BIOLOGICAL AND MICROBIAL CONTROL

Insecticidal Activity of a Destruxin-Containing Extract of

Metarhizium brunneum Against Ceratitis capitata
(Diptera: Tephritidae)
M. D. LOZANO-TOVAR,1 I. GARRIDO-JURADO,1 F. LAFONT,2 AND E. QUESADA-MORAGA1,3

J. Econ. Entomol. 108(2): 462–472 (2015); DOI: 10.1093/jee/tov041

ABSTRACT Tephritid fruit flies are major pests that limit fruit production around the world; they cause
important damages, increasing directly and indirectly annual costs, and their management is predomi-
nately based on the use of chemical insecticides. This research investigated the insecticidal activity of the
crude extract obtained of Metarhizium brunneum Petch EAMb 09/01-Su strain and its capacity to secrete
secondary metabolites including destruxins (dtx). Dtx A and A2 had insecticidal activity against Ceratitis
capitata (Wiedemann) when administered per os. The crude extract of seven Metarhizium and one Beau-
veria isolates were evaluated per os against medfly adults. The crude extracts of the isolate EAMb 09/01-
Su resulted in mortality ranging between 95 and 100% at 48 h. The high-pressure liquid chromatography
profile showed two active peaks (F5B and F6 subfractions) related with dtx A2 and dtx A, which caused
70 and 100% mortality on C. capitata at 48 h postfeeding, respectively. The LC50 was 104.92 ppm of dtx
A, contained in the F6 subfraction, and the LT50 was 4.16 h at a concentration of 400 ppm of dtx A con-
tained in the F6 subfraction. Moreover, the average survival time of adults exposed to this subfraction
was 12.6 h with only 1 h of exposure. The insecticide metabolites of the F6 subfraction of the EAMb
09/01-Su isolate retained >90% of its insecticidal activity after exposure to 60 C for 2 h and 120 C for
20 min. These results highlight the potential of this strain as a source of new insecticidal compounds of
natural origin for fruit fly control.

KEY WORDS bioinsecticide, biological control, fruit fly, insecticidal compound

The family Tephritidae, to which the fruit flies belong, and tropical regions of the Americas, making the pro-
comprises 4,500 species distributed in tropical, sub- duction of some varieties unprofitable (Steck 2001).
tropical, and temperate areas (Maddison and Bartlett The fruit flies cause direct physical damages to the
1989). The most important species of fruit flies around fruit and secondary damages by the entry of pathogens,
the world probably would be the olive fly, Bactrocera as well as the implications of quarantine measures and
oleae (Gmelin), which poses a severe economic threat postharvest treatments. Their management is based
for commercial olive growers and is largely responsible mainly on the use of chemical insecticides in bait
for 60% of the losses in the olive crop (Haniotakis sprays. However, increasing concerns on the effect of
2005, Quesada-Moraga et al. 2006). The medfly, Cera- such compounds on the environment and humans have
titis capitata (Wiedemann), is a cosmopolitan pest feed- prompted the search for their integrated pest
ing on a wide range of fruit crops in different climatic management based on nonchemical control methods
regions, and it annually produces important losses due (Magaña et al. 2007, Alaoui et al. 2010, Imoulan et al.
to infestation (Dimbi et al. 2009, Chueca et al. 2013, 2011).
De Villiers et al. 2013). Several species of Anastrepha Recent studies indicate the potential of entomopa-
genus are main pests in most fruit crops in tropical and thogenic fungi and their insecticidal compounds for ol-
subtropical countries, including Mexico, Central and ive fly (Yousef et al. 2013) and medfly control (Castillo
South America, and the Caribbean Islands (Rull et al. et al. 2000; Quesada-Moraga et al. 2006, 2008; Ortiz-
2012, Goncalves et al. 2013). Anastrepha obliqua (Mac- Urquiza et al. 2009, 2010). Entomopathogenic fungi
quart) is the most important pest of mangos in Mexico and their insecticidal compounds are considered safe
for humans, the environment and nontarget organisms
(Skrobek and Butt 2005, Boss et al. 2007, Skrobek
1
Laboratory of Agricultural Entomology, Department of Agricul-
et al. 2008, Garrido-Jurado et al. 2011). Active insecti-
tural and Forestry Sciences, ETSIAM, University of Cordoba, Cam- cidal proteins secreted by entomopathogenic fungi
pus de Rabanales. Edificio C4 Celestino Mutis. 14071 Cordoba, have been studied for activity against medfly and olive
Spain. fly (Ortiz-Urquiza et al. 2009, 2010), and recently, a
2
Mass Spectrometry & Chromatography Lab-SCAI, University of
Cordoba, Campus de Rabanales. Edificio Ramon y Cajal. 14071 Cór-
low-molecular weight fraction of a strain of a Meta-
doba, Spain. rhizium sp. has been found to have high insecticidal ac-
3
Corresponding author, e-mail: cr2qumoe@uco.es. tivity on medfly and olive fly (Yousef et al. 2013, 2014).
C The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
V
All rights reserved. For Permissions, please email: journals.permissions@oup.com
April 2015 LOZANO-TOVAR ET AL.: DESTRUXINS AGAINST Ceratitis capitata 463

Now, the extract of this strain has been characterized University of Innsbruck (Austria) and Prof. Richard
to determine the secondary metabolites responsible for Alan Humber from Cornell University (Ithaca, NY),
insecticidal activity against the fruit flies. respectively. The latter strain is known to produce a
Secondary metabolites secreted by the entomopatho- wide range of destruxins in culture media (Amiri-
genic fungi are a rich source of bioactive chemicals, in- Besheli et al. 2000, Hu et al. 2006, Wang et al. 2012).
cluding polyketides, depsipeptides, nonribosomal Insects. C. capitata flies, originally collected from
peptides, polyketide–peptide hybrid metabolites, and the stock colony of El Encı́n (INIA, Madrid, Spain),
terpenoids. These compounds display a wide array of have been maintained in the laboratory since 2004. To
biological activities, such as antitumoral, cytotoxic, im- reduce consanguinity, wild insects from infested orange
munosuppressant, antiproliferative, antiinflammatory, orchards at Cordoba (Spain) were introduced yearly.
antibiotic, antifungal, antiviral, insecticidal, and phyto- Flies were reared in the insectarium conditions at a
toxic activities (Ballard et al. 2002, Pedras et al. 2002, photoperiod of 16:8 (L:D) h with 50–60% relative
Sarabia et al. 2004, Peng et al. 2005, Uchida et al. humidity (RH) and at 26 6 2 C. Adult flies were pro-
2005, Lee et al. 2012, Liu and Tzeng 2012, Chun-Chi vided with water and a standard artificial diet consisting
et al. 2013, Lozano-Tovar et al. 2013). Likewise, ento- of yeast autolysate (ICN Biomedical, Aurora, OH) and
mopathogenic fungal strains of the genus Metarhizium sucrose (Panreac, Barcelona, Spain; 1:4 w/w). Larvae
are known to produce a series of biologically active me- were reared on a diet of wheat bran 300 g, sucrose
tabolites in vitro and in vivo (Skrobek et al. 2008, Liu 75 g, brewer’s yeast 38 g, nipagin 2 g, nipasol 2 g, and
and Tzeng 2012), such as destruxins (dtxs), cytochala- benzoic acid 2.4 g in 600 ml of water per kg of diet
sins C and D, helvolic acid, myroridins, swainsonine, (Albajes and Santiago-Álvarez 1980).
tyrosine betaine, serinocyclins A and B, aurovertins, Production of Fungal Crude Extracts in a Liquid
hydroxyovalicin, viridoxins, fungerins, metacytofilin, Medium. Fungal extracts were produced in G40P20
hydroxyfungerins A and B, and 12-hydroxyovalicin and medium (40 g of glucose and 20 g peptone per liter;
hydroxyfungerin, the 7-desmethyl analogs of fusarin C Ortiz-Urquiza et al. 2010). Briefly, a primary culture
and (8Z)-fusarin C (Fujii et al. 2000, Uchida et al. was inoculated with 107 conidia/ml and then cultivated
2005, Krasnoff et al. 2006, Zimmermann 2007, Azumi on a rotatory shaker at 110 rpm for 4 d at 25 6 1 C.
et al. 2008, Moon et al. 2008, Carollo et al. 2010). How- Subsequently, 2 ml of the primary culture was trans-
ever, strains of Metarhizium spp. differ considerably in ferred to a 1-liter flask containing 250 ml of the liquid
their metabolite profiles, with variation in destruxins medium. This secondary culture was cultivated for 7 d
production as a possible virulence or specificity factor under the former conditions. Cells were filtered with
(Amiri-Besheli et al. 1999, Wang et al. 2012). In fact, paper (Whatman No.3) and centrifuged at 9,000 rpm
destruxins are the most widespread of the secondary for 15 min; the supernatant was concentrated 10 times
metabolites produced by Metarhizium spp. and they their initial volume using a vertical flow chamber for
are, by far, the most exhaustively researched toxins of 24 h. Before performing the bioassay, the supernatants
this entomopathogenic fungus. were sterilized by filtration through 0.45- and 0.20-mm
In this study, we evaluated the insecticidal activity of filters.
crude extracts of entomopathogenic fungal isolates be- Insecticidal Activity of Crude Extracts. The
longing to genus Metarhizium and selected the most insecticidal activity of the concentrated supernatants
active of these extracts to elucidate both the molecular was assessed in a diet test. One-day-old male and
structure and production level of bioactive metabolites female flies were collected randomly and placed in 120
responsible for this activity. This crude supernantant by 51-mm2 (diameter  length) cages and fed with arti-
has been shown in previous studies to have insecticidal ficial diet and crude extract (test suspension: 0.4 mg of
activity on dipteran, lepidopteran, and coleopteran sucrose and 0.1 mg of protein–hydrolysate per ml of
pests [B. oleae, Spodoptera littoralis (Boisduval), and crude extract) on a 1.5-ml centrifuge tube cap. Controls
Rhynchophorus ferrugineus (Olivier); Resquin-Romero were treated with an equivalent volume of the G40P20
et al. 2012, Yousef et al. 2013]; however, the isolation liquid medium with the above proportions of sucrose
and characterization of bioactive compounds was not and yeast autolysate (control suspension). However,
previously conducted. two controls were established: one with the pH of the
G40P20 culture medium (5.2) and the other was acidi-
fied according to the average pH of the crude extracts
Materials and Methods (3.74). Four replicates of 10 insects each were used for
each group and for the controls. The experiment was
Entomopathogenic Fungi. The evaluated entomo- conducted and monitored at 26 6 2 C, 60 6 5% RH,
pathogenic fungal isolates are presented in Table 1. and a photoperiod of 16:8 (L:D) h. Adult mortality was
These isolates belong to the culture collection at the recorded at 24 and 48 h after treatment.
Agricultural and Forestry Sciences and Resources Obtaining of Fractions and Their Insecticidal
(AFSR) Department of the University of Cordoba Activity. In an initial attempt to elucidate the active
(Spain) and were obtained from soil and phylloplane fraction that was responsible for the insecticidal activity,
habitats. In addition, the reference strains BIPESCO 5 the concentrated supernatant of the isolate EAMb
(Metarhizium brunneum Petch) and ARSEF 23 (Meta- 09/01-Su was subjected to different fractionation proc-
rhizium robertsii J.F. Bisch., S.A. Rehner & Humber) esses. The concentrated supernatant was mixed with
were provided by Prof. Herman Strasser from the chloroform (1:1 v/v) and a chloroform fraction and
464 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 2

Table 1. Entomopathogenic fungal isolatesa assayed against C. capitata adult

Fungal species Isolate reference GeneBank Habitat Crop Site of origin

accession number
Metarhizium brunneum EAMb 09/01-Su KJ158746 Soil Holm oak forest Castiblanco de los Arroyos, Sevilla (Spain).
EAMb 09/02-Su – Soil Holm oak forest Castiblanco de los Arroyos, Sevilla (Spain).
EAMa 10/12-Su – Soil Olive Bobadilla, Malaga (Spain).
Metarhizium guizhouense EAMa 10/01-Fil – Phylloplane Olive Adamuz, Córdoba (Spain).
EAMa 10/02-Fil – Phylloplane Olive weed Adamuz, Córdoba (Spain).
EAMa 10/03-Fil – Phylloplane Olive weed Adamuz, Córdoba (Spain).
Beauveria bassiana EABb 09/16-Su KJ536056 Soil Olive Bobadilla, Malaga (Spain).
Metarhizium roberstii EAMr 09/01-Su KJ536064 Soil Holm oak forest Castiblanco de los Arroyos, Sevilla (Spain).
a
Isolates were obtained from the culture collection at AFSR Department of the University of Cordoba, Spain. In addition, there were refer-
ence strains BIPESCO 5 (M. brunneum) and ARSEF 23 (M. robertsii).

aqueous chloroform fraction were obtained. The Purification and Structural Elucidation of the
chloroform fraction was concentrated under reduced Active Compounds. The dialyzed fraction was sub-
pressure in a rotary evaporator Heidolph (Hei-VAP jected to a silica gel chromatography column (480 by
Advantaje) at 39 C and the aqueous fraction was dried 15 mm, Silica gel 60 Fluka, Sigma Aldrich, Steinheim,
in a vertical flow chamber at 28 C. Then the residue Germany) with four different eluent conditions:
was resuspended in water and mixed with methanol EtOAc:2-propanol (1:9 v/v), EtOAc:2-propanol:MeOH
(1:1 v/v), and placed at 20 C for 24 h to precipitate (5:45:50% v/v), EtOAc:2-propanol:MeOH:H2O
proteins and other compounds. The methanol fraction (5:45:45:5% v/v), and EtOAc:2-propanol:MeOH:H2O
was filtered at 4 C, and then the methanol was evapo- (5:45:40:10% v/v). All the reagents were supplied from
rated and resuspended in water (Konstantopoulou Panreac (Barcelona, Spain). Two milliliters of dialyzed
et al. 2006). fraction were eluted with 100 ml for each system elu-
The previous concentrated supernatant was dialyzed tion and carried at a flow rate of 0.7 ml/min. The sol-
against distilled water (1:20 fungal extract: water) in a vents of the four fractions obtained were evaporated
molecular porous membrane Spectra (VWR Interna- and resuspended in water and used in a bioassay
tional Eurolab S.L., Barcelona, Spain) with a cut-off of against newly emerged medfly adults.
3.5 kDa for globular proteins. The dialysis process was The more active fraction was submitted to high-pres-
carried out with constant agitation on a magnetic stirrer sure liquid chromatography (HPLC) reverse phase
for 48 h at 4 C, yielding two fractions, dialyzed and using a Biologic Duo Flow (Bio-Rad Inc., Richmond,
adialyzed (retained on the membrane). The adialyzates CA) with a QuadTec-4 detector and a RP-C-18 column
were concentrated at 4 C by embedding the mem- (Ascentis, Supelco, Bellefonte, PA; 10 by 10 mm by
brane in polyethylene glycol 20,000 (Merck-Schu- 10 mm) at a flow rate of 2 ml/min; a mobile phase linear
chardt, Hohenbrunn, Germany), while the dialyzates gradient was used (80/20 H2O/MeCN to 100% MeCN
were concentrated by evaporation at 28 C in a forced in 89.8 min). The injected volume was 500 ml, and the
air incubator (Selecta model; JP Selecta S.A., Barce- elution was monitored at four wavelengths: 190, 200,
lona, Spain; Lozano-Tovar et al. 2013). 204, and 210 nm simultaneously.
On the other hand, the crude soluble protein of con- The chromatographic spectrum of the eluted com-
centrated supernatant was precipitated with ammo- pounds was tested against C. capitata adults. Com-
nium sulfate (90% saturation; Panreac, Barcelona, pounds at each peak were collected and the solvent
Spain) and centrifugation at 10,000 g for 30 min. The was evaporated at 28 C in a forced air incubator and
pellets were dissolved in deionised water and desalted then resuspended in water and mixed with diet for
by dialyzing against 40 volumes of distilled water for feeding trials. The control treatments were prepared
24 h at 4 C using dialysis tubing with a 6–8 kDa cut-off according to the following criteria: control 1, distilled
membrane (Spectrum Europe, Breda, The Nether- water mixed with above described proportions of
lands). The desalted fractions were concentrated at sucrose and hydrolyzed protein; control 2, culture
4 C by embedding the membrane in polyethylene gly- medium acidified þ EtOAc:2-propanol (1:9 v/v) in a 1:1
col 20,000 (Merck-Schuchardt, Hohenbrunn Germany; (v/v) proportion; and control 3, culture medium
Ortiz-Urquiza et al. 2010). acidified þ MeCN in a 1:1 (v/v) proportion. The mix-
The fractions were sterilized by filtration by passing ture of acidified culture medium plus solvent was sub-
them through 0.45- and 0.20-mm filters successively. jected to drying, resuspended in water, mixed with the
The fractions were stored at 20 C until their use. All above-described proportions of sucrose, hydrolyzed
fractions were tested for biological activity on C. capi- protein and food was provided to C. capitata adults.
tata in the feeding bioassay. The fractions were mixed The eluted active peaks were analyzed by liquid
with the above-describe proportions of sucrose and chromatography–mass spectrometry (LC-MS) in a
hydrolyzed protein. Three controls were established: VARIAN 1200 LCMS equipped with electrospray
G40P20 culture medium and both aqueous chloroform ionization (ESI) and triple quadrupole (QqQ; Varian,
fraction and the methanol fraction obtained of G40P20 Inc. Walnut Creek, CA), and the identity of
culture medium. The experiments were repeated twice. destruxins was confirmed by ultra-high-pressure liquid
April 2015 LOZANO-TOVAR ET AL.: DESTRUXINS AGAINST Ceratitis capitata 465

chromatography (UHPLC) coupled with a high-resolu- Percentage of adult mortality was adjusted using
tion quadrupole–time of flight (qTOF) mass Abbott’s formula and values of corrected mortality (%)
spectrometer Bruker-MaXis Impact UHR-q TOF were processed for ANOVA. Comparisons and means
(Bruker International Corporation, Reutlingen, Ger- separations were performed using a Tukey’s studentized
many) working at 20.000 mass resolution. range honestly significant difference (HSD) test at
Biological activity of the Selected Fraction. P ¼ 0.05 with the analytical software Statistic 9 for Win-
Mean lethal concentration (LC50), mean lethal time dows (Analytical Software, Tallahassee, FL). LC50 and
(LT50), and the effect of exposure time on acute and LT50 values were calculated by probit analysis using
chronic insecticidal activity of the F6 active subfraction SPSS 12.0 for Windows (SPSS Inc., Chicago, IL).
against C. capitata were determined. Newly emerged
C. capitata adults were fed with microtube cups con- Results
taining a 100-ml of aliquot of the above-mentioned diet
containing F6 at five concentrations (25, 50, 100, 200, Insecticidal Activity of the Crude Extracts.
and 400 ppm of dtx A). Two controls were established: There were significant effects of the treatments with
liquid artificial diet and culture medium (G40P20) the fungal extracts on adult medfly mortality at 24 h
treated with 50% MeCN, the eluent was evaporated as (F7,24 ¼ 37.81; P < 0.0001) and 48 h postfeeding
previously described, resuspended in water, and mixed (F7,24 ¼ 52.46; P < 0.0001). At 48 h, crude supernatant
with the artificial diet. The mortality was evaluated at of EAMb 09/01-Su isolate, showed higher toxicity, caus-
18 h postfeeding. The concentration of dtx A in the ing 100% mortality and EAMr 09/01-Su isolate caused
active F6 subfraction was determined by comparison 86.8% mortality. The range of adult mortality caused by
with a dtx A standard (Sigma-Aldrich, Oakville, ON, the rest of the isolates varied from 0 to 21.2% at 24 h
Canada) using the VARIAN 1200 LCMS (with positive and from 10.0 to 47.4% at 48 h (Fig. 1). Crude superna-
electrospray ionization, ESI). tant of EAMb 09/01-Su isolate, therefore, was selected
To evaluate the effect of the exposure time on the for the next step of the prepurification process.
insecticidal activity of the F6 subfraction, adult flies Insecticidal Activity of Fractions Obtained of
were fed for 1, 3, 6, 9, and 12 h with active fraction EAMb 09/01-Su Isolate. The mortality rates caused
concentrated at 400 ppm of dtx A. Aliquots of the active by fractions obtained of EAMb 09/01-Su isolate
fraction (F6 subfraction) were mixed with sucrose and showed significant differences (F10,22 ¼ 55.67,86.25;
hydrolyzed protein for, as mentioned in the P < 0.0001). The mortality rate caused by the dialyzed
“Insecticidal Activity of Crude Extracts” subheading. fraction was >95% and was statistically equal to the
After each exposure time, adults were transferred to an crude supernatant and significantly higher than the
untreated artificial diet. The mortality was evaluated at mortality caused by the adialyzed fraction
the following time points: 0 h postfeeding, i.e., when (33.3–60.0%). For both fractions, the mortality rate
picking the microtube cap with treatment (acute mor- caused by aqueous chloroform fraction and methanol
tality) and 18 h postfeeding (chronic mortality). fraction was 60%. The range of adult mortality caused
The thermostability of the insecticidal activity was by the rest of the fractions varied from 0 to 20.0% at
determined. The crude supernatant, the EtOAc:2-prop- 48 h (Fig. 2).
anol fraction, the F6 subfraction and the F5B subfrac- Purification and Structural Elucidation of the
tion were subjected to two temperatures (20 min at Active Compounds of EAMb 09/01-Su Crude
120 C and 2 h at 60 C) and evaluated against C. capi- Extract. The mortality of newly emerged C. capitata
tata adults through a feeding bioassay. Three replicates adults, which were fed with diets containing different
of ten insects were used. The controls both water and fractions obtained than the the dialyzed fraction using
acidified G40P20 culture medium were mixed with pro- silica gel chromatography column under four eluent
portions of sucrose and yeast autolysate, as mentioned in conditions, showed significant differences among the
the “Insecticidal Activity of Crude Extracts” subheading. fractions at 24 and 48 h (F7,16 ¼ 63.66, 335.51;
Comparative Analysis of Insecticidal Activity of P < 0.0000). Only EtOAc:2-propanol:(1:9 v/v) fraction
both EAMb 09/01-Su Crude Extract and the F6 retained the insecticidal activity of the crude extract,
Sub-Fraction with Reference Strain. The insectici- with adult mortalities of 86.6 and 100.0% at 24 and
dal activity of the crude extract of EAMb 09/01-Su was 48 h, respectively (Fig. 3).
compared with the two reference strains BIPESCO 5 The EtOAc:2-propanol fraction obtained allowed for
and ARSEF 23. These strains were cultivated for 15 d the detection of >13 compounds with molecular
in G40P20, as described above, and the adult mortality masses between 453 and 611[M þ H]þ (ESI/Q1/MS
at 24 h and the average survival time (AST) were com- Varian 1200 LCMS); some of the compounds were
pared. Insecticidal activity of the F6 subfraction related to dtx A, A2, B, B2, and Desmethyl B (Fig. 4A).
obtained from the M. brunneum EAMb 09/01-Su strain When EtOAc:2-propanol fraction was submitted to
was compared with the F6 subfraction obtained from HPLC, the obtained subfractions were bioassayed
M. robertsii ARSEF 23 strain. The standard dtx A was against adult flies C. capitata adults and the insecticidal
using as control. activity was significantly retained in two subfractions,
Statistical Analyses. All bioassay tests were eval- F5B and F6, which caused mean mortality values of
uated under a randomized complete analysis of var- 40.0–75.0% and 75.0–100.0%, respectively (Fig. 4B).
iance (ANOVA) design with 3 and 4 replicates of 10 The analysis of the LC-MS mass spectrum of F6
insects each used for treatments and controls. showed a major molecular ion peak at m/z 578 at
466 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 2

Fig. 1. Activity of the crude supernatant of the entomopathogenic fungal isolates on C. capitata adults (mean 6 SE). Each
percentage is the average of 4 replications of 10 insects 6 SE of the mean. Percentage of adult mortality at 24 and 48 h
postfeeding of the crude extract was adjusted using Abbott’s formula [(corrected % ¼ mortality % in the treated
plot  mortality % in the control plot)/(100  mortality % in the control plot)  100]. Means with the same letter are not
significantly different according to Fisher’s protected or Tukey’s HSD tests at P ¼ 0.05. The mortality in the control treatment
ranged from 0 and 10%.

Fig. 2. Evaluation of fractions obtained of the crude extract of EAMb 09/01-Su isolate against C. capitata adults
(mean 6 SE). Each value is the average of 3 replications of 10 insects 6 SE of the mean. For each fractions, means with the
same letter are not significantly different according to Fisher’s protected or Tukey’s HSD tests at P ¼ 0.05.

8.3 min that correspond to dtx A. In addition, the analy- fraction, and in the F6 subfraction were 631.35, 660.96,
sis of the LC-MS mass spectrum of F5B showed a major and 505.92 ppm, respectively.
molecular ion peak at m/z 564 at 7.6 min, which was Chronic and Acute Biological Activity of the
related with dtx A2. Thus, the results of the mass spectral purified Active Compounds Against Newly
analysis were confirmed by UHPLC coupled to UHR- Emerged C. capitata Adults. There was a dose-
QTOF/ESI/MS (Maxis Impact, Bruker). The score of related mortality of newly emerged C. capitata adults
molecule formula C29H48N5O7 was 100%. The produc- fed with increasing concentrations of F6 subfraction
tion of dtx A was determined in crude concentrated (25, 50, 100, 200, and 400 ppm of dtx A in F6) with
supernatant, in the dialyzed fraction and in the F6 sub- mortality rates ranging between 10 and 70% at 18 h.
fraction by comparison with a dtx A standard (Sigma- The LC50 value was 104.97 ppm of dtx A in F6. The
Aldrich, Oakville, ON, Canada) using the VARIAN 1200 LC50 for control (standard dtx A) was similar with
LCMS (with positive ESI). The results obtained show 109.11 ppm (Table 2).
high efficiency in the process of extraction. The ranges The exposure time of newly merged C. capitata
of dtx A contained in crude supernatant, in the dialyzed adults to the F6 subfraction showed a significant effect
April 2015 LOZANO-TOVAR ET AL.: DESTRUXINS AGAINST Ceratitis capitata 467

Fig. 3. Mortality percentage of C. capitata caused by fractions of the M. brunneum EAMb 09/01-Su isolate obtained by
silica gel column chromatography. Mortality percentage at 24 and 48 h of exposure to fractions obtained under four different
eluent conditions: EtOAc:2-propanol:(1:9 v/v); EtOAc:2-propanol:MeOH (5:45:50% v/v); EtOAc:2-propanol:MeOH:H2O
(5:45:45:5% v/v); and EtOAc:2-propanol:MeOH:H2O (5:45:40:10% v/v). Means with the same letter are not significantly
different according to Fisher’s protected or Tukey’s HSD tests at P ¼ 0.05.

on the mortality rates at 0 h (F6,14 ¼ 47.30; P < 0.0001) was shorter (7.7 h) than the one of the ARSEF 23
and 18 h (F6,14 ¼ 143.42; P < 0.0001) after being trans- strain (9.2 h) and standard dtx A (10.4 h; Table 6).
ferred to untreated diet. One-hour exposure time was The dtx A production for both EAMb 09/01-Su and
statistically different from other exposure times both at ARSEF 23 strains was similar (830.5 and 884.5 ppm),
0 and 18 h postfeeding. The mortality rates were while the dtx A2 production was 21% higher in the
80–100% at 18 h for 3, 6, 9, and 12 h exposure times EAMb 09/01-Su strain than in the ARSEF 23 strain
(Table 3). There were also significant differences
among AST values that ranged from 2.8 h after 6 h of
Discussion
exposure to 12.7 h after 3 h of exposure (Table 3).
Effect of Temperature on the Insecticidal When investigating environmentally friendly biologi-
Activity of the Fractions Produced by the EAMb cal pest–control methods, microorganisms act as a
09/01-Su Strain. The insecticidal activity of the crude prominent reservoir of insecticidal compounds of natu-
supernatant, the EtOAc:2-propanol fraction, F6 sub- ral origin. The crude extract of the M. brunneum
fractions showed stability to temperature treatments to EAMb 09/01-Su strain contained secondary metabo-
which they were subjected. The mortality range was lites, including destruxins, which have insecticide activ-
maintained >90%. The insecticidal activity of F5B sub- ity against C. capitata when administered per os. The
fraction treated for 2 h at 60 C was not significantly entomopathogenic mitosporic ascomycete Metarhizium
affected. However, exposure to 120 C for 20 min signif- has been used worldwide for invertebrate pest control
icantly reduced the insecticidal activity of the F5B sub- as an alternative to chemical pesticides (Milner, 2000,
fraction by 60% (Table 4). Lomer et al. 2001, Zimmermann 2007, Khan et al.
Comparative Analysis of Insecticidal Activity of 2012). Numerous secondary metabolites have been iso-
Both the EAMb 09/01-Su Crude Extract and the lated from Metarhizium spp. through different method-
F6 Subfraction with the Reference Strains. Adult ologies and were evaluated on different insect (Pais
fly mortality caused by the EAMb 09/01-Su crude et al. 1981; Jegorov et al. 1992, 1998; Loutelier et al.
supernatant (96.6%) did not differ significantly from 1996; Hsiao and Ko 2001; Pedras et al. 2002; Uchida
that caused by the ARSEF 23 crude extract (93.0%), et al. 2005).
and both extracts caused higher mortality than one pro- Up to now, only one study has explored the activity
duced by the BIPESCO 5 strain (75.1%). A similar pat- of dichloromethane extracts from the entomopatho-
tern was observed for the AST of treated flies (Table 5). genic fungi on C. capitata (Castillo et al. 2000). How-
Moreover, the F6 subfractions of the EAMb 09/01-Su ever, our paper is the first one that characterizes a
and ARSEF 23 strains at 400 ppm of dtx A were Metarhizium crude extract and reports its activity on
equally toxic to the adult flies at 18 h with mortality val- C. capitata due to dtx A and dtx A2. In our study, the
ues of 90.0 and 97.0%, respectively, TL50 for F6 sub- insecticidal metabolites were obtained in the dialyzed
fraction of the EAMb 09/01-Su, ARSEF 23, and fraction and exhibited high efficiency and a range of
standard dtx A was similar with value of 4.13, 5.29, and mortalities against C. capitata, between 93.3 and
5.46 h, respectively, whereas the AST of the flies 100.0%; the metabolites corresponded to fractions
exposed to the F6 subfraction of EAMb 09/01-Su strain <3.5 kDa. Besides, the extract of EAMb 09/01-Su
468 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 2

Fig. 4. The HPLC profile of the EtOAc:2-propanol fraction of M. brunneum EAMb 09/01-Su and its evaluation. (A)
HPLC profile; mobile phase, H2O: MeCN 80:20 (v/v) with linear gradient in 89.8 min; reverse phase C18 (10 by 10 mm by
10 mm Ascentis), flow-rate 2 mL /min., (k) ¼ 210, 204, 200, 190 nm simultaneously. (B) Evaluation of HPLC subfractions
against C. capitata adults.

Table 2. Linear regression (Log-dose)

Treatment Probit regression Pearson Chi-square gl (2) LC50 95% Fiducial limits for dose
F6 subfraction Y ¼ 1.373X  2.775 0.053 5.879 104.97 0.678–2.069
Standard dtx A Y ¼ 2.895X  5.90 0.230 2.94 109.11 2.017–3.773
Percentage of adults mortality treated with five doses of F6 subfraction obtained of M. brunneum EAMb 09/01-Su isolate with corresponding
LC50 on adults of C. capitata.
The concentrations of F6-subfraction were established taking into account the content of dtx A. The concentration of dtx A in the active frac-
tion was determined for comparison to the standard dtx A (Sigma-Aldrich, Oakville, ON, Canada) used the VARIAN 1200 LCMS. The concen-
trations evaluated were 25, 50, 100, 200, and 400 ppm with relation to dtx A contained in F6 subfraction.

showed insecticidal activity when adialyzed and crude achieved an active fraction that produced 100% mortal-
protein fractions were offered, indicating that this ity on C. capitata 48 h postfeeding. The LC-MS analysis
strain is able to produce also insecticidal proteins but of the active fraction of the EAMb 09/01-Su crude
with less activity. extract obtained using EtOAc:2-propanol (1:9 v/v) found
The fractionation with the silica gel column using compounds with molecular masses between 453 and
EtOAc:2-propanol (1:9 v/v) as the eluent condition 611 [M þ H]þ. The HPLC-mediated fractionation of
April 2015 LOZANO-TOVAR ET AL.: DESTRUXINS AGAINST Ceratitis capitata 469

Table 3. Acute activity of the F6 subfraction from the crude extract of the EAMb 09/01-Su strain assayed at 400 ppm dtx A against
newly emerged C. capitata adults

Exposure time (h) Mortality at 0 h ( 6 SE) (%)a Mortality at 18 h ( 6 SE) (%)a Kaplan–Meier survival analysis
ASTb 95% Confidence interval
1 23.3 6 3.3b 70.0 6 5.7b 12.7c 10.3–15.1
3 70.0 6 11.5a 93.3 6 3.3a 5.6b 3.0–8.1
6 86.6 6 6.6a 100.0 6 0.0a 2.8a 1.1–4.5
9 80.0 6 5.7a 90.0 6 0.0a 4.2ab 1.8–6.5
12 83.3 6 3.3a 90.0 6 5.7a 3.6ab 1.5–5.7
Control- water 0.0b 0.0c 18d 18.0–18.0
Control-medium 0.0b 6.6 6 3.3c 18d 18.0–18.0
a
Means in each column with a common letter are not significantly different according to Fisher’s protected or Tukey’s HSD tests at P ¼ 0.05.
The concentration of dtx A in the active fraction was determined by comparison with the standard dtx A (Sigma-Aldrich, Oakville, ON, Canada)
using the VARIAN 1200 LC-MS.
b
Means with the same letter are not significantly different according to a log-rank test (P < 0.05). Average survival time (AST) limited to 18 h.
Adults were fed on contaminated diet for the selected exposure times and then transferred to untreated diet.

Table 4. Effect of temperature treatments on the insecticidal activity of the two active subfractions of the EAMb 09/01-Su crude
extract against newly emerged C. capitata adults

Treatment Crude supernatant EtOAc:2-propanol fraction F6 subfraction F5B subfraction

Control (25 C) 100a 96.6 6 3.3a 96.6 6 3.3a 80.6 6 5.2a
60 C to 2 h 90 6 10.0a 96.6 6 3.3a 93.3 6 3.3a 83.3 6 6.7a
120 C to 20 min 90 6 5.7a 100a 100a 53.3 6 12.0a
Data indicate mortality at 24 h (mean 6 SE).
a
Means in each column with the same letter are not significantly different according to Fisher’s protected or Tukey’s HSD tests at P ¼ 0.05.

Table 5. Acute mortality percentage (mean 6 SE) and AST of newly emerged C. capitata adults exposed to the crude supernatant of
the M. brunneum EAMb 09/01-Su isolate and the ARSEF 23 and BIPESCO5 reference strains

Strain Mortalitya (mean 6 SE (%)a Kaplan–Meier survival analysis

ASTb h 95% Confidence interval
BIPESCO5 75.1 6 5.9b 22.3b 20.4–24.1
EAMb 09/01-Su 96.6 6 3.3a 16.2c 12.9–19.4
ARSEF 23 93.0 6 3.5ab 15.3c 11.9–18.6
Control 3.3 6 3.3c 24.0a 24.0–24.0
a
In each column, mean values with the same letter are not significantly different according to Fisher’s protected or Tukey’s HSD test at
P ¼ 0.05.
b
Means with the same letter are not significantly different according to a log-rank test (P < 0.05). Average survival time (AST) was limited to
24 h. The crude supernatant was concentrated 10 times their initial volume.

Table 6. Linear regression (Log-time), lethal time and AST of newly emerged C. capitata adults exposed to the active F6 subfraction
of both the M. brunneum EAMb 09/01-Su isolate, ARSEF 23 reference strain, and standard dtx A

Treatment Linear regression LT50 h Kaplan–Meier survival analysis

AST* (h) 95% Confidence interval
F6 subfraction EAMb 09/01-Su Y ¼ 2.35X  1.458 gl(2); chi-square ¼ 3.161 4.13 7.7a 5.5–9.8
F6 subfraction ARSEF 23 Y ¼ 3.98X  2.888 gl(2); chi-square ¼ 1.584 5.29 9.2a 7.0–11.4
Standard dtx A Y ¼ 1.73X  1.272 gl(2); chi-square ¼ 0.949 5.46 10.4a 7.8–13.0
The concentration of dtx A in the active fractions (400 ppm) was determined by comparison with the dtx A standard (Sigma-Aldrich, Oakville,
ON, Canada) using the VARIAN 1200 LCMS.
*Means with the same letter are not significantly different according to a log-rank test (P < 0.05). Average survival time (AST) was limited to 18 h.

the active fraction using a linear gradient of H2O/MeCN the process of extraction. Our results are comparable
(80/20) to 100% MeCN showed the highest number of with the methodology used by Liu et al. (2005), who
compounds, 13 of which were related to destruxins. found that the MeCN/H2O ratio in the gradient elution
The ranges of dtx A contained in crude supernatant, buffer of the semipreparative HPLC yielded high
in the dialyzed fraction, and in the F6 subfraction were extraction efficiency of destruxins.
similar with 631.35, 660.96, and 505.92 ppm (mg dtx The insecticidal test of the F6 and F5B subfractions
A/ml), respectively, demonstrating high efficiency in resulting from the HPLC profile chromatogram
470 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 2

showed mortalities of C. capitata at 100 and 70% 48 h previous idea of using EAMb 09/01-Su extract in field
postfeeding, respectively. The toxicity test against under Mediterranean conditions.
C. capitata adults showed that the highly toxic com- When comparing the insecticidal activity of the F6
pounds of the EAMb 09/01-Su crude extract were con- subfraction of the EAMb 09/01-Su crude extract with
tained in these two subfractions. According to the mass the F6 subfraction of the ARSEF 23 crude extract
analysis, these compounds were related to dtx A and (reference strain), results were similar, with mortality
A2; therefore, C. capitata adults were found to be values of 90.0 and 97.0%, respectively. However, the
highly susceptible to these two destruxins. These results AST of the flies exposed to the F6 subfraction of the
are consistent with other research on the insecticidal EAMb 09/01-Su crude extract was shorter (7.7 h) than
properties of destruxins; dtx A has been referenced as that of the ARSEF 23 crude extract (9.2 h). The dtx A
an insecticide for several insect species, such as Spo- production was similar for both strains, but dtx A2 pro-
doptera litura (F.), with a mortality of 65% at 96 h with duction was higher for the EAMb 09/01-Su crude
300 ppm (Hu et al., 2007). The injection of dtx A into extract than for the ARSEF 23 crude extract.
Drosophila melanogaster Meigen adults yielded an The results of this research together with other
LC50 of 0.11 mM (Ruiz-Sánchez and O’Donnell 2012) previous investigations of the M. brunneum EAMb
and suppressed the innate immune response (Pal et al. 09/01-Su isolate that showed 100% C. capitata mortal-
2007). Furthermore, Plutella xylostella (L.) and Phae- ity with LC50 of 2.84  107 conidia/ml and LT50 of
don cochleariae (F.) larvae were highly susceptible to 5.6 d, additive effect with its crude extract, and stability
dtx A, with LC50 values of 30 and 17 ppm, respectively to environmental conditions (i.e., ultraviolet B radi-
(Amiri et al. 1999). The dtxs A and A2 have been shown ation; Yousef et al. 2013, Resquin-Romero et al. 2012,
to be toxic against Schistocerca gregaria (Forsskål) as Lozano-Tovar et al. 2012) demonstrate the potential of
well (James et al. 1993). this strain for the biological control of insects. With this
In this study, we investigated further into the biologi- strain, it is possible to generate a product with highly
cal activity of F6 subfraction against C. capitata adults. sought-after features, such as rapid action against target
The results showed that mortality was dose-related organisms and stability under adverse environmental
when exposed to treated diet. The LC50 was conditions (field and storage).
104.92 ppm, and the LT50 was 4.16 h with a 400 ppm
concentration of dtx A in F6-subfraction. At this con-
centration, chronic mortality reached 70% at 18 h post- Acknowledgments
feeding with only 1 h of exposure and an AST of 12.6 h.
This research was supported by the Spanish Ministry of
When C. capitata adults were exposed for 6 h to the
Science and Innovation Project AGL2011-27646. We thank
treated diet, the AST was reduced to 2.8 h. The Sandra Ma Castuera Santacruz and Carlos Campos Porcuna
relevance of this finding lies in that the major obstacle for their excellent technical assistance, and Dr. Hermann
of the commercial aspect limiting the marketing of Strasser (Institute of Microbiology, University of Innsbruck,
fungi as mycoinsecticides is their slow killing speed Austria) and Dr. Richard Humber (U.S. Department of
(Leger and Wang 2009, Khan et al. 2012). However, Agriculture–Agricultural Research Services [USDA-ARS],
the development of biopesticides through the obtaining Robert W. Holley Center Ithaca) for kindly providing
of secondary metabolites of entomopathogenic fungi is BIPESCO5 (Metarhizium brunneum) and ARSEF 23 (Meta-
a potential opportunity to protect agricultural crops rhizium robertsii) reference strains used in this investigation.
against insect pests by their rapid action against target
organisms. On the other hand, several fungi have been
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