NPs using a Hamilton injector to determine LD 50 value of SiO 2 NPs. Concentrations were determined according to previous studies. After treatment, the last instar larvae were placed in petri dishes ( n = 10). The total number of dead larvae in the treated groups was recorded 120 h after application of the SiO 2 NPs. Then, the LD 50 values of SiO 2 NPs for last instar of G. mellonella were determined using the probit analysis method by SPSS 21 statistical data software. 2.3. Experimental design and antioxidant enzyme activities For the experiment, LD 50 value of SiO 2 NPs (396 µg/ml ) , environmental concentration of CdSO₄ (10 µg/L), CuSO 4 (10 µg/L) and abamectin (Agrimec®EC, Syngenta®; analytical standard) (10 µg/L) were used singly and in mixtures. Experimental period was determined as 120h according to the LD 50 analysis. After the injection of SiO 2 NPs, CdSO 4 , CuSO 4 and abamectin singly and in mixtures, the larvae were placed in petri dishes for 120h. Then, they were put on ice for 2–3 minutes to slow their movements and cleaned with 95% ethyl alcohol. The larvae were dissected with micro scissors and the fat body and midgut were placed in Eppendorf tubes with cold homogenisation buffer (20 mM; pH 7.6) (Fig. 1 .) and homogenized at 4°C by Ultra Torrax. Fat body was homogenized with an ultrasonic homogenizer (Bandelin Sonoplus. HD 2070, Berlin, Germany) at 50 W, 40–50 s in homogenization buffer. The homogenates were centrifuged at 500×g for 15 min (+ 4°C) and supernatants recentrifuged at 12,000×g for 45 min (4°C) to participate the mitochondrial fraction. Cytosolic fraction was purified on a Sephadex® G-25 (PD10, Pharmacia) gel columns to remove low molecular weight proteins ( 34 ). The samples for biochemical assays were frozen − 80°C until use. The methods to be used for the determination of the antioxidant enzyme activities are described in Sezer Tuncsoy et al. ( 25 ). 2.4. GST activity GST activity was determined using the method developed by Habig et al. ( 26 ) based on conjugating CDNB with reduced glutathione ( 27 ). 2.5. AChE activity As for AChE activity, the midgut and fat body were homogenized on ice in five volumes of a Tris–HCl buffer (100 mM, pH 8.0) containing 10% Triton and centrifuged at 12,000 × g for 30 min (4°C). The AChE activity was assayed as described by Ellman et al. ( 28 ). 2.6. Cyt P450 activity 4-nitroanisole (PNOD) was utilized as a substrate to determine cytochrome P450 monooxygenase enzyme activity (29). 2.7. Phenoloxidase activity 8 µl of hemolymph and 400 µl of ice-cold phosphate buffered saline (PBS, pH 7.4) are mixed in an Eppendorf tube. Then, it was centrifuged at 10,000 g for 5 min at 4°C. The supernatant was mixed with 3,4-dihydroxy L-phenylalanine (L-DOPA) and the mixture was incubated for 20 minutes at 25°C. Then, read at 490 nm absorbance at 5 min intervals between 0 and 30 min in a UV spectrophotometer. The data obtained were determined as U/mg protein/min ( 30 ). 2.8. Protein content Protein content was measured according to the Bradford ( 31 ) method using bovine serum albumin as a substrate. 2.9. Total and Differential Hemocyte Counts (THC and DHC) After 120h, G. mellonella larvae were placed at -20°C for 15–20 seconds to stop their movements. The larvae were sterilised with 95% ethanol and cut from the first proleg to obtain hemolypmh. 4 µl of haemolymph was transferred to an Eppendorf tube containing 36 µl of anticoagulant (0.186 M NaCl, 0.017 M Na 2 EDTA, 0.098 M NaOH and 0.041 M citric acid, pH 4.5). 1:10 µL of the 10% diluted cell solution was added to the Neubauer haemocytometer. The haemocytes were counted using a Leica DM750 microscope and the count of haemocytes per ml of haemolymph was determined using the Jones method ( 32 ). As for DHC, 5 µl hemolymph was collected using a micropipette, then spread on slides and led to dry at room temperature. Air dried smear was then fixed in neat alcohol for 10 min, stained with Giemsa stain (MERCK Giemsa's Azure Eosin and Methylene Blue Solution) and mounted in Entellan (Sigma Aldrich, Darmstadt, Germany). The hemocyte types were identified and counted using a Leica DM750 microscope. 2.10. Apoptotic Index The larvae were held at -20°C for 15–20 seconds to inhibit movement for dissection of hemolypmph. After sterilization with 95% ethanol, 5 µl of the hemolymph was collected into the Eppendorf tubes contained 5 µl AO and 5 µl EB and mixed thoroughly. Then, 5 µl of the mixture was applied to slides cleaned with 70% ethyl alcohol. Slides were allowed to dry for 1–2 minutes before examination under a fluorescence microscope under a blue filter ( 33 ). 2.11. Data analysis Statistical data were compared between the control and experimental groups using the Student Newman Keul's (SNK) test in the SPSS 21 programme. A p-value of < 0.05 is considered statistically significant. 3. Results 3.1. Antioxidant Enzyme Activities The effects of SiO 2 NPs and CdSO 4 singly and in mixtures on the CAT, SOD and GPx enzyme activities in the fat body and midgut of G. mellonella are presented in Fig. 2 . There was an increase in CAT activity in the midgut in the mixture applied group according to the control and this increase was found to be statistically significant (37.85-fold) (p < 0.05). As for fat body, exposure to CdSO 4 and mixture resulted in a 1.94-fold and 1.55-fold decreases, respectively, while an increase occurred in the SiO 2 NPs applied group (1.22-fold) (p < 0.05). There was a significant decrease in SOD activity in the midgut in the SiO 2 NPs applied group compared to the control (1.25-fold; p < 0.05), while increases were observed in the CdSO 4 and mixture applied groups. This increases resulted in 1.5-fold and 1.7-fold, respectively (p < 0.05). As for fat body, a decrease in SOD activity was observed in the SiO 2 NPs applied group compared to the control, but increases were detected in the CdSO 4 and mixture applied groups (1.44-fold and 1.24-fold, respectively) (p < 0.05). As for GPx activity, there was a decrease in GPx activity in the midgut in the SiO 2 NPs applied groups compared to the control (2.13-fold), but an increase was observed in the mixture applied group (2.9-fold) (p < 0.05). As for fat body, a significant decrease in GPx activity was only detected in the CdSO 4 applied group compared to the control (1.76-fold) (p < 0.05). The effects SiO 2 NPs and CuSO 4 singly and in mixtures on CAT, SOD and GPx enzyme activities in the fat body and midgut of G. mellonella are presented in Fig. 3 . While no statistical difference was observed in SOD activity in the midgut and fat body in the SiO 2 NPs and CuSO 4 applied groups compared to the control (p > 0.05), a significant increase was observed in the mixture applied groups (3.03 fold and 2.78 fold, respectively; p < 0.05). As for CAT activity, an increase was observed in the midgut of all treatment groups compared to the control (1.39, 1.12, 1.20 fold, respectively; p < 0.05). In fat body, 1.26 and 1.62-fold increase in CAT enzyme activity was observed in SiO 2 NPs and mixture applied groups, respectively (p < 0.05). In GPx activity, while there was a decrease in enzyme activity in the CuSO 4 singly applied group in the midgut, a 1.19-fold increase was detected in the mixture applied group compared to the control (p < 0.05). In fat body, 1.47 and 1.40-fold increases in GPx enzyme activity was observed in SiO 2 NPs singly and mixture applied groups, respectively (p < 0.05). The effects SiO 2 NPs and Abamectin singly and in mixtures on CAT, SOD and GPx enzyme activities in the fat body and midgut of G. mellonella are presented in Fig. 4 . there was an increase in CAT activity in the midgut in the mixture applied group according to the control and this increase was found to be statistically significant (33.53-fold) (p < 0.05). The decreases in CAT activity in fat body was observed in the Abamectin and mixture applied groups, this decrease resulted in a 2,15-fold and 1.18-fold, respectively (p < 0.05). There was a significant increase in SOD activity in the midgut in the abamectin applied group compared to the control (1.8-fold), while a decrease was observed in the mixture group (2.5-fold) (p < 0.05). As for fat body, decreases in SOD activity were detected in the abamectin and mixture applied groups compared to the control (1.66-fold and 1.53-fold, respectively) (p < 0.05). As for GPx activity, there was a significant increase in GPx activity in the midgut in the mixture applied group compared to the control (4.68-fold). As for fat body, significant decreases in GPx activity were detected in the Abamectin and mixture applied groups compared to the control (1.40-fold and 1.39-fold, respectively) (p < 0.05). 3.3. AChE Enzyme Activity The effects of SiO 2 NPs, CdSO 4 , abamectin singly and in mixtures on AChE enzyme activity in the midgut and fat body of G. mellonella are presented in Fig. 5 . Increases were observed in midgut of SiO 2 NPs, CdSO 4 and mixture applied groups of according to the control (4.51-fold, 1.92-fold and 4.51-fold, respectively) (p < 0.05) (Fig. 5 -A). As for fat body, a significantincrease was only detected in the mixture applied group and this increase resulted in 3.50-fold (p < 0.05) (Fig. 5 -B). In midgut of the abamectin applied groups, there is no significant differences (Fig. 5 -C). Otherwise, significant increases were observed when larvae exposed to SiO 2 NPs and abamectin singly and in mixture in fat body of the larvae (Fig. 5 -D). In midgut of SiO 2 NPs and CuSO 4 singly and in mixture applied groups, AChE activities were significantly decreased according to the control (1.65-fold, 3.31-fold, 5.63-fold, respectively) (Fig. 5 -E). Also, in fat body of the SiO 2 NPs and CuSO 4 mixture applied group, it was determined that AChE activity was significantly decreased according to the control (1.79-fold) (Fig. 5 -F) 3.4. GST Enzyme Activity The effects of SiO 2 NPs, CuSO 4 , CdSO 4 , abamectin singly and in mixtures on the GST enzyme activity in the midgut and fat body of G. mellonella are presented in Figs. 6 . Decreases in GST activity in the midgut were detected in all applied groups compared to the control (1.6-fold, 1.6-fold and 2-fold, respectively; p < 0.05). Also, decreases in fat body were observed in all applied groups compared to the control (1.57-fold, 1.27-fold and 3.36-fold, respectively; p < 0.05). Increases in GST activity in the midgut were detected in the abamectin singly and in mixture with SiO 2 NPs applied groups compared to the control (3.88-fold and 7.75-fold, respectively; p < 0.05). A decrease in GST activity in fat body was observed in abamectin applied groups compared to the control and this decrease resulted in 1.27-fold (p < 0.05). ın midgut of the SiO 2 NPs and CuSO 4 singly and in mixture applied groups, it was determined that GST activities were increased in all applied groups according to the control (7.65-fold, 1.39-fold, 14.3-fold, respectively), otherwise in fat body the enzyme activity was significantly increased only in SiO 2 NPs and mixture applied groups (8.88-fold and 3.54-fold) (p < 0.05). 3.5. Immunocytotoxic effects of SiO 2 NPs, CuSO 4 , CdSO 4 , abamectin singly and in mixtures 3.5.1. Total Hemocyte Count To evaluate the cytotoxic effects of SiO 2 NPs and its mixture with environmental pollutants (CdSO 4 , CuSO 4 and abamectin), we determined total and diffencial hemocyte count of G. mellonella hemocytes. SiO 2 NPs and CdSO 4 singly and in mixtures on total hemocyte counts of G. mellonella last instar larvae are shown in Fig. 7 . Accordingly, it was noted that there was a significant decrease in SiO 2 and CdSO 4 singly applied groups compared to the control. However, an increase was detected in the mixture applied group compared to the control (p < 0.05). As for the effects of SiO 2 NPs and abamectin singly and in mixtures on total hemocyte counts, significant decreases occurred in all applied groups compared to the control (p < 0.05). Further, THC was significantly increased when larvae exposed to CdSO 4 and in mixture with SiO 2 NPs (Fig. 7 ) (p < 0.05). 3.5.2. Differential Hemocyte Count The effects of SiO 2 NPs and CdSO 4 singly and their mixtures on the differential hemocyte count in the hemolymph of G. mellonella last instar larvae are shown in Fig. 8 -A. In plasmatocyte count, increases were observed in SiO 2 NPs and CdSO 4 singly applied groups, while a significant decrease was detected in mixture applied group (p < 0.05). As for prohemocyte count, decreases were observed in SiO 2 NPs and CdSO 4 singly applied groups, while a significant increase was detected in mixture applied group (p < 0.05). A decrease in granulocyte counts were observed in all applied groups compared to the control (p < 0.05). When the spherulocyte counts were compared to the control, there was an increase in the application groups of CdSO 4 and mixture, but a decrease was observed in the SiO 2 group. When the oenocytoid counts were examined, an increase was detected in the CdSO 4 applied group compared to the control (p < 0.05). The effects of SiO 2 NPs and abamectin singly and their mixtures on the differential hemocyte counts in the hemolymph of G. mellonella last instar larvae are shown in Fig. 8 -B. In plasmatocyte count, it was detected that increases were observed in all applied groups compared to the control, otherwise in prohemocyte count decreases were detected in all applied groups (p < 0.05). Also, decreases were observed in granulocyte count in all applied groups (p < 0.05). On the other hand, in spherulocyte count there are increases in abamectin and mixture groups, while spehurolcyte count decreased in SiO 2 NPs applied groups (p < 0.05). In oenocytoid count, increases were only detected in abamectin and mixture groups (p < 0.05). In addition, it was noted that prohemocyte number was increased when larvae exposed to SiO 2 NPs in mixture with CuSO 4 , however it was determined that plasmatocytes and granulacytes were decreased. Further, it was found that spherulocyte number was increased when SiO 2 NPs applied in mixture with CuSO 4 . As for eunocytoid number, an increase occurred only in the CuSO 4 applied group (Fig. 8 -C) (p < 0.05). 3.5.3. Phenoloxidase Enzyme Activity in G. mellonella Larvae The effects of SiO 2 NPs and CdSO 4 singly and in mixture on the phenoloxidase enzyme activity in the hemolymph of G. mellonella last instar larvae are presented in Fig. 9 -A. In the study, increases occurred in all applied groups compared to the control and these increases resulted in 2.82-fold, 2.18-fold and 2.73-fold, respectively (p < 0.05). As for abamectin applied group, the increases were found in 2.82-fold, 2.60-fold and 3.13- fold, respectively (Fig. 9 -B). In addition, when larvae exposed to CuSO 4 singly and in mixture with SiO 2 NPs, phenoloxidase acitivities were also increased according to the control group (2.82-fold, 3.45- fold and 6.05-fold, respectively) (Fig. 9 -C) (p < 0.05). 3.5.4. Apoptotic Index Amount in Larvae In the hemolypmh of G. mellonella larvae, apoptotic index was determined according to the cells under microscope described below (Fig. 10 ); These cells are: Live cells. The nucleus is green, while the cytoplasm might be orange or red. Early apoptosis: The cell membrane remains intact, but chromatin condenses and fragments. Late apoptosis is sometimes known as secondary necrosis or apoptotic necrosis. Ethidium Bromide penetrates cells with compromised membrane integrity and turns the nucleus orange. Necrosis: the nucleus is orange. The effects of SiO 2 NPs and CdSO 4 sinlgy and in mixtures on the apoptotic index of G. mellonella are presented in Fig. 11 . There were decreases in the count of live cells and early apoptosis in all applied groups (SiO 2 , CdSO 4 , SiO 2 + CdSO 4 ) compared to the control (p < 0.05). Otherwise, significant increases in the late apoptosis was observed in all application groups compared to the control (Fig. 11 -A) (p < 0.05). As for abamectin groups, there were decreases in the count of live cells and early apoptosis in SiO 2 NPs and abamectin singly and in mixture groups compared to the control (p < 0.05). On the other hand, significant increases in the late apoptosis were observed in SiO 2 NPs and abamectin singly and in mixture groups compared to the control (Fig. 11 -B) (p < 0.05). In addition, significant decreases were observed in live cell and early apoptosis numbers in all applied groups according to the control, however it was determined that late apoptosis numbers were significantly increased in all applied groups (p < 0.05) (Fig. 11 -C). 4. Discussion In recent years, the unconscious use of nanoparticles and heavy metals in various environments, including industry and agriculture, has led to problems such as disruption of ecological balances and adverse effects on non-target organisms. Furthermore, these nanoparticles, heavy metals, pesticides, and their interactions have the potential to produce significant issues in organismal systems. In this study, the impacts of SiO 2 NPs, CdSO 4 , CuSO 4 and abamectin singly and in mixtures on the CAT, SOD and GPx, GST, and AChE enzyme activities in the midgut, and fat body as well as phenoloxidase activity, THC, DHC and apoptotic index in the hemolymph were investigated. In insects, xenobiotics such as nanoparticles, chemicals such as heavy metals, pesticides, some microorganisms and radiation that are taken into the body outside of normal metabolic activit ies cause an increase in free radicals and lead to oxidative stress. This stress causes damage to cellular components and interferes with cellular processes. Free radicals damage macromolecules in cells and activate their defensive mechanisms. SOD, CAT, and GPx are antioxidants that act as the first defence in neutralizing any molecule that has the potential to become a free radical or that might cause the generation of more radicals ( 35 ). In the study, it was detected that CAT activity increased in both mixture groups in midgut of the larvae. In fat body, CAT activity decreased apart from SiO 2 NPs applied group. When CuSO 4 was applied singly and in mixture with SiO 2 NPs in midgut of the larvae, it was determined that CAT activities were increased in both singly CuSO 4 and in mixture with SiO 2 NPs groups. On the other hand, in fat body, CAT activity was increased only in mixture applied group. As for the SOD activity in the midgut, it was found that there was a decrease in the groups where SiO 2 NPs were applied singly and in a mixture with abamectin, but an increase was observed in the groups where CdSO 4 and abamectin were applied singly and in the SiO 2 NPs + CdSO 4 mixture group. In fat body, it was also determined that SiO 2 NPs application decreased in SOD activity and increased in the groups in which CdSO 4 was applied singly and in mixture with SiO 2 NPs. When abamectin was applied singly and in mixture with SiO 2 NPs, it was determined that a decrease occurred in SOD activity in fat body. When CuSO 4 was applied singly and in mixture with SiO 2 NPs in midgut and fat body of the larvae, it was determined that only in mixture groups significant increases were observed. In GPx activity, it was determined that there was an increase in both groups in the midgut and a decrease in fat body. When CuSO 4 was applied singly and in mixture with SiO 2 NPs in midgut and fat body of the larvae, significant decreases in GPx activity were determined in both tissue of mixture applied groups. It is thought that the increased CAT activity may not have been sufficient to eliminate the H 2 O 2 formed as a result of the oxidative stress caused by the mixture application in the larvae, leading to an increase in GPx enzyme activity. In the previous studies, Tuncsoy et al. ( 27 ) found that TiO 2 and CuO NPs enhanced the total protein quantity and antioxidant enzyme activities in G. mellonella , indicating an increase in oxidative stress. In an another similar study, Emre ( 35 ) detected that when G. mellonella was exposed to an environmental pollutant and developed oxidative stress. These induced cellular stress and damage by raising intracellular reactive oxygen species (ROS) levels. They showed that heavy metals like CdSO 4 impair cellular redox equilibrium, resulting in increased oxidative stress. The GST enzymes are involved in the second step and are responsible for the modification and conjugation of polar compounds. GST plays a role in protecting cellular integrity, preventing oxidative stress reactions and DNA damage by catalysing endogenous and exogenous xenobiotics ( 36 ). In the present study, AChE enzyme activities were increased exposed to SiO 2 NPs and CdSO 4 singly and in mixture in the midgut and fat body of the larvae. Also, in the fat body, AChE enzyme activities increased in the SiO 2 NPs and Abamectin singly and in mixture applied groups. AChE activity was significantly reduced in all application groups of midgut and fat body when CuSO4 was applied alone and mixed with SiO2 NPs. As for GST activities, in all experimental groups GST activities were decreased apart from SiO 2 NPs and abamectin singly and in mixture applied groups. On the other hand, GST activity was found to be increased in all application groups of midgut and fat body exposed to CuSO 4 singly and in mixtures. As a result, it was observed that SiO 2 NPs increased the toxic effects of both abamectin, CdSO 4 and CuSO 4 , then led to alterations in detoxification enzymes, AChE and GST in the organism. Nanoparticles and heavy metals can accumulate in insects’ tissues, resulting in oxidative stress and cell membrane damage. It is known that environmental pollutants such as nanoparticles, pesticides and heavy metal can be found together in the environment and might be more hazardous if they were singly. Mese et al. ( 37 ), investigated the effects of Cu and Zn combinations on G. mellonella . Significant decreases in CAT activity were found in the Zn and mixture treated groups. The decreases were linked to the synergistic effects of metal combinations and elevated oxidative stress. Silica nanoparticles and other metal oxide nanoparticles can be incorporated into pesticide formulations, increasing their effectiveness in combating insects. These nanoparticles can kill insects by directly inflicting physical damage or by causing oxidative damage. Moreover, it was also detected that silica nanoparticles can induce synergistic effects when applied together with heavy metals. Guo et al. ( 38 ) determined that Cd accumulation in mouse liver increased as a result of Cd mixture with low concentrations of SiO 2 NPs and increased the hepatotoxic effect of cadmium. Moreover, Lu et al. ( 39 ) applied SiO 2 NPs and lead in a mixture and as a result, it was determined that while cellular oxidative stress and DNA damage did not occur in lung adrenocarcinoma (A549) cells at the non-toxic concentration of silica nanoparticles singly, when applied in a mixture with lead, oxidative stress and DNA damage in cells increased compared to the application of lead singly. As a result, they reported that the mixture application produced a synergistic effect. In an another study regarding synergistic effects of SiO 2 NPs, Yang et al. ( 40 ) reported that the effects of SiO 2 NPs and methyl mercury singly and in a mixture on human cardiac muscle cells (AC16) caused high toxic effects on cell viability and cell membrane damage. In addition, while ROS caused changes in MDA formation, it caused a decrease in SOD and GSH-Px activities. Moreover, they detected that it caused an increase in cellular apoptosis in heart muscle cells. Invertebrates have been used as an important model organism in toxicity studies, especially in recent years, due to their ability to be intermediate consumers in food chains. Mostly, the effects of environmental pollutants are measured by their effect on oxidative damage or mortality, but hemocytes are a more convenient tool. Hemocytes have a very important role in the immune system of invertebrates, and insect hemocytes have similar properties to blood cells in vertebrates, making them an important material for immunological studies ( 41 ). In this study, THC decreased when exposed to SiO 2 NPs, CdSO 4 and abamectin in singly. Nonetheless, it was determined that THC increased in the groups where CdSO 4 and abamectin were applied in mixture with SiO 2 NPs. By causing oxidative stress in the cells, both SiO 2 NPs, CdSO 4 and abamectin may have caused damage or death to the hemocytes. It was known that oxidative stress can cause damage to cell membranes, DNA and proteins and can lead to cell death. On the other hand, in the SiO 2 + CdSO 4 mixture group may have caused an increase in the count of hemocytes by increasing the activity of the immune system. This can be interpreted as the body's defence mechanism against toxic substances. Besides, when CuSO 4 applied both singly and in mixture with SiO 2 NPs, THC was significantly increased. Similar results were determined in our previous study when different concentrations of CuO NPs to the diet of G. mellonella , THC was significantly increased ( 37 ). As for DHC, it was determined that the count of plasmatocytes increased in the groups in which SiO 2 , CdSO 4 and abamectin were applied singly and in the groups in which abamectin was applied in mixture with SiO 2 NPs. On the other hand, it was analysed that plasmotocytes significantly diminished in the groups exposed to CuSO 4 singly and in mixture with SiO 2 NPs. Plasmatocytes are blood cells capable of phagocyte ( 42 ). It is thought that the increase in the count of plasmatocytes in these treated groups occurred in order to destroy foreign substances in the blood circulation. Moreover, it is thought that the reason for this increase is that plasmatocytes accumulate metals on the hemocoel wall with their ability to adhere, and this may be related to the blood cell's resistant effect against metals ( 43 ). Besides, the decrease in plasmotocytes when CuSO 4 applied, it might be due to the cellular lysis ( 44 ). It was also determined that the count of prohemocytes decreased in SiO 2 NPs and CdSO 4 applications singly, while the count of prohemocytes increased when applied as a mixture. This increase suggests that SiO 2 NPs increases the toxic effect of Cd and the count of prohemocytes, which are haematopoietic cells, increases for defence purposes. On the other hand, a decrease was observed in the groups in which abamectin was applied singly and in mixture with SiO 2 NP. In previous studies, it was reported that insecticides decreased the count of prohemocytes ( 45 ). It is thought that prohemocytes may have differentiated into plasmatocytes in order to phagocytise foreign substances entering the body and as a result, the percentage of prohemocytes in the blood circulation may have decreased (27; 46). In the study, decreasing prohemocyte and increasing plasmatocyte counts in the treatment groups support this. Granulocytes decreased in all applied groups. It is thought that this blood cell decreased because granulocytes are responsible for phagocytosis and can digest foreign substances taken in by hydrolytic enzymes. Besides, spherulocytes increased in all treatment groups. Although many comments have been made about the regulation of melanisation phagocytosis coagulation cell adhesion of spherulocytes, it has not been clarified yet ( 47 ). As for eunodytoids, it was determined that an increase occurred in the groups where CdSO 4 and abamectin were applied as a mixture with SiO 2 NPs. Eunocytoids contain the enzyme phenoloxidase, which is responsible for melanisation in the immune system. Studies have reported that phenoloxidase is synthesised in oenocytoids and released into plasma when they are lysed ( 48 ). It is suggested that the increase in oenocytoids in the groups treated with CdSO 4 and abamectin singly or in combination is due to the toxic effects of these substances. This is supported by the fact that the same application groups in the study showed an increase in phenoloxidase enzyme activity. In a study conducted by Çoğal et al. ( 49 ), it was determined that there was a visible decrease in the total hemocyte counts of G. mellonella last instar larvae applied with Al 2 O 3 NPs compared to the control group. Considering the important role of hemocytes in insect immunity, these results indicate that Al 2 O 3 NPs have suppressive effects on the immune system of G. mellonella . In addition, it was determined that there was a decrease in the count of granulocytes, prohemocytes, spherulocytes and oenocytoids, which are hemocyte types, while there was an increase in the count of plasmatocytes. It is thought that the reason for the increase in plasmatocytes is that these cells accumulate metals by adhering them to the hemocoel wall and tend to resist metals ( 50 ). Eskin ( 51 ) conducted a study with SiO 2 NPs and reported that it has a considerable influence on the insect's total hemocyte count and vitality. SiO₂ NPs were found to be 50% deadly at a dosage of 411.93 µg/10 µl. greater doses (100 and 180 µg/10 µl) resulted in considerably greater cell death rates than the control group. Adult growth time and longevity were considerably decreased in larvae fed low concentrations of SiO 2 NPs. Phenoloxidase (PO) is a typical metal enzyme, which requires metal ions as prosthetic groups to enable the full exertion of its activity and is essential for insect immunological system ( 52 ). It is known that physical injury or tissue damage can also stimulate phenoloxidase activity. In the event of an injury, the insect's immune system responds swiftly and activates defensive systems surrounding the affected region. This is linked to the insect's increased immunological response to speed up wound healing or cope with stress. Temperature fluctuations, environmental pollutants, and other stressors can all enhance phenoloxidase activity. Xu et al. ( 53 ) showed that ZnO NPs can trigger a protective immune response in G. mellonella . This response involves an increase in phenoloxidase activity. In an another research conducted by Wu and Yi ( 54 ), it was analysed that environmental pollutants such as chromium (Cr) and lead (Pb) have high toxicity on immune and antioxidant system of G. mellonella . Antioxidant enzymes (CAT, SOD, Peroxidase), THC and phenoloxidase activities were increased with increasing concentrations of dietary Cr and Pb. Based on our results, it can be concluded that in all mixture applied groups due to high levels of oenocytoids, phenoloxidase activities were increased. Thus, hemocytes may have disintegrated in the presence of SiO 2 NPs, abamectin and CuSO 4 , CdSO 4 and their mixtures, leading to the release of phenoloxidase enzyme into hemolymph. As for apoptotic index, there was a decrease in the count of living cells and early apoptosis in SiO 2 , CuSO 4 and CdSO 4 singly and in mixture applied groups compared to the control, and an increase in the count of late apoptosis was observed in the same groups. There was also a decrease in the count of live cells and early apoptosis in SiO 2 NPs and Abamectin singly and in mixture applied groups compared to the control, and an increase in the count of late apoptosis was observed in all application groups compared to the control. The reason for the gradual decrease in living cells is their transformation into early and late apoptosis due to the toxic effects of nanoparticles, heavy metals and pesticides. The reason for the gradual decrease in early apoptosis can be considered as its transformation into late apoptosis. In a research conducted by Eskin et al. ( 55 ) with CuO NPs, it was determined that there was an increase in apoptotic counts in G. mellonella and no necrotic death occurred. According to studies done with several insect species including G. mellonella larvae, blood cells in insects exposed to environmental contaminants such as nanoparticles, heavy metals, and pesticides change the morphological, histochemical, biochemical, and immunological defence systems. Knowing the observed toxic effect mechanism of silicon dioxide nanoparticle (SiO 2 NP), CdSO 4 , Abamectin, and their mixtures on insects is thought to allow the development of new chemical methods in the fight against harmful insects that have less adverse impact on non-target organisms and the environment. Due to the advancement of nanotechnologies, NPs are increasingly being employed and dispersed into the environment, either unintentionally or purposely. Consequently, humans may be exposed to increasingly large counts of these particles. Furthermore, NPs can enter the body through oral exposure as well as inhalation, and co-ingestion of NPs with other contaminants such as pesticides can also have a negative effect on human health, particularly on the gastrointestinal tract ( 56 – 58 ). Although they are constantly exposed to many pollution sources, the interactions between contaminants and the consequent cumulative toxicity have not received adequate attention in the literature. However, NPs discharged into the environment may interaction with other contaminants or absorb them on their surfaces, allowing them to enter the body ( 59 ). Even if the toxicity of individual compounds is well understood, it has been proposed that when such substances are combined, unanticipated detrimental impact may arise ( 60 – 61 ), implying a collective toxicity. A co-exposed contaminant may impact the cell membrane (in terms of fluidity, hydrophobicity, physical integrity or permeability,), increasing NP internalization and toxicity (3; 62). According to Deng et al. ( 63 ), NPs can be co-exposed to a wide range of compounds, including organic contaminants, metal/metalloid ions, inorganic ligands, and other NPs. The ability of NPs to adsorb a co-pollutant can have a significant impact on the toxicity of the NP or pollutant, particularly by promoting pollutant entrance into the body via the absorption of NPs-pollutant complexes. Once inside, complex contaminants can be released into the body, increasing their concentration and consequently bioavailability and toxicity. According to Lu et al. ( 39 ), harmful substances can be adsorbed on the surface of nanoparticles, allowing them to benefit from the carrier effect and reach the alveoli, causing more damage. Nonetheless, other pollutants can lessen NP toxicity by scavenging the ROS they create. Contaminants, on the other hand, have the potential to exacerbate the negative effects of nanoparticles by producing more ROS. ( 62 ). In conclusion, we determined that significant decreases and increases in the activities of Cyt P450, an important detoxifying enzyme, GST and AChE, enzymes responsible for neurotransmission, were detected in response to changes in the amount of silica nanoparticles, CdSO4 and abamectin, as well as CAT, SOD and GPx, indicators of oxidative stress. The use of sublethal doses in research is considered an ecologically acceptable method of pest control. The determination of LD 50 values helps to prevent the overuse of nanoparticles, leading to better results. Using nanoparticles with low LD 50 values in combination has several advantages, including minimising air pollution, slowing down the development of resistance and reducing costs. Although there were variations between tissues in the research, changes in antioxidant enzymes and detoxification enzymes were generally found as a result of the mixture. As a result, we concluded that SiO 2 NPs may be absorbed the CdSO 4 and abamectin and increased the toxicity of these environmental pollutants. Declarations Funding: Not applicable. Availability of data and materials : I do not have any research data outside the submitted manuscript file. Acknowledgements : Not applicable Author details : 1 Alparslan Turkes Science and Technology University, Engineering Faculty, Department of Bioengineering, Adana, Turkey, 2 Cukurova University, Science and Letter Faculty, Department of Biology, Adana, Turkey. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Murat İdikut and Mustafa Tuncsoy. The first draft of the manuscript was written by Murat Idikut and Benay Tuncsoy. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6653945","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":458633819,"identity":"820957b9-0ac6-471c-99cf-4540f2595dfa","order_by":0,"name":"Benay Tuncsoy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACAxiDH0IxE6GFDcqQbGCGamHDoxxFi8EBYrWYy/eYPfjxxyZx8/HzxyQYKqwTG+R7H+DVYtnGY27Y25aWuO1MMpsEw5n0xAY2dgO8WgyO8ZhJMzYcTtx2AKiFse0wUAsBl4G1MPw5nLi5/zFQyz+itbAdTtwgAbKlgQgtlm1pZZJAvxjPuPHY2CLhWLpxG1safi3mzIe3SQBDTLa/P/HhjQ811rL9zMfwa4EBxwYQmcBAOFrgwJ5YhaNgFIyCUTACAQDrEz6lbxMdpQAAAABJRU5ErkJggg==","orcid":"","institution":"Adana Alparslan Turkes Science and Technology University","correspondingAuthor":true,"prefix":"","firstName":"Benay","middleName":"","lastName":"Tuncsoy","suffix":""},{"id":458633820,"identity":"feee9ed4-474e-4184-8b32-afb70fe6ffad","order_by":1,"name":"Murat Idikut","email":"","orcid":"","institution":"Adana Alparslan Turkes Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Murat","middleName":"","lastName":"Idikut","suffix":""},{"id":458633824,"identity":"84c2048b-fffb-448d-b042-7cd86cded815","order_by":2,"name":"Mustafa Tuncsoy","email":"","orcid":"","institution":"Cukurova University","correspondingAuthor":false,"prefix":"","firstName":"Mustafa","middleName":"","lastName":"Tuncsoy","suffix":""}],"badges":[],"createdAt":"2025-05-13 09:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6653945/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6653945/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12011-025-04723-w","type":"published","date":"2025-06-26T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83119560,"identity":"6fda09d1-52b0-461b-9f68-cc1c74b85ab9","added_by":"auto","created_at":"2025-05-20 08:41:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":594137,"visible":true,"origin":"","legend":"\u003cp\u003eDissection of \u003cem\u003eG. mellonella\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/06acffbbe8d9619e9dcf5580.png"},{"id":83117849,"identity":"7042aca2-d227-4ccc-b93c-002216f5bbff","added_by":"auto","created_at":"2025-05-20 08:25:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90296,"visible":true,"origin":"","legend":"\u003cp\u003eCAT, SOD and GPx enzyme activities in the midgut and fat body of \u003cem\u003eG. mellonella \u003c/em\u003elarvae were exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly and in mixtures (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05)).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/bca305b90b8521981176fd73.png"},{"id":83117850,"identity":"6bffdf63-aa32-440d-96c4-b03faae00ed1","added_by":"auto","created_at":"2025-05-20 08:25:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87313,"visible":true,"origin":"","legend":"\u003cp\u003eCAT, SOD and GPx enzyme activities in the midgut and fat body of \u003cem\u003eG. mellonella \u003c/em\u003elarvae were exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs and CuSO\u003csub\u003e4\u003c/sub\u003e singly and in mixtures with SiO\u003csub\u003e2\u003c/sub\u003e NPs (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05)).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/2ade3875f2fd8a242a4444d1.png"},{"id":83118383,"identity":"b7a6ee96-a930-4329-b27b-f7173221a91d","added_by":"auto","created_at":"2025-05-20 08:33:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103114,"visible":true,"origin":"","legend":"\u003cp\u003eCAT, SOD and GPx enzyme activities in the midgut and fat body of \u003cem\u003eG. mellonella \u003c/em\u003elarvae were exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs and Abamectin singly and in mixtures with SiO\u003csub\u003e2\u003c/sub\u003e NPs (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/0471ca41b2009e8b00247fdf.png"},{"id":83117851,"identity":"888c5151-3a6b-4170-a1a6-56023d159bb7","added_by":"auto","created_at":"2025-05-20 08:25:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91913,"visible":true,"origin":"","legend":"\u003cp\u003eAChE enzyme activities in the midgut and fat body of \u003cem\u003eG. mellonella \u003c/em\u003elarvae were exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4 \u003c/sub\u003eand Abamectin singly and in mixtures with SiO\u003csub\u003e2\u003c/sub\u003e NPs (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05)).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/bf5c6ee418e25263b6acf125.png"},{"id":83117859,"identity":"1860c8fa-ece8-45a1-9811-79f973350d22","added_by":"auto","created_at":"2025-05-20 08:25:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":98964,"visible":true,"origin":"","legend":"\u003cp\u003eGST enzyme activities in the midgut and fat body of \u003cem\u003eG. mellonella \u003c/em\u003elarvae were exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4 \u003c/sub\u003eand abamectin singly and in mixtures with SiO\u003csub\u003e2\u003c/sub\u003e NPs (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05)).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/425287a6f3e92791665d224d.png"},{"id":83117855,"identity":"e400e649-39b7-4332-b15a-34f8e510bb5c","added_by":"auto","created_at":"2025-05-20 08:25:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":53802,"visible":true,"origin":"","legend":"\u003cp\u003eTotal hemocyte counts in the hemolymph of \u003cem\u003eG. mellonella\u003c/em\u003e larvae were exposed to SiO\u003csub\u003e2 \u003c/sub\u003eNPs, CdSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4 \u003c/sub\u003eand\u003csub\u003e \u003c/sub\u003eabamectin singly and in mixtures (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/b636db74f9438e2bde487f86.png"},{"id":83119562,"identity":"d789d15c-2ba8-4fa8-8a7a-2fbae346f812","added_by":"auto","created_at":"2025-05-20 08:41:33","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":125578,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential hemocyte counts in the hemolymph of \u003cem\u003eG. mellonella\u003c/em\u003e larvae were exposed to A. SiO\u003csub\u003e2 \u003c/sub\u003eNPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture B. SiO\u003csub\u003e2 \u003c/sub\u003eNPs and abamectin singly and in mixture C. SiO\u003csub\u003e2 \u003c/sub\u003eNPs and CuSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/edfab130982d69548c99db0c.png"},{"id":83117854,"identity":"bd1c8fca-46ac-4edd-8aef-0beb5e5eeda0","added_by":"auto","created_at":"2025-05-20 08:25:33","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":40100,"visible":true,"origin":"","legend":"\u003cp\u003ePhenoloxidase Activity in the hemolymph of \u003cem\u003eG. mellonella \u003c/em\u003elarvae were exposed to SiO\u003csub\u003e2 \u003c/sub\u003eNPs, CdSO\u003csub\u003e4\u003c/sub\u003e, CuSO4 and abamectin singly and in mixture abamectin singly and in mixtures (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05)).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/28a3f7b2a34d90a77f858dae.png"},{"id":83119565,"identity":"33aefe9e-fbfa-4334-a1df-05365c016d25","added_by":"auto","created_at":"2025-05-20 08:41:33","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":539400,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis in the hemolymph of \u003cem\u003eG. mellonella \u003c/em\u003elarvae\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/4c49864a548ee0242b6bf25c.png"},{"id":83118387,"identity":"3f94cc08-af86-4133-ab8e-066b1c6e7337","added_by":"auto","created_at":"2025-05-20 08:33:33","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":99149,"visible":true,"origin":"","legend":"\u003cp\u003eApoptotic Index in the hemolymph of \u003cem\u003eG. mellonella \u003c/em\u003elarvae were exposed to SiO\u003csub\u003e2 \u003c/sub\u003eNPs, CdSO\u003csub\u003e4\u003c/sub\u003e (A), abamectin (B) and CuSO\u003csub\u003e4\u003c/sub\u003e (C) singly and in mixtures (*SNK indicates that there is a statistically notable variation (P\u0026lt;0.05)).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/001f8e3be144a2c5e616e487.png"},{"id":85686349,"identity":"6ee3b771-ec48-4f62-9670-59c7c42e5088","added_by":"auto","created_at":"2025-06-30 16:05:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2838471,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6653945/v1/710c35f2-464a-4a71-9c71-771909b2cfe0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of the Effects of Silicon Dioxide Nanoparticles and Environmental Contaminants on Immunocytotoxic and Antioxidant Defence Systems in Model Organism Galleria mellonella L","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNowadays, nanotechnology has led to significant advances in many fields such as materials, electronic devices, medical applications and energy production. The physicochemical features of NPs and nanomaterials, such as shape, physicochemical stability, chemical composition, size, surface energy surface area, crystal structure and surface, all have an effect on their toxicity indicators (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Furthermore, although people are becoming more aware of the potential effects of nanoparticles on human health and the environment, their commercial use is increasing and concerns about their potential harmful effects are growing. Recently, several researches have focused on the toxicity impacts of diverse nanoparticles in order to discover different elements of nanotoxicity. Humans are continually exposed to a various of pollutants, including NPs, which are becoming more prevalent as a result of the rapid advancement of nanotechnologies and their unintentional or intentional discharge into the environment. Although these nanotechnological advances have enabled progress, their long-term effects on the environment and non-target organisms remain unclear. Moreover, their excessive use can lead to uncontrolled and excessive transfers to upper trophic levels (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Due to their special properties at the nanoscale, nanoparticles can interact physicochemical with organic chemicals or metals in the environment, changing their bioavailability and also causing other different reactions, including synergistic, antagonistic and potentiating effects (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The ability of nanoparticles to adsorb an environmental pollutant is critical to the toxicity of the NP or pollutant, as it facilitates the entry of the pollutant into the body, particularly through the absorption of nanoparticle-pollutant complexes. Complexed pollutants can be released into the body after entering the cell, raising their concentration and, as a result, bioavailability and toxicity. This process is known as the Trojan Horse effect (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). This idea encourages the entrance of hazardous substances adsorbed on NPs, causing an increase in intracellular pollutant concentration. In other cases, the complexes are effectively swallowed, and toxicity can be reduced if pollutants desorb from nanoparticle pollutant complexes in limited or partial amounts (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSilica nanoparticles (SiO\u003csub\u003e2\u003c/sub\u003e NPs) are considered an appropriate material for biomedical applications and are utilized in biosensors, biomarkers, cancer therapy, and DNA/drug delivery (\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) and is widely researched as enzyme immobilization (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Although SiO\u003csub\u003e2\u003c/sub\u003e NPs are often regarded as biocompatible materials for biomedical and biotechnological applications, they can aggregate and remain in the kidney, heart, spleen, liver and brain following ingestion, inhalation, or skin application (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). It is thought that it may also have insecticidal effects by blocking the digestive system of the insect and causing malformation in its external morphology (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe presence of organic and inorganic pollutants in the environment causes deterioration, a severe issue that endangers the global ecosystem (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Environmental pollutants can be defined as substances that harm the environment as a result of human activities and natural consequences. These pollutants include heavy metals and pesticides. Cadmium has become a pollutant in the environment because of industrial expansion and modern advances in technology (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The other material utilized in this study is abamectin. It is a natural substance derived from the soil bacterium \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e that is a widely used insecticide and anthelmintic. Abamectin affects the nervous system of insects, causing fatal consequences. It specifically targets glutamate-gated chloride channels and gamma-aminobutyric acid (GABA) gated chloride channels (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae have an immune system that is similar to mammals' innate immune response, and in recent years, they have been employed as model organisms to study the virulence mechanisms of human pathogens (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). \u003cem\u003eG. mellonella\u003c/em\u003e larvae have a wide surface area and are easy to isolate hemolymph, making them ideal for immunological research (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). They may be cultivated in great quantities in the laboratory without the need for specialized equipment (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Furthermore, \u003cem\u003eG. mellonella\u003c/em\u003e can live at temperatures ranging from 25 to 37\u0026deg;C, making it useful for infection investigations and allowing experiments to simulate mammalian systems (22; 23).\u003c/p\u003e \u003cp\u003ePollutant-nanoparticle interactions, as well as synergistic toxicity, are often overlooked. The goal of this research is to give a review of the combined toxicity of NPs and co-pollutants in order to emphasize the lack of evidence in the current literature and suggest that this issue deserves immediate attention. However, NPs released indiscriminately into the environment may interact with or absorb other pollutants on their surfaces, allowing them to enter the body (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). This may cause a significant alteration in the toxicological profile. As a result, while these interactions are difficult to characterize, they need serious consideration.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eThe following materials were purchased from Sigma Aldrich (St. Louis, MO, USA): SiO\u003csub\u003e2\u003c/sub\u003e NPs (nanopowder (spherical, porous), 5\u0026ndash;20 nm particle size (TEM), 99.5% trace metals basis), CdSO\u003csub\u003e4\u003c/sub\u003e (\u0026ge;\u0026thinsp;99.99% trace metals basis), 4-nitroanisole (PNOD), 3,4-dihydroxy L-phenylalanine (L-DOPA), Brillant Blue R Concentrate, entellan, ethidium bromide, Triton and CDNB. Giemsa's Azure Eosine and Methylene Blue Solution was purchased from MERCK and abamectin was purchased from Syngenta (Agrimec\u0026reg;EC, Syngenta\u0026reg;; analytical standard). Sephadex\u0026reg; G-25 (PD10, Pharmacia)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Determining of LD\u003csub\u003e50\u003c/sub\u003e value\u003c/h2\u003e \u003cp\u003e \u003cem\u003eG. mellonella\u003c/em\u003e larvae were reared at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH on a diet composed of bran, honey, glycerol, honeycomb and distilled water (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Newly hatched larvae were reared through the last instar at the artificial diet. The last instar \u003cem\u003eG. mellonella\u003c/em\u003e larvae were removed from the diet medium and separated into control and treatment groups. The larvae were injected with various concentrations of 50, 100, 250, 400, and 500 \u0026micro;g/ml SiO\u003csub\u003e2\u003c/sub\u003e NPs using a Hamilton injector to determine LD\u003csub\u003e50\u003c/sub\u003e value of SiO\u003csub\u003e2\u003c/sub\u003e NPs. Concentrations were determined according to previous studies. After treatment, the last instar larvae were placed in petri dishes (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10). The total number of dead larvae in the treated groups was recorded 120 h after application of the SiO\u003csub\u003e2\u003c/sub\u003e NPs. Then, the LD\u003csub\u003e50\u003c/sub\u003e values of SiO\u003csub\u003e2\u003c/sub\u003e NPs for last instar of \u003cem\u003eG. mellonella\u003c/em\u003e were determined using the probit analysis method by SPSS 21 statistical data software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design and antioxidant enzyme activities\u003c/h2\u003e \u003cp\u003eFor the experiment, LD\u003csub\u003e50\u003c/sub\u003e value of SiO\u003csub\u003e2\u003c/sub\u003e NPs (396 \u0026micro;g/ml\u003cb\u003e)\u003c/b\u003e, environmental concentration of CdSO₄ (10 \u0026micro;g/L), CuSO\u003csub\u003e4\u003c/sub\u003e (10 \u0026micro;g/L) and abamectin (Agrimec\u0026reg;EC, Syngenta\u0026reg;; analytical standard) (10 \u0026micro;g/L) were used singly and in mixtures. Experimental period was determined as 120h according to the LD\u003csub\u003e50\u003c/sub\u003e analysis. After the injection of SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4\u003c/sub\u003e and abamectin singly and in mixtures, the larvae were placed in petri dishes for 120h. Then, they were put on ice for 2\u0026ndash;3 minutes to slow their movements and cleaned with 95% ethyl alcohol. The larvae were dissected with micro scissors and the fat body and midgut were placed in Eppendorf tubes with cold homogenisation buffer (20 mM; pH 7.6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.) and homogenized at 4\u0026deg;C by Ultra Torrax. Fat body was homogenized with an ultrasonic homogenizer (Bandelin Sonoplus. HD 2070, Berlin, Germany) at 50 W, 40\u0026ndash;50 s in homogenization buffer. The homogenates were centrifuged at 500\u0026times;g for 15 min (+\u0026thinsp;4\u0026deg;C) and supernatants recentrifuged at 12,000\u0026times;g for 45 min (4\u0026deg;C) to participate the mitochondrial fraction. Cytosolic fraction was purified on a Sephadex\u0026reg; G-25 (PD10, Pharmacia) gel columns to remove low molecular weight proteins (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The samples for biochemical assays were frozen\u0026thinsp;\u0026minus;\u0026thinsp;80\u0026deg;C until use. The methods to be used for the determination of the antioxidant enzyme activities are described in Sezer Tuncsoy et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. GST activity\u003c/h2\u003e \u003cp\u003eGST activity was determined using the method developed by Habig et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) based on conjugating CDNB with reduced glutathione (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. AChE activity\u003c/h2\u003e \u003cp\u003eAs for AChE activity, the midgut and fat body were homogenized on ice in five volumes of a Tris\u0026ndash;HCl buffer (100 mM, pH 8.0) containing 10% Triton and centrifuged at 12,000 \u0026times; g for 30 min (4\u0026deg;C). The AChE activity was assayed as described by Ellman et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cyt P450 activity\u003c/h2\u003e \u003c/div\u003e\n\u003cp\u003e4-nitroanisole (PNOD) was utilized as a substrate to determine cytochrome P450 monooxygenase enzyme activity (29).\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Phenoloxidase activity\u003c/h2\u003e \u003cp\u003e8 \u0026micro;l of hemolymph and 400 \u0026micro;l of ice-cold phosphate buffered saline (PBS, pH 7.4) are mixed in an Eppendorf tube. Then, it was centrifuged at 10,000 g for 5 min at 4\u0026deg;C. The supernatant was mixed with 3,4-dihydroxy L-phenylalanine (L-DOPA) and the mixture was incubated for 20 minutes at 25\u0026deg;C. Then, read at 490 nm absorbance at 5 min intervals between 0 and 30 min in a UV spectrophotometer. The data obtained were determined as U/mg protein/min (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Protein content\u003c/h2\u003e \u003cp\u003eProtein content was measured according to the Bradford (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) method using bovine serum albumin as a substrate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Total and Differential Hemocyte Counts (THC and DHC)\u003c/h2\u003e \u003cp\u003eAfter 120h, \u003cem\u003eG. mellonella\u003c/em\u003e larvae were placed at -20\u0026deg;C for 15\u0026ndash;20 seconds to stop their movements. The larvae were sterilised with 95% ethanol and cut from the first proleg to obtain hemolypmh. 4 \u0026micro;l of haemolymph was transferred to an Eppendorf tube containing 36 \u0026micro;l of anticoagulant (0.186 M NaCl, 0.017 M Na\u003csub\u003e2\u003c/sub\u003eEDTA, 0.098 M NaOH and 0.041 M citric acid, pH 4.5). 1:10 \u0026micro;L of the 10% diluted cell solution was added to the Neubauer haemocytometer. The haemocytes were counted using a Leica DM750 microscope and the count of haemocytes per ml of haemolymph was determined using the Jones method (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). As for DHC, 5 \u0026micro;l hemolymph was collected using a micropipette, then spread on slides and led to dry at room temperature. Air dried smear was then fixed in neat alcohol for 10 min, stained with Giemsa stain (MERCK Giemsa's Azure Eosin and Methylene Blue Solution) and mounted in Entellan (Sigma Aldrich, Darmstadt, Germany). The hemocyte types were identified and counted using a Leica DM750 microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Apoptotic Index\u003c/h2\u003e \u003cp\u003eThe larvae were held at -20\u0026deg;C for 15\u0026ndash;20 seconds to inhibit movement for dissection of hemolypmph. After sterilization with 95% ethanol, 5 \u0026micro;l of the hemolymph was collected into the Eppendorf tubes contained 5 \u0026micro;l AO and 5 \u0026micro;l EB and mixed thoroughly. Then, 5 \u0026micro;l of the mixture was applied to slides cleaned with 70% ethyl alcohol. Slides were allowed to dry for 1\u0026ndash;2 minutes before examination under a fluorescence microscope under a blue filter (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Data analysis\u003c/h2\u003e \u003cp\u003eStatistical data were compared between the control and experimental groups using the Student Newman Keul's (SNK) test in the SPSS 21 programme. A p-value of \u0026lt;\u0026thinsp;0.05 is considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Antioxidant Enzyme Activities\u003c/h2\u003e \u003cp\u003eThe effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly and in mixtures on the CAT, SOD and GPx enzyme activities in the fat body and midgut of \u003cem\u003eG. mellonella\u003c/em\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. There was an increase in CAT activity in the midgut in the mixture applied group according to the control and this increase was found to be statistically significant (37.85-fold) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAs for fat body, exposure to CdSO\u003csub\u003e4\u003c/sub\u003e and mixture resulted in a 1.94-fold and 1.55-fold decreases, respectively, while an increase occurred in the SiO\u003csub\u003e2\u003c/sub\u003e NPs applied group (1.22-fold) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was a significant decrease in SOD activity in the midgut in the SiO\u003csub\u003e2\u003c/sub\u003e NPs applied group compared to the control (1.25-fold; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while increases were observed in the CdSO\u003csub\u003e4\u003c/sub\u003e and mixture applied groups. This increases resulted in 1.5-fold and 1.7-fold, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for fat body, a decrease in SOD activity was observed in the SiO\u003csub\u003e2\u003c/sub\u003e NPs applied group compared to the control, but increases were detected in the CdSO\u003csub\u003e4\u003c/sub\u003e and mixture applied groups (1.44-fold and 1.24-fold, respectively) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for GPx activity, there was a decrease in GPx activity in the midgut in the SiO\u003csub\u003e2\u003c/sub\u003e NPs applied groups compared to the control (2.13-fold), but an increase was observed in the mixture applied group (2.9-fold) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for fat body, a significant decrease in GPx activity was only detected in the CdSO\u003csub\u003e4\u003c/sub\u003e applied group compared to the control (1.76-fold) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effects SiO\u003csub\u003e2\u003c/sub\u003e NPs and CuSO\u003csub\u003e4\u003c/sub\u003e singly and in mixtures on CAT, SOD and GPx enzyme activities in the fat body and midgut of \u003cem\u003eG. mellonella\u003c/em\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. While no statistical difference was observed in SOD activity in the midgut and fat body in the SiO\u003csub\u003e2\u003c/sub\u003e NPs and CuSO\u003csub\u003e4\u003c/sub\u003e applied groups compared to the control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), a significant increase was observed in the mixture applied groups (3.03 fold and 2.78 fold, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for CAT activity, an increase was observed in the midgut of all treatment groups compared to the control (1.39, 1.12, 1.20 fold, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In fat body, 1.26 and 1.62-fold increase in CAT enzyme activity was observed in SiO\u003csub\u003e2\u003c/sub\u003e NPs and mixture applied groups, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In GPx activity, while there was a decrease in enzyme activity in the CuSO\u003csub\u003e4\u003c/sub\u003e singly applied group in the midgut, a 1.19-fold increase was detected in the mixture applied group compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In fat body, 1.47 and 1.40-fold increases in GPx enzyme activity was observed in SiO\u003csub\u003e2\u003c/sub\u003e NPs singly and mixture applied groups, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effects SiO\u003csub\u003e2\u003c/sub\u003e NPs and Abamectin singly and in mixtures on CAT, SOD and GPx enzyme activities in the fat body and midgut of \u003cem\u003eG. mellonella\u003c/em\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. there was an increase in CAT activity in the midgut in the mixture applied group according to the control and this increase was found to be statistically significant (33.53-fold) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The decreases in CAT activity in fat body was observed in the Abamectin and mixture applied groups, this decrease resulted in a 2,15-fold and 1.18-fold, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was a significant increase in SOD activity in the midgut in the abamectin applied group compared to the control (1.8-fold), while a decrease was observed in the mixture group (2.5-fold) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for fat body, decreases in SOD activity were detected in the abamectin and mixture applied groups compared to the control (1.66-fold and 1.53-fold, respectively) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for GPx activity, there was a significant increase in GPx activity in the midgut in the mixture applied group compared to the control (4.68-fold). As for fat body, significant decreases in GPx activity were detected in the Abamectin and mixture applied groups compared to the control (1.40-fold and 1.39-fold, respectively) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. AChE Enzyme Activity\u003c/h2\u003e \u003cp\u003eThe effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e, abamectin singly and in mixtures on AChE enzyme activity in the midgut and fat body of \u003cem\u003eG. mellonella\u003c/em\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Increases were observed in midgut of SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e and mixture applied groups of according to the control (4.51-fold, 1.92-fold and 4.51-fold, respectively) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-A). As for fat body, a significantincrease was only detected in the mixture applied group and this increase resulted in 3.50-fold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-B). In midgut of the abamectin applied groups, there is no significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-C). Otherwise, significant increases were observed when larvae exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs and abamectin singly and in mixture in fat body of the larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-D). In midgut of SiO\u003csub\u003e2\u003c/sub\u003e NPs and CuSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture applied groups, AChE activities were significantly decreased according to the control (1.65-fold, 3.31-fold, 5.63-fold, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-E). Also, in fat body of the SiO\u003csub\u003e2\u003c/sub\u003e NPs and CuSO\u003csub\u003e4\u003c/sub\u003e mixture applied group, it was determined that AChE activity was significantly decreased according to the control (1.79-fold) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-F)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. GST Enzyme Activity\u003c/h2\u003e \u003cp\u003eThe effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs, CuSO\u003csub\u003e4\u003c/sub\u003e, CdSO\u003csub\u003e4\u003c/sub\u003e, abamectin singly and in mixtures on the GST enzyme activity in the midgut and fat body of \u003cem\u003eG. mellonella\u003c/em\u003e are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Decreases in GST activity in the midgut were detected in all applied groups compared to the control (1.6-fold, 1.6-fold and 2-fold, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Also, decreases in fat body were observed in all applied groups compared to the control (1.57-fold, 1.27-fold and 3.36-fold, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Increases in GST activity in the midgut were detected in the abamectin singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs applied groups compared to the control (3.88-fold and 7.75-fold, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A decrease in GST activity in fat body was observed in abamectin applied groups compared to the control and this decrease resulted in 1.27-fold (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). ın midgut of the SiO\u003csub\u003e2\u003c/sub\u003e NPs and CuSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture applied groups, it was determined that GST activities were increased in all applied groups according to the control (7.65-fold, 1.39-fold, 14.3-fold, respectively), otherwise in fat body the enzyme activity was significantly increased only in SiO\u003csub\u003e2\u003c/sub\u003e NPs and mixture applied groups (8.88-fold and 3.54-fold) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Immunocytotoxic effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs, CuSO\u003csub\u003e4\u003c/sub\u003e, CdSO\u003csub\u003e4\u003c/sub\u003e, abamectin singly and in mixtures\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1. Total Hemocyte Count\u003c/h2\u003e \u003cp\u003eTo evaluate the cytotoxic effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs and its mixture with environmental pollutants (CdSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4\u003c/sub\u003e and abamectin), we determined total and diffencial hemocyte count of \u003cem\u003eG. mellonella\u003c/em\u003e hemocytes. SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly and in mixtures on total hemocyte counts of \u003cem\u003eG. mellonella\u003c/em\u003e last instar larvae are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Accordingly, it was noted that there was a significant decrease in SiO\u003csub\u003e2\u003c/sub\u003e and CdSO\u003csub\u003e4\u003c/sub\u003e singly applied groups compared to the control. However, an increase was detected in the mixture applied group compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for the effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs and abamectin singly and in mixtures on total hemocyte counts, significant decreases occurred in all applied groups compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Further, THC was significantly increased when larvae exposed to CdSO\u003csub\u003e4\u003c/sub\u003e and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2. Differential Hemocyte Count\u003c/h2\u003e \u003cp\u003eThe effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly and their mixtures on the differential hemocyte count in the hemolymph of \u003cem\u003eG. mellonella\u003c/em\u003e last instar larvae are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-A. In plasmatocyte count, increases were observed in SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly applied groups, while a significant decrease was detected in mixture applied group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for prohemocyte count, decreases were observed in SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly applied groups, while a significant increase was detected in mixture applied group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A decrease in granulocyte counts were observed in all applied groups compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When the spherulocyte counts were compared to the control, there was an increase in the application groups of CdSO\u003csub\u003e4\u003c/sub\u003e and mixture, but a decrease was observed in the SiO\u003csub\u003e2\u003c/sub\u003e group. When the oenocytoid counts were examined, an increase was detected in the CdSO\u003csub\u003e4\u003c/sub\u003e applied group compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs and abamectin singly and their mixtures on the differential hemocyte counts in the hemolymph of \u003cem\u003eG. mellonella\u003c/em\u003e last instar larvae are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-B. In plasmatocyte count, it was detected that increases were observed in all applied groups compared to the control, otherwise in prohemocyte count decreases were detected in all applied groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Also, decreases were observed in granulocyte count in all applied groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, in spherulocyte count there are increases in abamectin and mixture groups, while spehurolcyte count decreased in SiO\u003csub\u003e2\u003c/sub\u003e NPs applied groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In oenocytoid count, increases were only detected in abamectin and mixture groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, it was noted that prohemocyte number was increased when larvae exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs in mixture with CuSO\u003csub\u003e4\u003c/sub\u003e, however it was determined that plasmatocytes and granulacytes were decreased. Further, it was found that spherulocyte number was increased when SiO\u003csub\u003e2\u003c/sub\u003e NPs applied in mixture with CuSO\u003csub\u003e4\u003c/sub\u003e. As for eunocytoid number, an increase occurred only in the CuSO\u003csub\u003e4\u003c/sub\u003e applied group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-C) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.5.3. Phenoloxidase Enzyme Activity in \u003cem\u003eG. mellonella\u003c/em\u003e Larvae\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture on the phenoloxidase enzyme activity in the hemolymph of \u003cem\u003eG. mellonella\u003c/em\u003e last instar larvae are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e-A. In the study, increases occurred in all applied groups compared to the control and these increases resulted in 2.82-fold, 2.18-fold and 2.73-fold, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for abamectin applied group, the increases were found in 2.82-fold, 2.60-fold and 3.13- fold, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e-B). In addition, when larvae exposed to CuSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs, phenoloxidase acitivities were also increased according to the control group (2.82-fold, 3.45- fold and 6.05-fold, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e-C) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.5.4. Apoptotic Index Amount in Larvae\u003c/h2\u003e \u003cp\u003eIn the hemolypmh of \u003cem\u003eG. mellonella\u003c/em\u003e larvae, apoptotic index was determined according to the cells under microscope described below (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e);\u003c/p\u003e \u003cp\u003eThese cells are:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLive cells. The nucleus is green, while the cytoplasm might be orange or red.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eEarly apoptosis: The cell membrane remains intact, but chromatin condenses and fragments.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLate apoptosis is sometimes known as secondary necrosis or apoptotic necrosis. Ethidium Bromide penetrates cells with compromised membrane integrity and turns the nucleus orange.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eNecrosis: the nucleus is orange.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e sinlgy and in mixtures on the apoptotic index of \u003cem\u003eG. mellonella\u003c/em\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. There were decreases in the count of live cells and early apoptosis in all applied groups (SiO\u003csub\u003e2\u003c/sub\u003e, CdSO\u003csub\u003e4\u003c/sub\u003e, SiO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;CdSO\u003csub\u003e4\u003c/sub\u003e) compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Otherwise, significant increases in the late apoptosis was observed in all application groups compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e-A) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As for abamectin groups, there were decreases in the count of live cells and early apoptosis in SiO\u003csub\u003e2\u003c/sub\u003e NPs and abamectin singly and in mixture groups compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, significant increases in the late apoptosis were observed in SiO\u003csub\u003e2\u003c/sub\u003e NPs and abamectin singly and in mixture groups compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e-B) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, significant decreases were observed in live cell and early apoptosis numbers in all applied groups according to the control, however it was determined that late apoptosis numbers were significantly increased in all applied groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn recent years, the unconscious use of nanoparticles and heavy metals in various environments, including industry and agriculture, has led to problems such as disruption of ecological balances and adverse effects on non-target organisms. Furthermore, these nanoparticles, heavy metals, pesticides, and their interactions have the potential to produce significant issues in organismal systems. In this study, the impacts of SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4\u003c/sub\u003e and abamectin singly and in mixtures on the CAT, SOD and GPx, GST, and AChE enzyme activities in the midgut, and fat body as well as phenoloxidase activity, THC, DHC and apoptotic index in the hemolymph were investigated.\u003c/p\u003e \u003cp\u003eIn insects, xenobiotics such as nanoparticles, chemicals such as heavy metals, pesticides, some microorganisms and radiation that are taken into the body outside of normal metabolic activit\u003c/p\u003e \u003cp\u003eies cause an increase in free radicals and lead to oxidative stress. This stress causes damage to cellular components and interferes with cellular processes. Free radicals damage macromolecules in cells and activate their defensive mechanisms.\u003c/p\u003e \u003cp\u003eSOD, CAT, and GPx are antioxidants that act as the first defence in neutralizing any molecule that has the potential to become a free radical or that might cause the generation of more radicals (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In the study, it was detected that CAT activity increased in both mixture groups in midgut of the larvae. In fat body, CAT activity decreased apart from SiO\u003csub\u003e2\u003c/sub\u003e NPs applied group. When CuSO\u003csub\u003e4\u003c/sub\u003e was applied singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs in midgut of the larvae, it was determined that CAT activities were increased in both singly CuSO\u003csub\u003e4\u003c/sub\u003e and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs groups. On the other hand, in fat body, CAT activity was increased only in mixture applied group. As for the SOD activity in the midgut, it was found that there was a decrease in the groups where SiO\u003csub\u003e2\u003c/sub\u003e NPs were applied singly and in a mixture with abamectin, but an increase was observed in the groups where CdSO\u003csub\u003e4\u003c/sub\u003e and abamectin were applied singly and in the SiO\u003csub\u003e2\u003c/sub\u003e NPs\u0026thinsp;+\u0026thinsp;CdSO\u003csub\u003e4\u003c/sub\u003e mixture group. In fat body, it was also determined that SiO\u003csub\u003e2\u003c/sub\u003e NPs application decreased in SOD activity and increased in the groups in which CdSO\u003csub\u003e4\u003c/sub\u003e was applied singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs. When abamectin was applied singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs, it was determined that a decrease occurred in SOD activity in fat body. When CuSO\u003csub\u003e4\u003c/sub\u003e was applied singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs in midgut and fat body of the larvae, it was determined that only in mixture groups significant increases were observed. In GPx activity, it was determined that there was an increase in both groups in the midgut and a decrease in fat body. When CuSO\u003csub\u003e4\u003c/sub\u003e was applied singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs in midgut and fat body of the larvae, significant decreases in GPx activity were determined in both tissue of mixture applied groups. It is thought that the increased CAT activity may not have been sufficient to eliminate the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e formed as a result of the oxidative stress caused by the mixture application in the larvae, leading to an increase in GPx enzyme activity. In the previous studies, Tuncsoy et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) found that TiO\u003csub\u003e2\u003c/sub\u003e and CuO NPs enhanced the total protein quantity and antioxidant enzyme activities in \u003cem\u003eG. mellonella\u003c/em\u003e, indicating an increase in oxidative stress. In an another similar study, Emre (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) detected that when \u003cem\u003eG. mellonella\u003c/em\u003e was exposed to an environmental pollutant and developed oxidative stress. These induced cellular stress and damage by raising intracellular reactive oxygen species (ROS) levels. They showed that heavy metals like CdSO\u003csub\u003e4\u003c/sub\u003e impair cellular redox equilibrium, resulting in increased oxidative stress.\u003c/p\u003e \u003cp\u003eThe GST enzymes are involved in the second step and are responsible for the modification and conjugation of polar compounds. GST plays a role in protecting cellular integrity, preventing oxidative stress reactions and DNA damage by catalysing endogenous and exogenous xenobiotics (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In the present study, AChE enzyme activities were increased exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture in the midgut and fat body of the larvae. Also, in the fat body, AChE enzyme activities increased in the SiO\u003csub\u003e2\u003c/sub\u003e NPs and Abamectin singly and in mixture applied groups. AChE activity was significantly reduced in all application groups of midgut and fat body when CuSO4 was applied alone and mixed with SiO2 NPs. As for GST activities, in all experimental groups GST activities were decreased apart from SiO\u003csub\u003e2\u003c/sub\u003e NPs and abamectin singly and in mixture applied groups. On the other hand, GST activity was found to be increased in all application groups of midgut and fat body exposed to CuSO\u003csub\u003e4\u003c/sub\u003e singly and in mixtures. As a result, it was observed that SiO\u003csub\u003e2\u003c/sub\u003e NPs increased the toxic effects of both abamectin, CdSO\u003csub\u003e4\u003c/sub\u003e and CuSO\u003csub\u003e4\u003c/sub\u003e, then led to alterations in detoxification enzymes, AChE and GST in the organism.\u003c/p\u003e \u003cp\u003eNanoparticles and heavy metals can accumulate in insects\u0026rsquo; tissues, resulting in oxidative stress and cell membrane damage. It is known that environmental pollutants such as nanoparticles, pesticides and heavy metal can be found together in the environment and might be more hazardous if they were singly. Mese et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), investigated the effects of Cu and Zn combinations on \u003cem\u003eG. mellonella\u003c/em\u003e. Significant decreases in CAT activity were found in the Zn and mixture treated groups. The decreases were linked to the synergistic effects of metal combinations and elevated oxidative stress. Silica nanoparticles and other metal oxide nanoparticles can be incorporated into pesticide formulations, increasing their effectiveness in combating insects. These nanoparticles can kill insects by directly inflicting physical damage or by causing oxidative damage. Moreover, it was also detected that silica nanoparticles can induce synergistic effects when applied together with heavy metals. Guo et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) determined that Cd accumulation in mouse liver increased as a result of Cd mixture with low concentrations of SiO\u003csub\u003e2\u003c/sub\u003e NPs and increased the hepatotoxic effect of cadmium. Moreover, Lu et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) applied SiO\u003csub\u003e2\u003c/sub\u003e NPs and lead in a mixture and as a result, it was determined that while cellular oxidative stress and DNA damage did not occur in lung adrenocarcinoma (A549) cells at the non-toxic concentration of silica nanoparticles singly, when applied in a mixture with lead, oxidative stress and DNA damage in cells increased compared to the application of lead singly. As a result, they reported that the mixture application produced a synergistic effect. In an another study regarding synergistic effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs, Yang et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) reported that the effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs and methyl mercury singly and in a mixture on human cardiac muscle cells (AC16) caused high toxic effects on cell viability and cell membrane damage. In addition, while ROS caused changes in MDA formation, it caused a decrease in SOD and GSH-Px activities. Moreover, they detected that it caused an increase in cellular apoptosis in heart muscle cells.\u003c/p\u003e \u003cp\u003eInvertebrates have been used as an important model organism in toxicity studies, especially in recent years, due to their ability to be intermediate consumers in food chains. Mostly, the effects of environmental pollutants are measured by their effect on oxidative damage or mortality, but hemocytes are a more convenient tool. Hemocytes have a very important role in the immune system of invertebrates, and insect hemocytes have similar properties to blood cells in vertebrates, making them an important material for immunological studies (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). In this study, THC decreased when exposed to SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e and abamectin in singly. Nonetheless, it was determined that THC increased in the groups where CdSO\u003csub\u003e4\u003c/sub\u003e and abamectin were applied in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs. By causing oxidative stress in the cells, both SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e and abamectin may have caused damage or death to the hemocytes. It was known that oxidative stress can cause damage to cell membranes, DNA and proteins and can lead to cell death. On the other hand, in the SiO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;CdSO\u003csub\u003e4\u003c/sub\u003e mixture group may have caused an increase in the count of hemocytes by increasing the activity of the immune system. This can be interpreted as the body's defence mechanism against toxic substances. Besides, when CuSO\u003csub\u003e4\u003c/sub\u003e applied both singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs, THC was significantly increased. Similar results were determined in our previous study when different concentrations of CuO NPs to the diet of \u003cem\u003eG. mellonella\u003c/em\u003e, THC was significantly increased (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). As for DHC, it was determined that the count of plasmatocytes increased in the groups in which SiO\u003csub\u003e2\u003c/sub\u003e, CdSO\u003csub\u003e4\u003c/sub\u003e and abamectin were applied singly and in the groups in which abamectin was applied in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs. On the other hand, it was analysed that plasmotocytes significantly diminished in the groups exposed to CuSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs. Plasmatocytes are blood cells capable of phagocyte (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). It is thought that the increase in the count of plasmatocytes in these treated groups occurred in order to destroy foreign substances in the blood circulation. Moreover, it is thought that the reason for this increase is that plasmatocytes accumulate metals on the hemocoel wall with their ability to adhere, and this may be related to the blood cell's resistant effect against metals (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Besides, the decrease in plasmotocytes when CuSO\u003csub\u003e4\u003c/sub\u003e applied, it might be due to the cellular lysis (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). It was also determined that the count of prohemocytes decreased in SiO\u003csub\u003e2\u003c/sub\u003e NPs and CdSO\u003csub\u003e4\u003c/sub\u003e applications singly, while the count of prohemocytes increased when applied as a mixture. This increase suggests that SiO\u003csub\u003e2\u003c/sub\u003e NPs increases the toxic effect of Cd and the count of prohemocytes, which are haematopoietic cells, increases for defence purposes. On the other hand, a decrease was observed in the groups in which abamectin was applied singly and in mixture with SiO\u003csub\u003e2\u003c/sub\u003e NP. In previous studies, it was reported that insecticides decreased the count of prohemocytes (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). It is thought that prohemocytes may have differentiated into plasmatocytes in order to phagocytise foreign substances entering the body and as a result, the percentage of prohemocytes in the blood circulation may have decreased (27; 46). In the study, decreasing prohemocyte and increasing plasmatocyte counts in the treatment groups support this. Granulocytes decreased in all applied groups. It is thought that this blood cell decreased because granulocytes are responsible for phagocytosis and can digest foreign substances taken in by hydrolytic enzymes. Besides, spherulocytes increased in all treatment groups. Although many comments have been made about the regulation of melanisation phagocytosis coagulation cell adhesion of spherulocytes, it has not been clarified yet (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). As for eunodytoids, it was determined that an increase occurred in the groups where CdSO\u003csub\u003e4\u003c/sub\u003e and abamectin were applied as a mixture with SiO\u003csub\u003e2\u003c/sub\u003e NPs. Eunocytoids contain the enzyme phenoloxidase, which is responsible for melanisation in the immune system. Studies have reported that phenoloxidase is synthesised in oenocytoids and released into plasma when they are lysed (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). It is suggested that the increase in oenocytoids in the groups treated with CdSO\u003csub\u003e4\u003c/sub\u003e and abamectin singly or in combination is due to the toxic effects of these substances. This is supported by the fact that the same application groups in the study showed an increase in phenoloxidase enzyme activity. In a study conducted by \u0026Ccedil;oğal et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), it was determined that there was a visible decrease in the total hemocyte counts of \u003cem\u003eG. mellonella\u003c/em\u003e last instar larvae applied with Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e NPs compared to the control group. Considering the important role of hemocytes in insect immunity, these results indicate that Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e NPs have suppressive effects on the immune system of \u003cem\u003eG. mellonella\u003c/em\u003e. In addition, it was determined that there was a decrease in the count of granulocytes, prohemocytes, spherulocytes and oenocytoids, which are hemocyte types, while there was an increase in the count of plasmatocytes. It is thought that the reason for the increase in plasmatocytes is that these cells accumulate metals by adhering them to the hemocoel wall and tend to resist metals (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Eskin (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) conducted a study with SiO\u003csub\u003e2\u003c/sub\u003e NPs and reported that it has a considerable influence on the insect's total hemocyte count and vitality. SiO₂ NPs were found to be 50% deadly at a dosage of 411.93 \u0026micro;g/10 \u0026micro;l. greater doses (100 and 180 \u0026micro;g/10 \u0026micro;l) resulted in considerably greater cell death rates than the control group. Adult growth time and longevity were considerably decreased in larvae fed low concentrations of SiO\u003csub\u003e2\u003c/sub\u003e NPs.\u003c/p\u003e \u003cp\u003ePhenoloxidase (PO) is a typical metal enzyme, which requires metal ions as prosthetic groups to enable the full exertion of its activity and is essential for insect immunological system (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). It is known that physical injury or tissue damage can also stimulate phenoloxidase activity. In the event of an injury, the insect's immune system responds swiftly and activates defensive systems surrounding the affected region. This is linked to the insect's increased immunological response to speed up wound healing or cope with stress. Temperature fluctuations, environmental pollutants, and other stressors can all enhance phenoloxidase activity. Xu et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) showed that ZnO NPs can trigger a protective immune response in \u003cem\u003eG. mellonella\u003c/em\u003e. This response involves an increase in phenoloxidase activity. In an another research conducted by Wu and Yi (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), it was analysed that environmental pollutants such as chromium (Cr) and lead (Pb) have high toxicity on immune and antioxidant system of \u003cem\u003eG. mellonella\u003c/em\u003e. Antioxidant enzymes (CAT, SOD, Peroxidase), THC and phenoloxidase activities were increased with increasing concentrations of dietary Cr and Pb. Based on our results, it can be concluded that in all mixture applied groups due to high levels of oenocytoids, phenoloxidase activities were increased. Thus, hemocytes may have disintegrated in the presence of SiO\u003csub\u003e2\u003c/sub\u003e NPs, abamectin and CuSO\u003csub\u003e4\u003c/sub\u003e, CdSO\u003csub\u003e4\u003c/sub\u003e and their mixtures, leading to the release of phenoloxidase enzyme into hemolymph. As for apoptotic index, there was a decrease in the count of living cells and early apoptosis in SiO\u003csub\u003e2\u003c/sub\u003e, CuSO\u003csub\u003e4\u003c/sub\u003e and CdSO\u003csub\u003e4\u003c/sub\u003e singly and in mixture applied groups compared to the control, and an increase in the count of late apoptosis was observed in the same groups. There was also a decrease in the count of live cells and early apoptosis in SiO\u003csub\u003e2\u003c/sub\u003e NPs and Abamectin singly and in mixture applied groups compared to the control, and an increase in the count of late apoptosis was observed in all application groups compared to the control. The reason for the gradual decrease in living cells is their transformation into early and late apoptosis due to the toxic effects of nanoparticles, heavy metals and pesticides. The reason for the gradual decrease in early apoptosis can be considered as its transformation into late apoptosis. In a research conducted by Eskin et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) with CuO NPs, it was determined that there was an increase in apoptotic counts in \u003cem\u003eG. mellonella\u003c/em\u003e and no necrotic death occurred.\u003c/p\u003e \u003cp\u003eAccording to studies done with several insect species including \u003cem\u003eG. mellonella\u003c/em\u003e larvae, blood cells in insects exposed to environmental contaminants such as nanoparticles, heavy metals, and pesticides change the morphological, histochemical, biochemical, and immunological defence systems. Knowing the observed toxic effect mechanism of silicon dioxide nanoparticle (SiO\u003csub\u003e2\u003c/sub\u003e NP), CdSO\u003csub\u003e4\u003c/sub\u003e, Abamectin, and their mixtures on insects is thought to allow the development of new chemical methods in the fight against harmful insects that have less adverse impact on non-target organisms and the environment. Due to the advancement of nanotechnologies, NPs are increasingly being employed and dispersed into the environment, either unintentionally or purposely. Consequently, humans may be exposed to increasingly large counts of these particles. Furthermore, NPs can enter the body through oral exposure as well as inhalation, and co-ingestion of NPs with other contaminants such as pesticides can also have a negative effect on human health, particularly on the gastrointestinal tract (\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Although they are constantly exposed to many pollution sources, the interactions between contaminants and the consequent cumulative toxicity have not received adequate attention in the literature. However, NPs discharged into the environment may interaction with other contaminants or absorb them on their surfaces, allowing them to enter the body (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Even if the toxicity of individual compounds is well understood, it has been proposed that when such substances are combined, unanticipated detrimental impact may arise (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e), implying a collective toxicity. A co-exposed contaminant may impact the cell membrane (in terms of fluidity, hydrophobicity, physical integrity or permeability,), increasing NP internalization and toxicity (3; 62). According to Deng et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e), NPs can be co-exposed to a wide range of compounds, including organic contaminants, metal/metalloid ions, inorganic ligands, and other NPs. The ability of NPs to adsorb a co-pollutant can have a significant impact on the toxicity of the NP or pollutant, particularly by promoting pollutant entrance into the body via the absorption of NPs-pollutant complexes. Once inside, complex contaminants can be released into the body, increasing their concentration and consequently bioavailability and toxicity. According to Lu et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), harmful substances can be adsorbed on the surface of nanoparticles, allowing them to benefit from the carrier effect and reach the alveoli, causing more damage. Nonetheless, other pollutants can lessen NP toxicity by scavenging the ROS they create. Contaminants, on the other hand, have the potential to exacerbate the negative effects of nanoparticles by producing more ROS. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, we determined that significant decreases and increases in the activities of Cyt P450, an important detoxifying enzyme, GST and AChE, enzymes responsible for neurotransmission, were detected in response to changes in the amount of silica nanoparticles, CdSO4 and abamectin, as well as CAT, SOD and GPx, indicators of oxidative stress. The use of sublethal doses in research is considered an ecologically acceptable method of pest control. The determination of LD\u003csub\u003e50\u003c/sub\u003e values helps to prevent the overuse of nanoparticles, leading to better results. Using nanoparticles with low LD\u003csub\u003e50\u003c/sub\u003e values in combination has several advantages, including minimising air pollution, slowing down the development of resistance and reducing costs. Although there were variations between tissues in the research, changes in antioxidant enzymes and detoxification enzymes were generally found as a result of the mixture. As a result, we concluded that SiO\u003csub\u003e2\u003c/sub\u003e NPs may be absorbed the CdSO\u003csub\u003e4\u003c/sub\u003e and abamectin and increased the toxicity of these environmental pollutants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAvailability of data and materials\u003c/b\u003e: I do not have any research data outside the submitted manuscript file.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAcknowledgements\u003c/b\u003e: Not applicable\u003c/p\u003e \u003cp\u003e \u003cb\u003eAuthor details\u003c/b\u003e: \u003csup\u003e1\u003c/sup\u003eAlparslan Turkes Science and Technology University, Engineering Faculty, Department of Bioengineering, Adana, Turkey, \u003csup\u003e2\u003c/sup\u003eCukurova University, Science and Letter Faculty, Department of Biology, Adana, Turkey.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Murat İdikut and Mustafa Tuncsoy. The first draft of the manuscript was written by Murat Idikut and Benay Tuncsoy. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by Adana Alparslan Turkes Science and Technology University Scientific Research Coordination Unit. Project Number: 22103002.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthical Approval and consent to participate\u003c/b\u003e: No ethical approval is required for this study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConsent for publication\u003c/b\u003e: Not applicable.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCompeting interests\u003c/b\u003e: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGatoo, MA, Naseem, S, Arfat, MY, Mahmood Dar, A, Qasim, K, and Zubair, S (2014). Physicochemical properties of nanomaterials: implication in associated toxic manifestations. \u003cem\u003eBioMed research international\u003c/em\u003e, \u003cem\u003e2014\u003c/em\u003e(1), 498420.\u003c/li\u003e\n\u003cli\u003eCazenave, A., Hamlington, B., Horwath, M., Barletta, V. 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Toxicity to RAW264. 7 macrophages of silica nanoparticles and the E551 food additive, in combination with genotoxic agents. \u003cem\u003eNanomaterials\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(7), 1418.\u003c/li\u003e\n\u003cli\u003eShi, Y., Zhang, J. H., Jiang, M., Zhu, L. H., Tan, H. Q., and Lu, B. (2010). Synergistic genotoxicity caused by low concentration of titanium dioxide nanoparticles and p, p\u0026prime;‐DDT in human hepatocytes. \u003cem\u003eEnvironmental and Molecular Mutagenesis\u003c/em\u003e, \u003cem\u003e51\u003c/em\u003e(3), 192-204.\u003c/li\u003e\n\u003cli\u003eKhan, M., Khan, M. S. A., Borah, K. K., Goswami, Y., Hakeem, K. R., and Chakrabartty, I. (2021). 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Combined toxic exposures and human health: biomarkers of exposure and effect. \u003cem\u003eInternational journal of environmental research and public health\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(3), 629-647.\u003c/li\u003e\n\u003cli\u003eDeng, R., Lin, D., Zhu, L., Majumdar, S., White, J. C., Gardea-Torresdey, J. L., \u0026amp; Xing, B. (2017a). Nanoparticle interactions with co-existing contaminants: joint toxicity, bioaccumulation and risk. \u003cem\u003eNanotoxicology\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(5), 591-612.\u003c/li\u003e\n\u003cli\u003eDeng, Y., Zhang, Y., Lemos, B., and Ren, H. (2017b). Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. \u003cem\u003eScientific reports\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(1), 46687.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"G. mellonella, Silicon dioxide nanoparticle (SiO2 NPs), cadmium sulfate (CdSO4) and Abamectin","lastPublishedDoi":"10.21203/rs.3.rs-6653945/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6653945/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSilicon dioxide (SiO₂) nanoparticles are chemically stable, biocompatible, abundant and inexpensive. Moreover, they have highly surface-reactive. These properties make them suitable for environmental applications but also raise questions about their reactivity with environmental contaminants. In the study, enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), cytochrome P450 (Cyt P450), glutathione-s-transferase (GST), acetylcholinesterase (AChE) and total, differential haemocyte counts and apoptotic index were investigated in haemolymph, midgut and fat body of \u003cem\u003eGalleria mellonella\u003c/em\u003e exposed to LD\u003csub\u003e50\u003c/sub\u003e value of SiO\u003csub\u003e2\u003c/sub\u003e NP, environmental concentration of abamectin, cadmium sulphate (CdSO\u003csub\u003e4\u003c/sub\u003e) and copper sulphate (CuSO\u003csub\u003e4\u003c/sub\u003e) singly and in mixture.\u003c/p\u003e \u003cp\u003eThe total hemocyte count decreased in the SiO\u003csub\u003e2\u003c/sub\u003e and CdSO\u003csub\u003e4\u003c/sub\u003e singly applied groups however an increased were observed in the SiO\u003csub\u003e2\u003c/sub\u003e NPs\u0026thinsp;+\u0026thinsp;CdSO\u003csub\u003e4\u003c/sub\u003e mixture and a decreased were observed in the SiO\u003csub\u003e2\u003c/sub\u003e NPs\u0026thinsp;+\u0026thinsp;abamectin group compared with the control. As for differential hemocyte counts, prohemocytes, plasmatocytes, spherulocytes, granulocytes, and oenocytoids was altered following treatment with SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e, and abamectin singly and in mixture. As a result of this study, it was determined that SiO\u003csub\u003e2\u003c/sub\u003e NPs, CdSO\u003csub\u003e4\u003c/sub\u003e and Abamectin lead to toxic effects in \u003cem\u003eG. mellonella\u003c/em\u003e larvae as a result of single and mixture applications and it was also observed that SiO\u003csub\u003e2\u003c/sub\u003e NPs may increase the toxic effects of environmental pollutants on antioxidant defence and immune system depending on tissue differences as a result of mixture applications.\u003c/p\u003e","manuscriptTitle":"Investigation of the Effects of Silicon Dioxide Nanoparticles and Environmental Contaminants on Immunocytotoxic and Antioxidant Defence Systems in Model Organism Galleria mellonella L","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-20 08:25:28","doi":"10.21203/rs.3.rs-6653945/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-15T01:14:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T18:31:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T04:43:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-10T08:30:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-10T08:11:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306191592981169012219673195585198421586","date":"2025-06-10T07:48:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144852554346823495616627154329161152787","date":"2025-06-10T03:54:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65575973470578007072205173556508413967","date":"2025-06-09T20:13:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120220804760814117179345201765934025179","date":"2025-06-09T19:31:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17481735591053081534332886364770651567","date":"2025-05-19T10:04:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189162857537889349632952086257459048104","date":"2025-05-16T10:55:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-16T02:56:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-14T09:37:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-14T00:38:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Trace Element Research","date":"2025-05-13T09:30:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b8c73fbc-d87b-4f6c-8c09-fcc2a68d8267","owner":[],"postedDate":"May 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-30T16:04:53+00:00","versionOfRecord":{"articleIdentity":"rs-6653945","link":"https://doi.org/10.1007/s12011-025-04723-w","journal":{"identity":"biological-trace-element-research","isVorOnly":false,"title":"Biological Trace Element Research"},"publishedOn":"2025-06-26 15:56:51","publishedOnDateReadable":"June 26th, 2025"},"versionCreatedAt":"2025-05-20 08:25:28","video":"","vorDoi":"10.1007/s12011-025-04723-w","vorDoiUrl":"https://doi.org/10.1007/s12011-025-04723-w","workflowStages":[]},"version":"v1","identity":"rs-6653945","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6653945","identity":"rs-6653945","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}