Immunological mechanisms of synergy between fungus Metarhizium robertsii and bacteria Bacillus thuringiensis ssp. morrisoni on Colorado potato beetle larvae

Journal of Insect Physiology 96 (2017) 14–20

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Immunological mechanisms of synergy between fungus Metarhizium robertsii and bacteria Bacillus thuringiensis ssp. morrisoni on Colorado potato beetle larvae Olga N. Yaroslavtseva a,⇑, Ivan M. Dubovskiy a, Viktor P. Khodyrev a, Bahytzhan A. Duisembekov b, Vadim Yu. Kryukov a, Viktor V. Glupov a a b

Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630091, Russia Kazakh Research Institute for Plant Protection and Quarantine, Almaty, Kazakhstan

a r t i c l e

i n f o

Article history: Received 27 May 2016 Received in revised form 22 August 2016 Accepted 13 October 2016 Available online 14 October 2016 Keywords: Metarhizium robertsii Insect immunity Leptinotarsa decemlineata Esterases Glutathione-S-transferase Encapsulation Phenoloxidase Bt

a b s t r a c t The synergistic effect between the entomopathogenic fungus Metarhizium robertsii and a sublethal dose of the bacterium Bacillus thuringiensis ssp. morrisoni var. tenebrionis was studied in terms of immune defense reactions and detoxification system activity of the Colorado potato beetle, Leptinotarsa decemlineata, fourth instar larvae. Bacterial infection led to more rapid germination of fungal conidia on integuments. We found a significant decrease of cellular immunity parameters, including total hemocyte count and encapsulation response, under the influence of bacteria. Phenoloxidase activity in integuments was increased under bacteriosis, mycosis and combined infection compared to controls. However, phenoloxidase activity in the hemolymph was enhanced under bacteriosis alone, and it was decreased under combined infection. Activation of both nonspecific esterases and glutathione-S-transferases in the hemolymph was shown at the first day of mycosis and third day of bacteriosis. However, inhibition of detoxification enzymes was detected under combined infection. The suppression of cellular immunity and detoxification reactions in Colorado potato beetle larvae with a sublethal dose of bacteria is discussed as a reason for synergy between B. thuringiensis and M. robertsii. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction The Colorado potato beetle (CPB) is one of the economically important species in northern hemisphere. The high density of the pest has led to vast loss of potato yield in Eurasia and Northern America (Hare, 1980, 1990). At the present using of chemical insecticides remains by main method of CPB control. However fast development of resistance to main groups of insecticides has been reported (Alyokhin et al., 2007; Scott et al., 2015). Entomopathogenic bacteria and fungi are the most popular microbial groups for biological control of the CPB (Wraight and Ramos, 2002, 2005, 2015). However, the application of fungi or bacteria alone has an instability effect. Therefore, identifying different synergistic combinations is a promising approach for control (Kryukov et al., 2009, 2014). Both synergistic and additive effects between pathogens from different systematic group on pest insects are well known. For example, these effects were observed during combined fungal infection (Beauveria, Metarhizium) and bacteriosis ⇑ Corresponding author. E-mail address: [email protected] (O.N. Yaroslavtseva). http://dx.doi.org/10.1016/j.jinsphys.2016.10.004 0022-1910/Ó 2016 Elsevier Ltd. All rights reserved.

(Bacillus thuringiensis, Pseudomonas sp.) on Musca domestica (Mwamburi et al., 2009), Ostrinia furnacalis (Ma et al., 2008), Locusta migratoria (Lednev et al., 2008), Helicoverpa armigera (Wakil et al., 2013), Sesamia nonagrioides (Mantzoukas et al., 2013), Earias vittella (Ali et al., 2015), and Leptinotarsa decemlineata (Kryukov et al., 2009; Wraight and Ramos, 2005). The majority of mixed infection studies are related to identifying methods to enhance bioinsecticide efficacy. However, immune-physiological reasons for synergy remain poorly understood. Many studies are focused on the immune-physiological responses of insects under both fungal and bacterial monoinfections (reviewed by Gillespie et al., 2000; Hajek and Stleger, 1994; Kurata, 2006). The defense strategy of insects against fungi includes multi-factorial reactions, which are focused in integuments, as a primary insect antifungal barrier (Butt et al., 2016). Encapsulation and melanization leads to the isolation and elimination of fungus in the cuticle and hemocoel (Griesch and Vilcinskas, 1998; Schwarzenbach and Ward, 2007). Some intermediates of the phenoloxidase cascade (e.g., reactive oxygen species (ROS)) may be toxic for fungi (Nappi and Christensen, 2005). In addition, enzymes of the detoxification system (nonspecific esterases, glutathione-S-

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transferases (GST) and monooxygenases) participate in the defense of insects against toxins that are formed under fungal pathogeneses (Serebrov et al., 2006; Zibaee et al., 2009). The bacteria Bacillus thuringiensis (Bt) must be ingested to infect and kill its host. The insecticidal activity of Bt is primarily due to proteinaceous crystal endotoxins (Cry), which are produced during sporulation and activated in the host’s midgut (Bravo et al., 2007). Bt virulence factors also include enterotoxins, hemolysins, phospholipases and metalloproteases (Nielsen-LeRoux et al., 2012). Important insects defense mechanisms against Bt are related to enzymatic, regenerational and antimicrobial activities in the midgut (Gunning et al., 2005). Additionally, Bt infection also effects cellular and humoral insect immunity in the hemocoel (Dubovskiy et al., 2008; Grizanova et al., 2014). Moreover, sublethal Bt infection resulted in a decrease of hemocyte counts in the hemolymph of wax moth larvae (Grizanova et al., 2014). Therefore, the suppression of host cellular immunity by Bt can affect the susceptibility of insects to fungi. There are only few studies of the biochemical and physiological changes in insects under mixed infection (fungi and bacteria). In particular, Park and Kim (Park and Kim, 2011) found that metabolites of the bacteria Xenorhabdus nematophila have immunosuppressive effects on both cellular and humoral responses to B. bassiana infection in Spodoptera exigua. An analysis of the interactions in a three-component model of the CPB, Bt and entomopathogenic fungi (Beauveria bassiana s.l. or Metarhizium anisopliae s.l.) showed a stable synergistic effect under laboratory and field condition on all instars of larvae (Kryukov et al., 2009; Wraight and Ramos, 2005). The authors showed that bacteria arrested the nutrition of insects, delayed their growth, and increased the intermolt period. They proposed that these effects may assist the penetration of fungus through the integuments into the hemocoel leading to a more rapid development of fungus and killing of the host. However, the immune and detoxification reactions in Colorado potato beetle larvae under a combined infection with Bt and entomopathogenic fungi were not studied. This study explores the mechanisms of synergistic interactions between pathogens during a mixed infection of Metarhizium robertsii (Mr) and Bt. In this model, we estimated the total hemocyte count, encapsulation response, activity of phenoloxidases in the hemolymph and integuments, and detoxification system enzymes (nonspecific esterases, glutathione-S-transferases) in CPB larvae. 2. Materials and methods 2.1. Insects Colorado potato beetle (Leptinotarsa decemlineata) larvae were collected from farmer plantations of potato (Solanum tuberosum) in the Novosibirsk region (West Siberia; 53°730 5400 N; 77°640 4900 E) where there were no applications of chemical insecticides. Collected insects were maintained under laboratory conditions at LD 12:12 and at 25 °C. The larvae were kept in 300 mL plastic air containers (10 insects per 1 container). Larvae were fed fresh cut shoots of potato S. tuberosum. For experiments, we used fourth instars larvae no older than 10 h after molting. 2.2. Fungal and bacterial infections For infecting insects, we used strains from the collection of microorganisms of Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Science (ISEA SB RAS). The fungus Metarhizium robertsii (Mr) strain R-72 and Bacillus thuringiensis ssp. morrisoni var. thuringiensis (H8ab) (Bt) strain

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2495 were used to infect larvae. Conidia of Mr were grown on double autoclaved millet (Kryukov et al., 2009). Spores and crystals of bacterium were grown on meat peptone agar for 6 days and then washed off with distilled water. The titers of fungal conidia, spores and crystals of bacteria were counted in hemocytometer. The ratio of Bt spores and crystals was 50/50. Inoculation with fungi and bacteria was performed by single dipping (10 s) insects and potato leaves into water suspensions with fungal conidia and/or bacterial spore-crystal mix. The concentrations were 7  105 conidia/mL of Mr and 2  106 crystals and spores/mL of Bt because in preliminary experiments these concentrations led to sublethal bacteriosis and prolonged mycosis, and their combination gives stable synergistic effect in CPB mortality. Insects and potato leaves of the control group were treated with pure water. Treated leaves were replaced by untreated at 48 h after inoculation. Then foliages were changed daily during 12 days. The mortality rate of larvae was recorded daily over 12 days. 2.3. Conidia germination and colonization Determination of the extent of in vivo conidial germination on live larval cuticles was performed at 24 h post infection using a method adapted from Butt (Butt, 1997). Before microscopic investigation, larvae were dipped in an aqueous solution of 0.1% Calcofluor (Sigma) white stain for 25 s. The larvae were then airdried and the head and hemocoel contents were removed. The cuticle was observed using a fluorescent microscope Axioscope 40 (Zeiss, Germany). The stain allows the visualization of fungal cell walls such that it is possible to identify and count fungal germ tubes and hyphal penetration events. To establish the germination level, approximately 100 germinated conidia were observed for each larva. On the fourth day after infection, the hemolymph of larvae was collected (Dubovskiy et al., 2013) and plated onto Czapek’s medium with lactic acid (0.4%) in 90 mm Petri dishes. Dishes were incubated at 25 °C for 4 days and Mr colonies were detected, allowing the percentage of insects with systemic (hemolymph) fungal infections to be calculated. 2.4. Fat body, cuticle and hemolymph samples preparation Dissected fat body and hemolymph was sampled in 0.1 M sodium phosphate buffer pH 7.2 (PBS) in cooled tubes, to which 4-mg/mL phenylthiourea was added to prevent melanization. The fat body was ground with an ultrasonic homogenizer. Samples were centrifuged for 5 min at 500 g for hemolymph and for 10 min at 10,000g for fat bodies at 4 °C. Whole cuticles from 3 larvae were dissected in 500 ll PBS, washed 3 times by vortexing for 1 min in PBS, and homogenized in 300 ll PBS for 2 min at 6.5 M/s with a FastPrepÒ-24 homogenizer (MP Biomedicals, USA). The homogenates were centrifuged for 10 min at 10,000g at 4 °C. The supernatants (hemolymph, fat body, and cuticle) were used for spectrophotometric analysis of enzymatic activity and protein concentration. 2.5. Encapsulation rate and total hemocyte count The encapsulation rate and total hemocyte count were measured on the first day and third day after inoculation. Implants 2 mm long and 0.5 mm in diameter were injected into the larval hemocoel through the perforation of the parallel cuticle between 7–8, 8–9 segments ventral segment, not touching the internals. Implants were dissected out from the body cavity after 2 h of exposure and then photographed from three points of view. The encapsulation response was quantified by measuring the

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degree of melanization of the implants using the Image Pro software (Dubovskiy et al., 2013). A 10 ll volume of hemolymph was sampled in 30 ll cooled anticoagulant with PTU for a total hemocyte count (THC). The THC was immediately measured with a hemocytometer as the number of hemocytes per 1 ml of hemolymph. 2.6. Enzymatic activity The activity of enzymes were measured on the first day and third day after inoculation. Phenoloxidase (PO) enzymatic activity was estimated in a modification of the method described by Ashida and Soderhall (1984). Aliquots of 15 ll of hemolymph plasma or 50 ll of cuticle homogenate were added to 200 ll of 10 mM L-DOPA (L-3,4dihyroxyphenylalanine). After 15 min for hemolymph plasma, or 6 h for cuticle, at 28 °C, the absorbance was quantified at 490 nm. Nonspecific esterase (EST) activity was estimated by spectrophotometric analysis of the p-nitrophenyl acetate hydrolysis rate in accordance with Prabhakaran et al. (Prabhakaran and Kamble, 1995). Samples (5 ll) were incubated for 25 min (for hemolymph plasma) and 5 min (for fat body) with 200 ll pnitrophenyl acetate at 28 °C, then, the transmission density was measured at 410 nm. The activity of glutathione-S-transferase (GST) against 2-nitro5-thiobenzoic acid (DNTB) was estimated by the method of Habig (Habig et al., 1974). Incubation of a 10 ll sample (hemolymph plasma or fat body) was performed in 0.1 M potassium phosphate buffer (pH 6.5) with 1 mM glutathione and 1 mM DNTB at 25 °C for 20 min. The reaction was initiated by the addition of a DNTB acetone solution. The concentration of 5-(2,4-dinitrophenyl)-gluta thione generated by the reaction was estimated at a wavelength of 340 nm. The protein concentration in the hemolymph plasma or fat body was estimated using the Bradford method (Bradford, 1976). For the calibration curve, bovine serum albumin was used. EST, GST and PO activities were evaluated in units of transmission density (Da) of the incubation mixture during the reaction per 1 min and 1 mg of protein. 2.7. Statistical analysis Data on the survival rate of insects were analyzed using the Kaplan-Meyer method (Sigma-Stat 3). Analyses for additive and synergistic interactions were based on a binominal test, which involved comparing the expected and observed mortalities due to mixed treatment following the approach described in Tounou et al. (Tounou et al., 2008) on a daily basis. The activity of enzymes, THC and encapsulation rate are given as the arithmetic mean ± S.E. The normality of data was assessed using the Shapiro-Wilk W test and analyzed by two-way ANOVA, followed by the Tukey test (STATISTICA 6.0). At least 60 individuals were used for the survival bioassay, 30 for measurement of the encapsulation rate and PO activity, 8 and 20 for germination and colonization assays, respectively, and 10 for the measurement of THC, EST, and GST activity. 3. Results 3.1. Bioassay Fungal infection alone led to 86% mortality at 12 days and the median lethal time (LT50) was 8 ± 0.5 days (Fig. 1). The mortality of insects infected with Bt did not significantly differ from controls (v2 = 0.06; P = 0.80). Significantly faster mortality was observed under combined treatments, and the LT50 was 3 ± 0.2 days after

Fig. 1. Dynamics of survival of Colorado potato beetle IV instar larvae after treatment with the entomopathogenic fungus M. robertsii (Mr) (7  105 conidia/ml) and bacteria B. thuringiensis (Bt) (1  106 crystals/ml). ⁄ – synergistic effect (v2 > 4.91, P < 0.05).

mixed infection (Mr + Bt) and differed significantly from fungal infection alone (v2 = 25.9; P < 0.00001). A synergistic effect between bacteria Bt and fungus Mr was registered at 2–11 days after treatment of CPB larvae (v2 > 4.91; P < 0.05) (Fig. 1). Additionally, we recorded typical symptoms of bacteriosis and mycosis in larvae, including melanic spots after treatment with Mr and significant delay of growth during Bt infection (Figs. S1, S2). 3.2. Fungal germination and colonization An increase of the conidia germination level on the CPB cuticle was observed under mixed infection, and 85.8 ± 3.9% conidia were germinated under infection with fungus alone vs 95.0 ± 3.2% under mixed infection (t = 2.3; df = 10; P = 0.047). A tendency for a more rapid colonization of hemolymph with fungus was also observed in the mixed infection. At the fourth day of the experiment, fungus was detected in 45% of larvae infected with Mr and 65% larvae infected with combination of Mr and Bt (v2 = 1.6; P = 0.20). 3.3. Encapsulation rate and THC A significant changes in the intensity of encapsulation and THC were not observed after inoculation with fungus alone (P > 0.5). However, a week trend of decreasing cellular immunity parameters was observed on the third day of mycosis (Fig. 2A, B). Significant decrease of the encapsulation rate was observed under the influence of bacteria (F1, 93 = 17.4; P = 0.00007 and F1, 105 = 19.3; P = 0.00003 at the first and third days after treatment, respectively; Fig. 2A). In particular, we found a 1.5–2-fold decrease in the implant encapsulation intensity in the variants with bacteria at the first and third days after inoculation compared to control and fungal infection alone. Analogous changes were observed in the THC (Fig. 2B). Bacterial infection decreased the THC on the first and third days post infection (F1, 35 = 10.8; P = 0.002 and F1, 36 = 35.4; P = 0.00001, respectively). We did not revealed a significant interaction between investigated factors (fungal and bacterial infections) in cellular immunity parameters (F < 3.4; P > 0.08). Thus Bt had greater implication in inhibition of cellular immunity under development of the mixed infection.

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Fig. 2. Encapsulation response (A) and total hemocyte count (THC) (B) in the hemolymph of Colorado potato beetle larvae after treatment with the entomopathogenic fungus M. robertsii (Mr) and bacteria B. thuringiensis (Bt). The letters above the columns show significant (P < 0.05) differences: a – from control, b – from Mr infection, c – from Bt infection, d – from mixed infection, within day (HSD Tukey test).

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Fig. 3. Phenoloxidase (PO) activity in integuments (A) and hemolymph (B) of Colorado potato beetle larvae after treatment with entomopathogenic fungus M. robertsii (Mr) and bacteria B. thuringiensis (Bt). The letters above the columns show significant (P < 0.05) differences: a – from control, b – from Mr infection, c – from Bt infection, d – from mixed infection, within day (HSD Tukey test).

3.4. Phenoloxidase activity Fungal infection did not significantly change PO activity in integuments on the first day after treatment, but bacteria enhanced the activity of the enzime (F1, 36 = 4.7; P = 0.036) (Fig. 3A). On the third day of the experiment, both fungus and bacteria resulted in an increase in PO activity in integuments (F1, 33 = 34.3; P < 0.00001 and F1, 33 = 40.3; P < 0.00001, respectively) (Fig. 3A). Significantly increased PO was observed under mixed infection, but a significant interaction between bacteria and fungus was not revealed (F1, 33 = 1.7; P = 0.20). The PO activity in the hemolymph was not changed significantly under fungal infection on the first and third day after treatment (P > 0.3 compared to control), but it was increased under Bt monoinfection (P < 0.018 compared to control) (Fig. 3B). A significant decrease of enzyme activity was observed after combined treatment compared to mono-infections and control on the third day of the experiment (factors interaction: F1, 113 = 23.3; P = 0.00001).

3.5. Detoxifying enzymes We found a 1.6–1.9-fold (P < 0.05) increase of nonspecific esterases (EST) and GST activity in the hemolymph of larvae on the first day after inoculation with fungus compared to other groups (Fig. 4A, B). However, bacteria suppressed both EST and GST to the control level in larvae under combined infections (factors interaction: F1, 35 > 4.9; P < 0.03). By contrast, on the third day of the experiment, we observed enhanced EST and GST activity in the hemolymph under bacteriosis, but fungus restrained this increase in insects infected with Mr and Bt (factors interaction: F1, 35 > 9.5, P < 0.004). In the fat body, fungal infection did not change significantly EST and GST activity on the third day after treatment (F < 2.7; P > 0.20). However we observed a decrease of EST and GST activity under the influence of bacteria (F1, 35 > 5.9; P < 0.02) (Fig. 4C, D). On the third day of the experiment, a significant change in EST and GST activity was not detected.

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Fig. 4. Activity of nonspecific esterases (EST) and glutathione-S-transferases (GST) in the hemolymph and fat body of Colorado beetle larvae after treatment with the entomopathogenic fungus M. robertsii (Mr) and bacteria B. thuringiensis (Bt): A – EST in hemolymph, B – GST in hemolymph, C – EST in fat body, D – GST in fat body. The letters above the columns show significant (P < 0.05) differences: a – from control, b – from Mr infection, c – from Bt infection, d – from mixed infection, within day (HSD Tukey test).

4. Discussion This study shows that sublethal infection with Bt did not result in mortality of CPB larvae, but it led to a significant change of immune-physiological reactions and increased susceptibility to fungal infection. We found the bacteria, enhances the phenoloxidase activity, significantly inhibits cellular immunity and disrupts the response of detoxificative system to fungal infection. In previous works authors proposed that synergy between Bt and entomopathogenic fungi caused by the termination of insect feeding and delay of development under the influence of bacteria, that increases mortality due to fungi (Kryukov et al., 2009; Wraight and Ramos, 2005). We found an increase of conidia germination on insect cuticles and a trend of more rapid colonization of the hemocoel with fungus under the influence of Bt infection. We propose that a change in the immune status of CPB larvae during Bt infection also effects the cuticular composition via the activity of epidermal cells. Evidence for this is the increased PO activity in integuments after Bt sublethal dose. In addition, changes in integument structures may be connected to the systemic action of dam-

age associated molecular pattern molecules, which transfer damage signals from the midgut to other tissues (Krautz et al., 2014). Similar changes were identified for intestinal Bt infection in wax moth, Galleria mellonella, larvae (Dubovskiy et al., 2016). The elevated PO level under Bt infection may be also related to the termination of larval growth. We have shown early that highest level of cuticular PO activity was observed during 24–36 h post molting in IV instar, then (2–3 days post molting) the enzime level was declined significantly (Tyurin et al., 2016). Therefore, the PO level in integuments of intact larvae was highest at the beginning of IV instar and decreased to the third day after molting, such that changes of PO are observed simultaneously with a disturbance of ontogenesis under bacteriosis. Enhanced PO activity in integuments after Mr treatment indicates the active development of fungus in the cuticle. We previously showed the correlation between virulence of Metarhizium strains and PO response in the integument of CPB larvae (Tyurin et al., 2016). The strong activation of cuticular PO, which was observed at the third day of combined infection, indicates that fungus developed in integuments is more active under mixed infection. This observation is consistent with

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more active germination and rapid colonization of CPB larvae with fungus under Bt sublethal infection. We found a significant decrease of THC and encapsulation rate in the hemocoel of CPB larvae during Bt infection. The decrease of THC in Lymantria dispar larvae during bacterial infection with Bt was shown by Broderic et al. (Broderick et al., 2010). The decrease of cellular immunity reactions may be related to the decrease of hemocytes as result of bacterial intoxication (Grizanova et al., 2014). In addition, starvation, which is a symptom of Bt bacteriosis, may decrease the THC and encapsulation rate in insects (Lestes viridis, Spodoptera littoralis). This was shown by Block and Stoks (De Block and Stoks, 2008) and Lee et al. (Lee et al., 2006). Reactions of cellular immunity, such as encapsulation, have important functions against fungal infection (Griesch and Vilcinskas, 1998). Therefore, suppression of the cellular immune response in CPB under bacteriosis may be an important immunophysiological factor leading to high mortality due to fungus under sublethal Bt infection. Interestingly, during the development of acute pathological processes caused by M. robertsii, the encapsulation rate was decreased in CPB larvae (Dubovskiy et al., 2010). The decrease in phagocytosis and nodule formation occurs due to the action of pure fungal metabolites (Bandani, 2008) and under the development of fungal infections (Vilcinskas et al., 1997). The decrease of immunocompetent hemocyte count (plasmatocytes and granulocytes) at the initial stages of mycoses were observed after infection of CPB with different species of Metarhizium and the response correlated with the virulence of strains (Tyurin et al., 2016). In the present study, we observed a slight trend of decreased encapsulation intensity and THC under fungal infection alone. This is likely related to the low dose of conidia and long development of mycosis. Elevation of PO activity in the hemolymph under bacteriosis may be related to both the mobilization of the immune system and toxic effects of bacterial metabolites (Ayres and Schneider, 2008; Grizanova et al., 2014; Rahman et al., 2004). PO may be related to the wound repair process and melanization of pathogen crossing the midgut. Interestingly, we observed a reduction of PO activity on the third day of combined treatment with Bt and Mr that is evidence of strong suppression of CPB larvae immunity. We found that both monoinfections of fungus at the first day and Bt at the third day enhanced EST and GST activity in the hemolymph. The early activation of nonspecific esterases and GST in the hemolymph at the initial stage of acute mycosis was followed by a decrease to control levels (Dubovskiy et al., 2010; Serebrov et al., 2006; Zibaee et al., 2009). Clearly, this increase is related to the inactivation of fungal toxins (e.g., destruxins) and endobiotics produced during pathogenesis (lipid peroxidation products and ROS) (Slepneva et al., 2003; Sree et al., 2010). It is important to note that the increase of EST and GST activity in the hemolymph was not observed under mixed infection. Mixed infection likely led to restraint of the activation of enzymes. In addition, the decrease of EST and GST activity in the fat body as a main detoxifying organ was revealed under the influence of Bt. The suppression of the detoxification system under bacterial infection may enhance the susceptibility of insects to fungi and toxic metabolites formed during pathogenesis. In conclusion, we showed that sublethal Bt infection suppresses several defense systems related to the resistance of insects to fungi that leads to synergy between pathogens. This study confirms the hypothesis of the immunosuppressive influence of different stressfactors on insect susceptibility to low specificity pathogens, such as ascomycetes Metarhizium and Beauveria (Boomsma et al., 2014). These influences may not be lethal for insects, but the immunosuppressed insects-hosts becomes more susceptible to infections. Evidently, the mechanism of synergy between Bt and fungi occurs on multiple levels and includes disturbances in ontogenesis, changes

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of fungistatic properties of the cuticle, and suppression of cellular and humoral immunity and the detoxification system. We propose that the change in susceptibility of insects to pathogens under combined infection is determined by a range of interactions and is related to the general suppression of host resistance. The study confirms high availability to use combined preparations on the basis Bt and entomopathogenic fungi in biological control of CPB larvae. Acknowledgments This work was supported by the Russian Science Foundation (project No. 15-14-10014). The authors are grateful to Dr V.A. Shilo (Karasuk Station of the Institute of Systematics and Ecology of Animals, Siberian Branch, Russian Academy of Sciences) for help in organizing the experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinsphys.2016.10. 004. References Ali, K., Wakil, W., Zia, K., Sahi, S.T., 2015. Control of Earias vittella (Lepidoptera: Noctuidae) by Beauveria bassiana along with Bacillus thuringiensis. Int. J. Agric. Biol. 17, 773–778. Alyokhin, A., Dively, G., Patterson, M., Castaldo, C., Rogers, D., Mahoney, M., Wollam, J., 2007. Resistance and cross-resistance to imidacloprid and thiamethoxam in the Colorado potato beetle Leptinotarsa decemlineata. Pest Manag. Sci. 63, 32– 41. Ashida, M., Soderhall, K., 1984. The prophenoloxidase activating system in crayfish. Comp. Biochem. Phys. B 77, 21–26. Ayres, J.S., Schneider, D.S., 2008. A signaling protease required for melanization in Drosophila affects resistance and tolerance of infections. PLoS Biol. 6, 2764– 2773. Bandani, A.R., 2008. The effects of entomopathogenic fungus, Tolypocladium cylindrosporum on cellular defence system of Galleria mellonella. J. Agric. Sci. Technol. 10, 135–146. Boomsma, J.J., Jensen, A.B., Meyling, N.V., Eilenberg, J., 2014. Evolutionary interaction networks of insect pathogenic fungi. Annu. Rev. Entomol. 59 (2014), 467–485. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Bravo, A., Gill, S.S., Soberon, M., 2007. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49, 423–435. Broderick, N.A., Raffa, K.F., Handelsman, J., 2010. Chemical modulators of the innate immune response alter gypsy moth larval susceptibility to Bacillus thuringiensis. BMC Microbiol. 10, 129. Butt, T.M., 1997. In Manual of Techniques in Insect Pathology. Academic Press, London. Butt, T.M., Coates, C.J., Dubovskiy, I.M., Ratcliffe, N.A., 2016. Entomopathogenic fungi: new insights into host-pathogen interactions. Adv. Genet. 94, 307–364. De Block, M., Stoks, R., 2008. Compensatory growth and oxidative stress in a damselfly. Proc. R. Soc B Biol. Sci. 275, 781–785. Dubovskiy, I.M., Grizanova, E.V., Whitten, M.M., Mukherjee, K., Greig, C., Alikina, T., Kabilov, M., Vilcinskas, A., Glupov, V.V., Butt, T.M., 2016. Immuno-physiological adaptations confer wax moth Galleria mellonella resistance to Bacillus thuringiensis. Virulence, 1–11. Dubovskiy, I.M., Krukova, N.A., Glupov, V.V., 2008. Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis. J. Invertebr. Pathol. 98, 360–362. Dubovskiy, I.M., Whitten, M.A., Kryukov, V.Y., Yaroslavtseva, O.N., Grizanova, E.V., Greig, C., Mukherjee, K., Vilcinskas, A., Mitkovets, P.V., Glupov, V.V., Butt, T.M., 2013. More than a colour change: insect melanism, disease resistance and fecundity. Proc. R. Soc. B Biol. Sci. 280. Dubovskiy, I.M.K., Yu, V., Benkovskaya, G.V., Yaroslavtseva, O.N., Surina, E.V., Glupov, V.V., 2010. Activity of the detoxificative enzyme system and encapsulation rate in the Colorado potato beetle Leptinotarsa decemlineata (Say) larvae under organophosphorus insecticide treatment and entomopathogenic fungus Metharizium anisopliae. Euroasian Entomol. J. 9, 577–582. Gillespie, J.P., Bailey, A.M., Cobb, B., Vilcinskas, A., 2000. Fungi as elicitors of insect immune responses. Arch. Insect Biochem. 44, 49–68.

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