View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia Journal of Science: Vol. 11 | Article 138 Zibaee et al.

Cellular immune reactions of the , integriceps, to the entomopathogenic fungus, Beauveria bassiana and its secondary metabolites Arash Zibaee1a*, Ali Reza Bandani2b, Reza Talaei-Hassanlouei2c, and Davide Malagoli3d

1Department of Plant Protection, College of Agriculture, University of Guilan, Rasht 41635-1314, 2Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj 31584, Iran 3Department of Biology, University of Modena and Reggio Emilia, Via Campi 213/D, 41125, Modena, Italy Abstract In this study, five morphological types of circulating hemocytes were recognized in the hemolymph of the adult sunn pest, Eurygaster integriceps Puton (: ), namely prohemocytes, plasmatocytes, granulocytes, adipohemocytes, and oenocytoids. The effects of the secondary metabolites of the entomopathogenic fungus Beauveria bassiana on cellular immune defenses of Eurygaster integriceps were investigated. The results showed that the fungal secondary metabolites inhibited phagocytic activity of E. integriceps hemocytes and hampered nodule formation. A reduction of phenoloxidase activity was also observed. The data suggest that B. bassiana produce secondary metabolites that disable several immune mechanisms allowing the fungus to overcome and then kill its host. This characteristic makes B. bassiana a promising model for biological control of insect pests such as E. integriceps.

Keywords: fungi , hemocyte, nodule formation, phagocytosis Abbreviations: PO, prophenoloxidase; THC, total hemocyte count Correspondence: a [email protected], b [email protected], c [email protected], d [email protected], *Corresponding author Editor: Michael Strand was Editor of this paper. Received: 1 November 2010, Accepted: 17 April 2011 Copyright : This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed. ISSN: 1536-2442 | Vol. 11, Number 138 Cite this paper as: Zibaee A, Bandani AR, Talaei-Hassanlouei R, Malagoli D. 2011. Cellular immune reactions of the sunn pest, Eurygaster integriceps, to the entomopathogenic fungus, Beauveria bassiana and its secondary metabolites. Journal of Insect Science 11:138 available online: insectscience.org/11.138

Journal of Insect Science | www.insectscience.org 1 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al.

Introduction process by which cells engulf large particles from the environment, is essential for host live in different environments where defense against infectious microorganisms they are exposed to various potential invaders and for the clearance of apoptotic cells such as pathogens, parasites, and parasitoids. generated during development (Borges et al. However, insects are successful in colonizing 2008). Nodulation is a complex multi-step every niche on Earth. Thus, their success must process that occurs quickly after microbial also be attributed to their ability to neutralize infection. Nodule formation is initiated with pathogen invasions (Dunn 1986; Lowenberger the micro-aggregation of hemocytes, which 2001; Silva et al. 2002). Insects have a highly entrap large numbers of microorganisms. efficient immune system that is able to These micro-aggregates grow in size by withstand challenges from the majority of recruiting additional hemocytes (Ratcliffe and microorganisms present in the different Rowley 1976). Finally, the process ends with habitats where they live. Insect innate immune melanization into darkened nodules, which system is highly developed and it relies on the attach to the body wall or to various internal humoral and cellular components (Gillespie et organs (Franssens et al. 2006). In these al. 1997; Lavine and Strand 2002). Humoral processes, POs have an important role responses include the synthesis of a broad especially in nodule formation encapsulation. spectrum of antimicrobial proteins (Bulet et POs are present in the host cuticle and al. 1999) and the prophenoloxidase (PO) catalyze the hydroxylation of mono and cascade (Ashida and Brey 1998; Cerenius and diphenols to quinone intermediates. In insects, Söderhall 2004), as well as the production of the products of the PO cascade are believed to reactive intermediates of oxygen and nitrogen be involved in wound healing, sclerotisation (Lavine and Strand 2002). Antimicrobial of cuticle, and recognition and melanization of proteins and other immune-related molecules foreign particles (Soderhall and Aspan 1993; are mainly secreted by the larval hemocytes Sugumaran 1998). PO-derived quinone and and fat body (Bulet et al. 1999). Circulating melanin have been shown to have fungistatic hemocytes have important roles in immune and fungicidal activities in vitro (Söderhall mechanisms of insects against and Ajaxon 1982; St. Leger et al. 1988; microorganisms (Russo et al. 2001). Insects Gillespie et al. 2000). have several types of hemocytes that are commonly identified by morphological, Also, because of their highly efficient immune histochemical, and functional characteristics system, some insect pests prosper in human- (Gupta 1985). The most common types of influenced environments such as agro- hemocytes reported in the literature are ecosystems and cause sever economic prohemocytes, granulocytes, plasmatocytes, damage. As a consequence, pests are the adipohemocytes, and oenocytoids. These five target of different control procedures; hemocyte types have also been described in principally pesticides, and secondarily many insect species (Gupta 1985). They biological control agents like pathogens and additionally act through various processes parasitoids. Biological control of insect pests including phagocytosis, nodule formation, and is considered as a priority to decrease side encapsulation to entrap and clear pathogens effects due to the use of chemical pesticides. from the hemolymph. Phagocytosis, the Insect pathogens and entomopathogenic fungi

Journal of Insect Science | www.insectscience.org 2 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. have an ability to overcome the robust Materials and Methods immune systems of insects and reach successful pathogenesis (Gillespie et al. 1997; Insects Bandani 2005). Life cycles of The insects were collected from the Karaj entomopathogenic fungi are associated with farm and reared on seeds of the Fallat the synthesis and secretion of several numbers wheat cultivar in the laboratory at 27 ± 2 °C of toxic metabolites including extracellular and 14:10 L:D (Zibaee and Bandani 2010). enzymes, proteins, and low molecular weight Insects were fed seeds, and a piece of cotton compounds such as toxins (Bandani 2005). soaked with water was used as a water source. The growth of the entomopathogenic fungus Beauveria bassiana in the hemolymph of the B. bassiana culture host is associated with the secretion of Beauveria bassiana isolate B1 was cultured at metabolites, especially those originating from 25 ± 1 °C on Sabouraud Dextrose Agar (pH = proteins (Mazet et al. 1994; Clarkson and 5.6) amended with 1% yeast extract. After 14 Charnley 1996; Bandani et al. 2000; Bandani days, conidia of B. bassiana were washed off 2005). These peptides, such as destruxins and with a 0.01% aqueous solution of Tween 20 efrapeptins, are indicated as secondary (Sigma Aldrich, www.sigmaaldrich.com), and metabolites to differentiate them from the different concentrations of spores were cuticle-degrading protease that favors the prepared as required after several preliminary invasion of the pathogen. The secondary tests. metabolites are considered to be important pathogenicity determinants (Bandani et al. B. bassiana toxin extraction 2000; Bandani 2005; Zibaee et al. 2009). Conidia were harvested from 14-day-old sporulating cultures of B. bassiana by Studies on mechanisms of fungal pathogenesis scraping the surface with a spatula and and insect immune responses may provide suspending the conidia in sterile 0.01% v/v 6 strategies for the development of more aqueous Tween 20 and diluting to 10 conidia efficient mycoinsecticides for destructive per mL. One mL of conidial suspension was pests. One such insect, the sunn pest, then used to inoculate 100 mL of Czapek Dox Eurygaster integriceps Puton (Hemiptera: (Oxoid, www.oxoid.com) broth supplemented Scutelleridae), is a key constraint on wheat with 0.5% w/v Bactopetone (Oxoid) in 250 production in the wide area of the Near and mL Erlenmeyer flasks. The fungus was then Middle East, Eastern and Southern Europe cultured at 23 °C in a cooled orbital incubator and North Africa. E. integriceps causes severe at 1200 g for 12 days. The broth was filtered damages to the vegetative growth stage of through four layers of cheesecloth followed wheat, and significantly decreases both the by Whatman No. 1 filter paper (Whatman, quantity and quality of grains. Hence, the aims www.whatman.com) to ensure removal of of this study were the identification of distinct conidia and hyphal debris. Culture filtrates morphological types of hemocytes by light were extracted as described by Bandani et al. microscopy, and the determination of the (2000). This entailed extraction with effects of B. bassiana strain B1 and its chloroform, filtration of the solvent phase secondary metabolites on the cellular immune through Whatman No. 1 (phase separator) reactions of E. integriceps. filter paper to remove any aqueous residue, and removal of the solvent on a rotary

Journal of Insect Science | www.insectscience.org 3 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. evaporator. The residue was dissolved in were transferred to a 9 cm diameter Petri dish acetone, filtered through a cotton plug, and with wheat grain to follow the course of the concentrated under a stream of nitrogen at 40 assay. Secondary metabolite of B. bassiana °C. The residue was then weighed and stored was dissolved in dimethyl sulfoxide (DMSO) at 4 °C. (Sigma Aldrich) so that DMSO concentration in solution was 0.5% or less. Adults were Determination of hemocyte types by light injected with five concentrations of fungal microscopy secondary metabolite (2, 6, 15, 30, 50%) to For this purpose, hemolymph from 10 adult E. find lethal dose values to evaluate immune integriceps was collected carefully from responses. The DMSO (0.5%) injected adults severed front legs with a 50 L sterile glass were considered as control. capillary tube (Sigma Aldrich). The product was immediately diluted in an anticoagulant The effect of fungal spore and its secondary solution (0.01M ethylenediamine tetraacetic metabolite on hemocyte numbers acid, 0.1M glucose, 0.062M NaCl, 0.026M To determine if the injection of fungal citric acid, pH = 4.6) as described by secondary metabolite or conidia caused any Azambuja et al. (1991). Several samples were changes in the total and differentiate prepared, including 150 L hemolymph, 15 hemocyte counts (THC and DHC), adults L anticoagulant solution, and 80 L were injected laterally into the thorax with 1 phosphate buffer. 100 L of each sample were L of a 106 spores/mL concentration of B. then cytocentrifuged (Shandon Cytospin II, bassiana and 3, 7, and 12% concentrations of Thermo Scientific, fungal secondary metabolite, as well as www.thermoscientific.com) onto slides at 200 DMSO (0.5%) as a control. Hemolymph was rpm for 3 min. Nuclei of cells were stained collected 1, 3, 6, 12 and 24 hours after with Hoechst stain 33342, (Invitrogen, injection from the control group, spore- www.invitrogen.com) 10 mM for 10 min injected, and secondary-metabolite injected before being mounted with Mowiol (Merck, adults. Samples of hemolymph from 5 adults www.syngentacropprotection.com). Images of were bled into 1 mL of ice–cold anticoagulant cells were taken under an Eclipse 90i digital buffer in 1.5 mL plastic tubes. The tubes were microscope (Nikon, www.nikon.com). The gently inverted 5 to 7 times to facilitate microscope was equipped with a super high- mixing, and both total and different hemocyte pressure mercury lamp and connected to a DS numbers were counted using an improved cooled camera head DS-5Mc regulated by Neubauer hemocytometer. For each treatment, ACT-2U software (Nikon). 30 adults were used and the experiment was repeated twice. Injection of insects with spores and secondary metabolite Effects of fungal spores and secondary Adults (0.53 mg, No. 120) were chilled on ice metabolite on phagocytosis for 15 min, surface sterilized with 70% Thirty adults were injected laterally into the alcohol, and then injected with 1 L of five thorax with 1 L of a 106 spores/mL concentrations (104, 105, 106, 107, and 108 concentration of B. bassiana and 3, 7, and spore/mL) of fungal spores by a 10 L 12% concentrations of fungal secondary Burkard syringe (Burkard, metabolite. Hemolymph was collected in 30, www.burkard.co.uk). After injection, adults 60, and 120 min after injection. Phagocytic

Journal of Insect Science | www.insectscience.org 4 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. activity was determined by counting the cells containing spores in a Neubauer hemocytometer for spore-injected adults, secondary-metabolite injected adults, and DMSO (0.5%)-injected adults as control. Observations were made on an Olympus phase contrast microscope.

Effects of fungal spores and secondary metabolite on nodulation Injections were carried out according to the method described above. Nodulation was assessed at 1, 3, 6, 12, and 24-hour intervals. Adults were chilled on ice, hemolymph was gathered in a capillary tube, and then 200 L samples in three replicates were poured in a hemocytometer and nodules were counted (Franssens et al. 2006).

Phenoloxidase activity (PO) In order to test the effect of B. bassiana spores and its secondary metabolites on the PO system in adults of E. integriceps, a hemocyte lysate supernatant was prepared after injections. Hemolymph from adults was mixed with anticoagulant buffer and centrifuged at 12,000 rpm for 5 min; the supernatant was discarded and the pellet washed gently twice with a phosphate buffer (pH = 6.5, Leonard et al. 1985). Cells were homogenized in 500 mL of phosphate buffer centrifuged at 12,000 rpm for 15 min, and the hemocyte lysate supernatant was used in PO assays. Samples were pre-incubated with buffer at 30 °C for 30 min before the addition of 50 mL of 10 mM aqueous solution of L- dihydroxyphenylalanin. The mixture was incubated for an additional five min at 30 °C

and PO activity was measured in a Figure 1. (a-e) Light microscopy of Eurygaster integriceps hemocytes: spectrophotometer at 490 nm. One unit of PO (a) a prohemocyte with a large nucleus (thin arrow) and a thin cytoplasm; (b) a plasmatocyte exhibiting a spindle shape and activity represents the amount of enzyme cytoplasmic elaborations; (c) a granulocyte filled with the typical required to produce an increase in absorbance granules in the cytoplasm (arrow) and large nucleus (arrow); (d) an 1 adipohemocyte with lipid droplets spreading in the cytoplasm and of 0.01 min (Dularay and Lackie 1985). specific cytoplasm elaborations; (e) an oenocytoid with a round Activity in treated assays was compared with eccentric nucleus and crystalin inclusions. Magnification 40 with the exception of (b) (60). Bar = 5 μm, with the exception of (b) (3 μm). High quality figures are available online.

Journal of Insect Science | www.insectscience.org 5 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. that of controls. Assays were done in 3 After electrophoresis, gels were stained by a replicates (n = 3) and the whole experiment 10 mM solution of L-dihydroxyphenylalanine, was repeated twice. For measurement of PO washed with phosphate buffer, and then kinetic parameters, different concentrations of photographed. Molecular markers were L-dihydroxyphenylalanine; 3, 3.5, 4, 5, 6, 7, 8, stained by Comassie brilliant blue. 9, and 10 mM were mixed with 20 L of enzyme solution and read at 490 nm. The Protein determination Michaelis constant (Km) and the maximal Protein concentrations were measured velocity (Vmax) were estimated by SigmaPlot according to the method of Bradford (1976), v11 (Systat Software, www.sigmaplot.com) using bovine serum albumin (Bio-Rad, and the results of Km and Vmax were the means www.bio-rad.com/) as a standard. ± SE of 3 replicates (n = 3) for each concentration. Statistical analysis POLO-PC software (Leora 1987) was used in Isozyme electrophoresis assay of the determination of mortality and lethal phenoloxidase concentrations. All data were compared by Hemocyte lysate supernatants were prepared one-way analysis of variance (ANOVA), 12 h after injections in addition to a control followed by Tukey's studentized test and line sample using the procedure described by regression analysis when significant Leonard et al. (1985) and subjected to vertical differences were found at p  0.05 (SAS electrophoresis. Native-Polyacrylamide gel 1997). Differences between samplings were electrophoresis, 4% stacking and 8% considered statistically significant at a separating, was carried out at 100 mV probability more than 5% (p  0.05). constant current. In addition to provided samples, b-Galactosidase (116 kDa), bovine Results serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35.5 kDa), Identification of hemocytes by light restriction endonuclease Bsp 981 (25 kDa), b- microscopy lactoglobulin (18.4 kDa), and lysozyme (14.4 Five morphological types of the circulating kDa) were used as molecular mass standards.

Figure 2. Phagocytosis of Beauveria bassiana spores by Figure 3. Nodule formation in Eurygaster integriceps adults 12 hours plasmatocytes. In vivo effect of secondary metabolites on the E. after inoculation by Beauveria bassiana spores. Scale bar 10 μm. The integriceps phagocytic activity on B. bassiana spores. Mean ± standard magnification of images is 40. High quality figures are available error, N = 30, *p < 0.05 vs. control. High quality figures are available online. online. Journal of Insect Science | www.insectscience.org 6 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. hemocytes were recognized in the hemolymph intervals when compared with control, while of E. integriceps adult, including oenocytoids and adipohemocytes showed no prohemocytes, plasmatocytes, granulocytes, significant differences (Table 2). A significant adipohemocytes, and oenocytoids (Figure 1). increase in the number of plasmatocytes and Prohemocytes are small cells (10-12 m) with granulocytes was observed in injected adults a spherical shape. The nucleus is large, by fungal spores in all intervals after the centrally located, and fills the cell so that the inoculation, suggesting a possible role in cytoplasm occupies just a narrow area around phagocytosis or defense against fungal spores. the nucleus (Figure 1a). Plasmatocytes are The number of prohemocytes significantly spindle-like cells with an average size of 22 decreased in response to fungal infection after m length and 7 m width, with cytoplasmic 60 and 120 min, indicating a potential processes and granules in the cytoplasm conversion to plasmatocytes and granulocytes. (Figure 1b). Granulocytes (20-25 m in size) B. bassiana secondary metabolite showed a display an oval and regular shape, with a large negative effect on hemocytes of E. nucleus and numerous granules in the integriceps, most notably plasmatocytes and cytoplasm (Figure 1c). Adipohemocytes are granulocytes at different intervals after circular cells, 25-30 m in size, characterized injection (Table 3). Along with an increase of by droplets of lipid spread around the fungal secondary metabolite concentration, cytoplasm (Figure 1d). Oenocytoids display a the number of plasmatocytes and granulocytes round and regular shape (25-27 m) with a sharply decreased so that the lowest number small and eccentric nucleus and very few and was observed for the 15% concentration 30 small cytoplasmic granules and inclusions min after injection (Table 3). However, (Figure 1e). number of prohemocytes, oenocytoids, and adipohemocytes varied in different Effect of B. bassiana spores and secondary concentrations and intervals. metabolites on total and count of E. integricpes hemocytes In vivo effect of B. bassiana spores and Total and count of hemocytes of adult E. secondary metabolite on phagocytosis integriceps showed significant differences in Fungal injection increased phagocytic activity various intervals after injection of B. bassiana of E. integriceps so that it was two times spores and secondary metabolites in higher relative to the control (DMSO 0.5% comparison with control (Tables 1, 2, 3). injection, Figure 2). Fungal secondary Table 1 demonstrates that the highest number metabolite significantly decreased the amount of hemocytes was observed 6 h after DMSO of phagocytizing cells (Figure 2). The effect (0.5%), fungal spores, or secondary of B. bassiana metabolite was dose-related, as metabolites were injected. Increasing of indicated by the observation that 7 and 12% fungal secondary metabolite concentration concentrations had the most relevant effect on sharply decreased the total hemocyte number the number of phagocytized spores (Figure 2). of adults in all intervals after injection and demonstrated a dose-dependent relationship In vivo effect of B. bassiana spores and (Table 1). The profile of prohemocytes, secondary metabolite on nodule formation plasmatocytes, and granulocytes significantly Fungal spores and secondary metabolite changed after immune challenge by B. significantly affected nodule formation in bassiana spores after 30, 60, and 120 min adults of E. integriceps (Table 4, Figure 3).

Journal of Insect Science | www.insectscience.org 7 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al.

Figure 4. Double reciprocal plot to show the effect of different concentrations of Beauveria bassiana secondary metabolites on the phenoloxidase acyivity of Eurygaster integriceps adults (1/Vmax = intercept on the 1/V0 ordinate, 1/Km = intercept on the negative side of the 1/(S) abscissa). EtoH was used as control solution. High quality figures are available online.

Figure 5. Phenoloxidase isozyme profiles. (1) Molecular marker, (2) The highest number of formed nodules was control, (3) hemocyte lysate sample after 12-h injection of 15% observed three hours after spore injection, and concentration of secondary metabolite. Samples exposured to Beauveria bassiana secondary metabolite for 12 h versus control. High it was significantly higher than that observed quality figures are available online. in DMSO-injected insects. Injection of fungal secondary metabolite decreased the number of In addition, kinetic parameters of PO activity nodules; this decrease was dose–dependent, were also influenced significantly by the and the highest effect was observed in 12% different concentrations of B. bassiana concentration for all intervals (Table 3). secondary metabolites (Figure 4, Table 6). PO electrophoresis profiles are shown in Figure 5. In vivo effect of B. bassiana spores and Results of gel electrophoresis showed that secondary metabolite on PO activity protein bands in the control sample had Injecting conidia of B. bassiana into E. estimated molecular masses of 46 and 59 kDa. integriceps adults activated the PO system Twelve hours after the injection, enzyme during intervals after inoculation (Table 5). activity decreased to almost undetectable The activity increased during the first 6 hours, levels (Figure 5). at which time the maximum activity was observed. However, although PO activity Discussion decreased at 12 and 24 h post-inoculation, it remained significantly different from DMSO- This study provides novel information on the injected controls (Table 5). Fungal secondary hemocyte types of the sunn pest E. metabolite showed a dose-dependent effect, as integriceps. In addition to their identification, PO activity decreased along with the increase total and differentiate counts were performed of fungal secondary metabolite concentrations on hemocyte types. Finally, the effects of B. (Table 5). bassiana and its secondary metabolites on the cellular immune reactions of E. integriceps were also investigated. B. bassiana is the main

Journal of Insect Science | www.insectscience.org 8 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. entomopathogenic fungus in Iran, which has declined. Declines in THC of insects during been used against many insect pests across the fungal infection have been recorded globe (Wood and Way 1989). Recently, B. previously (Bidochka and Khachatourians bassiana was successfully used instead of 1987; Gunnarsson 1988; Hung and Boucias synthetic pesticides against E. integriceps, 1992). The subsequent decline in THC although the fungal capability to interfere with observed in this study may result in part from the immunity of the destructive insect pest the formation of nodules induced by soluble was not determined (Talaee and Kharrazi- fungal metabolites, since there was a Pakdel 2002). significant inverse correlation between THC and nodule counts. However, it is likely that Results of light microscopy photographing hemocyte aggregation does not fully account showed that the hemolymph of E .integriceps for the decline in THC, as cytotoxic fungal contains five different morphotypes of metabolites may play an important role. Mazet hemocytes (prohemocytes, plasmatocytes, et al. (1994) found that during infection of granulocytes, oenocytoids, adipohemocytes). moth larvae of Spodoptera exigua, B. Similar results were found by Borges et al. bassiana produced a toxic metabolite that (2008) on the blood-sucking bug Rhodnius reduced the activity of hemocytes in vitro. prolixus, where light and transmission Destruxins, produced in vitro by the fungus electron microscopy demonstrated the Metarhizium anisopliae (Samuels et al. 1988), presence of five hemocyte types are toxic to hemocytes (Huxham et al. 1989). (prohemocytes, plasmatocytes, oenocytoids, Studies on the interaction between M. adipohemocytes, granulocytes). anisopliae and the wax moth Galleria mellonella demonstrated that THC in treated E. integricpes plasmatocytes and granulocytes larvae was not significantly different from are the main actors in phagocytosis of B. control larvae until 24 h post-injection. bassiana spores, and are the most represented However, THC increased in the next few days among the circulating hemocytes, as observed (Sewify and Hashem 2001). Bandani (2005) also in R. prolixus (Borges et al. 2008). observed that THC of G. mellonella infected Injection of spores into the E. integricpes by entomopathogenic fungus Tolypocladium hemocyte induced changes in the relative cylindrosporum sharply decreased in percentage of prohemocytes, plasmatocytes, comparison with control in a dose-dependent and granulocytes, suggesting that these cells fashion. The count of different types of play an important role in cellular immune hemocytes indicated that there was an initial response. It has been observed that increase in plasmatocytes and granulocytes prohemocytes are located in hematopoietic during infection, followed by a decline that organs and the hemolymph of several insect included prohemocytes (Bandani 2005). A species (Borges et al. 2008). Prohemocytes decline in plasmatocyte number in immune- are considered by some authors to be stem challenged insects after initial elevation has cells, from which the other main types been noted before (Chain and Anderson 1983; differentiate (Yamashita and Iwabuchi 2001; Gunnarsson 1988; Pech and Strand 1996), and Ling et al. 2005). may reflect the involvement of plasmatocytes in nodule formation or a particular In this study, the total hemocyte count (THC) susceptibility to toxic fungal metabolites as during infection initially increased, then shown in our study. The decline in

Journal of Insect Science | www.insectscience.org 9 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. granulocytes observed may be due to their activity increased during infection while PO involvement in the latter stages of nodule declined. A similar phenomenon has been formation, as reported for other insects (Gotz observed in mosquitoes parasitized by and Boman 1985; Pech and Strand 1996; microfilariae (Cho et al. 1998). The activation Gillespie et al. 1997). of proPO system in E. integriceps is triggered within minutes after spores penetrate the In insects, different hemocytes may hemolymph, and the secretion of secondary participate in phagocytosis. This study found metabolites by fungus may play an important that plasmatocytes and granulocytes play a role to abortion of the activating process. critical role in phagocytosis of fungal spores. Also, analysis of Lineweaver-Burk plots Injection of fungal secondary metabolites provides information regarding to the mode of suppressed the hemocyte increase elicited by action of B. bassiana secondary metabolite fungal spores alone. B. bassiana secondary against PO activity of E. integriceps. The metabolites at different concentrations presence of secondary metabolites decreased suppress phagocytosis along with nodule the value of Vmax and increased Km. The effect formation and PO activity. Thus, it’s possible of secondary metabolites on the Vmax shows that fungal secondary metabolites interfere that it interferes with the rate of break down of with the ligand-receptor interactions, or may the enzyme-substrate complex. Thus, fungal cause ultrastructural alteration which secondary metabolite inhibits the enzymes by interferes with normal hemocyte function increasing the Km and decreasing affinity of (Chen et al. 1998; Vey et al. 2002; Hoffmann, the enzyme to the substrate. These results 1994; Hertu et al. 1998). show a mixed inhibition by fungal secondary metabolite on PO activity of the sunn pest. In Insect POs are synthesized as zymogens this type of inhibition, fungal secondary (prophenoloxidase, proPO), which are metabolites can bind to the enzyme at the activated by proteolytic cleavage at a specific same time as the enzyme binds to substrate. site in response to infection or wounding. This binding affects the binding of the Active PO catalyzes the formation of substrate, and vice versa. Although it is quinones, which undergo further reactions to possible for mixed-type inhibitors to bind in form melanin (Nappi and Christensen 2005). the active site, this type of inhibition generally After a microorganism penetrates into results from an allosteric effect, where the hemocoel, proPO is activated and causes inhibitor binds to a different site on an melanization of encapsulated parasites, which enzyme. Inhibitor binding to this allosteric site is thought to be an important defensive changes the conformation (i.e., tertiary response in insects. PO levels in mycosed structure) of the enzyme so that the affinity of insects can be consistently lower than in the substrate for the active site is reduced controls, and declined over the period of (Stryer 1995; Morris 1978). infection. In contrast, enhanced hemolymph PO was observed in the grasshopper Conclusions Melanoplus sanguinipes (Gillespie and Khachatourians 1992) and S. exigua (Hung Besides providing the first indications on the and Boucias 1992) injected with spores of B. E. integricpes hemocyte morphotypes, our bassiana. Our results are in line with these observations suggest that B. bassiana final observations, since E. integriceps proPO secondary metabolites strongly affect the

Journal of Insect Science | www.insectscience.org 10 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. cellular immune reaction and PO activity of E. Ballarin L, Cammarata M, Cima F, Grimaldi integriceps. Our experiments indicate that A, Lorenzon S, Malagoli D, Ottaviani E. upon an initial stimulation of the insect 2008. Immune-neuroendocrine biology of immune reactions, B. bassiana secretes a wide invertebrates: a collection of methods. range of secondary metabolites that could Invertebrate Survival Journal 5: 192-215. have an affect on the host’s immune system. Similar results have been demonstrated for Bandani A. 2005. Effects of Tolypocladium toxins of other entomogenous fungi cylindrosporum and its secondary metabolites, (Vilcinskas et al. 1997; Hung and Boucias efrapeptins, on the immune system of Galleria 1992; Huxham et al. 1989, Vey et al. 2002). mellonella larvae. Biocontrol Science and Entomopathogenic fungi are of special Technology 15: 67-79. importance because they are key regulatory Bandani AR, Khambay BPS, Faull L, Newton factors in insect pest populations. An R, Deadman M, Butt TM. 2000. Production of understanding of fungal-induced immune efrapeptins by Tolypocladium species responses and the identification of fungal (Deuteromycotina: Hyphomycetes) and virulence factors and their targets may reveal evaluation of their insecticidal and of significant utility in a biological control antimicrobial properties. Mycological scenario. Research 104: 537-544.

Acknowledgements Bidochka MJ, Khachatourians GG. 1987. Haemocytic defense responses to the This research was supported by a grant from entomopathogenic fungus Beauveria bassiana the University of Tehran. The authors would in the migratory grasshopper, Melanoplus like to thank Mr. Tork for his assistance. sanguinipes. Entomologia Experimentalis et Applicata 45: 151-156. References Borges AR, Santos PN, Furtado AF, Ashida M, Ishizaki Y, Iwahana H. 1983. Figueiredo RCB. 2008. Phagocytosis of latex Activation of pro-phenoloxidase by bacterial beads and bacteria by hemocytes of the cell walls or beta-1,3-glucans in plasma of the triatomine bug Rhodnius prolixus (Hemiptera: silkworm, Bombyx mori. Biochemistry and Reduvidae). Micron 39: 486-494. Biophysics Research Communications 113: Bradford M. 1976. A rapid and sensitive 562-568. method for quantitation of microgram Ashida M, Brey PT. 1998. Recent advances in quantities of protein utilizing the principle of research on the insect phenoloxidase cascade. protein-dye binding. Analytical Biochemistry In: Brey PT, Hultmark D, Editors. Molecular 72: 248-254. mechanisms of immune responses in insects. Bulet P, Hetru C, Dimarcq JL, Hoffmann D. pp.135-172. Chapman and Hall. 1999. Antimicrobialpeptides in insects: Azambuja P, Garcia ES, Ratcliffe NA. 1991. structure and function. Developmental and Aspects of classification of hemiptera Comparative Immunology 23: 329-344. hemocytes from six triatomine species. Cerenius L, Soderhall K. 2004. The Memórias do Instituto Oswaldo Cruz 86: 1- prophenoloxidase-activating system in 10.

Journal of Insect Science | www.insectscience.org 11 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. invertebrates. Immunological Review 198: Developmental and Comparative Immunology 116-126. 28: 689-700.

Chain B, Anderson RS. 1983. Observation on Dularay B, Lackie AM. 1985. Haemocytic the cytochemistry of the hemocytes of an encapsulation and the prophenoloxidase- insect, Galleria mellonella. Journal of activaion pathway in the locust Schistocerca Histochemistry and Cytochemistry 31: 601- gregaria. Journal of Insect Physiology 15: 607. 827-834.

Chen C, Rowley AF, Ratcliffe NA. 1998. Dunn PE. 1986. Biochemical aspects of insect Detection, purification by immunoaffinity immunity. Annual Review of Entomology 31: chromatography and properties of b-1,3- 321-339. glucan-specific lectins from the sera of several insect species. Insect Biochemistry and Franssens V, Smagghe G, Simonet G, Claeys Molecular Biology 28: 721-731. I, Breugelmans B, DeLoof A, Vanden Broeck J. 2006. 20-Hydroxy ecdysone and juvenile Cho WL, Liu HS, Lee CH, Kuo CC, Chang hormone regulate the laminarin-induced TY, Liu CT, Chen CC. 1998. Molecular nodulation reaction in larvae of the fleshfly, cloning, charaterization and tissue expression Neobellieria bullata. Developmental and of prophenoloxidase cDNA from mosquito Comparative Immunology 30: 735-740. Armigeres subalbatus inoculated with Dirofilaria immitis microfilariae. Insect Gillespie JP, Bailey AM, Cobb B, Vilcinskas Molecular Biology 7: 31-40. A. 2000. Fungi as elicitors of insect immune responses. Archives of Insect Biochemistry Clarkson JM, Charnley AK. 1996. New and Physiology 44: 49-68. insight into the mechanisms of fungal pathogenesis in insects. Trends in Gillespie JP, Kanost MR, Trenczek T. 1997. Microbiology 4: 197-203. Biological mediators of insect immunity. Annual Review of Entomology 42: 611-643. Da Silva JB, Albuquerque CM, Araju EC, Peixoto CA, Hurd H. 2000. Immune defence Gillespie JP, Khachatourians GG. 1992. mechanisms of Culex quinquefasciatus Characterization of the Melanoplus (Diptera: Culicida) against Candida albicans sanguinipes haemolymph after infection with infection. Journal of Invertebrate Pathology Beauveria bassiana. Comparative 76: 257-262. Biochemistry and Physiology - Part B 103: 455-463. Dean P, Potter U, Richards EH, Edwards JP, Charnley AK, Reynolds SE. 2004a. Gotz P, Boman HG. 1985. Insect immunity. Hyperphagocytocytic hemocytes in Manduca In: Kerkut GA, Gilbert L, Editors. sexta. Journal of Insect Physiology 20: 1027- Comprehensive Insect Physiology, 1036. Biochemistry and Pharmacology, volume 3. pp. 453–485. Pergamon Press. Dean P, Richards EH, Edwards JP, Reynolds SE, Charnley AK. 2004b. Microbial infection Green FJ. 1990. The Sigma-Aldrich handbook causes the appearance of hemocytes with of stains, dyes and indicators. Aldrich. extreme spreading ability in monolayer of the Tobacco hornworm, Manduca sexta.

Journal of Insect Science | www.insectscience.org 12 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. Gunnarsson SGS, Lackie AM. 1985. Kazzazi M, Bandani AR, Hosseinkhani S. Haemocytic aggregation in Schistocerca 2005. Biochemical characteristics of a- gregaria and Periplaneta americana as a amylase of the sunn pest, Eurygaster response to injected substances of microbial integriceps. Entomological Science 8: 371- origin. Journal of Invertebrate Pathology 46: 377. 312-319. Lackie AM, Vasta GR. 1988. The role of a Gunnarsson SGS. 1988. Infection of galactosyl-binding lectin in the cellular Schistocerca gregaria by the fungus, immune response of the cockroach Metarhizium anisopliae: Cellular reactions in Periplaneta americana (Dictyoptera). the integument studied by scanning electron Immunity 64: 353-357. and light microscopy. Journal of Invertebrate Pathology 52: 9-17. Lavine MD, Strand MR. 2002. Insect hemocytes and their role in immunity. Insect Gupta AP. 1985. Cellular elements in the Biochemistry and Molecular Biology 32: hemolymph. In: Kerkut GA, Gilbert LI, 1295-1309. Editors. Comprehensive Insect Physiology Biochemistry and Pharmacology, volume 3. Leonard C, Kenneth S, Ratcliffe NA. 1985. pp. 402-444. Pergamon Press. Studies on prophenoloxidase and protease activity of Blaberua craniifer haemocytes. Hertu C, Hoffmann D, Bulet P. 1998. Insect Biochemistry 15: 803-810. Antimicrobial peptides from insects. In: Brey PT, Hultmark D, Editors. Molecular LeOra Software. 1987. POLO-PC: A user’s Mechanisms of Immune Responses in Insects. guide to Probit Or LOgit analysis. LeOra pp. 40-66. Chapman and Hall.. Software Inc.

Hoffmann JA. 1994. Immune response of Ling E, Shirai K, Kanekatsu R, Kiguchi K. insects. Developmental and Comparative 2005. Hemocyte differentiation in the Immunology 18: 37-38. hematopoietic organs of the silkworm, Bombyx mori: prohemocytes have the function Hung SY, Boucias DG. 1992. Influence of of phagocytosis. Cell Tissue Research 320: Beauveria bassiana on the cellular defense 535-543. response of the beet armyworm, Spodoptera exigua. Journal of Invertebrate Pathology 60: Lowenberger C. 2001. Innate immune 152-158. response of Aedes aegypyi. Insect Biochemistry and Molecular Biology 31: 219- Huxham IM, Lackie AM, McCorkindale NJ. 229. 1989. Inhibitory effects of cyclodepsipeptides, destruxins, from the fungus Metarhizium Mazet I, Hung S-Y, Boucias DG. 1994. anisopliae, on cellular immunity in insects. Detection of toxic metabolites in the Journal of Insect Physiology 35: 97-105. hemolymph of Beauveria bassiana infected Spodoptera exigua larvae. Experentia 50: Kanost MR, Gorman MJ. 2008. 142-147. Phenoloxidases in Insect Immunity. In: Morris JG. 1978. A biologist's physical Beckage NE, Editor. Insect Immunity. pp. 69- nd 96. Academic press. chemistry, 2 edition. Edward Arnold publishing (Limited).

Journal of Insect Science | www.insectscience.org 13 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. Nappi AJ, Christensen BM. 2005. anisopliae for the tobacco hornworm Melanogenesis and associated cytotoxic Manduca sexta. Mycopathology 104: 51-58. reactions: Applications to insect innate immunity. Insect Biochemistry and Molecular SAS institute. 1997. SAS/STAT User’s Guide Biology 35: 443-459. for Personal Computers. SAS Institute.

Nayar JK, Knight JW. 1997. Hemagglutinind Sewify GH, Hashem MY. 2001. Effect of the in Anopheles quadrimaculatus strains entomopathogenic fungus Metarhizium susceptible and refractory to Brugia malayi anisopliae on cellular defense response and and their role in the immune response to oxygen uptake of the wax moth Galleria filarial parasites. Comparative Biochemistry mellonella L. (Lep., Pyralidae). Journal of and Physiology - Part B 116: 109-117. Applied Entomology 125: 533-536.

Pech LL, Strand MR. 1996. Granular cells are Silva JEB, Boleli IC, Simões ZLP. 2002. required for encapsulation of foreign targets Hemocytes types and total differential counts by insect haemocytes. Journal of Cell Science in unparasitized and parasitized Anastrepha 10 oblique (Diptera, Tephritidae) larvae. 9: 2053-2060. Brazilian Journal Biology 62: 689-699.

Ratcliffe NA, Rowley A. 1979. Role of Smith VJ, Soderall K, Hamilton M. 1984. b- hemocytes in defense against biological 1,3-Glucan induced cellular defence reaction agents. In: Gupta AP, Editor. Insect in the shore-crab Carcinus means. hemocytes. pp. 331-414. Cambridge Comparative Biochemistry and Physiology - University Press. Part A 77: 635-639.

Ratcliffe NA, Rowley AF. 1987. Insect Soderhall K, Aspan A. 1993. response to parasites and other pathogens. In: Prophenoloxidase activation system and its Soulsby EJL, Editor. Immunology, role in cellular communication. In: Pathak Immunoprophylaxis and Immunotherapy of JPN, Editor. Insect Immunity. pp. 113-129. Parasitic Infections. pp. 271-332. CRC Press. Oxford and IBH Publishing Company, Ltd.

Ratcliffe NA, Leonard C, Rowley AF. 1984. St. Leger PJ, Cooper RM, Charnley, RK. Prophenoloxidase activation: nonself 1988. The effect of melanisation of Manduca recognition and cell cooperation in insect sexta cuticle on growth and infection by immunity. Science 226: 557-559. Metarhizium anisopliae. Journal Invertebrate Pathology 52: 459-470.

Russo J, Brehelin M, Carton Y. 2001. th Haemocyte changes in resistant and Stryer L. 1995. Biochemistry, 4 edition. W. susceptible strains of D. melanogaster caused H. Freeman and Company. by virulent and avirulent strains of the Sugumaran M. 1998. Unified mechanism for parasitic wasp Leptopilina boulardi. Journal sclerotization of insect cuticle. Advances in of Insect Physiology 47: 167-172. Insect Physiology 27: 229-334.

Samuels RI, Charnley AK, Reynolds SE. Talaee R, Kharazzi-Pakdel A. 2002. 1988. The role of destruxins in the Evaluation of sunn pest, Eurygaster pathogenicity of 3 strains of Metarhizium integriceps susceptibility in different

Journal of Insect Science | www.insectscience.org 14 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. developmental stages to Beauvaria bassiana. 2nd international conference on alternative methods of plant pests and diseases control, 588-592.

Vey A, Matha V, Dumas C. 2002. Effects of the peptide mycotoxin destruxin E on insect haemocytes and on dynamics and efficiency of the multicellular immune reaction. Journal Invertebrate Pathology 80: 177-187.

Wood RKS, Way MJ. 1989. Biological control of pests, pathogens and weeds: development and prospects. Philosophical Transactions of the Royal Society, Series B, 318: 109-376.

Zibaee A, Bandani AR, Tork M. 2009. Effect of the entomopathogenic fungus, Beauveria bassiana, and its secondary metabolite on detoxifying enzyme activities and acetylcholinesterase (AChE) of the Sunn pest, Eurygaster integriceps (Heteroptera: Scutellaridae). Biocontrol Science Technology 19: 485-498.

Zibaee A, Bandani AR. 2010. Effects of Artemisia annua L. (Asteracea) on digestive enzymes profiles and cellular immune reactions of sunn pest, Eurygaster integriceps (Heteroptera: Scutellaridae), against Beauvaria bassiana. Bulletin Entomological Research 100: 185-196.

Table 1. The effect of Beauveria bassiana and its secondary metabolites on the total haemocyte count (cells 104/mL) of Eurygaster integriceps.

* Means (±SEM) followed by asterisks indicate significant differences (p < 0.05) versus control according to the Tukey’s test. Journal of Insect Science | www.insectscience.org 15 Journal of Insect Science: Vol. 11 | Article 138 Zibaee et al. Table 2. Number of hemocyte types 104/mL in Eurygaster integricpes injected with Beauveria bassiana spores.

* Means (±SEM) followed by the asterisk indicate significant differences versus control (p < 0.05) according to the Tukey’s test.

Table 3. Number of hemocyte types 104/mL from control and treated insects by secondary metabolites of Beauveria bassiana at different times after the immune challenge.

* Means (±SEM) followed by asterisks indicate significant differences (p < 0.05) versus control according to the Tukey’s test.

Table 4. Effects of Beauveria bassiana spores and secondary metabolites on the nodule formation of Eurygaster integriceps adults.

2Amount of nodules, Nodule104/mL * Means (±SEM) followed by asterisks indicate significant differences (p < 0.05) versus control according to the Tukey’s test.

Table 5. Effects of Beauveria bassiana spores and secondary metabolites on the phenoloxidase activity (μmol/min/mg protein) of Eurygaster integriceps adults.

3Means (±SEM) followed by asterisks indicate significant differences (p < 0.05) verss control according to the Tukey’s test.

Table 6. Km (mM) and Vmax (μmol/min/mg protein) for phenoloxidase of Eurygaster integriceps in the absence and presence of different concentrations of the Beauveria bassiana secondary metabolites.

1Concentration in percent, 2Mean±SE, 3Means (SEM±) followed by the same letters above bars indicate no significant difference (p < 0.05) according to the Tukey’s test.

Journal of Insect Science | www.insectscience.org 16