key: cord-0052097-4j3xoamb authors: Vengateswari, Govindaraj; Arunthirumeni, Murugan; Shivakumar, Muthugounder Subramanian title: Effect of food plants on Spodoptera litura (Lepidoptera: Noctuidae) larvae immune and antioxidant properties in response to Bacillus thuringiensis infection date: 2020-10-08 journal: Toxicol Rep DOI: 10.1016/j.toxrep.2020.10.005 sha: 49a48f31d59b6d25967c4bcf072690b55524f020 doc_id: 52097 cord_uid: 4j3xoamb The larvae of Spodoptera litura (Fabricius) were reared on five host plants, Brassica oleracea, Nicotiana tabacum, Ricinus communis, Gossypium hirsutum, and Arachis hypogaea. The larvae were immunized with Bacillus thuringiensis to observe the immune response. The results of total and differential hemocyte count were increased in B. oleracea, N. tabacum, and R. communis fed S. litura larval hemolymph. Similar results were observed in the parameter of nodulation, melanization, and phenoloxidase. Total protein was higher in R. communis fed larvae. Antioxidant levels like Catalase (CAT), Superoxide dismutase (SOD), Glutathione S- transferase (GST), Peroxidase (POX), Lipid peroxidase (LPO), and Esterase (EST) was found in moreover all plant-feeding insect. High CAT activity was observed 2–6 h in R. communis, G. hirsutum, and A. hypogaea fed S. litura larval midgut and fatbody samples. Increased SOD activity in both midgut and fatbody at 2–12 h of B. oleracea, G. hirsutum, and A. hypogaea fed. GST activity was increased initially 2−6 h in G. hirsutum and A. hypogaea. Increased POX activity was observed initially in all treated groups. Highest LPO observed at 6 h in N. tabacum in both midgut and fatbody. Whereas increased EST activity was observed in N. tabacum and B. oleracea. The results of the present study shows that nature of food influence the immunity against Bt infection. This information can be very useful for incorporating biological control program for insect pest. Insects are consumed varies types of host plant species and have varied types of nutrient quality in their food [1] . These plant nutrients play a vital role in insect development, reproductive, fecundity and also improve the immune defense mechanisms against their microbial infections [1, 2] . In previously reported the relationship between host plant food and insect immunity has concentrated on the quality and quantity of food nutrients [3, 4] . Insect immune system is aimed at eliminating the invading pathogens and parasites by the response of cellular and humoral immunity [5] . This activates the several immune parameters against the microbial infections like alteration of hemocytes, coagulation, microaggregation, increasing phenoloxidase, and formation of melanin surrounding with toxic particles [6, 7] . Humoral responses like, antimicrobial peptide production from fatbody cells have an effective role in killing the toxicants [8] . Including, the antioxidant enzymes as part of an immune response against the pathogens [9, 10] . This enzymes to control the ROS (Reactive oxygen species) generation from biotic and abiotic stress in insects [11, 12] . ROS including, free radicals, oxygen ions, and organic, inorganic molecules. These molecules are increased when the exposure to pathogens and damage the cell structure [13] [14] [15] . In many studies, the antioxidant system shows a defense mechanism against ROS production induced by pathogens [16, 17] . The major antioxidant enzymes in insects are catalase, glutathione-S-transferase, superoxide dismutases, peroxidases, and esterase [18] [19] [20] [21] [22] . These enzymes are mostly found in lepidopteran larvae on infection with pathogens [23] . Spodoptera litura (Lepidoptera: Noctuidae) is a notorious insect pest it causes severe economic loss in crop fields [24] . Insect pest management has become increasing difficult nowadays and most insecticides are ineffective. Insect control has mainly relied on chemical insecticides for decades, which had led to the development of insecticide resistance. Using microbes and their metabolites are cheap and safe methods for control the insects for example, Bacillus thuringiensis (Bt). Bt is spores forming gram-positive bacteria that produce several insecticidal activities [25] . This biopesticide is mostly used to control lepidopteran insect larvae [26] . Recently, the few studies reported that lepidopteran insects getting resistant to Bt by improving their immune status [27] [28] [29] . The ability of an insect to survive against any insecticide depends on its ability to produce a structural modification in receptors and to produce detoxification enzyme which can sequester/metabolize the toxins [30, 31] . For this, the insect diet may play a vital role in the supply of nutrients required for increased protein synthesis [32] . The composition or the types of diet may influence an insects ability to survive the infection. A further immune response is also related to improving the defense mechanism of the individual. Hence the present study understands the influence of different plant diet on the immunological and physiological mechanisms in a Bt challenged S. litura larvae. The tobacco cutworm, S. litura eggs were purchased from National Bureau of Agricultural Insect Resources (NBAIR), Bangalore, India. Purchased eggs were transferred into the plastic tray (8 cm in diameter and 10 cm in height) and allow to hatching. The newly hatched larvae transfer on another plastic tray (30 cm in diameter and 55 cm in height). Caster leaves were provided daily to larvae until the larvae reached pupae. Pre-pupae were collected and transfer into a big plastic tray (30 cm in diameter and 55 cm in height) containing soil for the complete pupal stage. Male and female pupae were identified to transfer into another plastic tray for adult formation. One male has two female pupae ware provide in a tray and covered with net cloth and allow to complete mating. The adults were fed with sugar solution through cotton balls and placed on top of the tray. Complete mating, the female adult were transferred to another plastic tray mentioned above and provide a 10 % sugar solution. The female adults produce the eggs masses were collected daily and hatched first instar larvae from this generation as used for testing. Newly hatched larvae were maintained with different plant leaves (Brassica oleracea (cabbage), Nicotiana tabacum (tobacco), Ricinus communis (castor), Gossypium hirsutum (cotton), and Arachis hypogaea (peanut)) up to 3 generations. Each treated plants have 30 larvae and each 3 replicates. The maintaining procedure of S. litura was mentioned above. Same procedure were applied to control experiment (artificial diet containing, chickpea flour (200 g), yeast powder (30 g), ascorbic acid (3.5 g), sorbic acid (1 g), formaldehyde solution (2.5 ml), methyl-p-hydroxybenzoate (2 g), agar (14 g) and distilled water (500 ml)). Bacillus thuringiensis (Bt) 4D1 strain was obtained from plant and microbial biotechnology laboratory, Periyar University, Salem. The culture was maintained on Luria Broth (LB) at 37 • C in a 50 ml conical flask with shaking at 200 rpm. 50 μl of mother culture transfer into 50 ml fresh LB medium and incubate at 37 • C for several hours. 10 ml of logphase culture was centrifuged for 10 min at 7000 rpm in 4 • C. Collected bacterial pellet washed with 10 ml of cold sterile 5 mM phosphate buffer saline (PBS). After a wash the pellet was dry for 10 min and stored at − 20 • C for further use. The 3rd generation, the 5th instar S. litura larvae were used for experimental study. Larvae were immunized with an injection of Bt 4D1 cells. The thin-micro syringe needle was dipped in bacterial pellet and injected to larval prolegs. Then the larvae were kept in 30 • C in dark and the hemolymph, midgut, and fatbody were collected at 2-24 h of exposure. The collected hemolymph sample were used for immunological assay and tissue samples, midgut and fatbody were used enzyme activity. The same procedure was applied for the control experiment except it was injected with PBS without bacteria. The experiments conducted by 3 replicates and each replicates having 10 larvae from the each treated plant groups. The hemocytes were counted by using a hemocytometer, neubauer chamber. Collected hemolymph sample form 2-24 h was diluted twenty times with phosphate buffer saline (PBS) containing, 3.8 g of Na 2 HPO 4, 5.47 g of K 2 HPO 4 , making at 1 L of distilled water, with pH 6.6. 20 μl of hemolymph sample was placed on coverslip edge of hemocytometer and counted four corners ruled squires under phase contrast microscope (PCM) at 40× magnification. The total hemocyte was counted by the method of Jones [33] . Formula: THC (Cells/mm 3 ) = X × dilution factor x depth factor Number of squares counted X = Total number of cells count, 20= Dilution factor, 10 = Depth factor, 4= Number of squares count Collected hemolymph sample was fixed on glacial acetic acid for 2− 3 min and the addition of PBS for 15 min to neutralize the hemocyte content. A drop of hemolymph sample was placed on a sterile glass slide to making a smear and added a few drops of Giemsa staining for 5 min. DH was counted under PCM with 40X magnification. The identification of DH count followed by Gupta [34] . Nodulation assay was followed by Franssens et al. [35] . A drop of collected hemolymph sample was immediately placed on hemocytometer for nodule/microaggregation count and the dark melanized nodules were observed under PCM with 40× magnification. Collected 2− 24 h of hemolymph sample was immediately centrifuged at 5000 rpm for 2 min to separate the plasma from the hemolymph. 100 μl of melanized plasma was added into 96 well plates and measured at 450 nm. 2.5 mM of phenylthiourea (PTU) was added into collected hemolymph and centrifuged. The PTU supplemented plasma used as a negative control. The melanization activity was calculated by Freitak et al. [36] . PO activity followed by the method of Harizanova et al. [37] . 10 μl of hemolymph sample was diluted in 1 ml of ice-cold PBS and frozen at 48 h. PO activity was measured by a frozen hemolymph sample was thawing at 37 • C for 5 min and centrifuged at 5000 rpm for 5 min. Collected the 100 μl of supernatant was added into 200 μl of 3 mM DL-dihydroxyphenylalanine (DL-DOPA) and incubated at 15 min in dark. The enzyme activity was measured at 490 nm for 45 min, using a spectrophotometer with dopamine as a substrate and enzyme expressed as unit/min/mg of protein. The protein was estimated by using the method of Lowry et al. [38] . The collected tissue samples (midgut and fatbody) were homogenized in 1 ml of ice-cold 50 mM PBS with 10 % glycerol. Sample mixtures centrifuge at 8000 rpm for 15 min in 4 • C. The collected supernatant was used for enzyme and metabolite assays. CAT activity was followed by the method of Luck [39] . The enzyme mixture contained, 0.4 ml of 10 mM H 2 O 2 , 2.6 ml of 50 mM ice-cold PBS, and 40 μl of an enzyme. Measurement of enzyme reaction at 240 nm using a spectrophotometer. CAT enzyme expressed as 1 μmol of H 2 O 2 decomposition/min/mg of protein. SOD enzyme activity was determined by using the method of Marklund and Marklund [40] . 3 ml of enzyme mixture contains, 2.8 ml of Tris-EDTA buffer, pH at 8.2 and 50 μl of enzyme extract. The enzyme mixture makes at final volume 2.9 ml of Tris-EDTA buffer and added 100 POX estimation was carried out using the method of Reddy et al. [42] . 0.5 μl of enzyme added into 2 ml of PBS, 1 ml of pyrogallol solution, 1 ml of 0.005 M hydrogen peroxide. The reaction mixture was incubated for 5 min at 25 • C and the enzyme reaction terminated by the addition of 1 ml sulphuric acid with 2.5 normality. The enzyme reaction was observed at 420 nm in a spectrophotometer and the rate of POX was calculated by Unit/min/mg of protein. LPO activity was estimated by the method of Esterbauer and Cheeseman [43] . The reaction mixture contains, 0.1 ml of an enzyme, 1.9 ml of PBS was incubated at 37 • C for 1 h and addition of 10 % trichloroacetic acid for precipitating the sample. The mixture was centrifuged at 5000 rpm for 15 min and collected supernatant into 1 ml of 1 % thiobarbituric acid (TBA). The samples allow to the boiled water bath for 10 min and after that, the collected supernatant allows it to cool. The formation of malondialdehyde (MDA) concentration was measured at 532 nm in a spectrophotometer and MDA concentration expressed as 1 μmol/min/mg of protein. EST activity was determined by using the method of Kranthi [44] . The reaction mixture contains, 0.2 ml of an enzyme into 0.1 ml of 0.3 mM a-naphthyl acetate was incubated for 20 min in 30 • C dark. The mixture sample added with 1.0 ml of fast BB salt and sodium dodecyl sulfate (SDS) solution at a 2:5 ratio. EST activity was measured at 590 nm using a spectrophotometer. The enzyme activity expressed as 1 μmol/min/mg of protein All experimental assays were performed using three replications, and the obtained assays data were analyzed by Bonferroni post-test (twoway ANOVA) using PRISM 5 software. The P-value (<0.05) was considered significant. Increased total hemocyte count was found in B. oleracea and N. tabacum fed insects after 6 h and thereafter the hemocyte level was reduced. R. communis fed larval hemocyte level was increased in 4 and 6 h of exposure and after that, the hemocyte level can decrease has come back to normal. Total hemocyte level was low in G. hirsutum and A. hypogaea fed larvae (Fig. 1) . Identify the five types of differential hemocytes based on morphology (Fig. S1 ). Prohemocyte (PR) is a small cell with a large central nucleus and thin cytoplasm (Fig. S1(A) ). Plasmatocyte (PL) is large and varies in size, with have a small nucleus and few granules in the cytoplasm (Fig. S1(B) ). Granulocyte (GR) is the ellipsoidal shape and large nucleus and a high granule present in the cytoplasm (Fig. S1(C) ). Oenocyte (OE) is an oval shape with a large unconventional nucleus within the cytoplasm (Fig. S1(D) ). Spherule cell (SP) has a circular cell which have several spherules in the cytoplasm (Fig. S1(E) ). Differential hemocyte count from 2− 24 h shows, Prohemocyte were increased after 12 h of B. oleracea, N. tabacum, and R. communis fed larvae. While decreased count was observed in G. hirsutum and A. hypogaea fed larvae when compared to the control. Increased Plasmotocyte were observed in 2, 12 and 24 h of B. oleracea and N. tabacum fed larvae. 2− 24 h of increased plasmatocyte were observed in R. communis. Increased Granulocytes were found in 2− 6 h of R. communis fed larvae and after 6 h the hemocyte levels were reduced and come back to normal. Oenocyte count was increased in 2 h of R. communis after that there was a reduction level observed in till 12 h. The increased spherule cells were observed in 2 and 24 h of R. communis fed larvae (Fig. S2) . B. oleracea and R. communis fed larvae were found to induce maximal nodule formation in 6 h and 12 h after the injection (Fig. S3) . Whereas the control group nodule formation was very low and after 6 h there were no nodules were found (Table 1) . Melanization was significantly increased from the 2 h infection in N. tabacum and 4 h infection B. oleracea and R. communis, while G. hirsutum and A. hypogaea showed the low level of melanization when compare to the control (Fig. 2) . Increased PO activity was observed between 2-12 h of infection and decreased activity was observed in after 12 h in all treatment groups (Fig. 3) . Total protein content was observed after Bt exposure in 2-24 h of S. litura. Increased protein level was observed after 2-6 h of all treated groups. Total protein content was highest in N. tabacum and R. communis (Fig. 4) . Increased CAT activity in R. communis, G. hirsutum, and A. hypogaea fed S. litura in 2-6 h of midgut and fatbody sample (Fig. 5) . SOD activity increased at 2-12 h of B. oleracea and 2, 4 and 12 h of G. hirsutum in midgut sample, whereas decreased activity was found in R. communis, N. tabacum, and A. hypogaea fed larvae. In fatbody, increased SOD activity was found in 4-12 h of G. hirsutum and 4-24 h of A. hypogaea when compare to other treatment groups (Fig. 6) . Increased GST activity in midgut, 2-24 h of G. hirsutum and A. hypogaea, while other treatments observed the decreased activity. In fatbody, increased activity was observed in all treated groups initially 2-4 h and after that, it decreased activity was observed (Fig. 7) . POX activity initially increased in 2-4 h of all treated groups and increased till 6 h of B. oleracea and G. hirsutum. After that, there was decreased in all groups. In fatbody, increased activity was in 6 h only of B. oleracea, N. tabacum and A. hypogaea (Fig. 8) . LPO activity was increased at 6 h in N. tabacum in both midgut and fatbody. After that there was a decreased activity was observed (Fig. 9) . Midgut, EST activity was increased in till 12 h of exposure in B. oleracea fed larva. Fatbody, increased in 2− 6 h of N. tabacum (Fig. 10 ). Lepidopteran insects are usually pests of crops. S. litura lepidopteran insect is a polyphagous pest feeding on several crops. In the present study, the S. litura larvae maintained on five different plants (diet) was analyzed after Bt exposure. Larval defense mechanism was assessed based on immunological, antioxidant and detoxification enzymes. Plant diet plays a vital role in the physiological mechanisms of vertebrate and invertebrate and also improve immunity [45, 46] . The quality and quantity of plant diet determine insect fitness. Insects have a robust immune system comprising of hemocytes, AMPs, phenoloxidase, lysozyme, and other mechanisms that are activated in response to infection [5, 7] . In this study, we found an increased THC in B. oleracea, and N. tabacum, and R. communis fed larvae when compared with control and other plant groups. This may the reason of food plants have sufficient nutrient value and high utilization of food. Therefore enhance the immunity of S. litura when compare to the other two plants, G. hirsutum and A. hypogaea (Fig. 1) . In other reports, the fluctuating hemocytes have seen in G. mellonella [47] , Reticulitermes flavipes [48] , Oxya japonica, Eurygaster integriceps [49] and Chilo suppressalis [50] after microbial exposure. While decreased THC was observed in Aspergillus flavus exposure on S. litura [51] . There is also a change in DH composition in insect hemolymph following microbial exposure. Identification of five hemocytes, prohemocyte, plasmatocyte, granulocyte, oenocyte, and spherule cells are usually found in lepidopteran insects [52, 53] . These hemocytes having a different functions in insect immunity, prohemocyte forms a putative stem cell population [54] , plasmatocyte participate in nodulation, encapsulation, and in wound healing [55] , granulocyte activates cell-mediated immunity. Oenocyte is involved in the melanization process [56] . Whereas spherule cells are not much involved in immune functions [53] . In our previous study reported the five hemocyte types are a response to pesticide exposure on S. litura larvae [57] . In this study, identify the DH type against Bt exposure on S. litura larvae. Increased granulocyte and plasmatocyte levels in B. oleracea, N. tabacum, and R. communis when compared with G. hirsutum, and A. hypogaea fed larvae which suggest an increased cell-mediated immunity and nodulation and encapsulation. Other previous studies was reported prohemocyte, granulocyte, and oenocyte were observed in the pea aphid, Acyrthosiphon pisum against parasite exposure [58] . Nodulation is one of the cellular responses of insects against microbes [47] . Bt toxin initiates nodule formation in S. litura larvae, the highest nodulation was observed in B. oleracea and R. communis fed larvae and no nodules were found in control group after 12 h and 24 h of treatment. A similar result was found in fungal pathogen exposure to Chilo suppressalis larvae [49] . Less number of the nodule was found in G. hirsutum, and A. hypogaea fed larvae, it may the reason of reduced THC. Gillespie et al. [59] has well established about the correlation between THC and nodule formation against pathogen infection. PO and melanization is a part of immune the response against the wounding/infection of invading microbes [60] . These enzymes hydrolyze the tyrosine to L-dihydroxyphenylalanine and oxidize the o-diphenol to form a quinone [61] . The conversion of melanin inhibits the Bt by forming of encapsulation was observed in S. exigua and Heliothis virescens [62] . In our experiments, the highest PO and melanization were observed in B. oleracea, N. tabacum, and R. communis fed larvae when compare to the control and other treated groups (Figs. 2 and 3) , which corresponds to the increasing of hemocyte and nodulation. These results may suggest that melanin deposition also is complementary to nodule formation. Therefore, in insects, PO, melanization, and nodulation are important immune functions against microbial toxins [63] [64] [65] 47, 48] . Based on the immune response the total protein level was higher in N. tabacum and R. communis fed larvae. These host plants have rich nutrients that enhance the protein content in the larval hemolymph. CAT enzyme is involved in oxidative stress and H 2 O 2 scavenging mechanism in insects [66] . Here, the highest CAT activity was observed in R. communis, G. hirsutum, and A. hypogaea fed larvae because this plant leaves may be containing high antioxidants levels when compared to the other plant leaves (Fig. 5) . In previous study, increased CAT activity was observed in silkworm (Bombyx mori) fat body [67] , and other lepidopteran insects, G. mellonella [17] , Helicoverpa armigera [68] , S. litura [50] , and including other hemiptera insect, Bemisia tabaci [69] . SOD and GST pay a protective role from oxidative stress [70] [71] [72] [73] . In this study, the highest SOD and GST activity was observed in G. hirsutum and A. hypogaea (Figs. 6 and 7). Similar results, the increasing SOD activity were observed in S. litura larvae [50] and increasing GST was seen in G. mellonella [67] . Therefore the results suggest that both enzymes play a vital role in the elimination of ROS. POX is an important antioxidant enzyme involved in the H 2 O 2 break down in insects [74] . Our study, the POX activity initially increased at all treated groups in midgut sample when compare to the control (Fig. 8) . Similar increased POX activity were observed in Apis mellifera against microbial exposure [75] . LPO is involved in the protection of cellular damage from toxin. In the present study, LPO activity was increased in 6-12 h of all treatment (Fig. 9) . Therefore, the insect suffers from cellular damage within 6 h of Bt exposed. These similar results were observed in other species, Bactrocera dorsalis [73] , and Scapharca broughtonii [76] . Insect EST is metabolized a wide range of xenobiotic. EST activity was increased in B. oleracea and N. tabacum fed larva in both midgut and fatbody when compare to the control (Fig. 10) . Similar results were observed in E. integriceps species with microbial exposure [77] . From this study, we suggest that esterase had an important role in the metabolism or detoxification of microbes in the insect. In this study, the effect of Bt on S. litura fed with five host plant were evaluated. R. communis, B. oleracea, and N. tabacum fed larvae S. litura improve their defense mechanisms against the microbial infection when compared with other plant treatment G. hirsutum, and A. hypogaea including control. Therefore the host plant and their nutrients may be regulating S. litura survival ability to overcome pathogenic infections. The presence of host plants in the field, the condition needs to be considered to avoid plant pest infecting species. Designed and performed the experiments, G. V.; Analysed the data, G.V.; M.A.; Manuscript correction, M.S.S. The authors report no declarations of interest. Natural host-plant quality affects immune defence of an insect herbivore Costs of resistance in insect-parasite and insect-parasitoid interactions Flexible diet choiceoffsets protein costs of pathogen resistance in a caterpillar Defoliating insect immune defense interacts with induced plant defense during a population outbreak Insect immune responses to nematode parasites Variation in the susceptibility of Drosophila to different entomopathogenic nematodes Damage signals in the insect immune response The immune response of Drosophila Innate immunity: an overview Recent advances in the innate immunity of invertebrate animals Comparative study of two thioredoxins from common cutworm (Spodoptera litura): cloning, expression, and functional characterization Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease, Free Radic Peroxiredoxin 5 from common cutworm (Spodoptera litura) acts as a potent antioxidant enzyme Reactive oxygen species as an initiator of toxic innate immune responses in retort to SARS-CoV-2 in an ageing population, consider Nacetylcysteine as early therapeutic intervention Active defense under oxidative stress. The antioxidant responsive element Level and activities of antioxidants in intestine of larvae Galleria mellonella L. (Lepidoptera, Pyralidae) at peroral infestation by bacteria Bacillus thuringiensis ssp. Galleriae Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae) Superoxide dismutase an enzymic function for erythrocuprein (hemocuprein) Comparison of catalase in diploid and haploid Rana rugosa using heat and chemical inactivation techniques Antioxidant enzymes in the honey bee, Apis mellifera Glutathione transferases: an overview, Method Enzymol Glutathione S-transferase activities in phytophagous insects: induction and inhibition by plant phototoxins and phenols Effect of juvenile hormone analogue, pyriproxyfen on antioxidant enzymes of greater wax moth Effect of host plants on life table parameters of Spodoptera litura Biochemistry and genetics of insect resistance to Bacillus thuringiensis Effect of Bacillus thuringiensis CAB109 on the growth, development, and generation mortality of Spodoptera exigua (Hubner) (Lepidoptera: Noctuidea) Pink bollworm resistance to Bt toxin Cry1Ac associated with an insertion in cadherin exon 20 An overview on resistance of insect pests against Bt crops Insect resistance to Bacillus thuringiensis toxin Cry2Ab is conferred by mutations in an ABC transporter subfamily a protein Bacillus thuringiensis insecticidal threedomain Cry toxins: mode of action, insect resistance and consequences for crop protection Lack of fitness costs and inheritance of resistance to Bacillus thuringiensis Cry1Ac toxin in a near-isogenic strain of Plutella xylostella (Lepidoptera: Plutellidae) Biosynthesis in Insects Current concepts concerning insect hemocytes Cellular elements in the hemolymph 20-Hydroxyecdysone and juvenile hormone regulate the laminarininduced nodulation reaction in larvae of the flesh fly, Neobellieria bullata Immune system responses and fitness costs associated with consumption of bacteria in larvae of Trichoplusia ni Developmental and organ-specific expression of transferrin in Drosophila melanogaster Protein measurement with the Folin phenol reagent Catalase, methods of enzymatic analysis Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase Glutathione S-transferase: the first enzymatic step in mercapturic acid formation Effect of light and benzyl adenine in dark treated growing leaves, changes in the peroxidase activity Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal Insecticide Resistance Monitoring Mechanisms and Management Manual A plant pathogen modulates the effects of secondary metabolites on the performance and immune function of an insect herbivore In vivo immunoprotective role of Indigofera tinctoria and Scoparia dulcis aqueous extracts against chronic noise stress induced immune abnormalities in Wistar albino rats Effect of the entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin on cellular defence response and oxygen uptake of the wax moth Galleria mellonella L. (Lep., Pyralidae) Cellular encapsulation in the eastern subterranean termite, Reticulitermes flavipes (Isoptera), against infection by the entomopathogenic fungus Metarhizium anisopliae Cellular immune reactions of the sunn pest, Eurygaster integriceps, to the entomopathogenic fungus, Beauveria bassiana and its secondary metabolites Immune response of Chilo suppressalis Walker (Lepidoptera: Crambidae) larvae to different entomopathogenic fungi Effect of Aspergillus flavus on the mortality and activity of antioxidant enzymes of Spodoptera lituraFab. (Lepidoptera: Noctuidae) larvae Insect hemocytes and their role in immunity Purification and characterization of silkworm hemocytes by flow cytometry Bombyx mori prohemocyte division and differentiation in individual microcultures Phagocytic Recognition: Bombyx mori. Immunology of Insects and Other Arthropods Biosynthesis of prophenoloxidase in hemocytes of larval hemolymph of the silkworm, Bombyx mori Protective effect of melatonin administration on abamectin-induced immunotoxicity in Spodoptera litura (Insecta: Lepidoptera) Characterisation of immune responses in the pea aphid, Acyrthosiphon pisum The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium anisopliae var acridum Phenoloxidases in insect immunity Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade Comparative evaluation of phenoloxidase activity in different larval stages of four lepidopteran pests after exposure to Bacillus thuringiensis Melanization immune responses in mosquito vectors Mosquito immunity against plasmodium Melanogenesis and associated cytotoxic reactions: applications to insect innate immunity Mechanisms for regulating oxygen toxicity in phytophagous insects, Free Radic Fat body catalase activity as a biochemical index for the recognition of thermotolerant breeds of mulberry silkworm, Bombyx mori L Ultraviolet light-induced oxidative stress: effects on antioxidant response of Helicoverpa armigera adults Antioxidant enzyme responses induced by whiteflies in tobacco plants in defense against aphids: catalase may play a dominant role Glutathione transferases Tolerance of spermatogonia to oxidative stress is due to high levels of Zn and Cu/ Zn superoxide dismutase The basic and applied aspects of superoxide dismutase Effects of thermal stress on lipid peroxidation and antioxidant enzyme activities of oriental fruit fly Characterization of a silkworm thioredoxin peroxidase that is induced by external temperature stimulus and viral infection Genes of the antioxidant system of the honey bee: annotation and phylogeny Activity of antioxidant enzymes and physiological responses in ark shell, Scapharca broughtonii, exposed to thermal and osmotic stress: effects on hemolymph and biochemical parameters Effect of the entomopathogenic fungus, Beauveria bassiana, and its secondary metabolite on detoxifying enzyme activities and acetylcholinesterase (AChE) of the Sunn pest We would like to thank the Department of Biotechnology, Periyar University, Salem, Tamil Nadu, India for providing the infrastructural facility for carrying out this research work. Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.toxrep.2020.10.005.