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Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions
Journal of Insect Physiology 55 (2009) 525–531
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Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions Iwona Wojda *, Patryk Kowalski, Teresa Jakubowicz Department of Invertebrate Immunology, Institute of Biology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 October 2008 Received in revised form 20 January 2009 Accepted 26 January 2009
Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 8C for 15 min extended their life time. Galleria mellonella larvae were injected with 104, 105 and 106 fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 105 and increased when 106 blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 8C for 30 min before infection extended their life time when 103 and 104 spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Galleria mellonella Beauveria bassiana Heat shock Antifungal peptides Lysozyme
1. Introduction Beauveria bassiana is an entomopathogenic fungus that naturally grows in soils and is used as biological insecticide to control a number of pests including lepidopterans and orthopterans (Wraight and Ramos, 2005). As an entomopathogen, it has evolved many mechanisms which break the defence response of the host. Upon contact with an insect, fungal cells bind to the cuticle and initiate a developmental program that includes the production of specialized infection structures, such as germ tubes and penetrant hyphae (Boucias and Pendland, 1991; Hajek and Eastburn, 2003; Fuguet et al., 2004). Fungal cell attachment to the cuticle may involve specific receptor–ligand recognition and also nonspecific hydrophobic and electrostatic interactions (Boucias et al., 1988; Doss et al., 1993). B. bassiana, depending on the culture conditions, produces a number of mononucleated single-cell types, including aerial conidia, blastospores, and submerged conidia which have different attachment properties (Holder and Keyhani, 2005). Among determinants which regulate penetration of host cuticle are: proteases, chitinases and lipases (Charnley and Leger, 1991; Charnley, 2003). After crossing the insect integument, the
* Corresponding author. Tel.: +48 815375050; fax: +48 815375050. E-mail address: [email protected] (I. Wojda). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.01.014
fungus grows within hemolymph releasing bioactive metabolites. Mazet et al. (1994) described a protein of molecular mass above 10 kDa, toxic to Spodoptera exigua larvae. B. bassiana also produces low molecular weight cyclic peptides such as beauvericin, enniatins, bassianolide, cyclosporins A and C, with insecticidal properties (Roberts, 1981; Weiser and Matha, 1988; Weiser et al., 1989; Vey et al., 2001). During infection Beauveria produces yeastlike cells that are surrounded by modified cell walls which limit recognition by the defense system. In general, the cell wall of the invading fungus contains a small amount of chitin and b-glucan, resulting in a much thinner structure in comparison to the in vitro growing fungus. Also, the cells lack detectable galactose residues, epitopes that are used by the circulating opsonins in non-selfrecognition (Tartar et al., 2005). The response of insects to infection comprises structural and passive barriers as well as cellular and humoral defence mechanisms. The cellular immune response is connected with rapid production and mobilization of hemocytes which engulf or surround invading organisms. Humoral immune response of insects is manifested by expression of antimicrobial peptide genes which are regulated by the Toll and Imd pathways discovered in Drosophila. Both pathways lead to the activation of Dif and Relish belonging to evolutionary conserved family of NF-kB transcription factors. As a result of the activation, antimicrobial peptides are synthesized mainly in the fat body and further exported to the
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hemolymph (for the review see Wang and Ligoxygakis, 2006; Marmaras and Lampropoulou, 2009). Antimicrobial peptides are mostly small, less than 10 kDa in size, and they act collectively against microbial infection (Hancock et al., 2006). The larvae of the greater wax moth Galleria mellonella is a useful model for studying such biochemical and physiological aspects of insect immunity as: testing the pathogenicity of bacteria and fungi, analyses of biochemical properties of hemolymph or fat body, and following the gene expression in a particular organ (Mylonakis, 2008). Many immune relevant proteins/peptides have already been found in this organism. For example, Seitz et al. (2003) identified 11 immune regulated genes by a subtractive hybridization approach. Some antimicrobial peptides such as cecropin, gallerimycin, galiomicin and lately, moricin-like peptides have been cloned (Schuhmann et al., 2003; Kim et al., 2004; Lee et al., 2004; Brown et al., 2008) and some other peptides exhibiting antibacterial and/or antifungal activity were purified (Cytryn´ska et al., 2007). Some proteins including apolipophorin III, an inducible inhibitor of metalloproteinases (IMPI), and gallysin are known to have immune relevant properties (Phipps et al., 1994; Halwani and Dunphy, 1999; Zakarian et al., 2002; Park et al., 2005; Wedde et al., 2007). We previously showed the dosage and time dependent appearance of antimicrobial activity in the hemolymph after immune challenge of G. mellonella larvae (Wojda et al., 2004). The humoral immune response was enhanced when the larvae were continuously reared at mild heat-shock temperature (38 8C) after injection with non-pathogenic mix of bacteria and yeast and we pointed to the role of Hsp90 derivatives in the stimulation of immune response (Wojda and Jakubowicz, 2007). In this paper we analyze the immune response of G. mellonella larvae after infection with an entomopathogenic fungus B. bassiana. We show the effect of short-time exposure to elevated temperature on the humoral immune response and survival rate, thus emphasizing physiological aspect of the temperature in group-living insects. 2. Materials and methods 2.1. Immunization of insects G. mellonella (Lepidoptera: Pyralidae) was reared on their natural diet of honeybee nest debris at 28 8C in darkness. Larvae of at least 200 mg weight were naturally infected by gentle rolling each larvae on the surface of sporulating fungal culture in a Petri dish (Lemaitre et al., 1997) or by injection of the indicated number of B. bassiana blastospores in the volume of 5 ml. In case of survival experiments groups of 20–30 larvae were infected with each dose/ treatment and every experiment was performed at least 3 times. In order to compare two means of survivals (Figs. 4 and 5), statistical analysis was performed using x2 test and significance (*) was established at P < 0.05. 2.2. Culture of B. bassiana and preparation of blastospores B. bassiana (Strain 80.2 a kind gift from Dr. B. Lemaitre, CNRS, SGM, Gif-sur Yvette, France) was grown on Sabouraud Dextrose Agar (1% polypeptone, 4% glucose, 1.5% agar) supplemented with 2% yeast extract. For preparation of blastospores, SL YPD medium (4% glucose, 2% bactopeptone, 0.5% yeast extract) was inoculated with B. bassiana and incubated for 3 days at 25 8C with gentle shaking. Afterwards, the culture was filtered through miracloth and centrifuged 8000 g for 10 min. The pelleted blastospores were washed with sterile apyrogenic water, counted using a Burker chamber, and kept at 20 8C until use.
2.3. Collection and preparation of hemocyte-free hemolymph The larvae were anesthetized by submerging in cold water, surface sterilized and injured with a sterile needle. The hemolymph from the group of 10 larvae was sampled in Eppendorf tubes containing a few crystals of phenylthiourea to prevent melanization and then centrifuged at 200 g for 5 min and 10,000 g for 10 min. Cell free hemolymph was kept at 20 8C until use. 2.4. Isolation of fat bodies and RNA extraction G. mellonella larvae were cooled down and sterilized in 70% ethanol. The fat bodies were isolated under ice-cold, cell-culture grade Ringer’s solution (172 mM KCl, 68 mM NaCl, 5 mM NaHCO3, pH 6.1, osmolarity 420 mOsm) and kept at 70 8C until RNA extraction. The organs from a minimum of five larvae were used for each time-point. The fat bodies were broken using Pellet Pestle Mixer (Sigma) and total RNA was isolated using GenElute Mammalian Total RNA Extraction Kit (Sigma), followed by DNase treatment (DNase kit, Sigma). 2.5. Reverse transcription and quantitative real-time PCR Reverse transcription was performed using 1 mg of total RNA using anchored oligo (dT)23 primers (Enhanced Avian HS PCR kit, Sigma) as described before (Wojda and Jakubowicz, 2007). Real Time qPCR from the obtained cDNA was performed by the Service (Genetic Modifications Analysis Laboratory, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland). The transcriptional levels of ribosomal protein S7e (housekeeping gene), gallerimycin, galiomicin and cecropin genes were determined with Real Time PCR (7500 Real Time PCR System, Applied Biosystems) with the use of SYBR Green, Sigma. The primers for S7e, gallerimycin and cecropin genes were as described before (Wojda and Jakubowicz, 2007). The primers for galiomicin were as follows: forward—50 -TCG TAT CGT CAC CGC AAA ATG-30 , reverse—50 -GCC GCA ATG ACC ACC TTT ATA-30 . All the products were of 131 bp length. The Real Time qPCR conditions were: 95 8C 3 min, 44 (95 8C 15 s, 60 8C 15 s, 72 8C 1 min). As a standard curve, PCR amplification was performed with several dilutions of DNA template from immunized larvae. The calculation was done by the deltadelta Ct method. The estimated error is given as an asymmetric range of values from four measurements as described in Livak and Schmittgen (2001). 2.6. Determination of defence activity in the hemolymph of G. mellonella larvae The antifungal activity was determined with the use of yeast S. cerevisiae W303, grown overnight at 37 8C and afterwards the culture was diluted and incubated with hemocyte-free hemolymph. After incubation, equal amounts of mixture from all the samples were spread on YPD plates and incubated at 28 8C. The number of CFU (colony forming units) was counted and antifungal activity of the hemolymph was quantified. The number of CFU for yeast incubated with the control hemolymph was taken as 100% (0% antifungal activity). Further details can be found in Wojda and Jakubowicz (2007). Antibacterial activity of the cell-free hemolymph was determined by an inhibition zone assay as described in Wojda et al. (2004). The streptomycin-resistant E. coli strain D31 (CGSC5165) obtained from E. coli genetic stock centre, new Haven, CT (Boman et al., 1974) was used. Antibacterial activity was quantified according to Hultmark (1998) using Cecropin B (Sigma, St. Louis MO) as a standard.
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2.7. Determination of lysozyme activity Lysozyme activity in the hemocyte-free hemolymph was detected using a radial-diffusion assay on Soerensen buffer (0.066 M KH2PO4, 0.066 M Na2HPO4, pH 6.4) agarose plates containing freeze-dried Micrococcus luteus (Sigma). Samples containing the cell-free hemolymph of equal amount of total protein in the volume of 5 ml, were applied on the plates and incubated at 28 8C for 18 h. Lysozyme activity was measured as a zone of digested M. luteus peptidoglycan and quantified using the standard curve made with EWL (Egg White Lysozyme, Sigma) according to Mohrig and Messner (1968) and expressed as an EWL equivalent (mg/ml). 3. Results 3.1. Natural infection of G. mellonella by B. bassiana induces antifungal but not antibacterial response G. mellonella was naturally infected by covering the surface of the larval bodies with B. bassiana spores as described in Section 2. After 72 h we observed first melanization spots (Fig. 1A, c) and this melanization extended on the whole body of animals resulting in completely dark but still alive animals 96 h after infection (Fig. 1A, d). We noticed the appearance of antifungal activity in the hemolymph, which increased in a time-dependent manner (Fig. 2A). No antibacterial activity (against E. coli) was detected in the hemolymph of G. mellonella larvae 24–72 h after infection
Fig. 2. Induction of humoral immune response in naturally infected G. mellonella larvae by B. bassiana measured as: antifungal (A) and lysozyme (C) activity in the hemolymph of the infected larvae and mRNA levels of genes encoding antimicrobial peptides: gallerimycin, galiomicin and cecropin (B). Antifungal and lysozyme activity was measured in hemolymph samples containing an equal amount of total protein and was quantified and described in Section 2. The results show the average values calculated from three experiments, SD. In each experiment 10 larvae were used for each time-point. Quantitative RT-PCR analysis of galiomicin (light grey bars), gallerimycin (white bars) and cecropin (dark grey bars) transcripts in the total RNA of G. mellonella fat bodies was performed as described in Section 2. The results are normalized to S7e mRNA, whose level did not change during the experiment (data not shown). A representative experiment is shown, bars represent the range of values as described in Section 2.
Fig. 1. (A) The phenotypes of naturally infected G. mellonella larvae: (a) healthy larvae, (b) larvae covered by fungal spores, (c) 72 h after infection: first melanization spots, (d) 96 h: dark but still alive larva, (e) 120 h and (f) 144 h: dead larvae, (g) 2 weeks after infection: dead larvae overgrown by fungus. (B) Survival rate of Galleria mellonella larvae naturally infected with Beauveria bassiana spores. The results show the average values calculated on the basis of three experiments, SD, with the total number of 80 larvae used.
(data not shown) suggesting that the immune response caused by natural infection with fungi is specifically directed against fungi. Indeed, RT-qPCR analysis of antimicrobial peptide gene expression confirmed this observation. We found only weak and transient expression of the gene encoding the antibacterial peptide cecropin 24 h after infection, whereas there was a time-dependent increase in the expression of mRNA encoding the two antifungal peptides galiomicin and gallerimycin, although with different kinetics. The induction of galiomicin could already be observed by 24 h postinfection and reached a maximum at 48 h, whereas, expression of
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Fig. 3. Induction of humoral immune response in G. mellonella larvae injected with various numbers of B. bassiana blastospores. Blastospores, 104, 105 and 106 were injected in the volume of 5 ml. Apyrogenic water was used as a control. After 24 h, hemolymph was obtained from the larvae. Samples containing an equal amount of total protein were used for estimation of antibacterial, antifungal and lysozyme activity as described in Section 2. The results show the average values, calculated from three experiments, SD. In each experiment the number of 10 larvae were used for each treatment.
gallerimycin was not evident at 24 h, gradually increased at 48– 72 h (Fig. 2B). We observed an increase in lysozyme activity 24 h after natural infection, but as infection progressed, the activity of lysozyme decreased even below the control (Fig. 2C). Finally, all the animals died within 216 h with 50% mortality after 160 h (Fig. 1A and B). About 2 weeks later, we could observe the dead bodies of larvae overgrown by B. bassiana (Fig. 1A, g).
3.4. Exposition of G. mellonella larvae to heat shock prior to infection enhances anti-fungal and lysozyme activities We investigated the anti-yeast activity in the larvae hemolymph 24 h after 104 blastospore injection. As can be seen in Fig. 6, anti-yeast activity was not detected either in control animals or in heat-shocked, non-infected larvae. Injection of B. bassiana blastospores led to the appearance of antifungal activity which was
3.2. Induction of humoral immune response after injection of B. bassiana blastospores into larvae hemocel Twenty-four hours after infection, an analysis of antimicrobial activity in the hemolymph of the animals injected with three dosages (104, 105 and 106) of B. bassiana blastospores was performed. We observed that antibacterial activity did not significantly exceed the level of control (injection with apyrogenic water) when the dosage of 104 blastospores was used, while in the case of higher blastospore dosages, the increase in the activity was proportional to the number of infecting spores (Fig. 3). A very high level of antifungal activity was observed in the case of all the three dosages used; also, lysozyme activity was increased by a factor of almost two at the lowest dose of 104 blastospores (Fig. 3). Interestingly, although both antifungal and lysozyme activities increased with blastospore dose from 104 to 105, there was no further increase at 106 blastospores, which may be a result of the ability of the entomopathogen to damage or suppress the immune defences of the infected larvae.
Fig. 4. The effect of short-term heat shock on the survival rate of G. mellonella larvae naturally infected with entomopathogenic fungus B. bassiana. The heat shock was performed for 15 min at 43 8C, 72 h after infection (indicated). Each day the live animals from the control group (not shocked, filled circles) and the heat-shocked animals (open circles) were counted. The bars represent SD. A total number of 80 larvae were used for each treatment. Statistically significant differences (P < 0.05) in the survival rate of shocked animals with respect to non-shocked larvae were calculated using x2 test and are indicated (*).
3.3. Short-term heat shock increases the survival rate of G. mellonella naturally infected as well as injected with B. bassiana spores On the third day after natural infection, G. mellonella larvae were subjected to heat shock for 15 min at 43 8C. The survival rate was higher in the shocked animals than in the control group. Time points at which the difference was significant were 180 and 192 h (Fig. 4). Furthermore, in order to eliminate the effect of temperature on the entomopathogen, we exposed G. mellonella larvae to 38 8C for 30 min prior to infection, let them cool down and then injected with B. bassiana blastospores. As can be seen in Fig. 5, the survival rate of the infected animals depended on the blastospore dosage. Fifty percent mortality was observed about 43 h post-injection when a dose of 104 blastospores was used, while in the case of 103 blastospores, 50% mortality was observed 1 day later. In both cases pre-incubation of the larvae at mild heatshock temperature resulted in prolonged life time but the difference was clearer when the lower dosage of blastospores was used.
Fig. 5. The survival rate of G. mellonella larvae pre-exposed to mild heat shock at 38 8C for 30 min followed by injection with 103 (circles) and 104 (squares) B. bassiana blastospores. Filled figures show the number of living, non-shocked animals and open figures show survivals exposed to mild heat shock prior to injection. The bars represent SD. A total number of 60 larvae were used for each treatment. Statistically significant differences (P < 0.05) in the survival rate of shocked animals with respect to non-shocked larvae were calculated using x2 test and are indicated (*).
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Fig. 6. Humoral immune response of G. mellonella larvae pre-exposed to heat shock prior to infection. The animals were exposed to 38 8C for 30 min (hs), part of them after heat shock were injected with 104 of B. bassiana blastospores (hs + Bb), and another group was injected with blastospores without prior heat shock (Bb). Antiyeast activity and lysozyme activity was assessed as described in Section 2 and the results show the average values from three experiments, SD. In each experiment 10 larvae were used for each treatment. Quantitative RT-PCR analysis of galiomicin transcripts in fat bodies 3 h after G. mellonella infection was performed. The bars represent the range of values as described in Section 2.
about 1.5 times higher in animals pre-exposed to heat shock in comparison to the non-pre-heated animals. The analysis of galiomicin gene expression with RT-qPCR confirmed this observation. The transcript level of gene encoding galiomicin was 1.3 times higher in the animals exposed to 30 min heat shock before blastospore injection than in the non-shocked animals (Fig. 6). We also followed the activity of lysozyme in the animals exposed to heat shock and to heat shock in combination with infection by B. bassiana. As shown in Fig. 6, exposition of larvae to 30 min heat shock did not significantly increase lysozyme activity in the hemolymph, while injection of B. bassiana spores caused a 2.3 times increase in lysozyme activity in comparison to the control. Interestingly, larvae which were exposed to heat shock prior to injection of blastospores had 1.3 times higher lysozyme activity in their hemolymph, compared to the hemolymph of animals that had been infected but not stressed by heat shock (Fig. 6). 4. Discussion Entomopathogenic fungi infect their host through the contact action on the cuticle and do not require to be ingested. One of the major responses of the host-insect is production of antimicrobial peptides. Most of these peptides exhibit both antibacterial and antifungal properties although there are some such as drosomycin in Drosophila melanogaster, galiomicin and gallerimycin in G. mellonella which act strongly against fungi (Fehlbaum et al., 1994; Schuhmann et al., 2003; Lee et al., 2004). The advantage of using B. bassiana to study innate immunity of G. mellonella is that insect larvae may be naturally infected, avoiding the artificial damage to larval tissues that is associated with injection. Such natural infection resulted in the death of animals within 120–216 h with 50% mortality after 160 h, despite induction of defence mechanisms in the infected animals such as transcriptional induction of antifungal peptide genes: galiomicin and gallerimycin. Among the
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induced antifungal defences galiomicin may be responsible for the detected anti-yeast activity, since gallerimycin has been shown to be inactive against yeast (Schuhmann et al., 2003). The appearance of antifungal activity in the larvae hemolymph was already detectable by 24 h after natural infection and increasing while infection progressed. The elevation of antifungal activity may be a result of the ongoing production of antifungal peptides by the infected insect, even though entomopathogens produce extracellular proteases that act to oppose the insect immune system (Bidochka and Khachatourians, 1987; Travis et al., 1995). It is also likely that at least part of this activity may come from entomopathogen B. bassiana, which ‘‘prevents’’ the larvae from being infected by other competitive fungi in a well-known mechanism of colonization (Lee et al., 2005). We did not observe antibacterial activity in the naturally infected G. mellonella larvae, which is in agreement with Vilcinskas and Matha (1997b); however, in that report the authors did not follow antifungal activity in the hemolymph. The presented results also point to the role of lysozyme in fighting fungal infection in G. mellonella. We observed an increase in lysozyme activity in the infected larvae 24 h after natural infection and also after injection of different amounts of B. bassiana blastospores. Lysozymes are widespread proteins occurring in insects, vertebrates, plants and microorganisms. They are muramidases that hydrolyze the 1-4-b-linkage between the N-acetylmuramic acid and N-acetyl glucosamine residues in the peptydoglycan layer of the bacterial cell and cause their lysis. The increased activity of lysozyme in infected insects indicates that in lepidopterans this protein is inducible, unlike its constitutively expressed vertebrate counterpart (Daffre et al., 1994). At present, increasing evidence points to the existence of a non-enzymatic mode of lysozyme action (Gandhe et al., 2007). There are also some reports indicating the ability of lysozyme to bind to chitin (Proctor and Cunningham, 1988) and its antifungal activity (Vilcinskas and Matha, 1997a; During et al., 1999). Additionally, it was shown that lysozyme acts synergistically with inducible antimicrobial peptides to fight off infection (Hultmark et al., 1982; Cytrynska et al., 2001). The two latter properties of lysozyme may explain its role upon infection with B. bassiana. The drop in lysozyme activity observed at the later time-points after infection may be due to the development of the above mentioned anti-defence mechanisms connected with, among others, production of extracellular proteases degrading insect cuticle and hemolymph proteins (Bidochka and Khachatourians, 1987). We previously reported that G. mellonella larvae injected with a mix of non-pathogenic bacteria and yeast showed an increased defence response when the animals were reared at mild heatshock temperature (38 8C) in comparison to those kept at 28 8C (Wojda and Jakubowicz, 2007). In the present work, we used a pathogenic strain of B. bassiana to study the effect of elevated temperature on insect immunity. When naturally infected G. mellonella larvae were exposed to short heat shock, 15 min at 43 8C, it had a positive effect on the survival rate, even though this temperature was lethal when the animals were reared continuously at 43 8C. In order to avoid these damaging effects of high temperature on entomopathogen we pre-exposed the larvae to elevated temperature before blastospore injection. This also resulted in a prolonged survival rate, when 103 and 104 blastospores were used. This finding is in agreement with Mowlds and Kavannah (2007), who showed that G. mellonella larvae preincubated for 24 h at elevated temperature were more resistant to infection caused by Candida albicans. There are also some other reports showing that physical stress may increase defence abilities in invertebrates (Singh and Aballay, 2006; Malagoli et al., 2007; Mowlds et al., 2008). Some papers show that keeping animals in
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lower (stressful) temperatures may have a similar effect (Mowlds and Kavannah, 2007; Linder et al., 2008). We found that the effect of short heat pre-treatment of larvae on the survival rate can be explained by enhanced immune response in the infected insects. It was manifested by increased antifungal activity in the hemolymph, as a result of increased transcription of e.g. defensin-like galiomicin in the fat bodies of the heat-shocked larvae, in comparison to the infected larvae not preexposed to elevated temperature. Interestingly, the role of lysozyme in fighting fungal infection was confirmed by investigation of the activity of this enzyme in the heat-shocked animals prior to infection. We observed increased activity of lysozyme in the infected animals, which was even higher when the infection was preceded by heat shock. The observations presented above seem to be important from a perspective that G. mellonella lives in colonies and accumulation of larvae produces high temperatures reaching even 40 8C (Schmolz and Schulz, 1995). Some authors point to the role of so-called ‘‘behavioural fever’’, which means that insects alter their temperature through thermoregulatory behaviour, thus increasing their ability to fight off pathogens (Watson et al., 1993; Blanford et al., 1998; Elliot et al., 2002; Thomas and Blanford, 2003). This correlates nicely with our observations showing that an increase in the temperature lasting even very shortly may have a physiological effect on their resistance to infection. This may be a part of the postulated ‘‘social immunity’’ (Cremer et al., 2007) and may be considered as one of the reasons why insects living in groups have become dominant species in many habitats. Acknowledgement This work was supported by the Ministry of Science and Higher Education, Poland, Grant No. 2P04 08029. References Bidochka, M.J., Khachatourians, G.G., 1987. Purification and properties of an extracellular protease produced by the entomopathogenic fungus Beauveria bassiana. Applied and Environmental Microbiology 53, 1679–1684. Blanford, S., Thomas, M.B., Langewald, J., 1998. Behavioural fever in the Senegalese grasshopper, Oedaleus senegalensis, and its implications for biological control using pathogens. Ecological Entomology 23, 9–14. Boman, H.G., Nilsson-Faye, I., Paul, K., Rasmuson Jr., T., 1974. Insect immunity. I. Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cythia pupae. Infection and Immunity 10, 136–145. Boucias, D.G., Pendland, J., 1991. Attachment of mycopathogens to cuticle. In: Cole, G.T., Hoch, H.C. (Eds.), The Fungal Spore and Disease Initiation in Plants and Animals. Plenum Press, New York, NY, pp. 101–127. Boucias, D.G., Pendland, J.C., Latge, J.P., 1988. Nonspecific factors involved in attachment of entomopathogenic deuteromycetes to host insect cuticle. Applied and Environmental Microbiology 54, 1795–1805. Brown, S.E., Howard, A., Kasprzak, A.B., Gordon, K.H., East, P.D., 2008. The discovery and analysis of a diverged family of novel antifungal moricin-like peptides in the wax moth Galleria mellonella. Insect Biochemistry and Molecular Biology 38, 201–212. Charnley, A.K., Leger, R.J.St., 1991. The role of cuticle-degrading enzymes in fungal pathogenesis in insects. In: Cole, E.T., Hoch, H.C. (Eds.), Fungal Spore Disease Initiation in Plants and Animals. Plenum Press, New York, NY, pp. 267–287. Charnley, A.K., 2003. Fungal pathogens of insects: cuticle degrading enzymes and toxins. Advances in Botanical Research 40, 241–321. Cremer, S., Armitage, S.A.O., Schmid-Hempel, P., 2007. Social immunity. Current Biology 17, R693–R702. Cytryn´ska, M., Mak, P., Zdybicka-Barabas, A., Suder, P., Jakubowicz, T., 2007. Purification and characterization of eight peptides from Galleria mellonella immune hemolymph. Peptides 28, 533–546. Cytrynska, M., Zdybicka-Barabas, A., Jabłon´ski, P., Jakubowicz, T., 2001. Detection of antibacterial polypeptide activity in situ after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Analytical Biochemistry 299, 274–276. Daffre, S., Kylsten, P., Samakovlis, C., Hultmark, D., 1994. The lysozyme locus in Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract. Molecular & General Genetics: MGG 242, 152–162. Doss, R.P., Potter, S.W., Chastagner, G.A., Christian, J.K., 1993. Adhesion of nongerminated Botrytis cinerea conidia to several substrata. Applied and Environmental Microbiology 59, 1786–1791.
During, K., Porsch, P., Mahn, A., Brinkmann, O., Gieffers, W., 1999. The non-enzymatic microbicidal activity of lysozymes. FEBS Letters 449, 93–100. Elliot, S.L., Blanford, S., Thomas, M.B., 2002. Host-pathogen interactions in a varying environment: temperature, behavioural fever and fitness. Proceedings of the Royal Society of London Series B: Biological Sciences 269, 1599–1607. Fehlbaum, P., Bulet, P., Michaut, L., Lagueux, M., Broekaert, W.F., Hetru, C., Hoffmann, J.A., 1994. Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. Journal of Biological Chemistry 269, 33159–33163. Fuguet, R., The´raud, M., Vey, A., 2004. Production in vitro of toxic macromolecules by strains of Beauveria bassiana, and purification of a chitosanase-like protein secreted by a melanizing isolate. Comparative Biochemistry and Physiology, Part C 138, 149–161. Gandhe, A.S., Janardhan, G., Nagaraju, J., 2007. Immune upregulation of novel antibacterial proteins from silkmoths (Lepidoptera) that resemble lysozymes but lack muramidase activity. Insect Biochemistry and Molecular Biology 37, 655–666. Hajek, A.E., Eastburn, C.C., 2003. Attachment and germination of Entomophaga maimaiga conidia on host and non-host larval cuticle. Journal of Invertebrate Pathology 82, 12–22. Halwani, A.E., Dunphy, G.E., 1999. Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity. Developmental and Comparative Immunology 23, 563–570. Hancock, R.E.W., Brown, K.L., Mookherjee, N., 2006. Host defence peptides from invertebrates—emerging antimicrobial strategies. Immunobiology 211, 315– 322. Holder, D.J., Keyhani, N.O., 2005. Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata. Applied and Environmental Microbiology 71, 5260–5266. Hultmark, D., 1998. Quantification of antimicrobial activity, using the inhibitionzone assay. In: Wiesner, A., Dunphy, A.G., Morishima, I., Sugumaran, M., Yamakawa, M. (Eds.), Techniques in Insect Immunology. SOS Publications, New Jersey, pp. 103–108. Hultmark, D., Engstrom, A., Bennich, H., Kapur, R., Boman, H.G., 1982. Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. European Journal of Biochemistry 127, 207– 217. Kim, C.H., Lee, J.H., Kim, I., Seo, S.J., Son, S.M., Lee, K.Y., Lee, I.H., 2004. Purification and cDNA cloning of a cecropin-like peptide from the great wax moth Galleria mellonella. Molecular Cell 17, 262–266. Lee, S.Y., Nakajima, I., Ihara, F., Kinoshita, H., Nihira, T., 2005. Cultivation of entomopathogenic fungi for the search of antibacterial compounds. Mycopathologia 160, 321–325. Lee, Y.S., Yun, E.K., Jang, W.S., Kim, I., Lee, J.H., Park, S.Y., Ryu, K.S., Seo, S.J., Kim, C.H., Lee, I.H., 2004. Purification, cDNA cloning and expression of an insect defensin from the great wax moth, Galleria mellonella. Insect Molecular Biology 13, 65–72. Lemaitre, B., Reichhart, J.M., Jules, A., Hoffmann, J.A., 1997. Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proceedings of the National Academy of Sciences United States of America 94, 14614–14619. Linder, J.E., Owers, K.A., Promislow, D.A.L., 2008. The effects of temperature on host– pathogen interactions in D. melanogaster: who benefits? Journal of Insect Physiology 54, 297–308. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCt method. Methods 25, 402–408. Malagoli, D., Casarini, L., Sacchi, S., Ottaviani, E., 2007. Stress and immune response in the mussel Mytilus galloprovincialis. Fish & Shellfish Immunology 23, 171–177. Marmaras, V.J., Lampropoulou, M., 2009. Regulators and signalling in insect haemocyte immunity. Cellular Signaling 21, 186–195. Mazet, I., Hung, S.-Y., Boucias, D.G., 1994. Detection of toxic metabolites in the hemolymph of Beauveria bassiana infected Spodoptera exigua larvae. Experientia 50, 142–147. Mohrig, W., Messner, B., 1968. Immunoreaktionen bei Insekten. I. Lysozym als grundlegender antibakterieller Faktor im humoralen Abwehrmechanismus der Insekten. Biologische Zentralblatt 87, 439–470. Mowlds, P., Barron, A., Kavanagh, K., 2008. Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans. Microbes and Infection 10, 628–634. Mowlds, P., Kavannah, K., 2007. Effect of pre-incubation temperature on susceptibility of Galleria mellonella larvae to infection by Candida albicans. Mycopathologia 165, 5–12. Mylonakis, E., 2008. Galleria mellonella and the study of fungal pathogenesis: making the case for another genetically tractable model host. Mycopathologia 165, 1–3. Park, S.Y., Kim, C.H., Jeong, W.H., Lee, J.H., Seo, S.J., Han, Y.S., Le, I.H., 2005. Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella. Developmental and Comparative Immunology 29, 43–51. Proctor, V.A., Cunningham, F.E., 1988. The chemistry of lysozyme and its use as a food preservative and a pharmaceutical. Critical Reviews in Food Science and Nutrition 26, 359–395. Phipps, D.J., Chadwick, J.S., Aston, W.P., 1994. Gallysin-1, an antibacterial protein isolated from hemolymph of Galleria mellonella. Developmental and Comparative Immunology 18, 13–23. Roberts, D.W., 1981. Toxins of entomopathogenic fungi. In: Burges, H.D. (Ed.), Microbial Control of Pests and Plant Diseases 1970–1980. Academic Press, New York, pp. 441–464.
I. Wojda et al. / Journal of Insect Physiology 55 (2009) 525–531 Schmolz, E., Schulz, O., 1995. Colorimetric investigations on thermoregulation and growth of wax moth larvae Galleria mellonella. Thermochimica Acta 251, 241– 245. Schuhmann, B., Seitz, V., Vilcinskas, A., Podsiadlowski, L., 2003. Cloning and expression of gallerimycin, an antifungal peptide expressed in immune response of greater wax moth larvae, Galleria melonella. Archives of Insect Biochemistry and Physiology 53, 125–133. Seitz, V., Clermont, A., Wedde, M., Hummel, M., Vilcinskas, A., Schlatterer, K., Podsiadlowski, L., 2003. Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by substractive hybridization approach. Developmental and Comparative Immunology 27, 207–215. Singh, V., Aballay, A., 2006. Heat-shock transcription factor (HSF)-1 pathway required for Caenorhabditis elegants immunity. Proceedings of the National Academy of Sciences United States of America 103, 13092–13097. Tartar, A., Shapiro, A.M., Scharf, D.W., Boucias, D.G., 2005. Differential expression of chitin synthase (CHS) and glucan synthase (FKS) genes correlates with the formation of a modified, thinner cell wall in in vivo-produced Beauveria bassiana cells. Mycopathologia 160, 303–314. Thomas, M.B., Blanford, S., 2003. Thermal biology in insect-parasite interactions. Trends in Ecology and Evolution 18, 344–350. Travis, J., Potempa, J., Maeda, H., 1995. Are bacterial proteinases pathogenic factors? Trends in Microbiology 3, 405–407. Vey, A., Hoagland, R., Butt, T.M., 2001. Toxic metabolites of fungal biocontrol agents. In: Butt, T.M., Jackson, C.W., Magan, N. (Eds.), Fungi as Biocontrol Agents: Progress, Problems and Potential. CABI Publishing, pp. 311–346. Vilcinskas, A., Matha, V., 1997a. Antimycotic activity of lysozyme and its contribution to antifungal humoral defence reactions in Galleria mellonella. Animal Biology 6, 19–29.
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Vilcinskas, A., Matha, V., 1997b. Effect of the entomopathogenic fungus Beauveria bassiana on the humoral immune response of Galleria mellonella larvae (Lepidoptera: Pyralidae). European Journal of Enthomology 94, 461–472. Wang, L., Ligoxygakis, P., 2006. Pathogen recognition and signaling in the Drosophila innate immune response. Immunobiology 211, 251–261. Watson, D.W., Mullens, B.A., Petersen, J.J., 1993. Behavioral fever response of Muscadomestica (Diptera, Muscidae) to infection by Entomophthora-Muscae (Zygomycetes, Entomophthorales). Journal of Invertebrate Pathology 61, 10–16. Wedde, M., Weise, C., Nuck, R., Altincicek, B., Vilcinskas, A., 2007. The insect metalloproteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two distinct inhibitors. Biological Chemistry 388, 119–127. Weiser, J., Matha, V., 1988. The insecticidal activity of cyclosporines on mosquito larvae. Journal of Invertebrate Pathology 51, 92–93. Weiser, J., Matha, V., Zizka, Z., Jegorov, A., 1989. Pathology of cyclosporin A in mosquito larvae. Cytobios 59, 143–150. Wojda, I., Jakubowicz, T., 2007. Humoral immune response upon mild heat-shock conditions in Galleria mellonella larvae. Journal of Insect Physiology 53, 1134– 1144. Wojda, I., Kowalski, P., Jakubowicz, T., 2004. JNK MAP kinase is involved in the humoral immune response of the greater wax moth larvae Galleria mellonella. Archives of Insect Biochemistry and Physiology 56, 143–154. Wraight, S.P., Ramos, M.E., 2005. Synergistic interaction between Beauveria bassiana- and Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of Colorado potato beetle larvae. Journal of Invertebrate Pathology 90, 139–150. Zakarian, R.J., Dunphy, G.B., Quiot, J.M., 2002. Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae. Journal of Insect Physiology 48, 715–723.
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Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions Iwona Wojda *, Patryk Kowalski, Teresa Jakubowicz Department of Invertebrate Immunology, Institute of Biology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 October 2008 Received in revised form 20 January 2009 Accepted 26 January 2009
Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 8C for 15 min extended their life time. Galleria mellonella larvae were injected with 104, 105 and 106 fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 105 and increased when 106 blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 8C for 30 min before infection extended their life time when 103 and 104 spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Galleria mellonella Beauveria bassiana Heat shock Antifungal peptides Lysozyme
1. Introduction Beauveria bassiana is an entomopathogenic fungus that naturally grows in soils and is used as biological insecticide to control a number of pests including lepidopterans and orthopterans (Wraight and Ramos, 2005). As an entomopathogen, it has evolved many mechanisms which break the defence response of the host. Upon contact with an insect, fungal cells bind to the cuticle and initiate a developmental program that includes the production of specialized infection structures, such as germ tubes and penetrant hyphae (Boucias and Pendland, 1991; Hajek and Eastburn, 2003; Fuguet et al., 2004). Fungal cell attachment to the cuticle may involve specific receptor–ligand recognition and also nonspecific hydrophobic and electrostatic interactions (Boucias et al., 1988; Doss et al., 1993). B. bassiana, depending on the culture conditions, produces a number of mononucleated single-cell types, including aerial conidia, blastospores, and submerged conidia which have different attachment properties (Holder and Keyhani, 2005). Among determinants which regulate penetration of host cuticle are: proteases, chitinases and lipases (Charnley and Leger, 1991; Charnley, 2003). After crossing the insect integument, the
* Corresponding author. Tel.: +48 815375050; fax: +48 815375050. E-mail address: [email protected] (I. Wojda). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.01.014
fungus grows within hemolymph releasing bioactive metabolites. Mazet et al. (1994) described a protein of molecular mass above 10 kDa, toxic to Spodoptera exigua larvae. B. bassiana also produces low molecular weight cyclic peptides such as beauvericin, enniatins, bassianolide, cyclosporins A and C, with insecticidal properties (Roberts, 1981; Weiser and Matha, 1988; Weiser et al., 1989; Vey et al., 2001). During infection Beauveria produces yeastlike cells that are surrounded by modified cell walls which limit recognition by the defense system. In general, the cell wall of the invading fungus contains a small amount of chitin and b-glucan, resulting in a much thinner structure in comparison to the in vitro growing fungus. Also, the cells lack detectable galactose residues, epitopes that are used by the circulating opsonins in non-selfrecognition (Tartar et al., 2005). The response of insects to infection comprises structural and passive barriers as well as cellular and humoral defence mechanisms. The cellular immune response is connected with rapid production and mobilization of hemocytes which engulf or surround invading organisms. Humoral immune response of insects is manifested by expression of antimicrobial peptide genes which are regulated by the Toll and Imd pathways discovered in Drosophila. Both pathways lead to the activation of Dif and Relish belonging to evolutionary conserved family of NF-kB transcription factors. As a result of the activation, antimicrobial peptides are synthesized mainly in the fat body and further exported to the
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hemolymph (for the review see Wang and Ligoxygakis, 2006; Marmaras and Lampropoulou, 2009). Antimicrobial peptides are mostly small, less than 10 kDa in size, and they act collectively against microbial infection (Hancock et al., 2006). The larvae of the greater wax moth Galleria mellonella is a useful model for studying such biochemical and physiological aspects of insect immunity as: testing the pathogenicity of bacteria and fungi, analyses of biochemical properties of hemolymph or fat body, and following the gene expression in a particular organ (Mylonakis, 2008). Many immune relevant proteins/peptides have already been found in this organism. For example, Seitz et al. (2003) identified 11 immune regulated genes by a subtractive hybridization approach. Some antimicrobial peptides such as cecropin, gallerimycin, galiomicin and lately, moricin-like peptides have been cloned (Schuhmann et al., 2003; Kim et al., 2004; Lee et al., 2004; Brown et al., 2008) and some other peptides exhibiting antibacterial and/or antifungal activity were purified (Cytryn´ska et al., 2007). Some proteins including apolipophorin III, an inducible inhibitor of metalloproteinases (IMPI), and gallysin are known to have immune relevant properties (Phipps et al., 1994; Halwani and Dunphy, 1999; Zakarian et al., 2002; Park et al., 2005; Wedde et al., 2007). We previously showed the dosage and time dependent appearance of antimicrobial activity in the hemolymph after immune challenge of G. mellonella larvae (Wojda et al., 2004). The humoral immune response was enhanced when the larvae were continuously reared at mild heat-shock temperature (38 8C) after injection with non-pathogenic mix of bacteria and yeast and we pointed to the role of Hsp90 derivatives in the stimulation of immune response (Wojda and Jakubowicz, 2007). In this paper we analyze the immune response of G. mellonella larvae after infection with an entomopathogenic fungus B. bassiana. We show the effect of short-time exposure to elevated temperature on the humoral immune response and survival rate, thus emphasizing physiological aspect of the temperature in group-living insects. 2. Materials and methods 2.1. Immunization of insects G. mellonella (Lepidoptera: Pyralidae) was reared on their natural diet of honeybee nest debris at 28 8C in darkness. Larvae of at least 200 mg weight were naturally infected by gentle rolling each larvae on the surface of sporulating fungal culture in a Petri dish (Lemaitre et al., 1997) or by injection of the indicated number of B. bassiana blastospores in the volume of 5 ml. In case of survival experiments groups of 20–30 larvae were infected with each dose/ treatment and every experiment was performed at least 3 times. In order to compare two means of survivals (Figs. 4 and 5), statistical analysis was performed using x2 test and significance (*) was established at P < 0.05. 2.2. Culture of B. bassiana and preparation of blastospores B. bassiana (Strain 80.2 a kind gift from Dr. B. Lemaitre, CNRS, SGM, Gif-sur Yvette, France) was grown on Sabouraud Dextrose Agar (1% polypeptone, 4% glucose, 1.5% agar) supplemented with 2% yeast extract. For preparation of blastospores, SL YPD medium (4% glucose, 2% bactopeptone, 0.5% yeast extract) was inoculated with B. bassiana and incubated for 3 days at 25 8C with gentle shaking. Afterwards, the culture was filtered through miracloth and centrifuged 8000 g for 10 min. The pelleted blastospores were washed with sterile apyrogenic water, counted using a Burker chamber, and kept at 20 8C until use.
2.3. Collection and preparation of hemocyte-free hemolymph The larvae were anesthetized by submerging in cold water, surface sterilized and injured with a sterile needle. The hemolymph from the group of 10 larvae was sampled in Eppendorf tubes containing a few crystals of phenylthiourea to prevent melanization and then centrifuged at 200 g for 5 min and 10,000 g for 10 min. Cell free hemolymph was kept at 20 8C until use. 2.4. Isolation of fat bodies and RNA extraction G. mellonella larvae were cooled down and sterilized in 70% ethanol. The fat bodies were isolated under ice-cold, cell-culture grade Ringer’s solution (172 mM KCl, 68 mM NaCl, 5 mM NaHCO3, pH 6.1, osmolarity 420 mOsm) and kept at 70 8C until RNA extraction. The organs from a minimum of five larvae were used for each time-point. The fat bodies were broken using Pellet Pestle Mixer (Sigma) and total RNA was isolated using GenElute Mammalian Total RNA Extraction Kit (Sigma), followed by DNase treatment (DNase kit, Sigma). 2.5. Reverse transcription and quantitative real-time PCR Reverse transcription was performed using 1 mg of total RNA using anchored oligo (dT)23 primers (Enhanced Avian HS PCR kit, Sigma) as described before (Wojda and Jakubowicz, 2007). Real Time qPCR from the obtained cDNA was performed by the Service (Genetic Modifications Analysis Laboratory, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland). The transcriptional levels of ribosomal protein S7e (housekeeping gene), gallerimycin, galiomicin and cecropin genes were determined with Real Time PCR (7500 Real Time PCR System, Applied Biosystems) with the use of SYBR Green, Sigma. The primers for S7e, gallerimycin and cecropin genes were as described before (Wojda and Jakubowicz, 2007). The primers for galiomicin were as follows: forward—50 -TCG TAT CGT CAC CGC AAA ATG-30 , reverse—50 -GCC GCA ATG ACC ACC TTT ATA-30 . All the products were of 131 bp length. The Real Time qPCR conditions were: 95 8C 3 min, 44 (95 8C 15 s, 60 8C 15 s, 72 8C 1 min). As a standard curve, PCR amplification was performed with several dilutions of DNA template from immunized larvae. The calculation was done by the deltadelta Ct method. The estimated error is given as an asymmetric range of values from four measurements as described in Livak and Schmittgen (2001). 2.6. Determination of defence activity in the hemolymph of G. mellonella larvae The antifungal activity was determined with the use of yeast S. cerevisiae W303, grown overnight at 37 8C and afterwards the culture was diluted and incubated with hemocyte-free hemolymph. After incubation, equal amounts of mixture from all the samples were spread on YPD plates and incubated at 28 8C. The number of CFU (colony forming units) was counted and antifungal activity of the hemolymph was quantified. The number of CFU for yeast incubated with the control hemolymph was taken as 100% (0% antifungal activity). Further details can be found in Wojda and Jakubowicz (2007). Antibacterial activity of the cell-free hemolymph was determined by an inhibition zone assay as described in Wojda et al. (2004). The streptomycin-resistant E. coli strain D31 (CGSC5165) obtained from E. coli genetic stock centre, new Haven, CT (Boman et al., 1974) was used. Antibacterial activity was quantified according to Hultmark (1998) using Cecropin B (Sigma, St. Louis MO) as a standard.
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2.7. Determination of lysozyme activity Lysozyme activity in the hemocyte-free hemolymph was detected using a radial-diffusion assay on Soerensen buffer (0.066 M KH2PO4, 0.066 M Na2HPO4, pH 6.4) agarose plates containing freeze-dried Micrococcus luteus (Sigma). Samples containing the cell-free hemolymph of equal amount of total protein in the volume of 5 ml, were applied on the plates and incubated at 28 8C for 18 h. Lysozyme activity was measured as a zone of digested M. luteus peptidoglycan and quantified using the standard curve made with EWL (Egg White Lysozyme, Sigma) according to Mohrig and Messner (1968) and expressed as an EWL equivalent (mg/ml). 3. Results 3.1. Natural infection of G. mellonella by B. bassiana induces antifungal but not antibacterial response G. mellonella was naturally infected by covering the surface of the larval bodies with B. bassiana spores as described in Section 2. After 72 h we observed first melanization spots (Fig. 1A, c) and this melanization extended on the whole body of animals resulting in completely dark but still alive animals 96 h after infection (Fig. 1A, d). We noticed the appearance of antifungal activity in the hemolymph, which increased in a time-dependent manner (Fig. 2A). No antibacterial activity (against E. coli) was detected in the hemolymph of G. mellonella larvae 24–72 h after infection
Fig. 2. Induction of humoral immune response in naturally infected G. mellonella larvae by B. bassiana measured as: antifungal (A) and lysozyme (C) activity in the hemolymph of the infected larvae and mRNA levels of genes encoding antimicrobial peptides: gallerimycin, galiomicin and cecropin (B). Antifungal and lysozyme activity was measured in hemolymph samples containing an equal amount of total protein and was quantified and described in Section 2. The results show the average values calculated from three experiments, SD. In each experiment 10 larvae were used for each time-point. Quantitative RT-PCR analysis of galiomicin (light grey bars), gallerimycin (white bars) and cecropin (dark grey bars) transcripts in the total RNA of G. mellonella fat bodies was performed as described in Section 2. The results are normalized to S7e mRNA, whose level did not change during the experiment (data not shown). A representative experiment is shown, bars represent the range of values as described in Section 2.
Fig. 1. (A) The phenotypes of naturally infected G. mellonella larvae: (a) healthy larvae, (b) larvae covered by fungal spores, (c) 72 h after infection: first melanization spots, (d) 96 h: dark but still alive larva, (e) 120 h and (f) 144 h: dead larvae, (g) 2 weeks after infection: dead larvae overgrown by fungus. (B) Survival rate of Galleria mellonella larvae naturally infected with Beauveria bassiana spores. The results show the average values calculated on the basis of three experiments, SD, with the total number of 80 larvae used.
(data not shown) suggesting that the immune response caused by natural infection with fungi is specifically directed against fungi. Indeed, RT-qPCR analysis of antimicrobial peptide gene expression confirmed this observation. We found only weak and transient expression of the gene encoding the antibacterial peptide cecropin 24 h after infection, whereas there was a time-dependent increase in the expression of mRNA encoding the two antifungal peptides galiomicin and gallerimycin, although with different kinetics. The induction of galiomicin could already be observed by 24 h postinfection and reached a maximum at 48 h, whereas, expression of
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Fig. 3. Induction of humoral immune response in G. mellonella larvae injected with various numbers of B. bassiana blastospores. Blastospores, 104, 105 and 106 were injected in the volume of 5 ml. Apyrogenic water was used as a control. After 24 h, hemolymph was obtained from the larvae. Samples containing an equal amount of total protein were used for estimation of antibacterial, antifungal and lysozyme activity as described in Section 2. The results show the average values, calculated from three experiments, SD. In each experiment the number of 10 larvae were used for each treatment.
gallerimycin was not evident at 24 h, gradually increased at 48– 72 h (Fig. 2B). We observed an increase in lysozyme activity 24 h after natural infection, but as infection progressed, the activity of lysozyme decreased even below the control (Fig. 2C). Finally, all the animals died within 216 h with 50% mortality after 160 h (Fig. 1A and B). About 2 weeks later, we could observe the dead bodies of larvae overgrown by B. bassiana (Fig. 1A, g).
3.4. Exposition of G. mellonella larvae to heat shock prior to infection enhances anti-fungal and lysozyme activities We investigated the anti-yeast activity in the larvae hemolymph 24 h after 104 blastospore injection. As can be seen in Fig. 6, anti-yeast activity was not detected either in control animals or in heat-shocked, non-infected larvae. Injection of B. bassiana blastospores led to the appearance of antifungal activity which was
3.2. Induction of humoral immune response after injection of B. bassiana blastospores into larvae hemocel Twenty-four hours after infection, an analysis of antimicrobial activity in the hemolymph of the animals injected with three dosages (104, 105 and 106) of B. bassiana blastospores was performed. We observed that antibacterial activity did not significantly exceed the level of control (injection with apyrogenic water) when the dosage of 104 blastospores was used, while in the case of higher blastospore dosages, the increase in the activity was proportional to the number of infecting spores (Fig. 3). A very high level of antifungal activity was observed in the case of all the three dosages used; also, lysozyme activity was increased by a factor of almost two at the lowest dose of 104 blastospores (Fig. 3). Interestingly, although both antifungal and lysozyme activities increased with blastospore dose from 104 to 105, there was no further increase at 106 blastospores, which may be a result of the ability of the entomopathogen to damage or suppress the immune defences of the infected larvae.
Fig. 4. The effect of short-term heat shock on the survival rate of G. mellonella larvae naturally infected with entomopathogenic fungus B. bassiana. The heat shock was performed for 15 min at 43 8C, 72 h after infection (indicated). Each day the live animals from the control group (not shocked, filled circles) and the heat-shocked animals (open circles) were counted. The bars represent SD. A total number of 80 larvae were used for each treatment. Statistically significant differences (P < 0.05) in the survival rate of shocked animals with respect to non-shocked larvae were calculated using x2 test and are indicated (*).
3.3. Short-term heat shock increases the survival rate of G. mellonella naturally infected as well as injected with B. bassiana spores On the third day after natural infection, G. mellonella larvae were subjected to heat shock for 15 min at 43 8C. The survival rate was higher in the shocked animals than in the control group. Time points at which the difference was significant were 180 and 192 h (Fig. 4). Furthermore, in order to eliminate the effect of temperature on the entomopathogen, we exposed G. mellonella larvae to 38 8C for 30 min prior to infection, let them cool down and then injected with B. bassiana blastospores. As can be seen in Fig. 5, the survival rate of the infected animals depended on the blastospore dosage. Fifty percent mortality was observed about 43 h post-injection when a dose of 104 blastospores was used, while in the case of 103 blastospores, 50% mortality was observed 1 day later. In both cases pre-incubation of the larvae at mild heatshock temperature resulted in prolonged life time but the difference was clearer when the lower dosage of blastospores was used.
Fig. 5. The survival rate of G. mellonella larvae pre-exposed to mild heat shock at 38 8C for 30 min followed by injection with 103 (circles) and 104 (squares) B. bassiana blastospores. Filled figures show the number of living, non-shocked animals and open figures show survivals exposed to mild heat shock prior to injection. The bars represent SD. A total number of 60 larvae were used for each treatment. Statistically significant differences (P < 0.05) in the survival rate of shocked animals with respect to non-shocked larvae were calculated using x2 test and are indicated (*).
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Fig. 6. Humoral immune response of G. mellonella larvae pre-exposed to heat shock prior to infection. The animals were exposed to 38 8C for 30 min (hs), part of them after heat shock were injected with 104 of B. bassiana blastospores (hs + Bb), and another group was injected with blastospores without prior heat shock (Bb). Antiyeast activity and lysozyme activity was assessed as described in Section 2 and the results show the average values from three experiments, SD. In each experiment 10 larvae were used for each treatment. Quantitative RT-PCR analysis of galiomicin transcripts in fat bodies 3 h after G. mellonella infection was performed. The bars represent the range of values as described in Section 2.
about 1.5 times higher in animals pre-exposed to heat shock in comparison to the non-pre-heated animals. The analysis of galiomicin gene expression with RT-qPCR confirmed this observation. The transcript level of gene encoding galiomicin was 1.3 times higher in the animals exposed to 30 min heat shock before blastospore injection than in the non-shocked animals (Fig. 6). We also followed the activity of lysozyme in the animals exposed to heat shock and to heat shock in combination with infection by B. bassiana. As shown in Fig. 6, exposition of larvae to 30 min heat shock did not significantly increase lysozyme activity in the hemolymph, while injection of B. bassiana spores caused a 2.3 times increase in lysozyme activity in comparison to the control. Interestingly, larvae which were exposed to heat shock prior to injection of blastospores had 1.3 times higher lysozyme activity in their hemolymph, compared to the hemolymph of animals that had been infected but not stressed by heat shock (Fig. 6). 4. Discussion Entomopathogenic fungi infect their host through the contact action on the cuticle and do not require to be ingested. One of the major responses of the host-insect is production of antimicrobial peptides. Most of these peptides exhibit both antibacterial and antifungal properties although there are some such as drosomycin in Drosophila melanogaster, galiomicin and gallerimycin in G. mellonella which act strongly against fungi (Fehlbaum et al., 1994; Schuhmann et al., 2003; Lee et al., 2004). The advantage of using B. bassiana to study innate immunity of G. mellonella is that insect larvae may be naturally infected, avoiding the artificial damage to larval tissues that is associated with injection. Such natural infection resulted in the death of animals within 120–216 h with 50% mortality after 160 h, despite induction of defence mechanisms in the infected animals such as transcriptional induction of antifungal peptide genes: galiomicin and gallerimycin. Among the
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induced antifungal defences galiomicin may be responsible for the detected anti-yeast activity, since gallerimycin has been shown to be inactive against yeast (Schuhmann et al., 2003). The appearance of antifungal activity in the larvae hemolymph was already detectable by 24 h after natural infection and increasing while infection progressed. The elevation of antifungal activity may be a result of the ongoing production of antifungal peptides by the infected insect, even though entomopathogens produce extracellular proteases that act to oppose the insect immune system (Bidochka and Khachatourians, 1987; Travis et al., 1995). It is also likely that at least part of this activity may come from entomopathogen B. bassiana, which ‘‘prevents’’ the larvae from being infected by other competitive fungi in a well-known mechanism of colonization (Lee et al., 2005). We did not observe antibacterial activity in the naturally infected G. mellonella larvae, which is in agreement with Vilcinskas and Matha (1997b); however, in that report the authors did not follow antifungal activity in the hemolymph. The presented results also point to the role of lysozyme in fighting fungal infection in G. mellonella. We observed an increase in lysozyme activity in the infected larvae 24 h after natural infection and also after injection of different amounts of B. bassiana blastospores. Lysozymes are widespread proteins occurring in insects, vertebrates, plants and microorganisms. They are muramidases that hydrolyze the 1-4-b-linkage between the N-acetylmuramic acid and N-acetyl glucosamine residues in the peptydoglycan layer of the bacterial cell and cause their lysis. The increased activity of lysozyme in infected insects indicates that in lepidopterans this protein is inducible, unlike its constitutively expressed vertebrate counterpart (Daffre et al., 1994). At present, increasing evidence points to the existence of a non-enzymatic mode of lysozyme action (Gandhe et al., 2007). There are also some reports indicating the ability of lysozyme to bind to chitin (Proctor and Cunningham, 1988) and its antifungal activity (Vilcinskas and Matha, 1997a; During et al., 1999). Additionally, it was shown that lysozyme acts synergistically with inducible antimicrobial peptides to fight off infection (Hultmark et al., 1982; Cytrynska et al., 2001). The two latter properties of lysozyme may explain its role upon infection with B. bassiana. The drop in lysozyme activity observed at the later time-points after infection may be due to the development of the above mentioned anti-defence mechanisms connected with, among others, production of extracellular proteases degrading insect cuticle and hemolymph proteins (Bidochka and Khachatourians, 1987). We previously reported that G. mellonella larvae injected with a mix of non-pathogenic bacteria and yeast showed an increased defence response when the animals were reared at mild heatshock temperature (38 8C) in comparison to those kept at 28 8C (Wojda and Jakubowicz, 2007). In the present work, we used a pathogenic strain of B. bassiana to study the effect of elevated temperature on insect immunity. When naturally infected G. mellonella larvae were exposed to short heat shock, 15 min at 43 8C, it had a positive effect on the survival rate, even though this temperature was lethal when the animals were reared continuously at 43 8C. In order to avoid these damaging effects of high temperature on entomopathogen we pre-exposed the larvae to elevated temperature before blastospore injection. This also resulted in a prolonged survival rate, when 103 and 104 blastospores were used. This finding is in agreement with Mowlds and Kavannah (2007), who showed that G. mellonella larvae preincubated for 24 h at elevated temperature were more resistant to infection caused by Candida albicans. There are also some other reports showing that physical stress may increase defence abilities in invertebrates (Singh and Aballay, 2006; Malagoli et al., 2007; Mowlds et al., 2008). Some papers show that keeping animals in
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lower (stressful) temperatures may have a similar effect (Mowlds and Kavannah, 2007; Linder et al., 2008). We found that the effect of short heat pre-treatment of larvae on the survival rate can be explained by enhanced immune response in the infected insects. It was manifested by increased antifungal activity in the hemolymph, as a result of increased transcription of e.g. defensin-like galiomicin in the fat bodies of the heat-shocked larvae, in comparison to the infected larvae not preexposed to elevated temperature. Interestingly, the role of lysozyme in fighting fungal infection was confirmed by investigation of the activity of this enzyme in the heat-shocked animals prior to infection. We observed increased activity of lysozyme in the infected animals, which was even higher when the infection was preceded by heat shock. The observations presented above seem to be important from a perspective that G. mellonella lives in colonies and accumulation of larvae produces high temperatures reaching even 40 8C (Schmolz and Schulz, 1995). Some authors point to the role of so-called ‘‘behavioural fever’’, which means that insects alter their temperature through thermoregulatory behaviour, thus increasing their ability to fight off pathogens (Watson et al., 1993; Blanford et al., 1998; Elliot et al., 2002; Thomas and Blanford, 2003). This correlates nicely with our observations showing that an increase in the temperature lasting even very shortly may have a physiological effect on their resistance to infection. This may be a part of the postulated ‘‘social immunity’’ (Cremer et al., 2007) and may be considered as one of the reasons why insects living in groups have become dominant species in many habitats. Acknowledgement This work was supported by the Ministry of Science and Higher Education, Poland, Grant No. 2P04 08029. References Bidochka, M.J., Khachatourians, G.G., 1987. Purification and properties of an extracellular protease produced by the entomopathogenic fungus Beauveria bassiana. Applied and Environmental Microbiology 53, 1679–1684. Blanford, S., Thomas, M.B., Langewald, J., 1998. Behavioural fever in the Senegalese grasshopper, Oedaleus senegalensis, and its implications for biological control using pathogens. Ecological Entomology 23, 9–14. Boman, H.G., Nilsson-Faye, I., Paul, K., Rasmuson Jr., T., 1974. Insect immunity. I. Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cythia pupae. Infection and Immunity 10, 136–145. Boucias, D.G., Pendland, J., 1991. Attachment of mycopathogens to cuticle. In: Cole, G.T., Hoch, H.C. (Eds.), The Fungal Spore and Disease Initiation in Plants and Animals. Plenum Press, New York, NY, pp. 101–127. Boucias, D.G., Pendland, J.C., Latge, J.P., 1988. Nonspecific factors involved in attachment of entomopathogenic deuteromycetes to host insect cuticle. Applied and Environmental Microbiology 54, 1795–1805. Brown, S.E., Howard, A., Kasprzak, A.B., Gordon, K.H., East, P.D., 2008. The discovery and analysis of a diverged family of novel antifungal moricin-like peptides in the wax moth Galleria mellonella. Insect Biochemistry and Molecular Biology 38, 201–212. Charnley, A.K., Leger, R.J.St., 1991. The role of cuticle-degrading enzymes in fungal pathogenesis in insects. In: Cole, E.T., Hoch, H.C. (Eds.), Fungal Spore Disease Initiation in Plants and Animals. Plenum Press, New York, NY, pp. 267–287. Charnley, A.K., 2003. Fungal pathogens of insects: cuticle degrading enzymes and toxins. Advances in Botanical Research 40, 241–321. Cremer, S., Armitage, S.A.O., Schmid-Hempel, P., 2007. Social immunity. Current Biology 17, R693–R702. Cytryn´ska, M., Mak, P., Zdybicka-Barabas, A., Suder, P., Jakubowicz, T., 2007. Purification and characterization of eight peptides from Galleria mellonella immune hemolymph. Peptides 28, 533–546. Cytrynska, M., Zdybicka-Barabas, A., Jabłon´ski, P., Jakubowicz, T., 2001. Detection of antibacterial polypeptide activity in situ after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Analytical Biochemistry 299, 274–276. Daffre, S., Kylsten, P., Samakovlis, C., Hultmark, D., 1994. The lysozyme locus in Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract. Molecular & General Genetics: MGG 242, 152–162. Doss, R.P., Potter, S.W., Chastagner, G.A., Christian, J.K., 1993. Adhesion of nongerminated Botrytis cinerea conidia to several substrata. Applied and Environmental Microbiology 59, 1786–1791.
During, K., Porsch, P., Mahn, A., Brinkmann, O., Gieffers, W., 1999. The non-enzymatic microbicidal activity of lysozymes. FEBS Letters 449, 93–100. Elliot, S.L., Blanford, S., Thomas, M.B., 2002. Host-pathogen interactions in a varying environment: temperature, behavioural fever and fitness. Proceedings of the Royal Society of London Series B: Biological Sciences 269, 1599–1607. Fehlbaum, P., Bulet, P., Michaut, L., Lagueux, M., Broekaert, W.F., Hetru, C., Hoffmann, J.A., 1994. Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. Journal of Biological Chemistry 269, 33159–33163. Fuguet, R., The´raud, M., Vey, A., 2004. Production in vitro of toxic macromolecules by strains of Beauveria bassiana, and purification of a chitosanase-like protein secreted by a melanizing isolate. Comparative Biochemistry and Physiology, Part C 138, 149–161. Gandhe, A.S., Janardhan, G., Nagaraju, J., 2007. Immune upregulation of novel antibacterial proteins from silkmoths (Lepidoptera) that resemble lysozymes but lack muramidase activity. Insect Biochemistry and Molecular Biology 37, 655–666. Hajek, A.E., Eastburn, C.C., 2003. Attachment and germination of Entomophaga maimaiga conidia on host and non-host larval cuticle. Journal of Invertebrate Pathology 82, 12–22. Halwani, A.E., Dunphy, G.E., 1999. Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity. Developmental and Comparative Immunology 23, 563–570. Hancock, R.E.W., Brown, K.L., Mookherjee, N., 2006. Host defence peptides from invertebrates—emerging antimicrobial strategies. Immunobiology 211, 315– 322. Holder, D.J., Keyhani, N.O., 2005. Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata. Applied and Environmental Microbiology 71, 5260–5266. Hultmark, D., 1998. Quantification of antimicrobial activity, using the inhibitionzone assay. In: Wiesner, A., Dunphy, A.G., Morishima, I., Sugumaran, M., Yamakawa, M. (Eds.), Techniques in Insect Immunology. SOS Publications, New Jersey, pp. 103–108. Hultmark, D., Engstrom, A., Bennich, H., Kapur, R., Boman, H.G., 1982. Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. European Journal of Biochemistry 127, 207– 217. Kim, C.H., Lee, J.H., Kim, I., Seo, S.J., Son, S.M., Lee, K.Y., Lee, I.H., 2004. Purification and cDNA cloning of a cecropin-like peptide from the great wax moth Galleria mellonella. Molecular Cell 17, 262–266. Lee, S.Y., Nakajima, I., Ihara, F., Kinoshita, H., Nihira, T., 2005. Cultivation of entomopathogenic fungi for the search of antibacterial compounds. Mycopathologia 160, 321–325. Lee, Y.S., Yun, E.K., Jang, W.S., Kim, I., Lee, J.H., Park, S.Y., Ryu, K.S., Seo, S.J., Kim, C.H., Lee, I.H., 2004. Purification, cDNA cloning and expression of an insect defensin from the great wax moth, Galleria mellonella. Insect Molecular Biology 13, 65–72. Lemaitre, B., Reichhart, J.M., Jules, A., Hoffmann, J.A., 1997. Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proceedings of the National Academy of Sciences United States of America 94, 14614–14619. Linder, J.E., Owers, K.A., Promislow, D.A.L., 2008. The effects of temperature on host– pathogen interactions in D. melanogaster: who benefits? Journal of Insect Physiology 54, 297–308. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCt method. Methods 25, 402–408. Malagoli, D., Casarini, L., Sacchi, S., Ottaviani, E., 2007. Stress and immune response in the mussel Mytilus galloprovincialis. Fish & Shellfish Immunology 23, 171–177. Marmaras, V.J., Lampropoulou, M., 2009. Regulators and signalling in insect haemocyte immunity. Cellular Signaling 21, 186–195. Mazet, I., Hung, S.-Y., Boucias, D.G., 1994. Detection of toxic metabolites in the hemolymph of Beauveria bassiana infected Spodoptera exigua larvae. Experientia 50, 142–147. Mohrig, W., Messner, B., 1968. Immunoreaktionen bei Insekten. I. Lysozym als grundlegender antibakterieller Faktor im humoralen Abwehrmechanismus der Insekten. Biologische Zentralblatt 87, 439–470. Mowlds, P., Barron, A., Kavanagh, K., 2008. Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans. Microbes and Infection 10, 628–634. Mowlds, P., Kavannah, K., 2007. Effect of pre-incubation temperature on susceptibility of Galleria mellonella larvae to infection by Candida albicans. Mycopathologia 165, 5–12. Mylonakis, E., 2008. Galleria mellonella and the study of fungal pathogenesis: making the case for another genetically tractable model host. Mycopathologia 165, 1–3. Park, S.Y., Kim, C.H., Jeong, W.H., Lee, J.H., Seo, S.J., Han, Y.S., Le, I.H., 2005. Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella. Developmental and Comparative Immunology 29, 43–51. Proctor, V.A., Cunningham, F.E., 1988. The chemistry of lysozyme and its use as a food preservative and a pharmaceutical. Critical Reviews in Food Science and Nutrition 26, 359–395. Phipps, D.J., Chadwick, J.S., Aston, W.P., 1994. Gallysin-1, an antibacterial protein isolated from hemolymph of Galleria mellonella. Developmental and Comparative Immunology 18, 13–23. Roberts, D.W., 1981. Toxins of entomopathogenic fungi. In: Burges, H.D. (Ed.), Microbial Control of Pests and Plant Diseases 1970–1980. Academic Press, New York, pp. 441–464.
I. Wojda et al. / Journal of Insect Physiology 55 (2009) 525–531 Schmolz, E., Schulz, O., 1995. Colorimetric investigations on thermoregulation and growth of wax moth larvae Galleria mellonella. Thermochimica Acta 251, 241– 245. Schuhmann, B., Seitz, V., Vilcinskas, A., Podsiadlowski, L., 2003. Cloning and expression of gallerimycin, an antifungal peptide expressed in immune response of greater wax moth larvae, Galleria melonella. Archives of Insect Biochemistry and Physiology 53, 125–133. Seitz, V., Clermont, A., Wedde, M., Hummel, M., Vilcinskas, A., Schlatterer, K., Podsiadlowski, L., 2003. Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by substractive hybridization approach. Developmental and Comparative Immunology 27, 207–215. Singh, V., Aballay, A., 2006. Heat-shock transcription factor (HSF)-1 pathway required for Caenorhabditis elegants immunity. Proceedings of the National Academy of Sciences United States of America 103, 13092–13097. Tartar, A., Shapiro, A.M., Scharf, D.W., Boucias, D.G., 2005. Differential expression of chitin synthase (CHS) and glucan synthase (FKS) genes correlates with the formation of a modified, thinner cell wall in in vivo-produced Beauveria bassiana cells. Mycopathologia 160, 303–314. Thomas, M.B., Blanford, S., 2003. Thermal biology in insect-parasite interactions. Trends in Ecology and Evolution 18, 344–350. Travis, J., Potempa, J., Maeda, H., 1995. Are bacterial proteinases pathogenic factors? Trends in Microbiology 3, 405–407. Vey, A., Hoagland, R., Butt, T.M., 2001. Toxic metabolites of fungal biocontrol agents. In: Butt, T.M., Jackson, C.W., Magan, N. (Eds.), Fungi as Biocontrol Agents: Progress, Problems and Potential. CABI Publishing, pp. 311–346. Vilcinskas, A., Matha, V., 1997a. Antimycotic activity of lysozyme and its contribution to antifungal humoral defence reactions in Galleria mellonella. Animal Biology 6, 19–29.
531
Vilcinskas, A., Matha, V., 1997b. Effect of the entomopathogenic fungus Beauveria bassiana on the humoral immune response of Galleria mellonella larvae (Lepidoptera: Pyralidae). European Journal of Enthomology 94, 461–472. Wang, L., Ligoxygakis, P., 2006. Pathogen recognition and signaling in the Drosophila innate immune response. Immunobiology 211, 251–261. Watson, D.W., Mullens, B.A., Petersen, J.J., 1993. Behavioral fever response of Muscadomestica (Diptera, Muscidae) to infection by Entomophthora-Muscae (Zygomycetes, Entomophthorales). Journal of Invertebrate Pathology 61, 10–16. Wedde, M., Weise, C., Nuck, R., Altincicek, B., Vilcinskas, A., 2007. The insect metalloproteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two distinct inhibitors. Biological Chemistry 388, 119–127. Weiser, J., Matha, V., 1988. The insecticidal activity of cyclosporines on mosquito larvae. Journal of Invertebrate Pathology 51, 92–93. Weiser, J., Matha, V., Zizka, Z., Jegorov, A., 1989. Pathology of cyclosporin A in mosquito larvae. Cytobios 59, 143–150. Wojda, I., Jakubowicz, T., 2007. Humoral immune response upon mild heat-shock conditions in Galleria mellonella larvae. Journal of Insect Physiology 53, 1134– 1144. Wojda, I., Kowalski, P., Jakubowicz, T., 2004. JNK MAP kinase is involved in the humoral immune response of the greater wax moth larvae Galleria mellonella. Archives of Insect Biochemistry and Physiology 56, 143–154. Wraight, S.P., Ramos, M.E., 2005. Synergistic interaction between Beauveria bassiana- and Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of Colorado potato beetle larvae. Journal of Invertebrate Pathology 90, 139–150. Zakarian, R.J., Dunphy, G.B., Quiot, J.M., 2002. Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae. Journal of Insect Physiology 48, 715–723.