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Different strategies to kill the host presented by

Metarhizium anisopliae and Beauveria bassiana

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2017-0517.R3

Manuscript Type: Article

Date Submitted by the Author: 09-Nov-2017

Complete List of Authors: Rustiguel, Cynthia; Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - USP, Biology Fernández-Bravo, María; University of Cordoba, Campus Rabanales, Spain Guimarães, Luis Henrique; Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - USP Quesada-Moraga , Enrique ; UNIVERSITY OF CORDOBA. SPAIN, C.R.A.F.

Is the invited manuscript for consideration in a Special

Issue? : N/A

Keyword: biological control, entomophatogenic fungi, Galleria mellonella, fungal pathogenicity, secondary metabolites

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Revised article 1

Different strategies to kill the host presented by Metarhizium anisopliae and 2

Beauveria bassiana 3

4 Cynthia Barbosa Rustiguel1, María Fernández-Bravo2, Luis Henrique Souza Guimarães1,*, Enrique 5

Quesada-Moraga2. 6

7

1Department of Biology, Faculty of Philosophy, Science and Letters from Ribeirão Preto, 8

University of São Paulo. Avenida Bandeirantes 3900, 14040-901 Ribeirão Preto, Monte Alegre, 9

São Paulo, Brazil. 10

2Department of Agricultural and Forestry Sciences, ETSIAM, University of Cordoba, Campus of 11

Rabanales, C4 Building, 14071 Cordoba, Spain. 12

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

*Corresponding author 34

Dr. Luis Henrique Souza Guimarães 35

Phone: +55 16 3602-4682; fax: +55 16 3602-4886 36

E-mail: [email protected] 37

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Abstract 38

39

Studies conducted over the last decades have shown the potential of entomopathogenic fungi 40

for the biocontrol of some insect pests. They infect their host through the cuticle, so they do not 41

need to be ingested to be effective. These fungi also secrete secondary metabolites and proteins that 42

are toxic to insect pests. In this context, we analyzed the pathogenicity of Metarhizium anisopliae 43

(Metschn.) strains IBCB 384 and IBCB 425 and Beauveria bassiana (Bals.-Criv.) Vuill. strains E 44

1764 and E 3158 against Galleria mellonella (Linn.) larvae, during pre-invasion and post-invasion 45

phases. The results showed that strains of M. anisopliae can be considered the most virulent in pre-46

invasion phase on G. mellonella especially IBCB 384, while strains of B. bassiana were in the post-47

invasion phase, especially the strain E 1764. During in vivo development as well as for production 48

of toxic serum, the strain B. bassiana E 3158 was the most virulent. Different fungal growth (or 49

toxin) strategies were observed for studied strains. The M. anisopliae strain IBCB 425 prioritizes 50

the growth strategy, while the strain IBCB 384 and B. bassiana strains E 1764 and E 3158 have a 51

toxic strategy. All strains have pathogenicity against G. mellonella, indicating their possible use for 52

biocontrol. 53

54

55 Keywords: biological control; entomophatogenic fungi; Galleria mellonella; fungal pathogenicity; 56

secondary metabolites. 57

58

Abbreviations: BSA, bovine serum albumin; SbmF, Submerged Fermentation; TRIS, Tris 59

(hydroxymethyl) – aminomethane; LT50, median lethal time. 60

61

62

63

64

65

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66

Introduction 67

Entomopathogenic fungi are insect killing fungi specialized in penetration through the insect 68

cuticle, configuring an advantage if compared to other pathogens that infect the insect using oral or 69

mesentery means (Alves 1998). They are important pathogens that are able to cause disease in 70

different insect species, including some species that promote serious problems in diverse plantations 71

around the world as, for example Diatrea sacchralis (Fabricius) (Lepdotera: Cambridae), 72

Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae), Euthinobothrus brasiliensis (Hambletom) 73

(Coleoptera: Curculionidae) and Aonidiella aurantii (Maskell) (Hemiptera: Diaspididae) (Alves 74

1998), Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Anopheles gambiae (Giles) (Diptera: 75

Culicidae) (Scholte et al. 2004; Scholte et al. 2005). 76

The interaction between pathogen (fungus) and its respective host is influenced by different 77

factors such as temperature, humidity, light and ultraviolet radiation, as well as by nutritional 78

conditions and host susceptibility. According to this, the infection cycle comprises the stages of 79

adhesion, germination, formation of appressoria, formation of penetration peg, penetration, 80

colonization hemolymph and fungal reproduction (Alves et al. 1998). Besides the spores, the main 81

propagules into fungi, enzymes play an important role during the penetration process of the fungus 82

into the host, by releasing enzymes that degrade the insect cuticle (St. Leger et al. 1988). Several 83

studies have related the production of enzymes and toxins to fungal pathogenicity and virulence 84

(Paris and Segretain 1978). According to Gimenez-Pecci et al. (2002), different strains of the same 85

species produce different levels of extracellular enzymes, as well as multi enzyme complexes 86

containing chitinases, proteases and lipases, among others, required for the degradation of the insect 87

cuticle to promote the infection (St. Leger et al. 1988). 88

Another important aspect that should be considered is the wide genetic variability of the 89

entomopathogenic fungi, which gives them important differences in virulence and pathogenicity to 90

specific groups of insects. Therefore, to select a good fungal strain, some parameters should be 91

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analyzed, such as vegetative growth and production of specific enzymes related to the infection 92

process (Paccola-Meirele and Azevedo, 1990). 93

The proteins/enzymes and toxins produced by entomopathogenic fungi have several roles during 94

all phases of the infection: i) they can be related to cell surface proteins that mediate attachment of 95

spores and join the host cuticle; ii) hydrolytic and detoxifying enzymes (lipase/esterase, catalase, 96

cytochrome P450, proteases (Pr1) and chitinases that degrade the cuticle of the insect body); iii) in 97

the formation of the mechanical pressure apressorium to facilitate the penetration throughout the 98

cuticle and degradation and iv) secondary metabolites or toxins that interfere with the physiological 99

and neurological systems reducing the host immune response (Amiri-Besheli et al. 2000; Quesada-100

Moraga and Vey 2004; Xu et al. 2008; Xu et al. 2009; Ortiz-Urquiza et al. 2013). 101

In general, all entomopathogenic fungi secrete a large variety of toxic metabolites when grown on 102

artificial media or when they infect their hosts (Vey et al. 2001). These secondary metabolites are 103

generally cyclic peptides with antibiotic and insecticidal properties (Vey et al. 2001; Strasser et al. 104

2000). The secondary metabolites and toxins produced and secreted by strains of B. bassiana or 105

Metarhizium anisopliae are beauvericins, bassianolides, oosporein, tenellin, destruxins, bassiacridin 106

and oxalic acid, among others. Unfortunately, the role of some of these compounds on the process 107

of infection is unknown (Quesada-Moraga and Vey 2004; Vey et al. 2001; Fargues et al. 1985; 108

Quesada-Moraga and Vey 2003; Kirkland et al. 2005). Entomopathogenic fungi can use multiple 109

strategies to infect their host. For this purpose, it is necessary i) specific detectors to recognize the 110

host surface; ii) setting mechanisms; iii) secretion of an enzyme complex; iv) penetration into the 111

host cuticle; v) development of hyphal body within the hemocoel; vi) melanization of the cuticle; 112

vii) paralyze the host and viii) sporulation over the dead host (Pedrini et al. 2013). 113

Entomopathogenic fungi to overcome the defense barriers found in their hosts can use different 114

strategies (Ortiz-Urquiza et al. 2013). 115

The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana are easily found 116

in the nature (Klingen and Haukeland 2006; Quesada-Moraga et al. 2007; Jaronski 2010; Garrido-117

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Jurado et al. 2015). Both have been investigated not only because of their abilities to target a large 118

number of insects (Alves 1998), but also because the possibility of the mass production (Kassa et al. 119

2008) and use of different application techniques (Farenhorst and Knols 2010). In general, M. 120

anisopliae infects and causes mortality of approximately 10% of D. saccharalis larvae (Coutinho 121

2009), while B. bassina causes mortality above 50% of Anthonomus grandis (Boheman) 122

(Coleoptera: Curculionidae) (Giometti et al. 2010). However, Zappelini et al. (2010) observed that 123

the strains M. anisopliae IBCB 384 and M. anisopliae IBCB 425 caused mortality of 96% and 82% 124

of D. saccharalis larvae, respectively. The endophytic strains B. bassina E 1764 and B. bassina E 125

3158 caused mortality of 40% and 60% on Spodoptera littoralis (Boisduval) (Lepdoptera: 126

Noctuidae), respectively (Resquín-Romero 2012). Despite the use of Galleria mellonella larvae in 127

the infection studies by M. anisopliae strains, the strategies used by M. anisopliae and B. bassiana 128

strains to kill these larvae were not previously reported. Then, this manuscript describes, 129

comparatively, the analysis of the strategies used by the entomopathogenic fungus M. anisopliae 130

(strains IBCB384 and IBCB 425, selected according to the mortality index on D. saccharalis 131

larvae) and the endophytic fungus B. bassiana (strains E 1764 and E 3158) to kill the G. melonella 132

larvae. 133

134

Material and Methods 135

Fungal origin 136

The strains of M. anisopliae IBCB 384 and IBCB 425 were provided by Dr. José Eduardo 137

Marcondes de Almeida (Collection of Entomopathogenic Microorganisms "Oldemar Cardim 138

Abreu," Biological Institute of Campinas, São Paulo, Brazil). The endophytic fungus B. bassiana 139

strains E 1764 and E 3158 were provided by Dr. Enrique Quesada Moraga (Soil and Endophytic B. 140

bassiana Collection of the Entomology Laboratory, School of Agricultural and Forestry Sciences of 141

the University of Cordoba, Spain). 142

143

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Insects 144

The bioassays were performed with 4th or 5th instar G. mellonella larvae from a healthy 145

colony established in our laboratory. G. mellonella was selected as an experimental host, as it 146

provides some unique advantages for the study of fungal pathogenesis (Mylonakis 2008). This order 147

of lepidopteran insects has been used by different authors to study the pathogenesis of 148

entomopathogenic fungi (Quesada-Moraga and Vey 2003; Fuguet and Vey 2004; Quesada-Moraga 149

et al. 2006). Larvae were reared in glass bottles of 28ºC and 40% relative humidity, and they were 150

fed an artificial diet (30.8 g of corn flour, 30.8 g wheat germ, bulgur 30.8 g, 21.1 g of milk powder 151

10.8 g yeast, 27 g of glycerol, 48.6 g of honey and 1.5 g of nipagin). 152

153

Maintenance of strains 154

The strains were grown on malt agar (MA) (Biokar Diagnostic, Beauvais, France) at 25°C in 155

the dark and then stored at 4°C. To prepare inoculums for the experiments, culture of the strains 156

were subcultured on MA medium in petri dishes. Conidia suspension were prepared scraping 157

conidial from 21 days-old slant cultures from petri dishes into sterile 0.1% Tween 80 aqueous 158

solution that was then sonicated for 5 min and filtered through several layers of cheesecloth to 159

remove mycelia mats and to collect pure conidia. The concentration of the conidial suspensions was 160

determined by Malassez chamber (Blau Brand, Wertheim, Germany). 161

162

Production of insecticidal fungal proteins in Adamek’s liquid medium 163

The liquid cultures were performed in two steps: i) 25 mL of Adamek`s medium (40 g 164

glucose, 40 g yeast extract, 30 g corn steep liquor and 1L of distilled water) (Adamek 1963) in 125 165

mL Erlenmeyer flask, previously autoclaved at 120°C and 1.5 atm for 20 minutes. The medium was 166

inoculated with 1 mL of a conidial suspension (previously adjusted to 107 conidia/mL) for each 167

fungal strain and then incubated for 4 days at 25°C under agitation (110 rpm); ii) After this period, 168

2 mL of each culture mentioned above were transferred to 250 mL a new medium with the same 169

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composition (Adamek`s medium) in 1-L Erlenmeyer flasks previously sterilized. The cultivation 170

was performed for 7 days at 26°C under agitation (110 rpm). 171

172

Topical application assays 173

Two different techniques were used for topical application, which were soaking and spray 174

applications. The M. anisopliae and B. bassiana conidial suspensions for treatments were prepared 175

as described above. The concentration of conidial suspensions was standardized at 1x108 176

conidia/mL for each strain. 177

178

Topical application using immersion 179 180

Ten larvae of G. mellonella at 4th instar were immersed in 5 mL of conidial suspension 181

(1x108 conidia/mL) for 30 seconds. This procedure was performed 3-times totaling 30 larvae for 182

each fungal strain. The control was performed using 10 larvae immersed in 0.1% Tween 80 aqueous 183

solution. After the immersion, each larva was placed individually in plastic Petri dishes (28 mm 184

diameter and 13 mm height) with damp cotton. The larvae were feed by an artificial diet as 185

described above (2.2 section). Mortality was monitored every 24 h for 15 days. Dead larvae were 186

removed daily and immediately surface-sterilized with 1% sodium hypochlorite solution for 1 min 187

and three rinses with sterile distilled water for 1 min. Each larva was then placed on sterile wet filter 188

paper in sterile petri plates that were sealed with Parafilm and kept at 25ºC to be inspected for 189

fungal outgrowth on the cadavers. 190

191 Topical application using sprayer 192 193

Ten larvae of G. mellonella at 4th instar were treated with 2 mL of a conidial suspension 194

(1x108 conidia/mL) using a Potter spray tower (Burkard Rickmans Worth Co. Ltd. UK) with a 195

standard tank of 1.54 ± 0.06 mg/cm2 and pressure of 0.7 bar. The control was performed using 10 196

larvae immersed in 0.1% Tween 80 aqueous solution. This procedure was performed 3-times 197

totalizing 30 larvae for each fungal strain. After treatment, each larva was placed individually in 198

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plastic Petri dishes (28 mm diameter and 13 mm height) with damp cotton. The larvae were feed by 199

an artificial diet, as described above. Mortality was monitored every 24 h for 15 days. Fungal 200

outgrowth were monitored, as described above. 201

202

Recovery of infected hemolymph and sera, and re-infection of larvae 203

Galleria mellonella larvae were injected with 8 µL conidia suspension (105 conidia/mL) 204

obtained for each M. anisopliae and B. bassiana strains, containing 800 conidia per dose. Six 205

possible treatments were injected into the larvae separated, as follow: negative control (without 206

fungus + water + 1% tween 80); positive control (without fungus + water + 1% tween 80 + 4% of 207

soil); IBCB 384 (water + 1% tween 80 + conidia 384); IBCB 425 (water + 1% tween 80 + conidia 208

425); E 1764 (water + 1% tween 80 + conidia 1764); E 3158 (water + 1% tween 80 + conidia 209

3158). The treated larvae received artificial diet as described previously and kept at 25°C until the 210

pre-lethal phase of infection. This phase is characterized by the presence of hyphae and blastopores 211

in the hemolymph. When the presence of these structures before the death of insects (around 96 h) 212

were observed, the hemolymph was collected in tubes containing 300 µL anticoagulant buffer, 213

consisting of 0.098 M NaOH, 0.18 M NaCl and 0.41 M citric acid (Wang et al., 2007), through a 214

cut in the false legs of the larvae. Then, the hemolymph was centrifuged at 13,500 rpm at 4°C for 215

10 min. to remove hemocytes and fungal structures, obtaining the serum. The proteins in the serum 216

were quantified according to Bradford (1976). This serum was sterilized by filtration through a 217

filter with 0.22 µm (Dynagard-Spectrum, Breda, Netherlands). After sterilization, 8 µL of each 218

serum obtained from each strain was injected into G. mellonella larvae. The treated larvae were 219

maintained at 25°C and artificial diet as cited above. Mortality was observed every 24 h for 9 days. 220

External and internal symptoms, such as melanization of the cuticle and trachea, were monitored 221

and compared to the symptoms caused by direct fungal infection. 222

223

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Obtainment of the crude extracts, fractions and toxicity test 224

The crude extracts for each strain was produced as describe above (2.4 section) and then 225

mycelia were removed from the liquid fermentations via vacuum filtration through a Whatman No. 226

3 Chr filter paper (Whatman, Kent, United Kingdom). Finally, proteinase inhibitor (Sigma, San 227

Louis, Mo) was added to crude extracts according to manufacturer’s instructions. The crude extracts 228

were standardized by protein concentration. For that, soluble proteins were determined by Bradford 229

assay using bovine serum albumin (Merck-Schuchardt) as standard. Absorbance at 280 nm was 230

used to estimate protein content in chromatographic fractions. Eight µL of filter-sterilized Dynagard 231

0.2 micrometer filter; Spectrum, Brenda, The Netherlands) crude extracts were injected in the 5th 232

instar G. mellonella larvae, using a micro-injector (Burkard manufacturing, Hertfordshire, UK), 233

between the second and the third abdominal segment. For control 8 µL of Adamek’s medium and 234

0.16 µL of protease inhibitor were injected in the control larvae. Assays were done in triplicate with 235

10 larvae each, totalizing 30 larvae for each crude extract tested. The bioassay was monitored daily 236

for 7 days. In a second stage, 50 mL crude extract of each strain was dialyzed using a membrane cut 237

off of 3.5 kDa (Spectrum Europe, Breda, The Netherlands) against 2 L distilled water for 24 h at 238

4°C. Thus, the fraction that was retained on the membrane was named non-dialyzed fraction while 239

the fraction related to 2 L of distilled water was named dialyzed fraction. Both fractions, for each 240

strain, were concentrated by lyophilization until the volume of 50 mL and injected into the larvae as 241

described above. The dead insects were dissected under a binocular microscope to observe signs of 242

toxicity on tissues, caused by toxic molecules present in the injected fractions (Ortiz-Urquiza et al., 243

2010a). 244

245

Statistical analyses 246

Mortality data were subjected to analysis of variance (ANOVA). LT50 significant difference 247

(LSD) for the comparison of means test was performed. The median lethal times (LT50) were 248

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calculated using the Kaplan-Meier survival test. Statistical analyzes were performed using Statistic 249

9.0 (Analytical Software, 2008) and SPSS 15.0 (SPSS Inc., 1989-2007) for Windows. 250

251 252

Results 253

Topical application of fungi on G. mellonella larvae 254

The penetration efficacy and the pathogenicity of strains M. anisopliae IBCB 384 and IBCB 255

425, and B. bassiana strains E 1764 and E 3158 against G. mellonella larvae using topical 256

application were analyzed (Table 1). The mortality caused by M. anisopliae strains IBCB 384 and 257

IBCB 425 and B. bassiana strains E 1764 and E 3158 , using spraying (F = 68.25; df = 4.14; p < 258

0.001) and immersion (F = 18.54; df = 4.14; p < 0.001) were significant when compared to the 259

control mortality. The strains IBCB 384 and IBCB 425 caused 100% larval mortality using conidia 260

spraying, while the B. bassiana strains caused 73-80% larval mortality. Considering the 261

pathogenicity for all strains, IBCB 384 provided the lowest median survival time (LT50) being 8.53 262

days. When the immersion procedure was used, the strains IBCB 384 and IBCB 425 also caused 263

100% larval mortality. For the B. bassiana strains, the best result was obtained for the strain E1764, 264

which promoted 86% larval mortality. Analyzing the pathogenicity of all strains using immersion, 265

the strain IBCB 425 was the most pathogenic (LT50 = 4.6 days), followed by IBCB 384. On the 266

other hand, when the mortality caused by mycosis was analyzed, it was observed low rates of 267

mortality using M. anisopliae strains under spraying. Under immersion procedure, M. anisopliae 268

strains were able to promote high larval mortality by mycosis compared to spraying procedure. 269

270

Determination of pathogenicity and mortality on G. mellonella larvae using conidia injection 271

The mortality caused by the strains IBCB 384 and IBCB 425 from M. anisopliae and the strains E 272

1764 and E 3158 from B. bassiana, using conidia injection were significant when compared to the 273

control mortality (F4.14 = 126.35; p < 0.001) (Table 2). According to the results presented in the 274

Table 2, M. anisopliae strains IBCB 384 and IBCB 425 promoted similar rate of mortality on G. 275

mellonella larvae (90-93%). For the LT50 values obtained for both strains IBCB 425 and IBCB 384 276

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there was no statistically significant difference if considered the absolute values. Considering only 277

the LT50 values, the strain IBCB 425 may be considered more pathogenic than the strain IBCB 384. 278

The strain E 1764 of B. bassiana promoted 100% larvae mortality while the strain E 3158 promoted 279

86% larval mortality. When the pathogenicity is analyzed considering the LT50 values for all strains, 280

the strain E 1764 was the most pathogenic. 281

282

Toxicity of serum obtained from G. mellonella larvae infected by M. anisopliae and B. 283

bassiana 284

The toxicity of serum obtained from G. mellonella larvae infected by M. anisopliae IBCB 384 and 285

IBCB 425 (SERUM-384 and SERUM-425, respectively), and B. bassiana E 1764 and E 3158 286

(SERUM-1764 and SERUM-3158, respectively) using microinjection technique was analyzed and 287

the mortality caused by serum from all strains are significant when compared to the negative control 288

(F5.17 = 2.71; p < 0.001) (Table 3). It was observed that the SERUM-384 was able to promote 61% 289

larval mortality with LT50 value of 6.56 days. For the others strains, the mortality was reduced (28-290

43%) and the LT50 values were higher than that obtained using strain IBCB 384. According to this, 291

it is clear that the SERUM-384 was the most toxic and pathogenic. Among the serum of B. bassiana 292

strains, the SERUM-3158 caused the higher larval mortality although the LT50 values for the serum 293

of both strains were similar. According to this, it is evident that the SERUM-3158 was the most 294

toxic. 295

Another important aspect that should be analyzed to verify the serum toxicity, the 296

melanization of the cuticle (outer part) and trachea (inside) can be observed in the Fig. 1. The 297

cuticle melanization showed different patterns for the fungal strains actions. There is no 298

melanization in control samples. The most pronounced cuticle melanization was observed for the 299

larvae that received the SERUM-425, while the trachea melanization was most evident for the 300

larvae that received SERUM-384 and SERUM-425. According to the Fig. 1, it is also possible to 301

observe the fungal development for the strains IBCB 384 and IBCB 425 in the hemolymph before 302

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the serum sterilization through the 0.22-µm membrane to be injected in the larvae, in contrast with 303

the results obtained for strains E 1764 and E 3158. 304

305

Analysis of the toxicity on G. mellonella larvae using microinjection 306

The toxicities of the crude extract, and dialyzed and not dialyzed fractions, obtained from the 307

fermentation of M. anisopliae and B. bassiana strains microinjected into the G. mellonella larvae 308

were analyzed and the results are presented in the Table 4. The crude extract obtained for M. 309

anisopliae IBCB 384, and for B. bassiana strains E 1764 and E 3158 promoted high larvae 310

mortality rates. The LT50 value obtained for the strain E 3158 was 1.43 day, indicating 311

pathogenicity 3.8-folds higher than that observed for IBCB 425 with LT50 value of 7.73 days (Table 312

4). 313

When analyzing the toxicity of the dialyzed fraction, it was observed that the higher mortality was 314

caused by B. bassiana strains, especially by the strain E 3158. On the other hand, the M. anisopliae 315

strains promoted similar larvae mortality. Regarding the toxicity of non-dialyzed fractions, B. 316

bassiana strains caused 100% larvae mortality, highlighting the strain E 3158 as the most virulent. 317

For the M. anisopliae strains, lower larvae mortality and high LT50 values were observed, indicating 318

that these fractions have low toxicity (Table 4). In general, it can be noted that B. bassiana E 3158 319

was 2.27-folds more pathogenic than M. anisopliae IBCB 425. In addition, the toxic effect for M. 320

anisopliae strains is present in the dialyzed fraction, while for the B. bassiana strains the toxic 321

effect can be observed for both fractions. However, non-dialyzed fraction has the lowest LT50 value, 322

indicating that the insecticidal activity is faster and more efficient when compared to the dialyzed 323

fraction. 324

325

Discussion 326

Topical application is a methodology that has been used as model for studies of insect pathogenesis 327

(Mylonakis 2008). Under this condition, it is possible to evaluate the performance of each fungal 328

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strain in the pre-invasion phase. In this phase, entomopathogenic fungi are able to produce enzymes 329

that degrade the insect cuticle (Fang et al. 2005; Shah et al. 2005). Among the strains analyzed in 330

this study, M. anisopliae IBCB 425 presented the best response for pre-invasion phase, promoting 331

100% larval mortality with low LT50 value (5 days) on G. mellonella larvae. This result differs from 332

that found by Zappelini et al. (2010), where the strain IBCB 425 was considered pathogenic for D. 333

saccharalis larvae, promoting 62% larval mortality with LT50 of 10 days, using Tower’s Potter. 334

However, it was evident that the strain IBCB 425 was not efficient in the pre-invasion phase on this 335

sugarcane borer, when compared to other strains, including the strain IBCB 384, what can be 336

explained by problems in the fungal adaptation to the host. The LT50 value obtained for the strain 337

IBCB 384 was very close to the value observed by Zappelini et al. (2010), being the most virulent 338

strain. The larval mortality by mycosis was checked observing the extrusion of strains. It was 339

observed low larval mortality using M. anisopliae strains under spraying. The B. bassiana strains E 340

1764 and E 3158 caused reduced mortality of G. mellonella larvae when compared to the M. 341

anisopliae strains, using both spraying and immersion procedures. In addition, the LT50 values were 342

higher for B. bassiana strains than that observed for M. anisopliae strains. This fact can be justified 343

because the B. bassiana strains have difficulty in the pre-invasion phase, while the fungus M. 344

anisopliae does not present difficulties growing inside the insect. Similar result was observed by 345

Resquín-Romero (2012) for the same B. bassiana strains on S. littoralis larvae. The differences 346

observed among the LT50 values for a same strain using immersion and spraying, both procedures 347

characterized as topical application, can be explained by the major contact between the larvae and 348

conidia when the former is used compared to the latter. 349

After checking the performance of the strains in the pre-invasion phase, conidia or crude 350

extracts produced by all strains were microinjected directly in the cavity of the hemocoel. Under 351

this condition, there is no barrier (cuticle) against the infection, allowing the observation of the 352

infection phase development. When conidia of M. anisopliae and B. bassiana strains were injected 353

in G. mellonella larvae, the fungal growth occurred in vivo. All strains caused similar percentage of 354

mortality. On the other hand, the lowest LT50 value was observed for B. bassiana strain E 1764, 355

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indicating that it is most virulent than others. Considering only the M. anisopliae strains, there is no 356

difference between the LT50 values. However, the absolute difference between the LT50 values for 357

these two strains is approximately 1 day, which in practice is very significant if considered the 358

negative impact that a phytophagous insect can cause on plantation for 24 h. Under this aspect, the 359

strain IBCB 425 can be considered more pathogenic than IBCB 384. This type of approach has 360

been used by different authors for different strains of B. bassiana (EABb 01/103-Su, EABb 01/12-361

Su, EABb 01/88-Su and EABb 01/110-Su) and M. anisopliae (EAMa 01/58-Su) (Ortiz-Urquiza et 362

al. 2010a; Ortiz-Urquiza et al. 2010b). 363

Analysis of the production of virulence factors under in vivo condition, assessed through the 364

larvae serum, revealed that the SERUM-384 was the most toxic promoting elevated mortality of G. 365

mellonella larvae with lowest LT50 value. Therefore, M. anisopliae strain IBCB 384 can be 366

considered as the most virulent strain then others analyzed here. Between the B. bassiana strains 367

used, the SERUM-3158 was the most toxic. After injection of the infected sera in healthy larvae, it 368

was observed melanization of the cuticle and trachea. The cuticle melanization was observed in all 369

larvae that received SERUM-384, SERUM-425, SERUM-3158 or SERUM-1764, but with different 370

intensities and patterns. According to Fuguet and Vey (2004), the melanization type A is the most 371

common and it is characterized by the formation of black spots, while melanization type B is called 372

Leopard, because the spots resemble the spots of the leopard (dark border and clear center), and the 373

melanization type C is characterized by large colored regions, covering most of the cuticle surface. 374

We observed melanization type A and C in the larvae that received SERUM-425 and melanization 375

type C in larvae that received SERUM-384, SERUM-3158 and SERUM-1764. However, it was 376

observed dark spots on larvae trachea in presence of SERUM-384 and SERUM-425, as also 377

described by Ortiz-Urquiza et al. (2013) for M. anisopliae EAMa 01/58-Su utilizing fractions of the 378

toxic proteins, demonstrating that it elicited defense-related responses including cuticle 379

melanization and tissue necrosis. Fuguet and Vey (2004) observed similar result for B. bassiana 380

EABb 90/2-Dm. 381

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Analysis of the larvae hemolymph obtained after infection with M. anisopliae IBCB 384 and 382

IBCB 425 revealed the presence of hyphal bodies with 72 h, as also observed by Zhang and Xia 383

(2009). On the other hand, no hyphal body was observed for B. bassiana strains. The toxicity of the 384

crude extract obtained from the fungal cultivations, dialyzed and non-dialyzed fractions on G. 385

mellonella larvae using microinjection was also analyzed. The B. bassiana strains were more 386

effectives to kill the G. mellonella larvae than M. anisopliae strains using crude extract, dialyzed 387

and non-dialyzed fractions, with reduced LT50 values. These results demonstrate that when the first 388

barrier to infection (cuticle) is overcome, the B. bassiana strains produce more toxic molecules, 389

effectives for the infection phase whereas M. anisopliae strains are more effectives in the pre-390

invasion phase. Considering the action of crude extract from strains IBCB 384 and IBCB 425, the 391

mortality was higher for the former, as well as, the pathogenicity as indicates by the lower LT50 392

value. On the other hand, another important effect observed that should be highlighted was the 393

paralysis of the larvae when the crude extract obtained from the cultivation of the strains IBCB 425 394

of M. anisopliae and E 3158 of B. bassiana was used. This paralysis effect was also reported by 395

Resquín-Romero (2012) and Fuguet and Vey (2004) for extracts obtained from different B. 396

bassiana strains and injected into G. mellonella larvae. To understand the toxicity of extracts and to 397

find the molecule responsible for the toxicity, all crude extracts (for each strain) were submitted to 398

dialysis. Two fractions were obtained, i) dialyzed fraction containing molecules smaller than 3.5 399

kDa and ii) non-dialyzed fraction with molecules higher than 3.5 kDa. Both fractions caused 400

mortality of G. mellonella larvae confirming the presence of toxic molecules in these fractions. In 401

general, the dialyzed fractions from M. anisopliae strains promoted 53-58% larvae mortality, higher 402

than that observed for non-dialyzed fractions, while the B. bassiana fractions (dialyzed and non-403

dialyzed) promoted 100% larvae mortality. The low mortality observed for non-dialyzed fractions 404

from M. anisopliae strains was confirmed by high LT50 values if compared to the dialyzed fractions 405

and B. bassiana fractions. According to this, it is evident that the dialyzed fractions from M. 406

anisopliae strains were the most toxic, in agreement with Wang et al. (2004) and Soledade et al. 407

(2002). The fungus M. anisopliae is able to produce different types of destruxin as, for example, 408

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destruxins A, B, C, D and E. These cyclic hexadepsipeptides consisting of α-hydroxyl acid and five 409

amino acid residues can be found in dialyzed fractions containing molecules with molecular mass 410

lower than 3.5 kDa. The destruxins described by Potterat et al. (2000) [44] have a molecular mass 411

of 577 Da, 593 Da, 623 Da and 593 Da. Destruxins have different biological activities such as 412

insecticidal and phytotoxic. Tetanus paralysis of insect larvae, as observed and related here, also has 413

been attributed to dextruxins (Samuels et al. 1988). 414

With regard to dialyzed and non-dialyzed fractions obtained from B. bassiana strains, they 415

were highly toxic for G. mellonella larvae, indicating that these strains are able to produce both 416

proteins and toxic secondary metabolites when cultivated in Adamek's medium. This result is 417

partially similar to that described by Quesada-Moraga and Vey (2003) for B. bassiana strains 90/2 418

and 90/11 which showed toxic molecules only in the non-dialyzed fraction when cultured in 419

medium Adamek's. Considering the results obtained for dialyzed and non-dialyzed factions for each 420

strain studied, it is evident that the non-dialyzed fraction from B. bassiana E 3158 was the most 421

toxic, in agreement with the results obtained by Resquín-Romero (2012) that used crude extract 422

produced by this same strain in S. littoralis larvae. Among the proteins in this fraction, bassiacridin 423

can be found as described by Quesada-Moraga and Vey (2004). Since dialyzed and non-dialyzed 424

fractions from B. bassiana strains presented toxicity, it is clear that these strains are able to produce 425

toxic cyclic peptides as beauvericin, bassionolide, oosporein, and oxalic acid (Vey et al. 2001; 426

Zimmermann, 2007) and toxic proteins as bassiacridin (Quesada-Moraga and Vey, 2004). 427

The development of the entomopathogenic fungi in laboratory is directly influenced by 428

culture medium composition and, consequently the production of virulence factors as well. So, 429

under different conditions, changes in the virulence can be observed. The influence of the medium 430

composition was analyzed by Ortiz-Urquiza et al. (2010a; 2010b) and for B. bassiana and M. 431

anisopliae, who found remarkable differences. Therefore, the verification of the fungal growth 432

under in vitro and in vivo conditions is important, as reported by Ortiz-Urquiza et al. (2010a) and 433

Funguet and Vey (2004). 434

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After evaluating the pathogenicity of the M. anisopliae and B. bassiana strains taking in 435

account the pre-invasion and post-invasion (investigated fungal growth and secretion of toxic 436

compounds in vitro and in vivo) phases, it is possible to conclude that all these strains were 437

pathogenic to G. mellonella. However, they presented different strategies to cause death of the host. 438

M. anisopliae strains were more efficient in the pre-invasion phase, while B. bassiana strains were 439

in the post-invasion phase. It is also evident that the strain IBCB 425 prioritizes the growth strategy 440

while the strain IBCB 384 and B. bassiana strains have a toxic strategy. Kershaw et al. (1999) also 441

demonstrate that different strains of M. anisopliae present different strategies to kill the host, as 442

observed in this work. 443

444

Conclusion 445 446

Pathogenicity should be studied case by case considering that many factors can interfere in 447

the infection process and in pathogen-host relationship. A specific microorganism may be 448

pathogenic for one species of host, but not for another. According to this, under the conditions used 449

in this work, the strains of M. anisopliae can be considered the most virulent in the pre-invasion 450

phase on G. mellonella larvae, while the B. bassiana strains were in the post-invasion phase. 451

Additionally, different strategies (growth or toxic) were observed for the fungal strains. Henceforth, 452

the entomopathogenic fungus M. anisopliae strains IBCB 384 and IBCB 425, and endophytic 453

fungus B. bassiana strains 1764 and 3158 are pathogenic and their actions should be investigated on 454

other hosts. 455

456

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Beauveria brongniartii. Biocontrol Sci. Technol. 17: 553-596. 585

586

587

588

589

590

591

592

593

594

595

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596

597

598

599

600

601

602

Legend 603

604

Fig. 1. Toxicity of the serum obtained from of Galleria mellonella larvae infected with Metarhizium 605

anisopliae IBCB 384 and IBCB 425, and Beauveria bassiana 1764 and 3158 after injection of 800 606

conidia, maintained at 25°C for 72 h. Negative control (without fungus + water + 1% tween 80); 607

positive control (without fungus + water +1% tween 80 + 4% of soil). In cuticle column, we 608

observe the cuticle of the staining characteristics of Galleria mellonella larvae after infected by 609

serum injection. In the trachea column it can be observed melanotic tissue (MT) after injection of 610

serum. In hemolymph column (before serum sterilization) it is verified the presence of hemocyte 611

(He), hemocyte aggregation melanotic (Ham) and blastospore (Bla). 612

613

614 615

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338x190mm (300 x 300 DPI)

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Table 1. Determination of percentage of mortality and Median Lethal Time of Metarhizium anisopliae IBCB 384 and IBCB 425, and Beauveria bassiana E

1764 and E 3158 applied topically on Galleria mellonella larvae

Spraying Immersion

Kaplan - Meier survival

analysis


analysis

Mortality a

Mortality

(mean ± SE) (%)a LT50

b 95% CI

Mortalitya

Mortality

(mean ± SE) (%)a LT50

b 95% CI

Treatments (mean ± SE) (%)

Fungal

outgrowth (day ± SE)

(mean ± SE)

(%) Fungal outgrowth (day ± SE)

Control 13.33 ± 5.77b 0.00 ± 0.00c 14.00 ± 0.46d 13.08-14.91

17.00 ± 3.3b 0.00 ± 0.00d 13.80± 0.52d 12.77-14.82

IBCB 384 100.00 ± 0.00a 23.33 ± 3.33b 8.53 ± 0.35c 7.64-9.43

100.00 ± 0.00a 83.33± 3.33a 7.00± 0.35b 6.64-8.02

IBCB 425 100.00 ± 0.00a 6.66 ± 3.33c 10.40 ± 0.48b 9.54-11.26

100.00 ± 0.00a 33.33± 3.33c 4.60± 0.48c 3.72-5.60

E 1764 73.00 ± 17.00a 20.00 ± 0.00b 12.16 ± 0.54a 11.26-13.07

86.66± 0.00a 46.43± 3.33b 9.00± 0.57a 7.89-10.03

E 3158 80.00 ± 10.00a 43.33 ± 3.33a 10.36 ±0.38ab 9.23-11.51 25.19± 5.77b 0.00 ± 0.00d 14.00±0.38d 13.14-14.65 a Columns with average mortality that have the same letters show no significant difference by LSD test (p ≤ 0.05).

b Columns average mortality with the same letters that show no significant difference by log -rank test (p ≤ 0.05). Median lethal time (LT50) was calculated for

15 days.

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Table 2. Determination of percentage of mortality and Median Lethal Time of Metarhizium

anisopliae IBCB 384 and IBCB 425, and Beauveria bassiana E 1764 and E 3158 on Galleria

mellonella larvae injected with conidia

Treatments

Mortalitya

Mortality

(mean ± SE) (%)a

Kaplan - Meier survival analysis

(mean ± SE) (%) Fungal outgrowth LT50b 95% CI

(day ± SE)

Control 3.33 ± 3.33c 0.00 ± 0.00c 6.93 ± 0.06c 6.80-7.06

IBCB 384 93.33 ± 3.33ab 100.00 ± 0.00a 5.90 ± 0.17a 5.57-6.23

IBCB 425 90.00 ± 5.77ab 100.00 ± 0.00a 5.50 ± 0.25a 5.00-5.99

E 1764 100.00 ± 0.00ab 100.00 ± 0.00a 5.00 ± 0.13b 4.77-5.36

E 3158 86.67 ± 3.33b 90.00 ± 10.00b 5.60 ± 0.15a 5.41-6.19 a Columns with average mortality that have the same letters show no significant difference by

LSD test (p ≤ 0.05). b Columns average mortality with the same letters that show no significant difference by log -rank

test (p ≤ 0.05). Median lethal time (LT50) was calculated for 7 days.

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Table 3. Toxicity of the sera extracted from Galleria mellonella larvae after

microinjection of conidia of Metarhizum anisopliae IBCB 384 and IBCB

425, and Beauvieria bassiana E 1764 and E 3158

Kaplan - Meier survival analysis

Mortality

ad LT50

bc 95% CI Protein

Treatments (mean ± SE) (%) (day ± SE)

mg by

larva

Control - 13.33 ± 0.00 a 8.70a 8.30-9.10 0.01

Control + 28.33 ± 7.23b 8.30b 7.88-8.78 0.01

IBCB 384 60.83 ± 6.2c 6.56c 5.71-7.42 0.01

IBCB 425 35.00 ± 8.63bc 7.73b 6.97-8.49 0.01

E 1764 28.83 ± 9.92b 7.86b 7.09-8.64 0.01

E 3158 43.66 ± 14.98bc 7.41bc 6.66-8.17 0.01 a Columns with average mortality that have the same letters show no

significant difference by LSD test (p ≤ 0.05). b Columns average mortality with the same letters that show no significant

difference by log -rank test (p ≤ 0.05). c Median lethal time (LT50) was calculated for 9 days.

d Mortality of control was 13%, TMS 8,73c and 0.01mg / 8µL protein.

Control - (without fungus + water + 1% tween 80).

Control + (without fungus + water + 1% tween 80 + 4% of soil).

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Table 4. Toxicity of different fractions obtained from Metarhizium anisopliae IBCB 384 and IBCB 425, and Beauveria bassiana E 1764 and E 3158 cultivated in

Adamek’s medium on Galleria mellonella larvae using micro-injection

Crude extract Dialyzed Non-dialyzed


analysis


analysis


analysis

Treatments

Mortalitya Protein LT50

bc 95% CI Mortality

a Protein LT50

bd 95% CI Mortality

a Protein LT50

bd 95% CI

(mean ± SE)

(%) mg/mL (day ± SE)

(mean ± SE)


(mean ± SE)


Control 11.66 ± 5.77d 0.75 6.40 ± 0.14d 6.36-7.03 13.33 ± 6.60c 0.30 7.66 ± 0.16d 5.25-6.11 13.33 ± 6.60c 0.75 7.66 ± 0.16c 7.36-7.99

IBCB 384 83.33 ± 5.77b 1.20 2.40 ± 0.40b 1.60-3.19 58.00 ± 11.00b 0.59 6.50 ± 0.41a 7.74-8.06 13.33 ± 6.60c 1.06 7.90 ± 0.08c 5.69-7.30

IBCB 425 46.66 ± 5.77c 1.14 5.46 ± 0.36c 4.75-6.17 53.00 ± 18.00b 0.49 6.63 ± 0.44a 6.25-7.75 33.33 ± 6.66b 0.58 7.00 ± 0.38c 7.27-8.12

E 1764 100.00 ± 0.00a 1.32 1.70 ± 0.19ab 1.32-2.07 100.00 ± 0.00a 1.09 4.80 ± 0.23b 3.15-4.29 100.00 ± 0.00a 0.882 3.72 ± 0.29a 4.34-5.25

E 3158 100.00 ± 0.00a 1.48 1.43 ± 0.16a 1.12-1.74 100.00 ± 0.00a 0.72 3.26 ± 0.24c 1.70-2.43 100.00 ± 0.00a 0.95 2.06 ± 0.18b 2.77-3.75

a Columns with average mortality that have the same letters show no significant difference by LSD test (p ≤ 0.05).

b Columns average mortality with the same letters that show no significant difference by log -rank test (p ≤ 0.05).

c Median lethal time (LT50) was calculated for 7 days.

d Median lethal time (LT50) was calculated for 8 days.

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