Synergistic Effects of Zn, Cu, and Ni and Bacillus Thuringiensis On the Hemocyte Count and the Antioxidant Activities of Hyphantria Cunea Drury (Lepidoptera: Arctiidae) Larvae

Synergistic Effects of Zn, Cu, and Ni and Bacillus Thuringiensis On the Hemocyte Count and the Antioxidant Activities of Hyphantria Cunea Drury (Lepidoptera: Arctiidae) Larvae | 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 Synergistic Effects of Zn, Cu, and Ni and Bacillus Thuringiensis On the Hemocyte Count and the Antioxidant Activities of Hyphantria Cunea Drury (Lepidoptera: Arctiidae) Larvae Oğuzhan Yanar, Elif F. Topkara, Fatma G. Solmaz, Sevcan Mercan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-399638/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Oct, 2021 Read the published version in Ecotoxicology → Version 1 posted 4 You are reading this latest preprint version Abstract Insects are model organisms for immunological studies. The cellular and the antioxidant enzyme responses of insects are major bioindicators against environmental stresses (metal exposure, infection, etc.). In our study, the differences in the hemocyte counts and the antioxidant enzyme activities of Hyphantria cunea larvae exposed to the different amounts of zinc, copper, and nickel and Bacillus thuringiensis infection were determined. With metal exposure, the superoxide dismutase, catalase, and glutathione peroxidase activities increased, but the hemocyte counts decreased. Additionally, both the hemocyte counts and the enzyme activities increased with Bacillus thuringiensis infection. As a result of this study, we found that the superoxide dismutase, catalase, and glutathione peroxidase and the hemocyte counts varied in response to both metal exposure and bacterial infection. Environmental Policy Toxicology Clinical Pharmacology Antioxidant enzyme Heavy metal Hemocyte Hyphantria cunea Infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Metals formed as a result of the natural processes and the anthropogenic activities are among the most important causes of water, soil, and plant pollution. While low amounts are essential for life, they show toxic effects at high concentrations (Cabassi 2007). Therefore, the balance of metals in the environment is very crucial. Metals affect the growth rate and the survival of herbivores (Ali et al. 2019), as well as immune function (Borowska and Pyza 2011; Pagliara and Stabili 2012). However, they can cause oxidative stress by increasing the amount of reactive oxygen species (ROS) (Koivula and Eeva 2010) (Fig. 1). To prevent ROS damage, the living organisms have complex defence mechanisms that contain antioxidants (Howe and Schilmiller 2002). Antioxidant enzymes are crucial in removing ROS from biological systems. The main antioxidant enzymes in insects are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (Mittapalli et al. 2007). SOD converts the superoxide radicals into molecular oxygen and H 2 O 2 , while both CAT and GSH-Px convert H 2 O 2 to oxygen and water (Ma et al. 2017). Also, the hemocytes are essential headliners of the insect immune system occurring at the cellular level, so they are important indicators in determining the cellular immune response. Hyphantria cunea Drury (Lepidoptera: Arctiidae) is an extreme polyphagous insect (Firidin et al. 2008). Its high fecundity, short generation time, and high starvation resistance facilitate its spread and potential to damage crops (Xu et al. 2019). The insect is a significant pest in many parts of the world (Ji et al. 2003) and also causes a loss of many crops in Turkey. In our study, we have chosen the Bacillus thuringiensis subsp. kurstaki ( Btk ), which is the most widely used microbial control agent (Saruhan et al. 2014). In this study, we selected zinc (Zn), copper (Cu), and nickel (Ni) from the most common and studied metals (van Ooik and Rantala 2010; Cheruiyot et al. 2013) in nature. We aimed to investigate how B. thuringiensis infection affected both the hemocyte counts and the antioxidant enzyme activities of H. cunea larvae, which consumed diets containing metals at different amounts. Materials And Methods Obtaining larvae and preparing artificial diets From Çarşamba District of Samsun Province of Turkey, H. cunea larvae were collected in 2020. They brought to the laboratory were kept at 25±2°C and 70% humidity (16 h light/8 h dark) and were let to feed a control diet (CD), developed by Yamamoto (1969) until they reached the pupal stage. The larvae of the 2nd generation obtained from the 1st generation were used for the experiment. Zn and Cu used in the study were purchased from Sigma-Aldrich (Darmstadt, Germany) and Ni was purchased from Merck (Darmstadt, Germany). By the purpose of the research, various diets were prepared by adding 0.788 g L -1 , 1.576 g L -1 , and 2.364 g L -1 zinc, copper, and nickel. We used the metal amounts we determined in our previous study (Topkara and Yanar 2019). With the addition of zinc, copper, and nickel to the control diet, a total of 20 different diets were obtained (Table 1). Bacterial culture conditions Btk was used in larval infection. The strain was obtained from culture collection of microbiology laboratory at Karadeniz Technical University. The Btk was grown overnight at 30°C in nutrient broth (AppliChem, Darmstadt, Germany). The optical density of the growing culture was measured at a wavelength of 600 nm and set to OD 600 = 1.89 (Danismazoglu et al. 2012). For infected groups, 1 mL of the bacterial suspension at this density was sprayed onto artificial diets. Experimental setups For both the control and the infected groups, 100 larvae were used to determine the enzyme activities whereas 50 larvae were used to determine the hemocyte counts. The larvae in the control groups were fed control diet for five days, and then the hemolymph of the larvae were taken by cutting third legs of the larvae. After five days, 1 mL of Btk suspension was sprayed into the diet of the larvae to be infected and continued to be fed for two more days. Then, the hemolymph of the larvae was taken, enzyme analyses were performed, and the hemocytes counted. Giemsa staining The insect hemolymphs to be used in the hemocyte counting were placed in the Eppendorf tubes, and 10 µl of the hemolymph was spread on the each slide. After drying the hemolymphs spread on the slide, staining steps were started with Giemsa. After staining, the fully protected preparations were obtained, and the hemocytes were counted with a microscope. Enzyme analysis The hemolymph samples taken from the larvae were homogenized with an ultrasonic processor (VCX 130 Sonics, Newtown, CT, USA). The homogenates, 20 mL each, were centrifuged for 20 minutes at 15000 rpm a refrigerated centrifuge (model 3500, Kubota, Tokyo, Japan). Protein determination in the study was made according to the method of Lowry et al. (1951). For this process, the intensity of the color caused by amino acids in the side chain of the reduced copper and proteins by reducing the Folin-Phenol reagent was measured spectrophotometrically at 595 nm. Superoxide dismutase activity was determined by the method of Flohé and Ötting (1984) and the spectrophotometric method of McCord and Fridovich (1969). To determine the SOD activity, the reduction of cytochrome c by the xanthine/xanthine oxidase system was spectrophotometrically measured at 550 nm. While catalase activity was determined by the Lück (1963) method, glutathione peroxidase activity determination was carried out by the method of Lawrence and Burk (1976). CAT activity was determined spectrophotometrically with the decrease in 240 nm absorbance due to H 2 O 2 degradation. The GSH-Px activity was measured spectrophotometrically at 340 nm under the cofactor of glutathione reductase and NADPH in the reaction medium. A UV/Vis spectrophotometer (model T70, Pharma Test Apparatebau, Hainburg, Germany) was used to determine enzyme activities. Statistical analyses Two independent sample t-tests were used to determine the relationship between the hemocyte counts and the enzyme activities depending on the diet content. SPSS 21.0 software (IBM Corp., Armonk, NY, USA) was used for these tests. Results Hemocyte counts Among the control groups, while the lowest hemocyte count was found to be in the larvae fed on the T diet (1269 ± 3.3, t = -6.6, p < 0.001), the highest hemocyte count was obtained in the larvae fed on the control (A) diet (2519 ± 17.1, t = -6.7, p < 0.001). The hemocyte counts of all groups infected with bacteria increased compared to the control ones. Among the infected groups, the lowest hemocyte count was in the larvae fed on the U diet (1336 ± 9.5, t = -6.6, p < 0.05), and the highest was in the B diet group (2778 ± 34.5, t = -6.7, p < 0.001) (Fig. 2). Superoxide dismutase activities Among the control groups, the highest SOD activity was in the group containing 1.576 g L -1 Zn (222 ± 1.3, t = 7.6, p < 0.001), while the lowest activity was in the A diet group (125 ± 2.7, t = 7.9, p < 0.001). In infected groups, it was determined that the highest SOD activity was in the F diet group (236 ± 1.4, t = 7.6, p < 0.001) whereas the lowest activity was in the larvae fed on the U diet (141 ± 1.3, t = 3.4, p < 0.05) (Fig. 3). Catalase activities Among the control groups, while the lowest CAT activity was found to be in the larvae fed on the A diet (222 ± 2.7, t = 3.2, p < 0.05), the highest one was obtained in the larvae fed on the E diet (288 ± 1.5, t = 7.3, p < 0.001). Among the infected groups, the lowest CAT activity was in the U diet group (230 ± 1.0, t = -5, p < 0.001) and the highest one was in the F diet group (304 ± 1.6, t = 7.3, p < 0.001) (Fig. 4). Glutathione peroxidase activities Among the control groups, the highest GSH-Px activity was in the G diet group (109±1.8, t = 3.5, p < 0.05), the lowest activity was in the control diet group (68 ± 0.8, t = 8.2, p < 0.001). In the infected groups, the highest GSH-Px activity was 124±1.5, t = 8.3, p < 0.001 at the F diet group, while the lowest one was 77±0.7, t = 8.2, p < 0.001 at the B diet group (Fig. 5). Discussion Differences in the hemocyte counts of insects can be used to measure the immuno-suppressive or -stimulating effects (Fallon et al. 2011; Browne et al. 2013). Environmental contaminants (such as metals and insecticides) can induce structural abnormalities in the hemocytes and/or change their counts. Results obtained from studies with different species have shown that, as a result of contaminants, the hemocyte counts change (Renwrantz 1990; Anderson et al. 1992; Coles et al. 1994). In our study, among the control groups, the highest hemocyte count was found in the larvae fed on the control diet. Studies showed that the hemocyte counts decreased with nickel and copper added to the diet (Sun et al. 2010; Kara et al. 2020). In our study, the result that the hemocyte count of the larvae decreased (except M) with increasing amounts of zinc, copper, and nickel in the diet among the control groups was consistent with the results of these studies. It was shown in various studies that the hemocytes could be affected by pathogens (Anderson et al. 1992; Oubella et al. 1993). In our study, we found that with the application of Btk , the hemocyte counts of all groups increased compared to the controls; this result coincided with the study by Dubovskiy et al. (2008) found that B. thuringiensis increased cellular immune response in Galleria mellonella . This increase may be due to the fact that the hemocytes fight bacteria in different ways (phagocytosis, nodulation) in response to the infection. Since superoxide dismutase is an enzyme involved in the reduction of superoxide radicals (Ali et al. 2017), the increase in the activity of this antioxidant enzyme is an indicator of oxidative stress. In our study, SOD activities increased with the presence of metals added to the diet. This situation proved that metals caused oxidative stress and consequently increases in SOD activities occurred. In a study with Spodoptera littoralis larvae (Abd El-Wahab and Anwar 2014), it was found that zinc and copper nanoparticles significantly increased SOD activity. In our study, it was determined that the groups with the highest SOD activities were the groups containing zinc and copper, and this result was consistent with the mentioned above. The reason for this increase is that the presence of zinc and copper is essential for SOD activity because these metals are the catalytic and structural components of the SOD enzyme. It was found that enzyme activities increased at 1.576 g L -1 of all three metals compared to 0.788 g L -1 in both the control and the infected groups, but the activities decreased in groups with the maximum metal amount. Studies have found that the SOD activities of Drosophila simulans fly infected with Wolbachia and G. mellonella larvae infected with B. thuringiensis were higher than controls (Brennan et al. 2012; Sezer-Tunçsoy and Ozalp 2016). The result that we found in our study that the SOD activities of all groups infected with Btk were higher compared to their controls coincides with these results. Hydrogen peroxide (H 2 O 2 ) can transform into a highly reactive hydroxyl radical in the presence of reduced metal atoms. In this case, CAT efficiently converts H 2 O 2 to water and oxygen (Tasaki et al. 2017). In our study, it was found that the highest CAT activity among the control groups was found in the larvae fed with a diet containing 1.576 g L -1 zinc (E diet). Similar to superoxide dismutase activities, it was found that CAT activities peaked at 1.576 g L -1 of all three metals in both the control and the infected groups, but the activities decreased in the groups with the maximum metal amount. Compared to the control group, the increase in CAT activities in parallel with the SOD activities with the presence of metal is the evidence that the H 2 O 2 , which is formed as a result of SOD, is reduced by CAT, that is, these two enzymes work in a complementary manner. It was found that the hemolymph CAT activities of G. mellonella larvae infected with B. thuringiensis were higher compared to the control (Sezer-Tunçsoy and Ozalp 2016). This result was consistent with what we found that all groups infected with bacteria had high CAT activities compared to the controls (except U). Metals can alter various aspects of immune function (Brousseau et al. 2000). Glutathione can prevent damage to important cellular components caused by ROS such as metals (Pompella et al. 2003), so it is crucial for cells. Sezer-Tunçsoy et al. (2019) found that copper oxide nanoparticles increased GSH-Px activities of G. mellonella larvae compared to the control. In our study, it was determined that the enzyme activities increased with the addition of copper, zinc, and nickel to the diet compared to the control groups. We found that the GSH-Px activity level was lower than SOD, suggesting that CAT may have a priority role in scavenging H 2 O 2 than GSH-Px (Meng et al. 2009). In our study, it was also found that the infection increased the GSH-Px activities compared to the control groups. The result that G. mellonella larvae infected with B. thuringiensis had higher GSH-Px activity compared to control larvae (Sezer-Tuncsoy and Ozalp 2016) was consistent with what we found in our study. Conclusions Insects have been successfully used as bioindicators of environmental pollution in industrial and even urban areas. The immune system of insects protects themselves against invasive microorganisms, pathogens, and toxins (Kingsolver et al. 2013). Antioxidant enzymes like SOD, CAT, and GSH-Px play a crucial role in oxidative stress defences of cells by eliminating ROS. In our study, the cellular and the enzymatic responses of H. cunea were determined after exposure to zinc, copper, and nickel and the bacterial infection. It was found that the enzyme activities increased, but the hemocyte counts decreased with metal exposure. Besides, both the hemocyte counts and the enzyme activities increased with the bacterial infection. As a result, it was concluded that the hemocyte counts and the antioxidant enzymes of H. cunea were affected by metal exposure and bacterial infection. In this context, our study will shed light on immunological studies with other species. Declarations Funding : Not applicable. Conflicts of interest/Competing interests : Not applicable. Availability of data and material : The data generated and/or analyzed during the current study are available from the corresponding author. Code availability: Not applicable. Authors’ contributions: OY contributed to the study design; EFT, FGS, and SM perform the data analyzes. All authors helped write the manuscript. Ethics approval: Not applicable. Consent to participate : Not applicable. Consent for publication : Not applicable. Acknowledgements: We thank for Prof. Dr. Mahmut Bilgener for his contributions. References Abd El-Wahab RA, Anwar EM (2014) The effect of direct and indirect use of nanoparticles on cotton leaf worm, Spodoptera littoralis . Int J Chem Biol Sci 1:17–24 Ali A, Rashid MA, Huang QY, Lei CL (2017) Influence of UV-A radiation on oxidative stress and antioxidant enzymes in Mythimna separata (Lepidoptera: Noctuidae). Environ Sci Pollut Res 24(9):8392–8398. https://doi.org/10.1007/s11356-017-8514-7 Ali S, Ullah MI, Saeed MF, Khalid S, Saqib M, Arshad M, Afzal M, Damalas CA (2019) Heavy metal exposure through artificial diet reduces growth and survival of Spodoptera litura (Lepidoptera: Noctuidae). Environ Sci Pollut Res 26:14426–14434. https://doi.org/10.1007/s11356-019-04792-0 Anderson DP, Paynter KT, Burreson EM, Anderson DP, Paynter KT, Burreson EM (1992) Increased reactive oxygen intermediate production by hemocytes withdrawn from Crassostrea v irginica infected with Perkinsus marinus . Biol Bull 183:476–481. https://doi.org/10.2307/1542024 Borowska J, Pyza E (2011) Effects of heavy metals on insect immunocompetent cells. J Insect Physiol 57:760–770. https://doi.org/10.1016/j.jinsphys.2011.02.012 Brennan LJ, Haukedal JA, Earle JC, Keddie B, Harris HL (2012) Disruption of redox homeostasis leads to oxidative DNA damage in spermatocytes of Wolbachia -infected Drosophila simulans . Insect Mol Biol 21:510–520. https://doi.org/10.1111/j.1365-2583.2012.01155.x Brousseau P, Pellerin J, Morin Y, Cyr D, Blakley B, Boermans H, Fournier M (2000) Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure to heavy metals. Toxicology 142:145–156. https://doi.org/10.1016/S0300-483X(99)00165-1 Browne N, Heelan M, Kavanagh K (2013) An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes. Virulence 4:597–603. https://doi.org/10.4161/viru.25906 Cabassi E (2007) The immune system and exposure to xenobiotics in animals. Vet Res Commun 31:115–120. https://doi.org/10.1007/s11259-007-0074-8 Cheruiyot DJ, Boyd RS, Moar WJ (2013) Exploring lower limits of plant elemental defense by cobalt, copper, nickel, and zinc. J Chem Ecol 39:666–674. https://doi.org/10.1007/s10886-013-0279-y Coles JA, Fairly SR, Pipe RK (1994) The effects of fluoranthene on the immunocompetence of the common marine mussel, Mytilus edulis . J Aquat Toxicol 30:367–379. https://doi.org/10.1016/0166-445X(94)00051-4 Danismazoglu M, Demir İ, Sevim A, Demirbag Z, Nalcacioglu R (2012), An investigation on the bacterial flora of Agriotes lineatus (Coleoptera: Elateridae) and pathogenicity of the flora members. Crop P 40:1–7. https://doi.org/10.1016/j.cropro.2012.04.012 Dubovskiy IM, Martemyanov VV, Vorontsova YL, Rantala MJ, Gryzanova EV, Glupov VV (2008) Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae). Comp Biochem Phys C: Toxicology Pharmacology 148:1–5. https://doi.org/10.1016/j.cbpc.2008.02.003 Fallon JP, Reeves EP, Kavanagh K (2011) The Aspergillus fumigatus toxin fumagillin suppresses the immune response of Galleria mellonella larvae by inhibiting the action of haemocytes. Microbiology 157:1481–1488. https://doi.org/10.1099/mic.0.043786-0 Firidin B, Bilgener M, Yanar O, Altun N, Ince A (2008) The effect of nutritional quality of some plant’s leaf on the feeding and development of Hyphantria cunea (Drury) (Lepidoptera: Arctiidae). IJONAS 2:61–68 Flohé L, Ötting F (1984) Superoxide dismutase assays. Method Enzymol 105:93–104. https://doi.org/10.1016/s0076-6879(84)05013-8 Howe GA, Schilmiller AL (2002) Oxylipin metabolism in response to stress. Curr Opin Plant Biol 5:230–236. https://doi.org/10.1016/S1369-5266(02)00250-9 Ji R, Xie BY, Li XH, Gao ZX, Li DM (2003) Research progress on the invasive species, Hyphantria cunea . Entomol Knowledg 40:13–18 Kara A, Tunçsoy B, Özalp P (2020) Effects of Cu, Zn and its mixtures on hemocyte types and counts of Galleria mellonella L. larvae. (Lepidoptera: Pyralidae) J Anatolian Env Anim Sciences 5:45–51. https://doi.org/10.35229/jaes.649536 Kingsolver MB, Huang Z, Hardy RW (2013) Insect antiviral innate immunity: pathways, effectors, and connections. J Mol Biol 425:4921–4936. https://doi.org/10.1016/j.jmb.2013.10.006 Koivula MJ, Eeva T (2010) Metal-related oxidative stress in birds. Environ Pollut 158:2359–2370. https://doi.org/10.1016/j.envpol.2010.03.013 Lawrence RA, Burk RF (1976) Glutathione peroxidase activity in selenium deficient rat liver. Biochem Biophys Res Commun 71:952–958. https://doi.org/10.1016/0006-291x(76)90747-6 Lowry OH, Rosebrough NT, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275 Lück H (1963) Catalase. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Weinheim and Academic Press, New York, pp 885–888 Ma X, Deng D, Chen W (2017) Inhibitors and activators of SOD, GSH-Px, and CAT. In: Şentürk M (ed) Enzyme inhibitors and activators. London, pp 207–224 McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic function for erithrocuprein (Hemocuprein). J Biol Chem 244:6049–6055 Meng JY, Zhang CY, Zhu F, Wang XP, Lei CL (2009) Ultraviolet light induced oxidative stress: effects on antioxidant response of Helicoverpa armigera adults. J Insect Physiol 55:588–592. https://doi.org/10.1016/j.jinsphys.2009.03.003 Mittapalli O, Neal JJ, Shukle RH (2007) Antioxidant defense response in a galling insect. Pnas 104:1889–1894. https://doi.org/10.1073/pnas.0604722104 Oubella R, Maes P, Paillard C, Auffret M (1993) Experimentally induced variation in hemocyte density for Ruditapes philippinarum and R . decussatus (Mollusca Bivalvia). Dis Aquat Org 15:193–197. https://doi.org/10.3354/dao015193 Pagliara P, Stabili L (2012) Zinc effect on the sea urchin Paracentrotus lividus immunological competence. Chemosphere 89:563–568. https://doi.org/10.1016/j.chemosphere.2012.05.052 Pompella A, Visvikis A, Paolicchi A, De Tata V, Casini AF ( 2003 ) The changing faces of glutathione, a cellular protagonist. Biochem Pharmacol 66:1499–1503. https://doi.org/10.1016/S0006-2952(03)00504-5 Renwrantz L (1990) Internal defence system of Mytilus edulis . In: Stefano GB (ed) Neurobiology of Mytilus edulis . Manchester University Press, Manchester, pp 256–275 Saruhan I, Akca I, Kushiyev R (2014) Toxicity of some biopesticides to the fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae). Egypt J Biol Pest Co 24:255–257 Sezer-Tuncsoy B, Ozalp P (2016) Combined effects of pyrıproxyfen and Bacillus thuringiensis on antioxidant activity of hemolymph, midgut and fat body of Galleria mellonella larvae. Fresenius Environ Bull 25:1660–1665 Sezer-Tuncsoy B, Tuncsoy M, Gomes T, Sousa V, Teixeira MR, Bebianno MJ, Ozalp P (2019) Effects of copper oxide nanoparticles on tissue accumulation and antioxidant enzymes of Galleria mellonella L. Bull Environ Contam Toxicol 102:341–346. https://doi.org/10.1007/s00128-018-2529-8 Sun HX, Xia Q, Tang WC, Zhou Q, Zhang GR, Dang Z (2010) Effects of dietary nickel on apoptosis of hemocytes of Spodoptera litura (Fabricius) larvae. Chin Sci Bull 55:390–397. https://doi.org/10.1007/s11434-009-0726-x Tasaki E, Kobayashi K, Matsuura K, Iuchi Y (2017) An efficient antioxidant system in a long-lived termite queen. Plos One 12(1):e0167412. https://doi.org/10.1371/journal.pone.0167412 Topkara EF, Yanar O (2019) Synergistic effects of some secondary compounds combined with some heavy metals on Hyphantria cunea Drury (Lepidoptera: Arctiidae) larvae. J Entomol Res Soc 21:85–95 van Ooik T, Rantala MJ (2010) Local adaptation of an insect herbivore to a heavy metal contaminated environment. Ann Zool Fenn 47:215–222. https://doi.org/10.5735/086.047.0306 Xu C, Wei H, Wang L, Yin T, Zhuge Q (2019) Optimization of the cry1Ah1 sequence enhances the hyper-resistance of transgenic poplars to Hyphantria cunea . Front Plant Sci 10:335. https://doi.org/10.3389/fpls.2019.00335 Yamamoto RT (1969) Mass rearing of tobacco hornworm. II. Larval rearing and pupation. J Econ Entomol 62:1427–1431. https://doi.org/10.1093/jee/62.6.1427 Tables Table 1 is not available with this version. Cite Share Download PDF Status: Published Journal Publication published 27 Oct, 2021 Read the published version in Ecotoxicology → Version 1 posted Reviewers invited by journal 21 Jun, 2021 Reviews received at journal 16 Apr, 2021 Editor invited by journal 04 Apr, 2021 First submitted to journal 04 Apr, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-399638","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":21917676,"identity":"1992177a-8f2a-4e5c-8a11-056432bfff60","order_by":0,"name":"Oğuzhan Yanar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYPACNjl+EJVQQLwWPmPJBpAWA+K1yCUaHADRxGjhl0h+9uDnHrME4/OrEz88MGCQ5xc7gF+L5Iw0c8OeZ2l5ZjfebpYAOsxw5uwE/FoMbiSYSfAcOFZsduPsBpCWBIPbBLWkf5P8c+B/4uYZZzf/IFJLjpk0zwG2xA38vduIs0Wy502ZtMwBNmOJG7zbLBIMJAj7hZ89fZvkmwPAqOw/u/nmjwobeX5pAloYBGAKJMAMCQLKwdYcQGeMglEwCkbBKEADAHE7RQ1PYYNJAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3457-0768","institution":"Ondokuz Mayıs University","correspondingAuthor":true,"prefix":"","firstName":"Oğuzhan","middleName":"","lastName":"Yanar","suffix":""},{"id":21917677,"identity":"12a42295-6672-4807-91e3-3b5d0b7239fc","order_by":1,"name":"Elif F. Topkara","email":"","orcid":"","institution":"Ondokuz Mayıs University","correspondingAuthor":false,"prefix":"","firstName":"Elif","middleName":"F.","lastName":"Topkara","suffix":""},{"id":21917678,"identity":"1bda678e-cc9b-4d7a-a717-02819b288b6c","order_by":2,"name":"Fatma G. Solmaz","email":"","orcid":"","institution":"Ondokuz Mayıs University","correspondingAuthor":false,"prefix":"","firstName":"Fatma","middleName":"G.","lastName":"Solmaz","suffix":""},{"id":21917679,"identity":"3ad38a4f-dec5-4816-98e0-895589dfebde","order_by":3,"name":"Sevcan Mercan","email":"","orcid":"","institution":"Ondokuz Mayıs University","correspondingAuthor":false,"prefix":"","firstName":"Sevcan","middleName":"","lastName":"Mercan","suffix":""}],"badges":[],"createdAt":"2021-04-06 19:45:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-399638/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-399638/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10646-021-02493-4","type":"published","date":"2021-10-27T14:13:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":8183855,"identity":"02ff41a1-791c-4917-a62b-272b57dd4bdd","added_by":"auto","created_at":"2021-04-19 19:40:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29958,"visible":true,"origin":"","legend":"Heavy metal toxicity for cells","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-399638/v1/3076b9da253f7747935ec945.png"},{"id":8183856,"identity":"51664a48-df18-4f5a-b55f-81d0ea88da09","added_by":"auto","created_at":"2021-04-19 19:40:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29279,"visible":true,"origin":"","legend":"Comparison of hemocyte counts of Hyphantria cunea larvae in the control and the infected groups. Data are expressed as mean ± S.E. Two independent samples t-test, p\u003c0.001","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-399638/v1/5ba21104314e10ed221ccb93.png"},{"id":8183236,"identity":"2ccf0ccc-b80e-4436-bd50-42eae6955b1e","added_by":"auto","created_at":"2021-04-19 19:37:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32686,"visible":true,"origin":"","legend":"Comparison of superoxide dismutase activities of Hyphantria cunea larvae in the control and the infected groups. Data are expressed as mean ± S.E. Two independent samples t-test, p \u003c0.001","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-399638/v1/c734de17ae1f0b8c35a75b30.png"},{"id":8183854,"identity":"5f5db52a-2f1e-4719-b030-85a2fbe4fbcc","added_by":"auto","created_at":"2021-04-19 19:40:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34968,"visible":true,"origin":"","legend":"Comparison of catalase activities of Hyphantria cunea larvae in the control and the infected groups. Data are expressed as mean ± S.E. Two independent samples t-test, p \u003c0.001","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-399638/v1/aaa8765df6dd06b516a185ab.png"},{"id":8183853,"identity":"00dd1f84-2fa4-46c5-a972-5bb7802dfca5","added_by":"auto","created_at":"2021-04-19 19:40:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29982,"visible":true,"origin":"","legend":"Comparison of glutathione peroxidase activities of Hyphantria cunea larvae in the control and the infected groups. Data are expressed as mean ± S.E. Two independent samples t-test, p \u003c0.001","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-399638/v1/c990c3edd55a84fbb39c00ea.png"},{"id":17204834,"identity":"42860a4b-3c11-4ced-ad92-80a6fb04f92c","added_by":"auto","created_at":"2022-01-11 14:13:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":429973,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-399638/v1/26f5b9e2-8c92-4e1c-9c40-4683c359ee4d.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSynergistic Effects of Zn, Cu, and Ni and Bacillus Thuringiensis On the Hemocyte Count and the Antioxidant Activities of Hyphantria Cunea Drury (Lepidoptera: Arctiidae) Larvae\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMetals formed as a result of the natural processes and the anthropogenic activities are among the most important causes of water, soil, and plant pollution. While low amounts are essential for life, they show toxic effects at high concentrations (Cabassi 2007). Therefore, the balance of metals in the environment is very crucial. Metals affect the growth rate and the survival of herbivores (Ali et al. 2019), as well as immune function (Borowska and Pyza 2011; Pagliara and Stabili 2012). However, they can cause oxidative stress by increasing the amount of reactive oxygen species (ROS) (Koivula and Eeva 2010) (Fig. 1). To prevent ROS damage, the living organisms have complex defence mechanisms that contain antioxidants (Howe and Schilmiller 2002). Antioxidant enzymes are crucial in removing ROS from biological systems. The main antioxidant enzymes in insects are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (Mittapalli et al. 2007). SOD converts the superoxide radicals into molecular oxygen and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, while both CAT and GSH-Px convert H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to oxygen and water (Ma et al. 2017). Also, the hemocytes are essential headliners of the insect immune system occurring at the cellular level, so they are important indicators in determining the cellular immune response.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHyphantria cunea\u003c/em\u003e Drury (Lepidoptera: Arctiidae) is an extreme polyphagous insect (Firidin et al. 2008). Its high fecundity, short generation time, and high starvation resistance facilitate its spread and potential to damage crops (Xu et al. 2019). The insect is a significant pest in many parts of the world (Ji et al. 2003) and also causes a loss of many crops in Turkey. In our study, we have chosen the \u003cem\u003eBacillus thuringiensis \u003c/em\u003esubsp.\u003cem\u003e kurstaki\u003c/em\u003e (\u003cem\u003eBtk\u003c/em\u003e), which is the most widely used microbial control agent (Saruhan et al. 2014). In this study, we selected zinc (Zn), copper (Cu), and nickel (Ni) from the most common and studied metals (van Ooik and Rantala 2010; Cheruiyot et al. 2013) in nature. We aimed to investigate how \u003cem\u003eB. thuringiensis\u003c/em\u003e infection affected both the hemocyte counts and the antioxidant enzyme activities of \u003cem\u003eH. cunea \u003c/em\u003elarvae, which consumed diets containing metals at different amounts.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eObtaining larvae and preparing artificial diets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom \u0026Ccedil;arşamba District of Samsun Province of Turkey, \u003cem\u003eH. cunea\u003c/em\u003e larvae were collected in 2020. They brought to the laboratory were kept at 25\u0026plusmn;2\u0026deg;C and 70% humidity (16 h light/8 h dark) and were let to feed a control diet (CD), developed by Yamamoto (1969) until they reached the pupal stage. The larvae of the 2nd generation obtained from the 1st generation were used for the experiment. Zn and Cu used in the study were purchased from Sigma-Aldrich (Darmstadt, Germany) and Ni was purchased from Merck (Darmstadt, Germany). By the purpose of the research, various diets were prepared by adding 0.788 g L\u003csup\u003e-1\u003c/sup\u003e, 1.576 g L\u003csup\u003e-1\u003c/sup\u003e, and 2.364 g L\u003csup\u003e-1\u003c/sup\u003e zinc, copper, and nickel. We used the metal amounts we determined in our previous study (Topkara and Yanar 2019). With the addition of zinc, copper, and nickel to the control diet, a total of 20 different diets were obtained (Table 1).\u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial culture conditions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBtk\u003c/em\u003e was used in larval infection. The strain was obtained from culture collection of microbiology laboratory at Karadeniz Technical University. The \u003cem\u003eBtk\u003c/em\u003e was grown overnight at 30\u0026deg;C in nutrient broth (AppliChem, Darmstadt, Germany). The optical density of the growing culture was measured at a wavelength of 600 nm and set to OD\u003csub\u003e600 \u003c/sub\u003e= 1.89 (Danismazoglu et al. 2012). For infected groups, 1 mL of the bacterial suspension at this density was sprayed onto artificial diets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental setups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;For both the control and the infected groups, 100 larvae were used to determine the enzyme activities whereas 50 larvae were used to determine the hemocyte counts. The larvae in the control groups were fed control diet for five days, and then the hemolymph of the larvae were taken by cutting third legs of the larvae. After five days, 1 mL of \u003cem\u003eBtk\u003c/em\u003e suspension was sprayed into the diet of the larvae to be infected and continued to be fed for two more days. Then, the hemolymph of the larvae was taken, enzyme analyses were performed, and the hemocytes counted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGiemsa staining\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe insect hemolymphs to be used in the hemocyte counting were placed in the Eppendorf tubes, and 10 \u0026micro;l of the hemolymph was spread on the each slide. After drying the hemolymphs spread on the slide, staining steps were started with Giemsa. After staining, the fully protected preparations were obtained, and the hemocytes were counted with a microscope.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The hemolymph samples taken from the larvae were homogenized with an ultrasonic processor (VCX 130 Sonics, Newtown, CT, USA). The homogenates, 20 mL each, were centrifuged for 20 minutes at 15000 rpm a refrigerated centrifuge (model 3500, Kubota, Tokyo, Japan). Protein determination in the study was made according to the method of Lowry et al. (1951). For this process, the intensity of the color caused by amino acids in the side chain of the reduced copper and proteins by reducing the Folin-Phenol reagent was measured spectrophotometrically at 595 nm. Superoxide dismutase activity was determined by the method of Floh\u0026eacute; and \u0026Ouml;tting (1984) and the spectrophotometric method of McCord and Fridovich (1969). To determine the SOD activity, the reduction of cytochrome c by the xanthine/xanthine oxidase system was spectrophotometrically measured at 550 nm. While catalase activity was determined by the L\u0026uuml;ck (1963) method, glutathione peroxidase activity determination was carried out by the method of Lawrence and Burk (1976). CAT activity was determined spectrophotometrically with the decrease in 240 nm absorbance due to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e degradation. The GSH-Px activity was measured spectrophotometrically at 340 nm under the cofactor of glutathione reductase and NADPH in the reaction medium. A UV/Vis spectrophotometer (model T70, Pharma Test Apparatebau, Hainburg, Germany) was used to determine enzyme activities.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwo independent sample t-tests were used to determine the relationship between the hemocyte counts and the enzyme activities depending on the diet content. SPSS 21.0 software (IBM Corp., Armonk, NY, USA) was used for these tests.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHemocyte counts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Among the control groups, while the lowest hemocyte count was found to be in the larvae fed on the T diet (1269 \u0026plusmn; 3.3, t = -6.6, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), the highest hemocyte count was obtained in the larvae fed on the control (A) diet (2519 \u0026plusmn; 17.1, t = -6.7, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The hemocyte counts of all groups infected with bacteria increased compared to the control ones. Among the infected groups, the lowest hemocyte count was in the larvae fed on the U diet (1336 \u0026plusmn; 9.5, t = -6.6, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), and the highest was in the B diet group (2778 \u0026plusmn; 34.5, t = -6.7, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuperoxide dismutase activities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Among the control groups, the highest SOD activity was in the group containing 1.576 g L\u003csup\u003e-1\u003c/sup\u003e Zn (222 \u0026plusmn; 1.3, t = 7.6, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), while the lowest activity was in the A diet group (125 \u0026plusmn; 2.7, t = 7.9, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). In infected groups, it was determined that the highest SOD activity was in the F diet group (236 \u0026plusmn; 1.4, t = 7.6, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) whereas the lowest activity was in the larvae fed on the U diet (141 \u0026plusmn; 1.3, t = 3.4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCatalase activities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Among the control groups, while the lowest CAT activity was found to be in the larvae fed on the A diet (222 \u0026plusmn; 2.7, t = 3.2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), the highest one was obtained in the larvae fed on the E diet (288 \u0026plusmn; 1.5, t = 7.3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Among the infected groups, the lowest CAT activity was in the U diet group (230 \u0026plusmn; 1.0, t = -5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and the highest one was in the F diet group (304 \u0026plusmn; 1.6, t = 7.3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlutathione peroxidase activities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Among the control groups, the highest GSH-Px activity was in the G diet group (109\u0026plusmn;1.8, t = 3.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), the lowest activity was in the control diet group (68 \u0026plusmn; 0.8, t = 8.2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). In the infected groups, the highest GSH-Px activity was 124\u0026plusmn;1.5, t = 8.3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 at the F diet group, while the lowest one was 77\u0026plusmn;0.7, t = 8.2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 at the B diet group (Fig. 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDifferences in the hemocyte counts of insects can be used to measure the immuno-suppressive or -stimulating effects (Fallon et al. 2011; Browne et al. 2013). Environmental contaminants (such as metals and insecticides) can induce structural abnormalities in the hemocytes and/or change their counts. Results obtained from studies with different species have shown that, as a result of contaminants, the hemocyte counts change (Renwrantz 1990; Anderson et al. 1992; Coles et al. 1994). In our study, among the control groups, the highest hemocyte count was found in the larvae fed on the control diet. Studies showed that the hemocyte counts decreased with nickel and copper added to the diet (Sun et al. 2010; Kara et al. 2020). In our study, the result that the hemocyte count of the larvae decreased (except M) with increasing amounts of zinc, copper, and nickel in the diet among the control groups was consistent with the results of these studies. It was shown in various studies that the hemocytes could be affected by pathogens (Anderson et al. 1992; Oubella et al. 1993). In our study, we found that with the application of \u003cem\u003eBtk\u003c/em\u003e, the hemocyte counts of all groups increased compared to the controls; this result coincided with the study by Dubovskiy et al. (2008) found that \u003cem\u003eB. thuringiensis\u003c/em\u003e increased cellular immune response in \u003cem\u003eGalleria mellonella\u003c/em\u003e. This increase may be due to the fact that the hemocytes fight bacteria in different ways (phagocytosis, nodulation) in response to the infection.\u003c/p\u003e\n\u003cp\u003eSince superoxide dismutase is an enzyme involved in the reduction of superoxide radicals (Ali et al. 2017), the increase in the activity of this antioxidant enzyme is an indicator of oxidative stress. In our study, SOD activities increased with the presence of metals added to the diet. This situation proved that metals caused oxidative stress and consequently increases in SOD activities occurred. In a study with \u003cem\u003eSpodoptera littoralis\u003c/em\u003e larvae (Abd El-Wahab and Anwar 2014), it was found that zinc and copper nanoparticles significantly increased SOD activity. In our study, it was determined that the groups with the highest SOD activities were the groups containing zinc and copper, and this result was consistent with the mentioned above. The reason for this increase is that the presence of zinc and copper is essential for SOD activity because these metals are the catalytic and structural components of the SOD enzyme. It was found that enzyme activities increased at 1.576 g L\u003csup\u003e-1\u003c/sup\u003e of all three metals compared to 0.788 g L\u003csup\u003e-1\u003c/sup\u003e in both the control and the infected groups, but the activities decreased in groups with the maximum metal amount. Studies have found that the SOD activities of \u003cem\u003eDrosophila simulans\u003c/em\u003e fly infected with \u003cem\u003eWolbachia\u003c/em\u003e and \u003cem\u003eG. mellonella\u003c/em\u003e larvae infected with \u003cem\u003eB. thuringiensis\u003c/em\u003e were higher than controls (Brennan et al. 2012; Sezer-Tun\u0026ccedil;soy and Ozalp 2016). The result that we found in our study that the SOD activities of all groups infected with \u003cem\u003eBtk\u003c/em\u003e were higher compared to their controls coincides with these results.\u003c/p\u003e\n\u003cp\u003eHydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) can transform into a highly reactive hydroxyl radical in the presence of reduced metal atoms. In this case, CAT efficiently converts H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003eto water and oxygen (Tasaki et al. 2017). In our study, it was found that the highest CAT activity among the control groups was found in the larvae fed with a diet containing 1.576 g L\u003csup\u003e-1\u003c/sup\u003e zinc (E diet). Similar to superoxide dismutase activities, it was found that CAT activities peaked at 1.576 g L\u003csup\u003e-1\u003c/sup\u003e of all three metals in both the control and the infected groups, but the activities decreased in the groups with the maximum metal amount. Compared to the control group, the increase in CAT activities in parallel with the SOD activities with the presence of metal is the evidence that the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which is formed as a result of SOD, is reduced by CAT, that is, these two enzymes work in a complementary manner. It was found that the hemolymph CAT activities of \u003cem\u003eG. mellonella\u003c/em\u003e larvae infected with \u003cem\u003eB. thuringiensis\u003c/em\u003e were higher compared to the control (Sezer-Tun\u0026ccedil;soy and Ozalp 2016). This result was consistent with what we found that all groups infected with bacteria had high CAT activities compared to the controls (except U).\u003c/p\u003e\n\u003cp\u003eMetals can alter various aspects of immune function (Brousseau et al. 2000). Glutathione can prevent damage to important cellular components caused by ROS such as metals (Pompella et al. 2003), so it is crucial for cells. Sezer-Tun\u0026ccedil;soy et al. (2019) found that copper oxide nanoparticles increased GSH-Px activities of \u003cem\u003eG. mellonella \u003c/em\u003elarvae compared to the control. In our study, it was determined that the enzyme activities increased with the addition of copper, zinc, and nickel to the diet compared to the control groups. We found that the GSH-Px activity level was lower than SOD, suggesting that CAT may have a priority role in scavenging H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e than GSH-Px (Meng et al. 2009). In our study, it was also found that the infection increased the GSH-Px activities compared to the control groups. The result that \u003cem\u003eG. mellonella\u003c/em\u003e larvae infected with \u003cem\u003eB. thuringiensis\u003c/em\u003e had higher GSH-Px activity compared to control larvae (Sezer-Tuncsoy and Ozalp 2016) was consistent with what we found in our study.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eInsects have been successfully used as bioindicators of environmental pollution in industrial and even urban areas. The immune system of insects protects themselves against invasive microorganisms, pathogens, and toxins (Kingsolver et al. 2013). Antioxidant enzymes like SOD, CAT, and GSH-Px play a crucial role in oxidative stress defences of cells by eliminating ROS. In our study, the cellular and the enzymatic responses of \u003cem\u003eH. cunea\u003c/em\u003e were determined after exposure to zinc, copper, and nickel and the bacterial infection. It was found that the enzyme activities increased, but the hemocyte counts decreased with metal exposure. Besides, both the hemocyte counts and the enzyme activities increased with the bacterial infection. As a result, it was concluded that the hemocyte counts and the antioxidant enzymes of \u003cem\u003eH. cunea\u003c/em\u003e were affected by metal exposure and bacterial infection. In this context, our study will shed light on immunological studies with other species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eNot applicable. \u003cstrong\u003eAvailability of data and material\u003c/strong\u003e: The data generated and/or analyzed during the current study are available from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability: \u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u003c/strong\u003e OY contributed to the study design; EFT, FGS, and SM perform the data analyzes. All authors helped write the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval: \u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements: \u003c/strong\u003eWe thank for Prof. Dr. Mahmut Bilgener for his contributions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbd El-Wahab RA, Anwar EM (2014) The effect of direct and indirect use of nanoparticles on cotton leaf worm, \u003cem\u003eSpodoptera littoralis\u003c/em\u003e. Int J Chem Biol Sci 1:17\u0026ndash;24\u003c/li\u003e\n\u003cli\u003eAli A, Rashid MA, Huang QY, Lei CL (2017) Influence of UV-A radiation on oxidative stress and antioxidant enzymes in \u003cem\u003eMythimna separata\u003c/em\u003e (Lepidoptera: Noctuidae). Environ Sci Pollut Res 24(9):8392\u0026ndash;8398. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-017-8514-7\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eAli S, Ullah MI, Saeed MF, Khalid S, Saqib M, Arshad M, Afzal M, Damalas CA (2019) Heavy metal exposure through artificial diet reduces growth and survival of \u003cem\u003eSpodoptera litura\u003c/em\u003e (Lepidoptera: Noctuidae). Environ Sci Pollut Res 26:14426\u0026ndash;14434. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-019-04792-0\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eAnderson DP, Paynter KT, Burreson EM, Anderson DP, Paynter KT, Burreson EM (1992) Increased reactive oxygen intermediate production by hemocytes withdrawn from \u003cem\u003eCrassostrea\u003c/em\u003e v\u003cem\u003eirginica\u003c/em\u003e infected with \u003cem\u003ePerkinsus marinus\u003c/em\u003e. Biol Bull 183:476\u0026ndash;481. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/1542024\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eBorowska J, Pyza E (2011) Effects of heavy metals on insect immunocompetent cells. J Insect Physiol 57:760\u0026ndash;770. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2011.02.012\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eBrennan LJ, Haukedal JA, Earle JC, Keddie B, Harris HL (2012) Disruption of redox homeostasis leads to oxidative DNA damage in spermatocytes of \u003cem\u003eWolbachia\u003c/em\u003e-infected \u003cem\u003eDrosophila simulans\u003c/em\u003e. Insect Mol Biol 21:510\u0026ndash;520. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2583.2012.01155.x\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eBrousseau P, Pellerin J, Morin Y, Cyr D, Blakley B, Boermans H, Fournier M (2000) Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam \u003cem\u003eMya arenaria\u003c/em\u003e hemocytes following in vitro exposure to heavy metals. Toxicology 142:145\u0026ndash;156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0300-483X(99)00165-1\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eBrowne N, Heelan M, Kavanagh K (2013) An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes. Virulence 4:597\u0026ndash;603. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4161/viru.25906\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eCabassi E (2007) The immune system and exposure to xenobiotics in animals. Vet Res Commun 31:115\u0026ndash;120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11259-007-0074-8\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eCheruiyot DJ, Boyd RS, Moar WJ (2013) Exploring lower limits of plant elemental defense by cobalt, copper, nickel, and zinc. J Chem Ecol 39:666\u0026ndash;674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10886-013-0279-y\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eColes JA, Fairly SR, Pipe RK (1994) The effects of fluoranthene on the immunocompetence of the common marine mussel, \u003cem\u003eMytilus edulis\u003c/em\u003e. J Aquat Toxicol 30:367\u0026ndash;379. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0166-445X(94)00051-4\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eDanismazoglu M, Demir İ, Sevim A, Demirbag Z, Nalcacioglu R (2012), An investigation on the bacterial flora of \u003cem\u003eAgriotes lineatus\u0026nbsp;\u003c/em\u003e(Coleoptera: Elateridae) and pathogenicity of the flora members. Crop P 40:1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cropro.2012.04.012\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eDubovskiy IM, Martemyanov VV, Vorontsova YL, Rantala MJ, Gryzanova EV, Glupov VV (2008) Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of \u003cem\u003eGalleria mellonella\u003c/em\u003e L. larvae (Lepidoptera, Pyralidae). Comp Biochem Phys C: Toxicology Pharmacology 148:1\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cbpc.2008.02.003\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eFallon JP, Reeves EP, Kavanagh K (2011) The \u003cem\u003eAspergillus fumigatus\u003c/em\u003e toxin fumagillin suppresses the immune response of \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae by inhibiting the action of haemocytes. Microbiology 157:1481\u0026ndash;1488. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/mic.0.043786-0\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eFiridin B, Bilgener M, Yanar O, Altun N, Ince A (2008) The effect of nutritional quality of some plant\u0026rsquo;s leaf on the feeding and development of \u003cem\u003eHyphantria cunea\u003c/em\u003e (Drury) (Lepidoptera: Arctiidae). IJONAS 2:61\u0026ndash;68\u003c/li\u003e\n\u003cli\u003eFloh\u0026eacute; L, \u0026Ouml;tting F (1984) Superoxide dismutase assays. Method Enzymol 105:93\u0026ndash;104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0076-6879(84)05013-8\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eHowe GA, Schilmiller AL (2002) Oxylipin metabolism in response to stress. Curr Opin Plant Biol 5:230\u0026ndash;236. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1369-5266(02)00250-9\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eJi R, Xie BY, Li XH, Gao ZX, Li DM (2003) Research progress on the invasive species, \u003cem\u003eHyphantria cunea\u003c/em\u003e. Entomol Knowledg 40:13\u0026ndash;18\u003c/li\u003e\n\u003cli\u003eKara A, Tun\u0026ccedil;soy B, \u0026Ouml;zalp P (2020) Effects of Cu, Zn and its mixtures on hemocyte types and counts of \u003cem\u003eGalleria mellonella\u003c/em\u003e L. larvae. (Lepidoptera: Pyralidae) J Anatolian Env Anim Sciences 5:45\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.35229/jaes.649536\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eKingsolver MB, Huang Z, Hardy RW (2013) Insect antiviral innate immunity: pathways, effectors, and connections. J Mol Biol 425:4921\u0026ndash;4936. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jmb.2013.10.006\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eKoivula MJ, Eeva T (2010) Metal-related oxidative stress in birds. Environ Pollut 158:2359\u0026ndash;2370. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envpol.2010.03.013\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eLawrence RA, Burk RF (1976) Glutathione peroxidase activity in selenium deficient rat liver. \u003cem\u003eBiochem Biophys\u003c/em\u003e Res \u003cem\u003eCommun\u003c/em\u003e 71:952\u0026ndash;958. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0006-291x(76)90747-6\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eLowry OH, Rosebrough NT, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265\u0026ndash;275\u003c/li\u003e\n\u003cli\u003eL\u0026uuml;ck H (1963) Catalase. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Weinheim and Academic Press, New York, pp\u0026nbsp;885\u0026ndash;888\u003c/li\u003e\n\u003cli\u003eMa X, Deng D, Chen W (2017) Inhibitors and activators of SOD, GSH-Px, and CAT. In: Şent\u0026uuml;rk M (ed) Enzyme inhibitors and activators. London, pp\u0026nbsp;207\u0026ndash;224\u003c/li\u003e\n\u003cli\u003eMcCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic function for erithrocuprein (Hemocuprein). J Biol Chem 244:6049\u0026ndash;6055\u003c/li\u003e\n\u003cli\u003eMeng JY, Zhang CY, Zhu F, Wang XP, Lei CL (2009) Ultraviolet light induced oxidative stress: effects on antioxidant response of \u003cem\u003eHelicoverpa armigera\u003c/em\u003e adults. J Insect Physiol 55:588\u0026ndash;592. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2009.03.003\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eMittapalli O, Neal JJ, Shukle RH (2007) Antioxidant defense response in a galling insect. Pnas 104:1889\u0026ndash;1894. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.0604722104\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eOubella R, Maes P, Paillard C, Auffret M (1993) Experimentally induced variation in hemocyte density for \u003cem\u003eRuditapes philippinarum\u003c/em\u003e and \u003cem\u003eR\u003c/em\u003e. \u003cem\u003edecussatus\u003c/em\u003e (Mollusca Bivalvia). Dis Aquat Org 15:193\u0026ndash;197. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3354/dao015193\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003ePagliara P, Stabili L (2012) Zinc effect on the sea urchin \u003cem\u003eParacentrotus lividus\u003c/em\u003e immunological competence. Chemosphere 89:563\u0026ndash;568. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2012.05.052\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003ePompella A, Visvikis A, Paolicchi A, \u003cem\u003eDe\u003c/em\u003e Tata V, Casini AF \u003cem\u003e(\u003c/em\u003e2003\u003cem\u003e)\u003c/em\u003e The changing faces of glutathione, a cellular protagonist. Biochem Pharmacol 66:1499\u0026ndash;1503. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0006-2952(03)00504-5\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eRenwrantz L (1990) Internal defence system of \u003cem\u003eMytilus edulis\u003c/em\u003e. In: Stefano GB (ed) Neurobiology of \u003cem\u003eMytilus edulis\u003c/em\u003e. Manchester University Press, Manchester, pp\u0026nbsp;256\u0026ndash;275\u003c/li\u003e\n\u003cli\u003eSaruhan I, Akca I, Kushiyev R (2014) Toxicity of some biopesticides to the fall webworm, \u003cem\u003eHyphantria cunea\u003c/em\u003e Drury (Lepidoptera: Arctiidae). Egypt J Biol Pest Co 24:255\u0026ndash;257\u003c/li\u003e\n\u003cli\u003eSezer-Tuncsoy B, Ozalp P (2016) Combined effects of pyrıproxyfen and \u003cem\u003eBacillus thuringiensis\u003c/em\u003e on antioxidant activity of hemolymph, midgut and fat body of \u003cem\u003eGalleria mellonella\u003c/em\u003e larvae. Fresenius Environ Bull 25:1660\u0026ndash;1665\u003c/li\u003e\n\u003cli\u003eSezer-Tuncsoy B, Tuncsoy M, Gomes T, Sousa V, Teixeira MR, Bebianno MJ, Ozalp P (2019) Effects of copper oxide nanoparticles on tissue accumulation and antioxidant enzymes of \u003cem\u003eGalleria mellonella\u003c/em\u003e L. Bull Environ Contam Toxicol 102:341\u0026ndash;346. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00128-018-2529-8\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eSun HX, Xia Q, Tang WC, Zhou Q, Zhang GR, Dang Z (2010) Effects of dietary nickel on apoptosis of hemocytes of \u003cem\u003eSpodoptera litura\u003c/em\u003e (Fabricius) larvae. Chin Sci Bull 55:390\u0026ndash;397. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11434-009-0726-x\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eTasaki E, Kobayashi K, Matsuura K, Iuchi Y (2017) An efficient antioxidant system in a long-lived termite queen. Plos One 12(1):e0167412. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0167412\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eTopkara EF, Yanar O (2019) Synergistic effects of some secondary compounds combined with some heavy metals on \u003cem\u003eHyphantria cunea\u003c/em\u003e Drury (Lepidoptera: Arctiidae) larvae. J Entomol Res Soc 21:85\u0026ndash;95\u003c/li\u003e\n\u003cli\u003evan Ooik T, Rantala MJ (2010) Local adaptation of an insect herbivore to a heavy metal contaminated environment. Ann Zool Fenn 47:215\u0026ndash;222. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5735/086.047.0306\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eXu C, Wei H, Wang L, Yin T, Zhuge Q (2019) Optimization of the cry1Ah1 sequence enhances the hyper-resistance of transgenic poplars to \u003cem\u003eHyphantria cunea\u003c/em\u003e. Front Plant Sci 10:335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2019.00335\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eYamamoto RT (1969) Mass rearing of tobacco hornworm. II. Larval rearing and pupation. J Econ Entomol 62:1427\u0026ndash;1431. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jee/62.6.1427\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Antioxidant enzyme, Heavy metal, Hemocyte, Hyphantria cunea, Infection","lastPublishedDoi":"10.21203/rs.3.rs-399638/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-399638/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInsects are model organisms for immunological studies. The cellular and the antioxidant enzyme responses of insects are major bioindicators against environmental stresses (metal exposure, infection, etc.). In our study, the differences in the hemocyte counts and the antioxidant enzyme activities of \u003cem\u003eHyphantria cunea\u003c/em\u003e larvae exposed to the different amounts of zinc, copper, and nickel and \u003cem\u003eBacillus thuringiensis\u003c/em\u003e infection were determined. With metal exposure, the superoxide dismutase, catalase, and glutathione peroxidase activities increased, but the hemocyte counts decreased. Additionally, both the hemocyte counts and the enzyme activities increased with \u003cem\u003eBacillus thuringiensis\u003c/em\u003e infection. As a result of this study, we found that the superoxide dismutase, catalase, and glutathione peroxidase and the hemocyte counts varied in response to both metal exposure and bacterial infection.\u003c/p\u003e","manuscriptTitle":"Synergistic Effects of Zn, Cu, and Ni and Bacillus Thuringiensis On the Hemocyte Count and the Antioxidant Activities of Hyphantria Cunea Drury (Lepidoptera: Arctiidae) Larvae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-19 19:37:56","doi":"10.21203/rs.3.rs-399638/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2021-06-22T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-04-17T00:00:00+00:00","index":0,"fulltext":""},{"type":"editorInvited","content":"Ecotoxicology","date":"2021-04-05T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Ecotoxicology","date":"2021-04-04T05:41:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8adfa138-bd93-40b9-9f12-d260312090df","owner":[],"postedDate":"April 19th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":3733857,"name":"Environmental Policy"},{"id":3733858,"name":"Toxicology"},{"id":3733859,"name":"Clinical Pharmacology"}],"tags":[],"updatedAt":"2022-01-11T14:13:04+00:00","versionOfRecord":{"articleIdentity":"rs-399638","link":"https://doi.org/10.1007/s10646-021-02493-4","journal":{"identity":"ecotoxicology","isVorOnly":false,"title":"Ecotoxicology"},"publishedOn":"2021-10-27 14:13:04","publishedOnDateReadable":"October 27th, 2021"},"versionCreatedAt":"2021-04-19 19:37:56","video":"","vorDoi":"10.1007/s10646-021-02493-4","vorDoiUrl":"https://doi.org/10.1007/s10646-021-02493-4","workflowStages":[]},"version":"v1","identity":"rs-399638","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-399638","identity":"rs-399638","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}