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

In nature, insects are constantly exposed to various environmental stressors. Heavy metals are one of the important factors of environmental pollution. Heavy metals can cause adverse effects on the growth rate and the survival of herbivores, as well as immune function. In addition to heavy metals, another factor that insects are exposed to in nature is entomopathogens. The cellular and the antioxidant enzyme responses of insects are major bioindicators against the stressors. In this 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. Although heavy metal exposure decreased the hemocyte counts and increased the antioxidant enzyme activities, the increase in the hemocyte counts with bacterial infection and the increased antioxidant enzyme activities demonstrated that the response to infection in the insect was stronger and the synergistic effect was occurred. As a result of this study, we found that the activities of superoxide dismutase, catalase, and glutathione peroxidase and the hemocyte counts varied in response to both metal exposure and bacterial infection.


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). To prevent ROS damage, the living organisms have complex defense 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 . 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 used 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) among 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
Rearing larvae and preparing artificial diets Hyphantria cunea larvae were collected in field surveys in the borders of Çarşamba District of Samsun, Turkey, in 2020. They were brought to the laboratory, kept at 25 ± 2°C and 70% humidity (16 h light/8 h dark), and fed an artificial diet, developed by Yamamoto (1969) until they reached the pupal stage. The larvae of the second generation obtained from the first 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, 1.576, and 2.364 g/L zinc, copper, and nickel to artificial diet. Totally, 20 different diets were prepared (Table 1).

Bacterial culture conditions
Bacillus thuringiensis subsp. kurstaki (Btk) 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 infected and non-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 noninfected groups were fed for 5 days. After sterilizing the larvae with 95% ethanol, hemolymph was collected using a microcapillary tube by puncturing the third legs of the larvae with a fine-tipped dissecting needle and transferred to Eppendorf tubes containing N-phenylthiourea (Sigma-Aldrich) . After 5 days, 1 mL of Btk suspension was sprayed into the diet of the larvae to be infected and they continued to be fed for 2 more days. Then, the hemolymph of the infected larvae was also taken, and enzyme analyses were performed, and the hemocytes were counted.

Total hemocyte count
A total of 50 larvae were used for all groups. The last instar larvae of H. cunea were pierced on the third legs for the hemocyte counting. The insect hemolymphs to be used in the hemocyte counting were placed in Eppendorf tubes. Ten microliter of hemolymph was spread on a glass slide and allowed to air-dry for 20-30 min to allow the hemocytes to adhere to the glass. The cells were fixed in methanol:acetic acid solution (3:1) for 10 min. The slides were stained with Giemsa (Merck) for 10 min and then washed with distilled water. After air-drying, the slides were treated with xylene and then mounted in Entellan. The hemocytes were counted under a Zeiss Primo Star microscope. For each slide, the hemocytes were counted in twenty randomly selected areas. The hemocyte counts were calculated by multiplying the mean cell counts by the microscope factor calculated from the microscope sight field (Fitts and Laird 2004).

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 transferred to Eppendorf tubes and centrifuged for 20 min at 15,000 rpm in a refrigerated centrifuge (model 3500, Kubota, Tokyo, Japan) at +4°C. The supernatant obtained after centrifugation was kept at −80°C until total protein determination and enzyme activity analyses were performed. Protein determination was made according to the method of Lowry et al. (1951). Solution A containing 2% Na 2 CO 3 , 1% CuSO 4 , and 2% Na-K-Tartrate mixture and solution B containing 1:1 diluted Folin ciocalteuis phenol reagent were prepared for protein determination. Ten microliter of sample was added to 2.5 mL of the solution A and vortexed. Two hundred and fifty microliter of the solution B was then added and vortexed again. This mixture was incubated in the dark for 30-60 min. Spectrophotometric measurement was performed at 595 nm. Standards were prepared with Bovine Serum Albumin (BSA) and plotted. The absorbance values obtained from the samples were calculated by proportioning by the standard. Superoxide dismutase activity was determined by the method of Flohé and Ötting (1984) and the spectrophotometric method of McCord and Fridovich (1969). A 0.76 mg (5 μL) xanthine solution in 10 mL 0.001 N NaOH was mixed with a 24.8 mg (2 µmol) cytochrome c solution in 100 mL 50 Mm pH 7.8 phosphate buffer containing 0.1 M EDTA. A freshly prepared 0.2 U/mL xanthine oxidase solution in 0.1 mM EDTA was used in this experiment. To determine the SOD activity, the reduction of cytochrome c by the xanthine/xanthine oxidase system was spectrophotometrically measured at 550 nm. Catalase activities were determined by the Lück (1963) method. Then, 1/15 M Na 2 HPO 4 .H 2 O-KH 2 PO 4 buffer was prepared at pH 7 to determine CAT activities; 160 μL of H 2 O 2 was added to 100 mL of Na-K buffer for each activity measurement. When the samples were added to this mixture, CAT activities were determined spectrophotometrically with the decrease in absorbance at 240 nm due to H 2 O 2 degradation. Glutathione peroxidase activity determination was carried out by the method of Lawrence and Burk (1976). GSH-Px catalyzes the oxidation of glutathione by Cumene hydroperoxide. NADP + oxidation occurs during the conversion of oxidized glutathione to reduced glutathione in the reaction medium with the cofactors glutathione reductase and NADPH. A 50 mM potassium phosphate buffer was prepared at pH 7 to measure GSH-Px activities. The decreases in absorbance were measured using a spectrophotometer at 340 nm. The activities were measured in terms of µmoles of oxidized NADPH per minute. A UV/Vis spectrophotometer (model T70, Pharma Test Apparatebau, Hainburg, Germany) was used to determine enzyme activities.

Statistical analyses
The ANOVA-Dunnet test was used to compare the activities of superoxide dismutase, catalase, and glutathione peroxidase, as well as hemocyte count, between the noninfected and infected groups. To best demonstrate the synergistic effect, the larvae fed in 10 different diet groups and their infected larvae groups were compared within themselves, and then the differences in hemocyte counts and enzyme activities were determined using an independent two-sample t-test. SPSS 21.0 software (IBM Corp., Armonk, NY, USA) was used for these tests.

Hemocyte counts
Among the non-infected groups, the lowest hemocyte count was found to be in the larvae fed diet T (1269 ± 3.3, t = −6.6, p < 0.001), and the highest hemocyte count was obtained in the larvae fed non-infected diet A (2519 ± 17.1, t = −6.7, p < 0.001). The hemocyte counts of all groups infected with bacteria increased compared to the noninfected ones. Among the infected groups, the lowest hemocyte count was in the larvae fed diet U (1336 ± 9.5, t = −6.6, p < 0.05), and the highest one was in diet B (2778 ± 34.5, t = −6.7, p < 0.001) (Fig. 1).

Superoxide dismutase activities
Among the non-infected groups, the highest SOD activity was in the group containing 1.576 g/L Zn (222 ± 1.3, t = 7.6, p < 0.001) while the lowest activity was in diet A (125 ± 2.7, t = 7.9, p < 0.001). In infected groups, it was determined that the highest SOD activity was in diet F (236 ± 1.4, t = 7.6, p < 0.001), whereas the lowest activity Fig. 1 Comparison of hemocyte counts of Hyphantria cunea larvae in the non-infected and the infected groups. Error bars are standard errors (SE) of three independent trials was in the larvae fed diet U (141 ± 1.3, t = 3.4, p < 0.05) (Fig. 2).

Catalase activities
Among the non-infected groups, the lowest CAT activity was found to be in the larvae fed diet A (222 ± 2.7, t = 3.2, p < 0.05), the highest one was obtained in the larvae fed diet E (288 ± 1.5, t = 7.3, p < 0.001). Among the infected groups, the lowest CAT activity was in diet U (230 ± 1.0, t = −5, p < 0.001), and the highest one was in diet F (304 ± 1.6, t = 7.3, p < 0.001) (Fig. 3).

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 noninfected 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;. Also, the hemocyte count of the larvae decreased (except diet M) with increasing amounts of zinc, copper, and nickel in the diet among the non-infected 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 non-infected ones; 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. Furthermore, the fact that the infected heavy metal groups had higher hemocyte counts than their own non-infected ones, despite the fact that the heavy metal added to the diet decreased the hemocyte count, demonstrates the power of cellular immunity in infected insects. 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, it was found that zinc and copper nanoparticles significantly increased SOD activity (Abd El-Wahab and Anwar 2014). 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 result of the study 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 of all three metals compared to 0.788 g/L in both the non-infected and the infected groups, but the activities decreased in groups with the maximum metal amount. Studies 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-Tuncsoy and Ozalp 2016). The result we obtained from our study that the SOD activities of all groups infected with Btk were higher compared to their non-infected ones coincides with these results. In this study, it was found that heavy metals added to the diet increased SOD activities, and that these enzyme activities increased even more with infection. This result demonstrates the synergistic effect.
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 non-infected groups was found in the larvae fed with a diet containing 1.576 g/L zinc (diet E). Similar to superoxide dismutase activities, it was found that CAT activities peaked at 1.576 g/L of all three metals in both the non-infected 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 group (Sezer-Tuncsoy and Ozalp 2016). This result is consistent with the finding of our study that all groups infected with bacteria had high CAT activities compared to the noninfected ones (except diet U). In this study, it is evidence of the synergistic effect that the three heavy metals added to the diet caused an increase in CAT activities, and a further increase in these enzyme activities occurred with the addition of bacterial infection.
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-Tuncsoy et al. (2019) found that copper oxide nanoparticles increased GSH-Px activities of G. mellonella larvae compared to the control group. 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 non-infected groups. We found that the GSH-Px activity level was lower than SOD, suggesting that CAT may have a more 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 noninfected 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. Similar to SOD and CAT enzyme activity results, it was determined that GSH-Px activities increased with metals added to the diets, but there was a significantly higher increase in the enzyme activities with infection.

Conclusions
Insects have been successfully used as bioindicators of environmental pollution in industrial and even urban areas. The immune system of insects protects 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 defenses 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. Both the hemocyte counts and the enzyme activities increased with the bacterial infection. Although heavy metal exposure decreased the hemocyte counts and increased the antioxidant enzyme activities, the increase in the hemocyte counts with bacterial infection and the increased antioxidant enzyme activities demonstrated that the response to infection in the insect was stronger and synergistic. 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.

Data availability
The data generated and/or analyzed during the current study are available from the corresponding author.