RESEARCH REPORT ISJ 11: 11-21, 2014 ISSN 1824-307X RESEARCH REPORT Immune reactions of the lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) to the entomopathogenic fungus, Beauveria bassiana (Bals.- Criv.) Vuill and two developmental hormones R Khosravi1, JJ Sendi1, A Zibaee1, MA Shokrgozar2 1Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht 41635-1314, Iran 2Pasteur Institute of Iran (IPI), NO. 69, Pasteur Ave, Tehran, 1316943551, Iran Accepted December 27, 2013 Abstarct Effects of Beauveria bassiana (Bals.-Criv.) spores on immune functions of Glyphodes pyloalis Walker larvae were studied. Total and differential hemocyte counts revealed that infection by B. bassiana caused a dramatic change in hemocyte number. In vivo and in vitro studies demonstrated that increase in time exposure to fungal pathogen resulted in elevated phagocytic activity. Nodules were formed in response to spore injection and their numbers were maximal 6 h post injection. The phenoloxidase (PO) activity in treated larvae changed significantly 3 and 6 h after spore injection compared with the control larvae. The reduction in PO activity was observed 12 and 24 h post injection. Effect of two developmental hormones, juvenile hormone (JH) and ecdysone on cellular immune response of G. pyloalis were also evaluated. Larval treatment with JH prior to B. bassiana spore injection reduced nodulation while ecdysone enhanced it. These results demonstrated that ecdysone and JH play an important regulatory role in the immune response in the studied insect. Key Words: hemocyte; nodulation; juvenile hormone (JH); ecdysone; cellular immunity   Introduction Entomopathogens are important regulatory factors of insect populations. At present, several species of entomopathogenic fungi are used as biocontrol agents in insect pests (Bogus et al., 2007). The entomopathogenic fungus, Beauveria bassiana (Bals.-Criv.) shows great potential for the control of a broad range of insect species. This fungus has been developed for use as a biological pesticide (Ansari and Butt, 2012). The process of infection begins with attachment of the conidia to the cuticle. The fungus then germinates, grows and penetrates the integument. In addition to being able to reach the cuticle barrier, this pathogen possesses the ability to replicate in the insect hemocel (Xiong et al., 2013). Upon entering the hemocel, fungal cells interact with insect hemocytes. They then, induce peptides and proteins that mediate humoral immunity (Borges et al., 2008). Insects defend themselves from infection by a variety of potential pathogens including; bacteria, ___________________________________________________________________________ Corresponding author: Roya Khosravi Department of Plant Protection Faculty of Agriculture University of Guilan Rasht 41635-1314, Iran E-mail: khosravi.roya@yahoo.com fungi and parasites in natural habitats. They have therefore evolved efficient host-defense mechanisms to survive (Yamauchi, 2001). The immunity system in insects consists of cellular and humoral defense responses. Humoral defenses refer to antimicrobial peptides, cell adhesion molecules, lysozyme, lectins, and the prophenoloxidase (proPO) (Hoffmann, 2003; Kanost et al., 2004). Cellular defenses include responses such as phagocytosis, encapsulation, and clotting that with hemocytes acting as important mediators of such processes (Lavine and Strand, 2002). Several morphologically distinct hemocyte cell types work together in the immune reactions. Cellular reaction include phagocytosis in which individual hemocyte ingest large particles that enter the hemocel from the environment. This process is essential for host defense in higher eukaryotes against infectious microorganisms and for the elimination of apoptic cells generated during development. This is regarded as the main cellular reaction of innate immunity in vertebrates and invertebrates (Borges et al., 2008). Foreign particles are identified by phagocytic cells via recognition by a series of receptors on cell membranes that bind to pathogen-associated molecules (Rosales, 2011). Two types of hemocytes involved in phgocytosis are the granulocytes and the plasmatocytes. However, 11   http://en.wikipedia.org/wiki/Giuseppe_Gabriel_Balsamo-Crivelli http://en.wikipedia.org/wiki/Giuseppe_Gabriel_Balsamo-Crivelli http://en.wikipedia.org/wiki/Jean_Paul_Vuillemin mailto:khosravi.roya@yahoo.com Fig. 1 Total hemocyte count (cells ×104/ml) of G. pyloalis infected with B. bassiana spores. *Means ± SE followed by the asterisk indicate significant differences versus control (p < 0.05) according to the Tukey’s test. their contribution in this process varies between insect species (Moushumi et al., 2008). After entering the hemocel, micro-aggregations of hemocytes (granulocytes and plasmatocytes) are initiated on microorganisms. This process is initiated by changing of circulating hemocytes from non- adhesive to adhesive cells that are able to bind to microorganisms (Lavine and Strand, 2002). These micro-aggregation will eventually lead to the formation of nodules. In the later stages of nodule formation, melanization takes place within the nodules. Finally, foreign particles almost die. Several factors such as asphyxiation, production of toxic quinones or hydroquinones via the proPO cascade and antibacterial peptides have been proposed to function as killing agents (Nappi and Christensen, 2005). Phenoloxidases (POs) are vital enzymes involved in a number of crucial processes, such as defense, wound healing, coagulation, sclerotization, and melanization (Bogus et al., 2007). POs are present as inactive precursor, the proPO in insect hemolymph and are activated in response to wounding or infection as a part of the innate immune response (Cerenius et al., 2008). These enzymes are similar to mammalian tyrosinases in their ability to use reactive sites containing copper atoms to catalyze two types of reactions that require molecular oxygen as a substrate. The cytotoxic quinones that are produced during oxidation are placed over the surface of large foreign materials in order to form melanized layers, which help to kill the encapsulated fungi, bacteria or parasites (Ling et al., 2005). Table 1 Number of hemocyte types from control and treated larvae of G. pyloalis with B.bassiana at different times after the immune challenge. Control Treatment 1 h 3h 6h 12 h 24 h 1 h 3 h 6 h 12 h 24 h Pr 20.28±2.02 17±2.9 18±1.89 18.6±2.03 15.6±1.69 20.2±1.76 17.4±1.74 19.6±1.44 15±1.49 18±1.89 Pl 107.6±3.36 124.6±3.59 112.4±2.85 119±2.07 123±3.8 185.2±3.64* 214±1.81* 183±3.22* 99.8±4.41* 86±4.84* Gr 52.4±2.4 57.2±1.06 65±3.48 62±2.47 68.2±2.26 67±3.61* 110±3.96* 72±3.02* 49±4.13* 41±3.42* Sp 7.2±0.91 8.6±1.28 11.2±1.28 10±1.25 9±1.1 7.6±1.06 9±1.31 9±1.0* 8.2±1.21* 8.2±0.91 Oe 10.6±1.39 9.8±2.12 10±1.23 9.8±1.21 8.6±0.94 13.4±1.29* 12±1.45 11.2±1.43 13±1.49* 9.6±1.23 Pr= Prohemocyte; Pl= Plasmatocyte; Gr= Granulocyte; Sp= Spherulocyte; Oe= Oenocytoid 12   Fig. 2 In vivo and in vitro phagocytosis of B. bassiana spores by plasmatocytes and granulocytes. Endocrine factors are involved in the immune system and mediate immune signals. The eicosanoids (Stanley, 2000), biogenic amines (Baines et al., 1992) adipokinetic hormone (Goldsworthy et al., 2003), juvenile hormone (JH) and 20-hydroxyecdysone (20E) (Franssens et al., 2006) change insect immune response to pathogenic agents. Inhibition of hemocyte encapsulation response was observed in Tenebrio molitor after injection of JH (Rantala et al., 2003). The 20E promotes nodule formation of Neobelliera bullata in response to injection of laminarin, the components of bacterial cell wall (Franssens et al., 2006). The lesser mulberry pyralid, Glyphodes pyloalis (Lepidoptera: Pyralidae) is a specialist insect on mulberry, and is widely distributed throughout Asia and the northern province of Iran. This pest has caused severe damage to mulberry plantations in northern Iran and has posed a serious concern to silkworm growers. Fifth instar larvae feed on the whole leaf until only the ribs remain (Khosravi and Jalali Sendi, 2010). Populations of most pests are usually regulated by density dependent factors involving pathogens and parasites. Thus, understanding the interactions of pest with their pathogens and parasites and hence, the cellular defense responses are needed for developing best methods of pests' control. Hemocytes perform certain vital activities in insects, and thus hematological studies are fundamental in the field of insect physiology (El-Aziz and Awad, 2010). An important necessity in the study of the fungal pathogen host interaction is the ability to detect mechanisms that are used by pathogens to overcome the insect’s immune defense system. Table 2 THC and number of plasmatocytes and granulocytes (×104 cell/mL) of G. pyloalis larvae at 3, 6 and 12 h after JH treatment prior to B. bassiana spore injection. THC Number of plasmatocytes Number of granulocytes 3 h 6 h 12 h 3 h 6 h 12 h 3 h 6 h 12 h K 347.67±4.21a 337.67± 4.78a 292.33± 3.53a 215± 6.13a 241.33± 3.01a 239.66± 2.22a 85.66± 2.74a 87.3±3. 16a 84.00± 1.62a A 323.67±3.6a 278.00± 4.6b 251.67± 3.64b 220±2. 08a 181.00± 3.04c 166.00± 1.58c 68.33± 2.24b 69.66± 2.42b 69.33± 2.21b B 332.47±3.09a 287.33± 4.3b 256.33± 3.53b 224±2. 95a 193.00± 2.56bc 173.33± 2.12c 69.66± 2.24b 64.3± 1.74b 67.00± 2.46b C 330.00±3.92a 307.00± 2.79ab 283.33± 3.96ab 213.6± 1.87a 191.33± 2.42bc 213.33± 2.58ab 73.00± 2.64ab 75.00± 1.89ab 66.4±1. 44b D 327.43±2.38a 309.67± 3.53ab 276.33± 2.53ab 224.5± 2.88a 201.00± 3.14b 198.00± 3.08bc 75.65± 1.87ab 74.66± 2.85b 74.32± 2.17ab K = Control; A = JH 0.5 mg/mL; B = JH 0.25 mg/mL, C = JH 0.125 mg/mL; D = JH 0.062 mg/mL. Means ± SE within the same column followed by the same letter are not significantly different (p ≤ 0.05 Tukey test). 13   This study was undertaken to investigate the effects of B. bassiana isolate Fashand on cellular immune responses and the PO activity of G. pyloalis. Secondly, in order to understand the role of insect hormones in immune responses, the effects of JH and ecdysone, two key insect hormones on immune reactions of this pest were evaluated. Materials and methods Insects rearing Larvae of G. pyloalis were collected from mulberry orchards (Kenmuchi Var.) in Rasht (37°16′51″N 49°34′59″E), north of Iran. They were maintained in the laboratory in a rearing chamber of constant temperature (25 ± 1 ˚C), relative humidity (75 ± 5 %) and photoperiod (16:8 h light:dark). Larvae were placed in plastic jars 10×20 cm and were daily provided with fresh mulberry leaves (Kenmuchi Var.). On adult emergence, they were transferred to 18×7 cm transparent jars and were provided with fresh leaves for egg laying and cotton wool soaked in 10 % honey for feeding. Beauveria bassiana culture B. bassiana isolate Fashand was grown in sterile petri dishes containing potato dextrose agar (PDA) and were incubated at 25 ± 1 ˚C in complete darkness. Spores were harvested from PDA plates with a sterile scalpel after 14 days. The final concentration was adjusted to 1×105 spores/ml using a hemocytometer in distilled water containing 0.01 % of Tween 20. Injection of insects with spores Fifth instar larvae of G. pyloalis were immobilized on ice for 5 min and were surface sterilized with 70 % ethanol. This experiment was replicated three times and ten insects were used in each replicate. After injection with 1×105 spores/ml Fig. 3 Induction of nodule formation in G. pyloalis larvae with 2 μLB. bassiana spores injection. (2 µL) by a 10 µL Hamilton syringe, the larvae were transferred to rearing jars provided with fresh mulberry leaves, to follow the course of the assay for further observation. The control larvae were injected with distilled water containing 0.01 % of Tween 20 (2 µL) alone. Effect of fungal spores of B. bassiana on hemocyte number To determine, whether injection of spores of B. bassiana could cause any changes in the levels of total and differential hemocyte numbers, the fifth instar larvae were injected with 2 µL of 1×105 spores/mL concentration between the second and third prolegs. Similarly, the controls were injected with 2 µL of sterile distilled water containing 0.01 % Fig. 4 Effect of B. bassiana spores on nodule formation in fifth instar larvae of G. pylalis. *Means ± SE followed by the asterisk indicate significant differences versus control (p < 0.05) according to the Tukey’s test. 14   http://tools.wmflabs.org/geohack/geohack.php?pagename=Rasht¶ms=37_16_51_N_49_34_59_E_type:city_region:IR Fig. 5 Effect of B. bassiana spores on phenoloxidase (PO) activity in fifth instar larvae of G. pylalis. *Means ± SE followed by the asterisk indicate significant differences versus control (p < 0.05) according to the Tukey’s test of Tween 20. Hemolymph was collected by cutting one of the prologs 1, 3, 6, 12, and 24 h after injection from chilled, surface sterilized (70 % ethanol) larvae. This experiment was replicated three times and ten insects were used in each replicate. Samples of hemolymph from each larva were diluted 5-fold with a cold anticoagulant buffer (0.098 M NaOH, 0.186 M NaCl, 0.017 M EDTA and 0.041 M citric acid, pH 4.5). Then, the total and differential hemocyte numbers were counted on an improved Neubauer hemocytometer for each treatment (El-Aziz and Awad, 2010). Labeling of B. bassiana spores The spores of B. bassiana for labeling were obtained from the 10 - 14 day culture on PDA medium. The spores were re-suspended in 10 mL of phosphate buffered saline (PBS) (0.13 M NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4 and 1.47 mM KH2PO4, pH 7.4, autoclaved), were then washed and re- suspended in a sterile CO3-HCO3 buffer at pH 9.4 (9.5 mL 0.2 M Na2CO3 was mixed with 41.5 ml 0.2 M NaHCO3). The solution was made up to 200 ml and was labeled by mixing this solution with 1mg of FITC (Fluorescein Isothiocyanate, Sigma) on a shaker for 30 min at room temperature in complete darkness (Rohloff et al., 1994).The spores were rinsed by phosphate buffered saline four times, pelletized, and the pellets from the last wash were re-suspended in 1 mL of Grace’s insect medium (GIM, Gibco) and stored at -20 ˚C until needed. Phagocytosis assay The phagocytic activity of hemocytes was assessed using the FITC-labeled B. bassiana spores. Larvae were injected with 2 µL of 1×105 FITC- labeled B. bassiana spores/mL and phagocytic activity of hemocytes was investigated 15, 30, and 60 min post injection. This experiment was replicated three times and ten insects were used in each replicate. The body surface of each larva was sterilized with 70 % ethanol before extracting the blood. The larvae were bled by cutting one of the prolegs. The hemolymph was then collected into ice-cold anticoagulant solution and poured over microscopic slide. The slide was incubated in a moist dark chamber at room temperature for 5min with 10 µL of trypan blue solution (2 mg/mL) in order to quench spores that were not ingested. For the in vitro phagocytosis assays, 10 µL of FITC-labeled B. bassiana spores were mixed with 10 µL of freshly collected hemolymph on a microscopic slide and were then incubated in a moist dark chamber at room temperature for 60 and 120 min. Then, 10 µL of trypan blue solution was added and the mixture was incubated for another 5 min. In both the tests the phagocytic activity was determined by counting hemocytes with or without ingested spores under a fluorescence microscope (Leica, Wetzlar, Germany). Ten photo-frames per microscope slide were counted and the average was calculated. Each experiment was repeated for 3 times (Tseng et al., 2008). Phagocytosic activity was calculated as the number of phagocytosing cells×(number of total cells)-1×100. Effect of fungal spore on nodulation Injections were carried out as mentioned above. The number of nodules formed at 1, 3, 6, 12, and 24 h post injection were determined. Hemolymph was collected from each larva, then samples in 5 replicates were poured into a hemocytometer, and the number of nodules was counted (Franssens et al., 2006). Assay for PO activity PO activity was measured according to the procedure of Catalan et al. (2012) with slight 15   modification. In order to test the effect of B. bassiana spores on the PO system in G. pyloalis larvae, 10 µL of hemolymph was diluted with 90 µL of ice-cold sterile phosphate buffered saline and then were vortexed. Samples were frozen at -20 ˚C for 48 h. For assay of PO activity, L-DOPA was used as a substrate (Wilson et al., 2001; Cotter et al., 2004). Then samples were centrifuged at 5,000g at 4 ˚C for 5 min. Fifty microliters of hemolymph- buffer supernatant was mixed with 150 µL of L- DOPA (10 mM). PO activity was measured (in 3 replicates) at 490 nm for 30 min in 5 min intervals using a microplate reader (Awareness Technology Inc, Florida, USA). Protein determination The method of Bradford (1976) was used for determining total protein, using bovine serum albumin (Bio-Rad, Munchen, Germany) as the standard. Effect of ecdysone and Juvenile hormone (JH) on immune responses Ecdysone was dissolved in Ringer’s solution (0.123 M NaCl, 1.53 M CaCl2, 4.96 M KCl, pH 7.4) at a concentration of 5 mg/mL. Initially, preliminary tests were performed to find the effective dose ranges of ecdysone and JH on the development of G. pyloalis. Then the stock solution of ecdysone was diluted with ringer to a concentration of 0.5, 0.25, 0.125, and 0.062 mg/mL. JH was dissolved in acetone at 5, 2.5, 1.25 and 0.625 mg/mL and 2 µL of this solution was topically applied onto the metathoracic tergum of each larva. Four hours after topical application of JH on metathoracic tergum or injection of Ecdysone, a suspension of B. bassiana spores (1×105 spores/mL) was prepared and 2 µL of which was injected to the larva by a Hamilton syringe. Then, after 3, 6, and 12 h total and differentiated hemocyte number and nodules were counted. Control specimens were either first topically treated with 2µL of acetone or were first injected with Ringer’s solution and then with B. bassiana spores. Three replicates were used for each concentration (N = 30) and totally 120 insects were used for immunological assays. Statistical analysis All data obtained from the experiments were subjected to analysis of variance (ANOVA) (p < 0.05). Means were compared by Tukey’s studentized range test, accepting significant differences at p ≤ 0.05 (SAS Institute, 1997). Results Effect of B. bassiana spores on hemocyte number, phagocytosis and nodulation Total number of circulating hemocytes in lesser mulberry pyralid, G. pyloalis fifth instar larvae exhibited major changes after B. bassiana spore injection (Fig. 1). A significant increase in total hemocyte count (THC) was recorded 3 and 6 h post injection (365 and 481×104 cell/mL respectively), while in control THC was recorded only 295 and 305×104 cell/mL, respectively (p ≤ 0.05). As shown in Table 1, significant changes in the profile of five hemocyte types were observed in various intervals post-injection of B. bassiana spore compared with the control. The number of plasmatocytes and granulocytes increased 1, 3, and 6 h post- injection, and then decreased after 12 and 24 h. Fungal infection generally increases the number of plasmatocytes in first interval after the inoculation. The data revealed that the numbers of oenocytoids were increased significantly after 1 and 12 h pos-injection compared with the control. The number of prohemocytes did not change significantly but the number of spherulocytes decreased 6 and 12 h post injection of B. bassiana spores. Hemocytes of G. pyloalis showed a basic phagocytosis activity against B. bassiana spores. Results of this study showed that plasmatocytes and granulocytes of G. pyloalis have an important role in phagocytosis of foreign particles. The most phagocytic activity was observed 30 and 60 min after injection of spores. The phagocytic potential of G. pyloalis was higher in vivo than in vitro (Fig. 2). Nodule formation (Fig. 3) in G. pyloalis larvae was significant after injection of B. bassiana spores. Most of the nodules occurred 3, 6 and 12 h after inoculation of fungi. The highest number of nodules could be observed 6 h post injection, and then decreased 24 h post-injection (Fig. 4). Effect of B. bassiana spores on phenoloxidase activity When fifth instar larvae of G. pyloalis were injected with B. bassiana spores (1×105), the PO system was activated during intervals after inoculation (Fig. 5). The highest PO activity was observed 3 and 6 h after the injection, and then decreasing after 12 and 24 h but not significant statistically. Effect of exogenous ecdysone and JH on immune responses in G. pyloalis Quantitative analysis of THC of insects treated with exogenous JH is shown in Table 2. By increasing the concentration of exogenous JH from 0.625 to 5 mg/mL a dose-dependent decrease in total number of hemocytes was observed compared with the control. Plasmatocyte numbers sharply decreased along with increase in the concentrations. The number of granulocytes decreased at higher concentrations compared with the control. The changes of total hemocyte count in the fifth instar G. pyloalis larvae were affected by injection of exogenous ecdysone. As shown in Table 3, total hemocyte count significantly increased with time and concentration. Higher concentrations of exogenous ecdysone seemed to play a strong facilitating role in promoting the increase in granulocyte numbers. Treatment of larvae with exogenous JH significantly inhibited the nodule formation in larvae injected with B. bassiana spores (Fig. 6A). While, increasing concentrations of exogenous ecdysone (0.5 and 0.25 mg/mL), enhanced nodulation 3 and 6 h after B. bassiana injection (Fig. 6B). 16   a) b) Fig. 6 Influence of different concentrations of JH (A) and ecdysone (B) on nodule formation of G. pyloalis larvae at 3, 6 and 12 h after injection with B. bassiana spores. Data of each treatment at any time were compared with control at the same time. Discussion To understand the role of hemocytes and their involvement in the defense reactions of lesser mulberry pyralid larvae a pathogenic agent was used. It was found that, total hemocyte count (THC) first increased during infection, then declined 12 and 24 h post-injection. As the infection progressed the THC and more specifically the granulocyte number were reduced. Similar reduction in circulating hemocytes was reported for Spodoptera exigua infected by B. bassiana (Hung and Boucias, 1992). Previous studies by other researchers showed a major effect of pathogenesis on THC in insects (Moushumi et al., 2008; Ajamhassani et al., 2013). Bandani (2008) reported no significant reduction in the total hemocyte count (THC) when Galleria mellonella larvae were injected with Tolypocladium cylindrosporum spores until 24 hours post injection. However, THC was suppressed 48 h post-treatment of larvae with spores. Meshrif et al. (2011) also indicated that population of S. littoralis hemocytes 48 h post injection of B. bassiana spores were significantly lower than the control, but 72 h post injection they were increased. The decline in THC in the later stages of infection could partly be attributed to the hemocyte aggregation (nodule formation), induced by soluble wall components released from circulating fungus (Gillespie et al., 2000). Furthermore, cytotoxic fungal metabolites may play a key role in the reduction of hemocyte numbers. The differential hemocyte count (DHC) showed an initial increase in plasmatocytes (PLs) and granulocytes (GRs) during infection, followed by a decline in these cell numbers. This result was in accordance to other reports (Gunnarsson, 1987; Pech and Strand, 1995; Gillespie et al., 2000; Meshrif et al., 2011). The total granulocyte and plasmotocyte numbers also increased 12 h after treatment with Metarhizium anisopliae in Oxya japonica (Anggraeni and Putra, 2011). PLs and GRs are known to give out cytoplasmic processes in retaliation to any invading foreign material (Sharma et al., 2008). 17   Table 3 THC and number of plasmatocytes and granulocytes (×104 cell/mL) of G. pyloalis larvae at 3, 6 and 12 h after ecdysone treatment prior to B. bassiana spore injection THC Number of plasmatocytes Number of granulocytes 3 h 6 h 12 h 3 h 6 h 12 h 3 h 6 h 12 h K 341.33± 4.26a 326.00± 3.88c 292.23± 3.53c 215.00± 4.13ab 241.3± 3.01b 239.66± 2.22ab 79.00± 2.74b 87.26± 3.16c 84± 1.62c A 373.64± 3.17a 423.0± 3.18a 386.00± 3.43a 257.22± 3.53a 304.43± 2.60a 254.33± 4.26a 108.00± 3.08a 143.66± 3.16a 110.4± 3.6b B 362± 4.28a 392.65± 3.45ab 354.3± 4.32ab 232± 2.56ab 270 ±3.48b 240± 3.47ab 80.3± 2.76b 131± 2.92ab 124.33± 1.72a C 358.33± 6.36a 363.2± 6.62bc 323.67± 6.23bc 221± 5.46ab 236.12± 4.98b 259± 3.48a 106± 3.6a 119.42± 4.09b 120± 1.44ab D 334.43± 2.73a 337.5± 3.17bc 329.± 2.49bc 207.35± 4.93b 243.67± 5.36b 221.27± 3.08b 91.2± 3.6ab 110.1± 3.53b 123.3± 2.24a K = Control; A = ecdysone 0.5 mg/mL; B = ecdysone 0.25 mg/mL, C = ecdysone 0.125 mg/mL; D = ecdysone 0.062 mg/mL. Means ± SE within the same column followed by the same letter are not significantly different (p ≤ 0.05 Tukey test). In the current study we have demonstrated that oenocytoides' (OE) number changed in response to B. bassiana spores. Changes in the numbers of OEs following fungal injection may be attributed to the beginning of humoral activity or the active transformation of GRs into SPs and OEs as suggested by Gupta (1985). The decrease of PLs and GRs can be attributed principally to their involvement in nodule formation and partially to programmed cell death induced by toxins secreted by the growing fungi. The decline in GRs observed may be due to their involvement in the latter stages of nodule formation, as has been reported in other insects (Gotz and Boman, 1985; Pech and Strand, 1995; Gillespie et al., 1997). A significant increase in the percentage of GRs was observed in latex bead-treated insects 60 and 120 min after the inoculation. The percentage of oenocytoides significantly varied in response to Staphylococcus aureus infection, with an initial increase at 30 min followed by accentuated decrease 60 and 120 min post-inoculation (Borges et al., 2008). In the process of phagocytosis cells recognize, bind and ingest relatively large elements and protect insects. Several authors have indicated that PLs are the major cell type involved in phagocytosis (Ratcliffe et al., 1985; Anggraeni and Ratcliffe, 1991; Rohloff et al., 1994). Immune reactions of G. pyloalis against B. bassiana showed that PLs and GRs are the main factors in phagocytosis and nodulation. In this study, most of phagocytotic activity occurred 30 and 60 min after injection. This study showed that nodules are formed in response to injection of B. bassiana spores. The maximal number of nodules were observed 3, 6 and 12 h post-injection, but declined later. Similar results were recorded by other researchers (Gillespie et al., 2000; Meshrif et al., 2011; Ajamhassani et al., 2013). It is generally agreed that the formation of nodules is initiated by degranulation of GRs, and the contents released act as an opsonin in the employment of other hemocytes, mainly PLs (Ratcliffe et al., 1985). Contact of GRs with the fungus, or the release of β-1, 3 glucan of other soluble material from the fungus could be the cause of this initial degranulation (Gillespie et al., 2000). The subsequent decline in nodule numbers may be due to their running out of circulation and attaching to the body wall when they attain a stable size (Brookman et al., 1989). It is likely that the immunosuppressive effects of fungal metabolites might have a role in their decline. The dark color of the nodules demonstrates the synthesis of melanin and at least a localized activation of proPO, which is indicative of their formation by hemocytes (Lavin and Strand, 2002). PO is responsible for the activation of melanogenesis in invertebrates. The main role of PO in melanogenesis is to convert phenols to quinones which subsequently polymerize to form melanin (González-Santoyo and Cόrdoba-Aguilar, 2011). Furthermore, recent research has documented PO as an important tool used against several pathogens (Cerenius and Söderhäll, 2004). Wounding or infection activates PO system as part of immune response (Kanost and Gorman, 2008). In the present study we observed high level of PO activity in hemolymph of larvae injected with B. bassiana spores 3, 6 and 12 h post-injection. Gillespie et al. (2000) demonstrated that a topical application of conidia from M. anisopliae var. acridum led to significant elevation of proPO in the hemolymph. We investigated the effect of different concentrations of JH and ecdysone on immune parameters including total hemocyte counts and nodule formation. We found that high concentrations of JH reduced total hemocyte number. Also both plasmatocyte and granulocyte numbers sharply 18   decreased in a time and concentration-dependent manner. Zibaee et al. (2012) demonstrated that Pyriproxyfen (pyridine-based pesticide with JH mimicking activity) caused significant reduction in THC, plasmatocyte and granulocyte population in Eurygaster integriceps adults. Kim et al. (2008) showed a dose response relationship for 20E on the number of plasmatocytes in Spodoptera exigua. Treatment of G. pyloalis larvae with JH caused reduction in the ability of the larvae to form nodules in response to injection of B. bassiana. Similar results have been reported by Rantala et al. (2003) and Zibaee et al. (2012). We have also assessed the influence of ecdysone on total hemocyte count and nodulation. It was observed that ecdysone stimulated nodule formation in a dose dependent manner. When larvae of N. bullata were treated with 20E prior to laminarin (a storage glucan) injection increased the nodulation response in a dose-dependent manner. Contrary to ecdysone treated larvae with JH or juvenile hormone analogs (JHA), fenoxycarb and pyriproxyfen, significantly reduced the formation of nodules in response to laminarin (Franssens et al., 2006). The phagocytic activity of plasmatocytes in larvae of N. bullata was enhanced after 20E injection (Lanot et al., 2001). It was also shown that 20E signaling was needed for Drosophila lymph gland growth and hematopoiesis, both of which are required for pathogen encapsulation (Sorrentino et al., 2002). In the grey flesh fly larvae N. bullata, 20E promotes cell-mediated nodulation response (Franssens et al., 2006). In the tobacco hornworm Manduca sexta JH acts as inhibitor for PO synthesis and thus cuticular melanization did not occur (Hiruma and Riddiford, 1988). Similarly in the mealworm beetle T. molitor, JH reduced immune parameters such as PO levels and encapsulation (Rolff and Siva-Jothy, 2002; Rantala et al., 2003). From these results, it is well established that 20E positively regulate the innate immune system of insects, while JH works as an immuno-suppressor (Flatt et al., 2005). Flatt and Kawecki (2007) showed that JH and 20E have hostile effects on the induction of antimicrobial peptide genes in fruit fly, Drosophila melanogaster. The present study demonstrated that the entomopathogenic fungus, B. bassiana has properties that allow for its successful replication in lesser mulberry pyralid and strongly affect the immune responses of this pest. The understanding of interaction between entomopathogenic fungus and the insect is an important step in fungal disease propagation. 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