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Quantification of fungal growth and destruxin A during infection of Galleria mellonella larvae by Metarhizium brunneum
Accepted Manuscript Quantification of fungal growth and destruxin A during infection of Galleria mellonella larvae by Metarhizium brunneum A. Ríos-Moreno, I. Garrido-Jurado, M.C. Raya-Ortega, E.Quesada-Moraga PII: DOI: Reference:
S0022-2011(16)30247-6 http://dx.doi.org/10.1016/j.jip.2017.06.007 YJIPA 6961
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
12 December 2016 31 May 2017 15 June 2017
Please cite this article as: Ríos-Moreno, A., Garrido-Jurado, I., Raya-Ortega, M.C., E.Quesada-Moraga, Quantification of fungal growth and destruxin A during infection of Galleria mellonella larvae by Metarhizium brunneum, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.06.007
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Title: Quantification of fungal growth and destruxin A during infection of Galleria mellonella larvae by Metarhizium brunneum
Authors: A. Ríos-Morenoa, I. Garrido-Juradoa,M. C. Raya-Ortegab and E.Quesada-Moragaa*
Address: aDepartment
of Agricultural and Forestry Sciences, ETSIAM, University of Cordoba, C4Building, Campus of Rabanales, 14071 Cordoba, Spain.
bDepartment
of Agronomy, ETSIAM, University of Cordoba, C4Building, Campus of Rabanales, 14071 Cordoba, Spain.
*Corresponding author: tel.: + 34-957218475; fax: +34-957218440. E-mail address:[email protected] (E. Quesada-Moraga)
Abstract Destruxin A is among the major secondary metabolites produced by the entomopathogenic ascomycete Metarhizium sp., and the lack of studies concerning production of destruxin A by the fungus is most likely the biggest obstacle for the registration of new fungal strains. Although several studies focus on the production of destruxin A in culture media, few studies examine destruxin A in vivo during host infection. In the current work, Galleria mellonella was used as an insect model to develop for the first time in vivo real-time PCR- and HPLC-MS-based quantification of fungal growth and metabolite production, respectively, during infection by two strains of M. brunneum. Total mortality of sixth instar G. mellonella larvae that were immersed in a suspension of 1.0 x 108 conidia mL-1 of M. brunneum EAMa 01/58-Su or BIPESCO5 strains reached 85.5 % and 78.8 %, respectively, and the percentage of cadavers with fungal outgrowth was low at 12.2 % and 4.4 %, respectively. The average survival time of treated larvae was 5.5 days for both fungal strains. Using EAMa 01/58-Su and BIPESCO5 specific primer set, real-time PCR showed that the patterns of fungal growth were different for the two strains, whereas no significant differences were detected in the number of fungal sequence copies recovered from the infected larvae. EAMa 01/58-Su and BIPESCO5 strains secreted destruxin A from days 2 to 6 andfrom days 2 to 5 post treatment, respectively. For
EAMa 01/58-Su and BIPESCO5, the maximum titer of destruxin A in the host was on day 4 at 0.369 and 0.06 µ g/larva, respectively, and throughout the pathogenic process, the total production was 0.6 and 0.09 µg/larva, respectively. These results demonstrated that the strains pose a low hazard, if any, to humans and the environment. The methods used in this study to quantify fungal growth and metabolite production provided valuable data to better understand the role of destruxin A during the growth of M. brunneum in the host larvae and to monitor the fate of destruxin A in food chains.
Keywords: Real-time PCR, HPLC-MS, BIPESCO5, EAMa 01/58-Su, mode of action, risk assessment 1. Introduction The effectiveness of Metarhizium sp. in controlling many insect pests in agriculture makes the fungus an attractive alternative to conventional pesticides, with many commercial products on the market or in development (Zimmermann, 2007; Lacey et al., 2015). This fungus is considered safe for humans and the environment, but the production and the evaluation of safety data for registration of new entomopathogenic fungal strains are expensive and time-consuming (Zimmermann, 2007; Strauch et al., 2011). Indeed, the requirement by regulatory authorities for risk assessment studies with detailed information concerning the production of secondary metabolites is the biggest obstacle for registration (Skrobek et al., 2006; Strauch et al., 2011). Concerning secondary metabolites, Metarhizium sp. produces a family of cyclic peptide toxins, destruxins, with the focus on destruxin A as the most likely major secondary metabolite produced by this genus (Zimmermann, 2007). Indeed, several studies focus on
the production of destruxin A in culture media (Skrobek and Butt, 2005; Ríos-Moreno et al., 2016a). More recently, the necessity to better understand the association between entomopathogenic fungi and plants has also been emphasized, which includes determination of whether the fungi produce metabolites in plants (Vega et al., 2009). Hence, destruxins A, B and E were detected in cowpea plants inoculated with M. robertsii (Golo et al., 2014), destruxin A was quantified in melon and tomato plants sprayed with M. brunneum (Garrido-Jurado et al., 2016a; Resquín-Romero et al., 2016), and using an extraction procedure based on QuEChERS, destruxins were quantified in strawberry and corn (Taibon et al., 2015). A reliable optimization method based on a modified version of QuEChERS and HPLC-MS to detect and monitor destruxins in potato plants has also been proposed (Carpio et al., 2016; Ríos-Moreno et al., 2016b). Although studies have examined the role of destruxins in the pathogenicity and virulence of Metarhizium sp. (Pal et al., 2007; Hu et al., 2009; Donzelli et al., 2012), studies are lacking on the dynamics of fungal growth and secretion of destruxin A during the infection of the insect host by the fungal biocontrol agent.Only one study has reported on in vivo detection and quantification of destruxin A (Skrobek et al., 2008), andthe study used HPLC-DAD. However, the detection and quantification limits and recovery levels can be improved using HPLC-MS, which is generally a more sensitive detection method for such metabolites (Bogusz and Carracedo, 2004). Recent studies show that quantitative real-time polymerase chain reaction (qPCR) can be used to determine the amount of DNA from fungi in a sample (Arquiza-Apollo and Hunter, 2014), demonstrating that this technique is a powerful, specific, accurate and sensitive tool for detection and quantification of fungi (Malvick and Impullitti, 2007; Tae and Knudsen, 2016). Real-time PCR has been used to
monitor and quantify M. anisopliae and Beauveria bassiana during Anopheles infection in the presence or absence of malaria parasites (Bell et al., 2009) and to examine the relationship between fungal growth kinetics and virulence in the housefly (Anderson et al., 2011). Studying in vivo secretion of destruxin A during infection of the insect host by M. brunneum could increase understanding of the role of destruxin A as a virulence factor. Additionally, the total amount of metabolite secreted per insect host could be quantified, which is the first step to monitor whether destruxin A enters the food chain and poses a risk to humans and the environment. Therefore, the aim of the current study was to use G. mellonella as an insect model to monitor the dynamics of fungal growth and secretion of destruxin A by two strains of M. brunneum using real-time PCR and HPLC-MS, respectively. Among model insects, G. mellonella is used extensively for the isolation of entomopathogenic fungi and in studies of their mode of action, among others. 2. Materials and methods 2.1 Insect material Galleria mellonella larvae used in this study were obtained from a stock colony at the Department of Agricultural and Forestry Sciences of the University of Cordoba (Spain). Larvae were maintained in an environmental chamber programmed at 26 ± 2 ºC and 70 ± 5 % RH, with a photoperiod of 16:8 (L:D) h. The larvae were reared on an artificial diet consisting of a mixture of 30.8 g of corn flour, 30.8 g of wheat germ, 30.8 g of wheat bran, and 10.8 g of brewer’s yeast, with 27 mL of glycerol and 48.6 mL of honey per 200 g of artificial diet.
2.2. Fungal isolates and preparation Two M. brunneum strains were evaluated in this study. EAMa 01/58-Su strain (formally M. anisopliae) was from the culture collection at the Department of Agricultural and Forestry Sciences and Resources of the University of Cordoba (Spain) and was originally isolated from the soil of a wheat crop at Hinojosa del Duque (Cordoba, Spain) in 2001. This strain was deposited with accession number CECT 20764 in the Spanish collection of culture types (CECT) located at the University of Valencia (Spain). BIPESCO5 strain (=V275=F52) was from the BIPESCO Team Innsbruck culture collection and was originally isolated in Austria from Cydia pomonella L. (Lepidoptera: Tortricidae) in 1967. To prepare inoculum for the experiments, isolates were subcultured on Sabouraud dextrose CAF500 (Biolife, Milan, Italy)for 15 d at 25 ºC in darkness. The petri plates were sealed with Parafilm® (Pechiney Plastic Packaging Co., Chicago, IL, USA). Conidial suspensions were prepared by scraping conidia from the petri plates into a sterile aqueous solution of 0.1 % Tween 80. These initial suspensions were sonicated for 5 min to homogenize the conidial suspension and then filtered through several layers of cheesecloth to remove mycelial structures. The conidial suspensions used for the bioassays were adjusted by diluting the conidia with 0.1 % Tween 80 to a final concentration of 108 conidia mL-1. The number of conidia was estimated using a Malassez chamber. 2.3. Inoculation of insects
Fungal virulence per strain was tested in fifteen sixth instar larvae of the greater wax moth G. mellonella, which were immersed in a suspension of 108 conidia mL-1 for 20 s and then placed in petri dishes with diet at 25 ºC. Larval mortality was recorded daily for 7 days. The dead larvae were immediately surface sterilized with 1 % sodium hypochlorite, followed by three rinses with sterile distilled water. A 100 µL sample of the final water rinse was plated on selective culture medium SDA CAF 500 to determine the effectiveness of the disinfection. The dead larvae were then placed on sterile wet filter paper in petri plates sealed with Parafilm® and maintained at room temperature (25 ± 2 ºC). After seven days, fungal outgrowth on the surface of the insect cuticle was observedusing a light microscope (Nikon, Japan). Control larvae were immersed for 20 s in 0.1 % Tween 80 in sterile water and evaluated as described above. Each treatment was replicated three times. An additional 300 larvae were inoculated as previously described, and live larvae were sampled daily and frozen immediately at -4 ºC until used for extraction of DNA (three replicates with 1 larvae/replicate) and destruxin A (three replicates with 3 larvae/replicate). Larvae preserved frozen were washed to remove conidia attached to the cuticle as described previously. Larvae were subsequently frozen at -80 ºC and lyophilized for 48 h. Each treatment was replicated three times. Both assays were repeated twice. 2.4. DNA extraction DNA was extracted from one larvae, approximately 200 mg (wet weight), each day for 7 days using the DNeasy® Plant Mini kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. Samples were disrupted in a FastPrep®-24 (M.P. Biomedicals, Santa Ana, California, USA). Pulverized tissue was suspended in 400 µL of Buffer AP1 (preheated to 65 ºC), and 4 µL of RNaseA was
added. Tissues were incubated at 65 ºC for 10 min. The mixture was then incubated in digital dry bath (Labnet International, Edison, USA) for 10 min at 65 ºC and was mixed three times during the incubation by inverting the tube. The lysate was then added to 130 µL of P3 buffer, mixed and incubated for 5 min on ice. The lysate was applied to the QIAshredder minispin column placed in a 2 mL collection tube and centrifuged for 5 min at 14,000 × g in a PrismR (Labnet International, Edison, USA). The flow-through fraction was transferred to a new tube without disturbing the remaining pellet, and then 1.5 volumes of AW1 buffer was added,with mixing by pipetting. A total of 650 µL of the mixture was transferred to the DNeasy mini spin column placed in a 2 mL collection tube and centrifuged for 1 min at 6000 × g. The DNeasy mini spin column was placed in a new 2 mL collection tube, with 500 µL of AW2 buffer added, and centrifuged for 1 min at 6000 × g. The DNeasy mini spin column, with 500 µL of AW2 buffer added, was centrifuged for 3 min at 14,000 × g to dry the membrane. The DNeasy mini spin column was then transferred to a 2 mL microcentrifuge tube. A total of 100 µL of AE buffer (twice) was pipetted directly onto the DNeasy membrane, which was incubated for 5 min at room temperature and then centrifuged for 1 min at 6000 × g to elute. DNA was also extracted from non-inoculated larvae and from 0.026 g of BIPESCO5 and 0.024g of EAMa 01/58-Su conidia, which were the amounts required for aliquots (1 mL) from a suspension of 10 8 conidia mL-1 2.5. Primer design A specific primer pair for targeting the ITS1 region of the rRNA gene of EAMa 01/58-Su and BIPESCO5 strains of M. brunneum, forward primer (5'-GCCGGGGACCCAAACCTTCTGA-3') and reverse primer (5'-ATACTGACGGGCGCAATGTGCG-3'), was designed using the software Primer3 (http://frodo.wi.mit.edu/primer3/). Primers were commercially synthesized by Sigma-Aldrich (St. Louis,
Missouri, USA). To determine whether the primer pair was specific for the amplification of M. brunneum strains used in this study, DNA was extracted as described in section 2.4 and the samples tested in the following conditions. All PCR amplifications were performed in a total reaction volume of 50 µL consisting of 10 µL of 5x My Taq buffer, 1.5 µL of DNA template, 1 µL of each primer, 0.5 µL of my Taq DNA polymerase (Bioline, London UK) and water for molecular biology up to 50 µL (Panreac, Barcelona, Spain). PCR was conducted using a Veriti™ 96-Well Thermal Cycler (Applied Biosystems, Foster City, California, USA), and the conditions were as follow: 95 ºC for 3 min, followed by 40 cycles of 95 ºC for 15 s, 47 ºC for 10 s and 72 ºC for 10 s, with a final extension at 68 ºC for 7 min. PCR products were loaded onto 1.5 % agarose gels stained with SYBR®safe DNA gel (Thermo Fisher Scientific, USA). Electrophoresis was conducted at 80v for 60 min.The gels were photographed in UV-light using a Gel Doc™ EZ Imager (BIO-RAD, Hercules, CA, USA). 2.6. Real-time PCR assays Quantitative real-time PCR (qPCR) amplifications were performed and results analyzed using a Light Cycler® Nano System, software version 1.0 (Roche, Mannheim, Germany) in a total volume of 20 µL. The reaction mixture was composed of 10 µL of Sensi Fast SYBR & Fluorescein mix (2x) (Bioline, London UK), 5.2 µL of sterile water, 0.4 µL (100 µmol/L) of forward and reverse primer and 4 µL of genomic DNA. Each reaction was run in triplicate according to the following: initial desnaturation at 90 ºC for 180 s, followed by 40 cycles each consisting of 95 ºC for 10 s, 68 ºC for 20 s and 72 ºC for 20 s, annealing at 95 ºC for 60 s, final extension at 60 ºC for 60s and as the final step, the dissociation curves at 60 ºC to 95 ºC at 0.1 ºC/s. After amplification, to confirm that only one product was amplified
from the samples that contained the DNA of M. brunneum strains and that no amplification products were in the negative controls, the samples were loaded onto a 1.5 % agarose electrophoresis gel as previously described. 2.7. Quantification assay of M. brunneum in G. mellonella A standard curve was generated from a 10-fold serial dilution of the DNA extracted from the initial conidia inoculum (108 conidia mL-1). In each run of the real-time quantitative PCR assay, at least five of these dilutions with three replicates were included. The amount of genomic DNA in the samples was calculated from threshold cycle (Ct) values using the standard curve and was automatically calculated by the Light Cycler® Nano System, software version 1.0. Fungal kinetics was defined by the time course evolution of the number of fungal sequence copies recovered from inoculated larvae (Bell et al., 2009). 2.8. Destruxin A extraction and quantification The extraction of destruxin A (each day for seven days) was conducted following the sample treatment proposed by Skrobek et al. (2008), with some modifications. Lyophilized larvae (three replicates with 3 larvae/replicate) were crushed in a porcelain mortar and mixed with 5 mL of dichloromethane:ethyl acetate (1:1 v/v) for 2.5 h at 100 rpm, followed by sonication for 30 min and evaporation in a flow chamber. The extract was then re-suspended in 1mL of methanol: acetonitrile (1:1 v/v). The samples were filtered through a 0.2 µm filter and analyzed using an Agilent Technologies 1200-HPLC tandem mass spectrometry Q Trap AB Sciex 5500 (AB SCIEX, Darmstadt, Germany) with electrospray ionization (ESI). A Phenomenex C18 (150 mm
Kinetex x 2.10 mm, 2.7 µm) column was used for the separation, and the data were collected using Analyst®software version 1.6.2 with MS/MS in MRM mode (AB SCIEX) following the analytical method previously described by Carpio et al. (2016) with some modifications. A mobile phase consisting of 0.01 % aqueous formic acid solution (solvent A) and MeOH (solvent B) at a flow of 0.25 mL/min was used. The eluent gradient profile was as follows: 0 min, 5 % B; 15 min, 65 % B; and 15.50 min, 90 % B. The eluent was returned to 5 % B after 0.5 min and maintained for 2 min to allow column equilibration. The column temperature was set at 35 ºC, and the injection volume was 10 µL. The MS/MS was working with ESI in positive mode. Calibration was performed with external standards. The calibration curve was obtained using five concentrations of destruxin A obtained from Sigma Aldrich (St. Louis, MO, USA), ranging between 0.1 and 10 µg/kg. The calibration curve was y = 4.94x + 004 (R2 = 0.9992), the limit of detection (LOD) was 0.7 µg/kg and the limit of quantification (LOQ) was 1.8 µg/kg. A recovery experiment was used to evaluate the extraction efficiency; 0.2 g samples from lyophilized larvae were spiked with 10 µL from a solution of 4 mg/kg of destruxin A and processed as described previously. The results showed very good recovery of 95.3 ± 4.73 %. 2.9. Statistical Analyses Mortality data were analyzed by ANOVA using Statistix 9.0 [Analytical Software, 2008]), and Tukey test was used to compare the means. The values of average survival time (AST) obtained by the Kaplan-Meier method andcompared using the log-rank test were calculated with the SPSS 24statistical software package for Windows (IBM company, 2015). The areas under the curves for M. brunneum
secreted destruxin A were calculated using the trapezoidal integration method of the SAS statistical software package (Campbell and Madden, 1990). Then, the values were analyzed by ANOVA, as previously indicated. 3. Results Virulence, fungal growth kinetics and destruxin A production in G. mellonella larvae were monitored over a 7-day-period after the larvae were treated with the two strains of M. brunneum. 3.1. Fungal virulence Total mortality of sixth instar G. mellonella larvae treated with 1.0 x 108 conidia mL-1 of M. brunneum EAMa 01/58-Su and BIPESCO5 strains reached 85.5 % and 78.8 %, respectively (Table 1). Mortality in the treated insects began approximately 3 days after treatment with BIPESCO5 and 4 days after treatment with EAMa 01/58-Su strains. Four days after treatment, the survival ratio steadily decreased until the conclusion of the study (Fig. 1). No significant differences in mortality rates were detected between the two strains of M. brunneum (P > 0.05). No mortality was observed in the controls. Notably, the percentages of dead insects in which sporulation did not occur on the cadavers were very high, reaching 73.3 % for EAMa 01/58-Su and 74.4 % for BIPESCO5 strains. Additionally, the percentage of cadavers with fungal outgrowth was low, 12.2 % and 4.4 %, respectively (Table 1). Average survival time (AST ± SE) of treated larvae was 5.5 days for both fungal strains (Table 1). But, fungal growth within the insect was observed with microscopy at 72 and 96 h after treatment (Supplementary material).
3.2. Fungal growth kinetics The standard curve, which related Ct values and the logarithm of template copies recovered from the inoculated larvae, is shown in Fig. 2. Fungal DNA was not recovered from either the control larvae or the non-template controls that contained water in place of DNA. A good linear correlation was obtained, with R2 values of 0.998 for EAMa 01/58-Su and 0.999 for BIPESCO5. The slopes of the linear regression curves were -3.37 for EAMa 01/58-Su and -3.39 for BIPESCO5 strains, and the amplification efficiency (E) values were 1.98 and 2.0 for EAMa 01/58-Su and BIPESCO5, respectively. Thus, the precision and accuracy of the qPCR in quantifying the amount of M. brunneum genomic DNA within G. mellonella larvae were confirmed (Fig. 2). Additionally, analyses of melting curves resulted in single dissociation curve peaks with a specific melting temperature of 81.5 °C. Furthermore, the qPCR products loaded in 1.5 % agarose electrophoresis gels resulted in single bands, and the specificity of the selected primers was initially assessed by Basic Local Alignment Search Tool (BLAST) to exclude similar sequences in other microorganisms within the NCBI GenBank databases. Fungal kinetics were represented by the time course evolution curves of the number of fungal sequence copies recovered from G. mellonella colonized by EAMa 01/58-Su and BIPESCO5 strains, which are shown in fig. 1. The two fungal strains had different patterns of growth. For strain EAMa 01/58-Su, the fungal burden increased from day 1 to 4 post exposure and then decreased until the end of the assay. By contrast, for BIPESCO5 strain, the number of fungal sequence copies recovered increased steadily until day 2 post exposure,
then decreased until day 4 and finally increased progressively until the end of evaluation period. The number of fungal sequence copies recovered was not significantly affected by the fungal strain (F = 0.62, P > 0.05). 3.3. Detection and quantification of destruxin A HPLC-MS analyses confirmed production of destruxin A for both strains in infected G. mellonella larvae. The titer of destruxin A differed significantly between strains (P < 0.01). EAMa 01/58-Su strain produced measurable amounts of the peptide from day 2 to 6 postexposure (Fig. 1) and BIPESCO5 from day 2 to 5 (Fig. 1), with both strains producing the maximum concentration on day 4 with 0.369 and 0.06 µg/insect for EAMa 01/58-Su and BIPESCO5, respectively. Destruxin A was not detected in either EAMa 01/58-Su-or BIPESCO5-infected larvae from day 6 after treatment until the end of the study. Notably, the Tukey/ANOVA analysis revealed that EAMa 01/58-Su-infected larvae contained a 6.7-fold higher titer of destruxin A than that of BIPESCO5-infected ones (F = 147.23; P < 0.001). The total mean titer of destruxin A per infected larva was 0.612 ± 0.02 and 0.091 ± 0.01 µg/larva for EAMa 01/58-Su and BIPESCO5 strains, respectively. The kinetics of fungal growth and production of destruxin A followed similar patterns for EAMa 01/58-Su strain; however, with BIPESCO5 strain, not only was the secretion of destruxin A 6.7-fold lower but also the kinetics of secretion and that of fungal growth were uncoupled.
4. Discussion
The greater wax moth G. mellonella has emerged as a powerful model to study fungal virulence and pathogenesis (Vilcinskas, 2010; Kavanagh and Fallon, 2010). In the present investigation, the aim was to provide data for risk assessment and determination of the mode of action of Metarhizium sp. by using, for the first time, tools developed to quantify fungal growth and metabolite production during the infection process of G. mellonella larvae by this entomopathogenic fungus. Based on the methods of qPCR and HPLC-MS used in this study, the two M. brunneum strains secreted destruxin A in infected larvae, with secretion parallel to that of the fungal burden for EAMa 01/58-Su but uncoupled with that for BIPESCO5. Notably, in both strains, mortality from other causes was much higher than mortality with fungal outgrowth, which might bean indication of the toxic strategy proposed by Kershaw et al. (1999). Similarly, BIPESCO5 strain follows this strategy when infecting housefly adults (Anderson et al., 2011), as does EAMa 01/58-Su strain when infecting Bactrocera oleae Gmel. and cotton leaf worm Spodoptera littoralis (Yousef et al., 2013; Resquín-Romero et al., 2016). Whether destruxin A is the major Metarhizium sp. virulence factor (Vilcinskas et al., 1997; Pedras et al., 2002; Meng et al., 2011) or only one among others (Shah et al., 2005; Golo et al., 2015) remains uncertain, with this work revealing intraspecific differences. The fungal burden reached similar and maximum levels for both M. brunneum strains on day four after treatment, which has also been reported for Zoophthora radicans (Brefeld) Batko, Pandora blunckii (Bose and Mehta) and BIPESCO5 (Anderson et al., 2011; Guzman-Franco et al., 2011). However, destruxin A secretion was 6.7-fold higher for EAMa 01/58-Su than that for BIPESCO5. These results suggested that
the virulence of EAMa 01/58-Su was highly related to destruxin A secretion, whereas for BIPESCO5, the virulence could require the involvement of other factors in addition to destruxin A during the infection process. In the present study, the mean destruxin A secretion per infected larva was 0.6 and 0.09 µg/insect for EAMa 01/58-Su and BIPESCO5 strains, respectively. By using HPLC coupled to diode array detection, Skrobek et al. (2008) did not detect destruxin A in V275=BIPESCO5-infected G. mellonella larvae, with a recovery rate of 84 %, LOD of 1.2 ppm and LOQ of 3.4 ppm. The HPLC-MS method used in the present work was more sensitive than HPLC-DAD and measured destruxin A; the recovery rate was higher, 95.3 %, and the LOD, 0.7 µg/kg = ppb, and LOQ, 1.8 µg/kg = ppb, were lower. Thus, the HPLC-MS method successfully detected and monitored destruxin A at low levels in Galleria larvae, which suggest that this approach can be applied to samples from other insects. The LD100 of G. mellonella larvae injected with destruxin A is 11 µg/larvae, with a high recovery ratio when injected with 8 µg/larva (Jegorov et al., 1992). These values are 29.8- and 183.3-fold higher than the total mean titer of destruxin A per infected larva secreted by EAMa 01/58-Su and BIPESCO5, respectively, in this study. Therefore, these results suggest that in vivo secretion of destruxin A by EAMa 01/58-Su and BIPESCO5 strains was not sufficient to kill the host, although this secondary compound could contribute and interfere with both cellular and humoral immune responses, like morphological and cytoskeletal changes in insect plasmatocytes in vitro, and this adversely affects insect cellular immune responses, such as encapsulation and phagocytosis (Vilcinskas et al. 1997; Gao et al., 2011; Wang et al., 2012; Meng et al., 2013; Mudgal et al., 2013). Extremely low doses of destruxins surrounding fungal cells will become an effective weapon to defeat host’s hemocytes (Fan et al., 2013). The ability to secrete secondary metabolites may be maintained by the need to
employ chemicals to withstand, or attack and overcome, the hosts’ immune system (Rohlfs and Churchill, 2011). Also destruxin A influence development of insects by disturbing the metabolism of juvenile hormone, might cause lack of amino acid to accomplish life history and suppression of genes related to the Toll pathway (Han et al., 2013). Similarly, Metarhizium sp. strains likely differ in destruxin production depending on the presence of the destruxin S1 gene cluster; the pathogenicity of generalist Metarhizium sp. strains is related to this gene, whereas the gene is absent in specialist strains (Wang et al., 2012). The LD50 in small mammalian systems for destruxin A is 1.0-1.35 mg/kg (Kodaira, 1961; Mudgal et al., 2013). Therefore, the risk of poisoning is very low for destruxin A because approximately 2800 or 17000 larvae of G. mellonella infected with EAMa 01/58-Su or BIPESCO5, respectively, would have to be ingested by a mouse. Indeed, the total mean titer of destruxin A per infected larva secreted by EAMa 01/58-Su and BIPESCO5 would have no cytotoxic effect, considering destruxins A, B, and E had no effect on the viability of human leukemic cells at 500 mg/kg (Skrobek and Butt, 2005). Destruxins also influence the survival of aquatic invertebrates, including insects such as mosquitoes and crustaceans such as Daphnia and Artemia, by triggering caspase-mediated apoptosis (Garrido-Jurado et al., 2016b). Favilla et al. (2006) assessed the acute toxicity of destruxinA in crustaceans and showed a lethal concentration (LC50) of 0.20 and 2.92 µg/mL in Daphnia magna and Artemia salina at 24 h, respectively. These values have a much wider range (1000-10000-fold) compared with the amounts quantified in Galleria larvae in our study. In conclusion, very low concentrations of destruxin A were secreted by the two M. brunneum strains during a short period (2-6 days after treatment), indicating little potential hazard to human and animal health and the environment. The present study
provides valuable data to better understand the role of destruxin A as a virulence factor and to monitor the fate of destruxin A in food chains.
Acknowledgments Ríos-Moreno gratefully acknowledges “SENACYT and IFARHU from Panama” for a doctoral grant. We thank the Central Service of Research Support from the University of Cordoba (SCAI) for analyses of destruxin A.
Conflict of interest The authors declare that there are no conflicts of interest.
Funding information A grant from the European Community’s Seventh Framework Program (FP7-ENV.2011.3.1.9-1 ECO-INNOVATION, INBIOSOIL,Grant Agreement No. 282767) and a grant from the Ministerio de Economía y Competitividad, Spain, Project AGL 2016-80483-R, supported this research.
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Table 1 Pathogenicity of M. brunneum strains to sixth instar G. mellonella larvae after treatment with a suspension of 1.0 x 108 conidia mL-1. Fifteen larvae were used per replicate (three replicates), and the entire experiment was repeated twice. Combined data per strain, n=90. Mortality (mean ± SE)%* Strain
Kaplan-Meier survival analysis AST** 95% CI (d, mean ± SE)
Total Mortality
Fungal outgrowth
Other causes
EAMa 01/58-Su
85.5±5.8a
12.2±5.1a
73.3±3.3a
5.45±0.15a
5.16-5.79
BIPESCO5
78.8±1.1a
4.4±1.9a
74.4±3.8a
5.47±0.16a
5.15-5.75
*
Control mortality was zero and was not included in the analysis. ** AST: Average survival time, limited to 7 days. *** Means within columns with the same letter are not significantly different (P ≤ 0.05) according toTukey HSD test.
Figure captions Fig. 1. Cumulative survival ratio (mean ± SE, n=90), marker gene expression recovered over the course of the infection (n= 42), and destruxin A titer (n= 126) in sixth instar Galleria mellonella larvae inoculated with a suspension of 108 conidia mL-1 of M. brunneum. A. EAMa 01/58-Su strain; B. BIPESCO5 strain. Data are presented as the mean ± SEM. Destruxin A was not detected in either EAMa 01/58Su-or BIPESCO5-infected larvae from day 6 after treatment until the end of the study.
Fig. 2. Standard curves generated using qPCR analyses of five 10-fold serial dilutions (108-104 conidia mL-1) of M. brunneum by plotting Ct against log10. The average efficiency value (E) and correlation factor (R2) are indicated in the graph. A. EAMa 01/58-Su strain; B. BIPESCO5 strain.
Supplementary figure Figure. S1. Haemolymph samples from Galleria mellonella infected with Metarhizium brunneum strain EAMa 01/58-Su: A) and B) 72h after treatment, C) and D) 96 h after treatment. Haemocytes (he), fungal structures, including hyphae and hyphal bodies (fs)
Figure. S1
Graphical abstract
Research Highlights •
Metarhizium brunneum destruxin A and fungal growth during Galleria mellonella infection were quantified by HPLC-MS and qPCR, respectively.
•
EAMa 01/58-Su and BIPESCO5 strains secreted destruxin A in infected larvae, with secretion parallel to that of the fungal burden for EAMa 01/58-Su but uncoupled with that for BIPESCO5.
•
Very low concentrations of destruxin A were secreted, indicating little potential hazard to human and animal health and the environment.
S0022-2011(16)30247-6 http://dx.doi.org/10.1016/j.jip.2017.06.007 YJIPA 6961
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
12 December 2016 31 May 2017 15 June 2017
Please cite this article as: Ríos-Moreno, A., Garrido-Jurado, I., Raya-Ortega, M.C., E.Quesada-Moraga, Quantification of fungal growth and destruxin A during infection of Galleria mellonella larvae by Metarhizium brunneum, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.06.007
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Title: Quantification of fungal growth and destruxin A during infection of Galleria mellonella larvae by Metarhizium brunneum
Authors: A. Ríos-Morenoa, I. Garrido-Juradoa,M. C. Raya-Ortegab and E.Quesada-Moragaa*
Address: aDepartment
of Agricultural and Forestry Sciences, ETSIAM, University of Cordoba, C4Building, Campus of Rabanales, 14071 Cordoba, Spain.
bDepartment
of Agronomy, ETSIAM, University of Cordoba, C4Building, Campus of Rabanales, 14071 Cordoba, Spain.
*Corresponding author: tel.: + 34-957218475; fax: +34-957218440. E-mail address:[email protected] (E. Quesada-Moraga)
Abstract Destruxin A is among the major secondary metabolites produced by the entomopathogenic ascomycete Metarhizium sp., and the lack of studies concerning production of destruxin A by the fungus is most likely the biggest obstacle for the registration of new fungal strains. Although several studies focus on the production of destruxin A in culture media, few studies examine destruxin A in vivo during host infection. In the current work, Galleria mellonella was used as an insect model to develop for the first time in vivo real-time PCR- and HPLC-MS-based quantification of fungal growth and metabolite production, respectively, during infection by two strains of M. brunneum. Total mortality of sixth instar G. mellonella larvae that were immersed in a suspension of 1.0 x 108 conidia mL-1 of M. brunneum EAMa 01/58-Su or BIPESCO5 strains reached 85.5 % and 78.8 %, respectively, and the percentage of cadavers with fungal outgrowth was low at 12.2 % and 4.4 %, respectively. The average survival time of treated larvae was 5.5 days for both fungal strains. Using EAMa 01/58-Su and BIPESCO5 specific primer set, real-time PCR showed that the patterns of fungal growth were different for the two strains, whereas no significant differences were detected in the number of fungal sequence copies recovered from the infected larvae. EAMa 01/58-Su and BIPESCO5 strains secreted destruxin A from days 2 to 6 andfrom days 2 to 5 post treatment, respectively. For
EAMa 01/58-Su and BIPESCO5, the maximum titer of destruxin A in the host was on day 4 at 0.369 and 0.06 µ g/larva, respectively, and throughout the pathogenic process, the total production was 0.6 and 0.09 µg/larva, respectively. These results demonstrated that the strains pose a low hazard, if any, to humans and the environment. The methods used in this study to quantify fungal growth and metabolite production provided valuable data to better understand the role of destruxin A during the growth of M. brunneum in the host larvae and to monitor the fate of destruxin A in food chains.
Keywords: Real-time PCR, HPLC-MS, BIPESCO5, EAMa 01/58-Su, mode of action, risk assessment 1. Introduction The effectiveness of Metarhizium sp. in controlling many insect pests in agriculture makes the fungus an attractive alternative to conventional pesticides, with many commercial products on the market or in development (Zimmermann, 2007; Lacey et al., 2015). This fungus is considered safe for humans and the environment, but the production and the evaluation of safety data for registration of new entomopathogenic fungal strains are expensive and time-consuming (Zimmermann, 2007; Strauch et al., 2011). Indeed, the requirement by regulatory authorities for risk assessment studies with detailed information concerning the production of secondary metabolites is the biggest obstacle for registration (Skrobek et al., 2006; Strauch et al., 2011). Concerning secondary metabolites, Metarhizium sp. produces a family of cyclic peptide toxins, destruxins, with the focus on destruxin A as the most likely major secondary metabolite produced by this genus (Zimmermann, 2007). Indeed, several studies focus on
the production of destruxin A in culture media (Skrobek and Butt, 2005; Ríos-Moreno et al., 2016a). More recently, the necessity to better understand the association between entomopathogenic fungi and plants has also been emphasized, which includes determination of whether the fungi produce metabolites in plants (Vega et al., 2009). Hence, destruxins A, B and E were detected in cowpea plants inoculated with M. robertsii (Golo et al., 2014), destruxin A was quantified in melon and tomato plants sprayed with M. brunneum (Garrido-Jurado et al., 2016a; Resquín-Romero et al., 2016), and using an extraction procedure based on QuEChERS, destruxins were quantified in strawberry and corn (Taibon et al., 2015). A reliable optimization method based on a modified version of QuEChERS and HPLC-MS to detect and monitor destruxins in potato plants has also been proposed (Carpio et al., 2016; Ríos-Moreno et al., 2016b). Although studies have examined the role of destruxins in the pathogenicity and virulence of Metarhizium sp. (Pal et al., 2007; Hu et al., 2009; Donzelli et al., 2012), studies are lacking on the dynamics of fungal growth and secretion of destruxin A during the infection of the insect host by the fungal biocontrol agent.Only one study has reported on in vivo detection and quantification of destruxin A (Skrobek et al., 2008), andthe study used HPLC-DAD. However, the detection and quantification limits and recovery levels can be improved using HPLC-MS, which is generally a more sensitive detection method for such metabolites (Bogusz and Carracedo, 2004). Recent studies show that quantitative real-time polymerase chain reaction (qPCR) can be used to determine the amount of DNA from fungi in a sample (Arquiza-Apollo and Hunter, 2014), demonstrating that this technique is a powerful, specific, accurate and sensitive tool for detection and quantification of fungi (Malvick and Impullitti, 2007; Tae and Knudsen, 2016). Real-time PCR has been used to
monitor and quantify M. anisopliae and Beauveria bassiana during Anopheles infection in the presence or absence of malaria parasites (Bell et al., 2009) and to examine the relationship between fungal growth kinetics and virulence in the housefly (Anderson et al., 2011). Studying in vivo secretion of destruxin A during infection of the insect host by M. brunneum could increase understanding of the role of destruxin A as a virulence factor. Additionally, the total amount of metabolite secreted per insect host could be quantified, which is the first step to monitor whether destruxin A enters the food chain and poses a risk to humans and the environment. Therefore, the aim of the current study was to use G. mellonella as an insect model to monitor the dynamics of fungal growth and secretion of destruxin A by two strains of M. brunneum using real-time PCR and HPLC-MS, respectively. Among model insects, G. mellonella is used extensively for the isolation of entomopathogenic fungi and in studies of their mode of action, among others. 2. Materials and methods 2.1 Insect material Galleria mellonella larvae used in this study were obtained from a stock colony at the Department of Agricultural and Forestry Sciences of the University of Cordoba (Spain). Larvae were maintained in an environmental chamber programmed at 26 ± 2 ºC and 70 ± 5 % RH, with a photoperiod of 16:8 (L:D) h. The larvae were reared on an artificial diet consisting of a mixture of 30.8 g of corn flour, 30.8 g of wheat germ, 30.8 g of wheat bran, and 10.8 g of brewer’s yeast, with 27 mL of glycerol and 48.6 mL of honey per 200 g of artificial diet.
2.2. Fungal isolates and preparation Two M. brunneum strains were evaluated in this study. EAMa 01/58-Su strain (formally M. anisopliae) was from the culture collection at the Department of Agricultural and Forestry Sciences and Resources of the University of Cordoba (Spain) and was originally isolated from the soil of a wheat crop at Hinojosa del Duque (Cordoba, Spain) in 2001. This strain was deposited with accession number CECT 20764 in the Spanish collection of culture types (CECT) located at the University of Valencia (Spain). BIPESCO5 strain (=V275=F52) was from the BIPESCO Team Innsbruck culture collection and was originally isolated in Austria from Cydia pomonella L. (Lepidoptera: Tortricidae) in 1967. To prepare inoculum for the experiments, isolates were subcultured on Sabouraud dextrose CAF500 (Biolife, Milan, Italy)for 15 d at 25 ºC in darkness. The petri plates were sealed with Parafilm® (Pechiney Plastic Packaging Co., Chicago, IL, USA). Conidial suspensions were prepared by scraping conidia from the petri plates into a sterile aqueous solution of 0.1 % Tween 80. These initial suspensions were sonicated for 5 min to homogenize the conidial suspension and then filtered through several layers of cheesecloth to remove mycelial structures. The conidial suspensions used for the bioassays were adjusted by diluting the conidia with 0.1 % Tween 80 to a final concentration of 108 conidia mL-1. The number of conidia was estimated using a Malassez chamber. 2.3. Inoculation of insects
Fungal virulence per strain was tested in fifteen sixth instar larvae of the greater wax moth G. mellonella, which were immersed in a suspension of 108 conidia mL-1 for 20 s and then placed in petri dishes with diet at 25 ºC. Larval mortality was recorded daily for 7 days. The dead larvae were immediately surface sterilized with 1 % sodium hypochlorite, followed by three rinses with sterile distilled water. A 100 µL sample of the final water rinse was plated on selective culture medium SDA CAF 500 to determine the effectiveness of the disinfection. The dead larvae were then placed on sterile wet filter paper in petri plates sealed with Parafilm® and maintained at room temperature (25 ± 2 ºC). After seven days, fungal outgrowth on the surface of the insect cuticle was observedusing a light microscope (Nikon, Japan). Control larvae were immersed for 20 s in 0.1 % Tween 80 in sterile water and evaluated as described above. Each treatment was replicated three times. An additional 300 larvae were inoculated as previously described, and live larvae were sampled daily and frozen immediately at -4 ºC until used for extraction of DNA (three replicates with 1 larvae/replicate) and destruxin A (three replicates with 3 larvae/replicate). Larvae preserved frozen were washed to remove conidia attached to the cuticle as described previously. Larvae were subsequently frozen at -80 ºC and lyophilized for 48 h. Each treatment was replicated three times. Both assays were repeated twice. 2.4. DNA extraction DNA was extracted from one larvae, approximately 200 mg (wet weight), each day for 7 days using the DNeasy® Plant Mini kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. Samples were disrupted in a FastPrep®-24 (M.P. Biomedicals, Santa Ana, California, USA). Pulverized tissue was suspended in 400 µL of Buffer AP1 (preheated to 65 ºC), and 4 µL of RNaseA was
added. Tissues were incubated at 65 ºC for 10 min. The mixture was then incubated in digital dry bath (Labnet International, Edison, USA) for 10 min at 65 ºC and was mixed three times during the incubation by inverting the tube. The lysate was then added to 130 µL of P3 buffer, mixed and incubated for 5 min on ice. The lysate was applied to the QIAshredder minispin column placed in a 2 mL collection tube and centrifuged for 5 min at 14,000 × g in a PrismR (Labnet International, Edison, USA). The flow-through fraction was transferred to a new tube without disturbing the remaining pellet, and then 1.5 volumes of AW1 buffer was added,with mixing by pipetting. A total of 650 µL of the mixture was transferred to the DNeasy mini spin column placed in a 2 mL collection tube and centrifuged for 1 min at 6000 × g. The DNeasy mini spin column was placed in a new 2 mL collection tube, with 500 µL of AW2 buffer added, and centrifuged for 1 min at 6000 × g. The DNeasy mini spin column, with 500 µL of AW2 buffer added, was centrifuged for 3 min at 14,000 × g to dry the membrane. The DNeasy mini spin column was then transferred to a 2 mL microcentrifuge tube. A total of 100 µL of AE buffer (twice) was pipetted directly onto the DNeasy membrane, which was incubated for 5 min at room temperature and then centrifuged for 1 min at 6000 × g to elute. DNA was also extracted from non-inoculated larvae and from 0.026 g of BIPESCO5 and 0.024g of EAMa 01/58-Su conidia, which were the amounts required for aliquots (1 mL) from a suspension of 10 8 conidia mL-1 2.5. Primer design A specific primer pair for targeting the ITS1 region of the rRNA gene of EAMa 01/58-Su and BIPESCO5 strains of M. brunneum, forward primer (5'-GCCGGGGACCCAAACCTTCTGA-3') and reverse primer (5'-ATACTGACGGGCGCAATGTGCG-3'), was designed using the software Primer3 (http://frodo.wi.mit.edu/primer3/). Primers were commercially synthesized by Sigma-Aldrich (St. Louis,
Missouri, USA). To determine whether the primer pair was specific for the amplification of M. brunneum strains used in this study, DNA was extracted as described in section 2.4 and the samples tested in the following conditions. All PCR amplifications were performed in a total reaction volume of 50 µL consisting of 10 µL of 5x My Taq buffer, 1.5 µL of DNA template, 1 µL of each primer, 0.5 µL of my Taq DNA polymerase (Bioline, London UK) and water for molecular biology up to 50 µL (Panreac, Barcelona, Spain). PCR was conducted using a Veriti™ 96-Well Thermal Cycler (Applied Biosystems, Foster City, California, USA), and the conditions were as follow: 95 ºC for 3 min, followed by 40 cycles of 95 ºC for 15 s, 47 ºC for 10 s and 72 ºC for 10 s, with a final extension at 68 ºC for 7 min. PCR products were loaded onto 1.5 % agarose gels stained with SYBR®safe DNA gel (Thermo Fisher Scientific, USA). Electrophoresis was conducted at 80v for 60 min.The gels were photographed in UV-light using a Gel Doc™ EZ Imager (BIO-RAD, Hercules, CA, USA). 2.6. Real-time PCR assays Quantitative real-time PCR (qPCR) amplifications were performed and results analyzed using a Light Cycler® Nano System, software version 1.0 (Roche, Mannheim, Germany) in a total volume of 20 µL. The reaction mixture was composed of 10 µL of Sensi Fast SYBR & Fluorescein mix (2x) (Bioline, London UK), 5.2 µL of sterile water, 0.4 µL (100 µmol/L) of forward and reverse primer and 4 µL of genomic DNA. Each reaction was run in triplicate according to the following: initial desnaturation at 90 ºC for 180 s, followed by 40 cycles each consisting of 95 ºC for 10 s, 68 ºC for 20 s and 72 ºC for 20 s, annealing at 95 ºC for 60 s, final extension at 60 ºC for 60s and as the final step, the dissociation curves at 60 ºC to 95 ºC at 0.1 ºC/s. After amplification, to confirm that only one product was amplified
from the samples that contained the DNA of M. brunneum strains and that no amplification products were in the negative controls, the samples were loaded onto a 1.5 % agarose electrophoresis gel as previously described. 2.7. Quantification assay of M. brunneum in G. mellonella A standard curve was generated from a 10-fold serial dilution of the DNA extracted from the initial conidia inoculum (108 conidia mL-1). In each run of the real-time quantitative PCR assay, at least five of these dilutions with three replicates were included. The amount of genomic DNA in the samples was calculated from threshold cycle (Ct) values using the standard curve and was automatically calculated by the Light Cycler® Nano System, software version 1.0. Fungal kinetics was defined by the time course evolution of the number of fungal sequence copies recovered from inoculated larvae (Bell et al., 2009). 2.8. Destruxin A extraction and quantification The extraction of destruxin A (each day for seven days) was conducted following the sample treatment proposed by Skrobek et al. (2008), with some modifications. Lyophilized larvae (three replicates with 3 larvae/replicate) were crushed in a porcelain mortar and mixed with 5 mL of dichloromethane:ethyl acetate (1:1 v/v) for 2.5 h at 100 rpm, followed by sonication for 30 min and evaporation in a flow chamber. The extract was then re-suspended in 1mL of methanol: acetonitrile (1:1 v/v). The samples were filtered through a 0.2 µm filter and analyzed using an Agilent Technologies 1200-HPLC tandem mass spectrometry Q Trap AB Sciex 5500 (AB SCIEX, Darmstadt, Germany) with electrospray ionization (ESI). A Phenomenex C18 (150 mm
Kinetex x 2.10 mm, 2.7 µm) column was used for the separation, and the data were collected using Analyst®software version 1.6.2 with MS/MS in MRM mode (AB SCIEX) following the analytical method previously described by Carpio et al. (2016) with some modifications. A mobile phase consisting of 0.01 % aqueous formic acid solution (solvent A) and MeOH (solvent B) at a flow of 0.25 mL/min was used. The eluent gradient profile was as follows: 0 min, 5 % B; 15 min, 65 % B; and 15.50 min, 90 % B. The eluent was returned to 5 % B after 0.5 min and maintained for 2 min to allow column equilibration. The column temperature was set at 35 ºC, and the injection volume was 10 µL. The MS/MS was working with ESI in positive mode. Calibration was performed with external standards. The calibration curve was obtained using five concentrations of destruxin A obtained from Sigma Aldrich (St. Louis, MO, USA), ranging between 0.1 and 10 µg/kg. The calibration curve was y = 4.94x + 004 (R2 = 0.9992), the limit of detection (LOD) was 0.7 µg/kg and the limit of quantification (LOQ) was 1.8 µg/kg. A recovery experiment was used to evaluate the extraction efficiency; 0.2 g samples from lyophilized larvae were spiked with 10 µL from a solution of 4 mg/kg of destruxin A and processed as described previously. The results showed very good recovery of 95.3 ± 4.73 %. 2.9. Statistical Analyses Mortality data were analyzed by ANOVA using Statistix 9.0 [Analytical Software, 2008]), and Tukey test was used to compare the means. The values of average survival time (AST) obtained by the Kaplan-Meier method andcompared using the log-rank test were calculated with the SPSS 24statistical software package for Windows (IBM company, 2015). The areas under the curves for M. brunneum
secreted destruxin A were calculated using the trapezoidal integration method of the SAS statistical software package (Campbell and Madden, 1990). Then, the values were analyzed by ANOVA, as previously indicated. 3. Results Virulence, fungal growth kinetics and destruxin A production in G. mellonella larvae were monitored over a 7-day-period after the larvae were treated with the two strains of M. brunneum. 3.1. Fungal virulence Total mortality of sixth instar G. mellonella larvae treated with 1.0 x 108 conidia mL-1 of M. brunneum EAMa 01/58-Su and BIPESCO5 strains reached 85.5 % and 78.8 %, respectively (Table 1). Mortality in the treated insects began approximately 3 days after treatment with BIPESCO5 and 4 days after treatment with EAMa 01/58-Su strains. Four days after treatment, the survival ratio steadily decreased until the conclusion of the study (Fig. 1). No significant differences in mortality rates were detected between the two strains of M. brunneum (P > 0.05). No mortality was observed in the controls. Notably, the percentages of dead insects in which sporulation did not occur on the cadavers were very high, reaching 73.3 % for EAMa 01/58-Su and 74.4 % for BIPESCO5 strains. Additionally, the percentage of cadavers with fungal outgrowth was low, 12.2 % and 4.4 %, respectively (Table 1). Average survival time (AST ± SE) of treated larvae was 5.5 days for both fungal strains (Table 1). But, fungal growth within the insect was observed with microscopy at 72 and 96 h after treatment (Supplementary material).
3.2. Fungal growth kinetics The standard curve, which related Ct values and the logarithm of template copies recovered from the inoculated larvae, is shown in Fig. 2. Fungal DNA was not recovered from either the control larvae or the non-template controls that contained water in place of DNA. A good linear correlation was obtained, with R2 values of 0.998 for EAMa 01/58-Su and 0.999 for BIPESCO5. The slopes of the linear regression curves were -3.37 for EAMa 01/58-Su and -3.39 for BIPESCO5 strains, and the amplification efficiency (E) values were 1.98 and 2.0 for EAMa 01/58-Su and BIPESCO5, respectively. Thus, the precision and accuracy of the qPCR in quantifying the amount of M. brunneum genomic DNA within G. mellonella larvae were confirmed (Fig. 2). Additionally, analyses of melting curves resulted in single dissociation curve peaks with a specific melting temperature of 81.5 °C. Furthermore, the qPCR products loaded in 1.5 % agarose electrophoresis gels resulted in single bands, and the specificity of the selected primers was initially assessed by Basic Local Alignment Search Tool (BLAST) to exclude similar sequences in other microorganisms within the NCBI GenBank databases. Fungal kinetics were represented by the time course evolution curves of the number of fungal sequence copies recovered from G. mellonella colonized by EAMa 01/58-Su and BIPESCO5 strains, which are shown in fig. 1. The two fungal strains had different patterns of growth. For strain EAMa 01/58-Su, the fungal burden increased from day 1 to 4 post exposure and then decreased until the end of the assay. By contrast, for BIPESCO5 strain, the number of fungal sequence copies recovered increased steadily until day 2 post exposure,
then decreased until day 4 and finally increased progressively until the end of evaluation period. The number of fungal sequence copies recovered was not significantly affected by the fungal strain (F = 0.62, P > 0.05). 3.3. Detection and quantification of destruxin A HPLC-MS analyses confirmed production of destruxin A for both strains in infected G. mellonella larvae. The titer of destruxin A differed significantly between strains (P < 0.01). EAMa 01/58-Su strain produced measurable amounts of the peptide from day 2 to 6 postexposure (Fig. 1) and BIPESCO5 from day 2 to 5 (Fig. 1), with both strains producing the maximum concentration on day 4 with 0.369 and 0.06 µg/insect for EAMa 01/58-Su and BIPESCO5, respectively. Destruxin A was not detected in either EAMa 01/58-Su-or BIPESCO5-infected larvae from day 6 after treatment until the end of the study. Notably, the Tukey/ANOVA analysis revealed that EAMa 01/58-Su-infected larvae contained a 6.7-fold higher titer of destruxin A than that of BIPESCO5-infected ones (F = 147.23; P < 0.001). The total mean titer of destruxin A per infected larva was 0.612 ± 0.02 and 0.091 ± 0.01 µg/larva for EAMa 01/58-Su and BIPESCO5 strains, respectively. The kinetics of fungal growth and production of destruxin A followed similar patterns for EAMa 01/58-Su strain; however, with BIPESCO5 strain, not only was the secretion of destruxin A 6.7-fold lower but also the kinetics of secretion and that of fungal growth were uncoupled.
4. Discussion
The greater wax moth G. mellonella has emerged as a powerful model to study fungal virulence and pathogenesis (Vilcinskas, 2010; Kavanagh and Fallon, 2010). In the present investigation, the aim was to provide data for risk assessment and determination of the mode of action of Metarhizium sp. by using, for the first time, tools developed to quantify fungal growth and metabolite production during the infection process of G. mellonella larvae by this entomopathogenic fungus. Based on the methods of qPCR and HPLC-MS used in this study, the two M. brunneum strains secreted destruxin A in infected larvae, with secretion parallel to that of the fungal burden for EAMa 01/58-Su but uncoupled with that for BIPESCO5. Notably, in both strains, mortality from other causes was much higher than mortality with fungal outgrowth, which might bean indication of the toxic strategy proposed by Kershaw et al. (1999). Similarly, BIPESCO5 strain follows this strategy when infecting housefly adults (Anderson et al., 2011), as does EAMa 01/58-Su strain when infecting Bactrocera oleae Gmel. and cotton leaf worm Spodoptera littoralis (Yousef et al., 2013; Resquín-Romero et al., 2016). Whether destruxin A is the major Metarhizium sp. virulence factor (Vilcinskas et al., 1997; Pedras et al., 2002; Meng et al., 2011) or only one among others (Shah et al., 2005; Golo et al., 2015) remains uncertain, with this work revealing intraspecific differences. The fungal burden reached similar and maximum levels for both M. brunneum strains on day four after treatment, which has also been reported for Zoophthora radicans (Brefeld) Batko, Pandora blunckii (Bose and Mehta) and BIPESCO5 (Anderson et al., 2011; Guzman-Franco et al., 2011). However, destruxin A secretion was 6.7-fold higher for EAMa 01/58-Su than that for BIPESCO5. These results suggested that
the virulence of EAMa 01/58-Su was highly related to destruxin A secretion, whereas for BIPESCO5, the virulence could require the involvement of other factors in addition to destruxin A during the infection process. In the present study, the mean destruxin A secretion per infected larva was 0.6 and 0.09 µg/insect for EAMa 01/58-Su and BIPESCO5 strains, respectively. By using HPLC coupled to diode array detection, Skrobek et al. (2008) did not detect destruxin A in V275=BIPESCO5-infected G. mellonella larvae, with a recovery rate of 84 %, LOD of 1.2 ppm and LOQ of 3.4 ppm. The HPLC-MS method used in the present work was more sensitive than HPLC-DAD and measured destruxin A; the recovery rate was higher, 95.3 %, and the LOD, 0.7 µg/kg = ppb, and LOQ, 1.8 µg/kg = ppb, were lower. Thus, the HPLC-MS method successfully detected and monitored destruxin A at low levels in Galleria larvae, which suggest that this approach can be applied to samples from other insects. The LD100 of G. mellonella larvae injected with destruxin A is 11 µg/larvae, with a high recovery ratio when injected with 8 µg/larva (Jegorov et al., 1992). These values are 29.8- and 183.3-fold higher than the total mean titer of destruxin A per infected larva secreted by EAMa 01/58-Su and BIPESCO5, respectively, in this study. Therefore, these results suggest that in vivo secretion of destruxin A by EAMa 01/58-Su and BIPESCO5 strains was not sufficient to kill the host, although this secondary compound could contribute and interfere with both cellular and humoral immune responses, like morphological and cytoskeletal changes in insect plasmatocytes in vitro, and this adversely affects insect cellular immune responses, such as encapsulation and phagocytosis (Vilcinskas et al. 1997; Gao et al., 2011; Wang et al., 2012; Meng et al., 2013; Mudgal et al., 2013). Extremely low doses of destruxins surrounding fungal cells will become an effective weapon to defeat host’s hemocytes (Fan et al., 2013). The ability to secrete secondary metabolites may be maintained by the need to
employ chemicals to withstand, or attack and overcome, the hosts’ immune system (Rohlfs and Churchill, 2011). Also destruxin A influence development of insects by disturbing the metabolism of juvenile hormone, might cause lack of amino acid to accomplish life history and suppression of genes related to the Toll pathway (Han et al., 2013). Similarly, Metarhizium sp. strains likely differ in destruxin production depending on the presence of the destruxin S1 gene cluster; the pathogenicity of generalist Metarhizium sp. strains is related to this gene, whereas the gene is absent in specialist strains (Wang et al., 2012). The LD50 in small mammalian systems for destruxin A is 1.0-1.35 mg/kg (Kodaira, 1961; Mudgal et al., 2013). Therefore, the risk of poisoning is very low for destruxin A because approximately 2800 or 17000 larvae of G. mellonella infected with EAMa 01/58-Su or BIPESCO5, respectively, would have to be ingested by a mouse. Indeed, the total mean titer of destruxin A per infected larva secreted by EAMa 01/58-Su and BIPESCO5 would have no cytotoxic effect, considering destruxins A, B, and E had no effect on the viability of human leukemic cells at 500 mg/kg (Skrobek and Butt, 2005). Destruxins also influence the survival of aquatic invertebrates, including insects such as mosquitoes and crustaceans such as Daphnia and Artemia, by triggering caspase-mediated apoptosis (Garrido-Jurado et al., 2016b). Favilla et al. (2006) assessed the acute toxicity of destruxinA in crustaceans and showed a lethal concentration (LC50) of 0.20 and 2.92 µg/mL in Daphnia magna and Artemia salina at 24 h, respectively. These values have a much wider range (1000-10000-fold) compared with the amounts quantified in Galleria larvae in our study. In conclusion, very low concentrations of destruxin A were secreted by the two M. brunneum strains during a short period (2-6 days after treatment), indicating little potential hazard to human and animal health and the environment. The present study
provides valuable data to better understand the role of destruxin A as a virulence factor and to monitor the fate of destruxin A in food chains.
Acknowledgments Ríos-Moreno gratefully acknowledges “SENACYT and IFARHU from Panama” for a doctoral grant. We thank the Central Service of Research Support from the University of Cordoba (SCAI) for analyses of destruxin A.
Conflict of interest The authors declare that there are no conflicts of interest.
Funding information A grant from the European Community’s Seventh Framework Program (FP7-ENV.2011.3.1.9-1 ECO-INNOVATION, INBIOSOIL,Grant Agreement No. 282767) and a grant from the Ministerio de Economía y Competitividad, Spain, Project AGL 2016-80483-R, supported this research.
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Table 1 Pathogenicity of M. brunneum strains to sixth instar G. mellonella larvae after treatment with a suspension of 1.0 x 108 conidia mL-1. Fifteen larvae were used per replicate (three replicates), and the entire experiment was repeated twice. Combined data per strain, n=90. Mortality (mean ± SE)%* Strain
Kaplan-Meier survival analysis AST** 95% CI (d, mean ± SE)
Total Mortality
Fungal outgrowth
Other causes
EAMa 01/58-Su
85.5±5.8a
12.2±5.1a
73.3±3.3a
5.45±0.15a
5.16-5.79
BIPESCO5
78.8±1.1a
4.4±1.9a
74.4±3.8a
5.47±0.16a
5.15-5.75
*
Control mortality was zero and was not included in the analysis. ** AST: Average survival time, limited to 7 days. *** Means within columns with the same letter are not significantly different (P ≤ 0.05) according toTukey HSD test.
Figure captions Fig. 1. Cumulative survival ratio (mean ± SE, n=90), marker gene expression recovered over the course of the infection (n= 42), and destruxin A titer (n= 126) in sixth instar Galleria mellonella larvae inoculated with a suspension of 108 conidia mL-1 of M. brunneum. A. EAMa 01/58-Su strain; B. BIPESCO5 strain. Data are presented as the mean ± SEM. Destruxin A was not detected in either EAMa 01/58Su-or BIPESCO5-infected larvae from day 6 after treatment until the end of the study.
Fig. 2. Standard curves generated using qPCR analyses of five 10-fold serial dilutions (108-104 conidia mL-1) of M. brunneum by plotting Ct against log10. The average efficiency value (E) and correlation factor (R2) are indicated in the graph. A. EAMa 01/58-Su strain; B. BIPESCO5 strain.
Supplementary figure Figure. S1. Haemolymph samples from Galleria mellonella infected with Metarhizium brunneum strain EAMa 01/58-Su: A) and B) 72h after treatment, C) and D) 96 h after treatment. Haemocytes (he), fungal structures, including hyphae and hyphal bodies (fs)
Figure. S1
Graphical abstract
Research Highlights •
Metarhizium brunneum destruxin A and fungal growth during Galleria mellonella infection were quantified by HPLC-MS and qPCR, respectively.
•
EAMa 01/58-Su and BIPESCO5 strains secreted destruxin A in infected larvae, with secretion parallel to that of the fungal burden for EAMa 01/58-Su but uncoupled with that for BIPESCO5.
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Very low concentrations of destruxin A were secreted, indicating little potential hazard to human and animal health and the environment.