Larvicidal Activity of Beauveria bassiana Extracts against Aedes aegypti and Identification of Beauvericins

Beauveria bassiana is an entomopathogenic fungus that has been well known for its capacity to act as biopesticide on various disease vectors. The analysis of organic extracts of strains CG71 and UNI40 led to identification of cyclodepsipeptides beauvericin, beauvericin A or F, beauvericin E and bassianolide by ultra-high pressure liquid chromatography-high resolution mass spectrometry in tandem mode (UHPLC-HRMS/MS). The larvicidal activity on 3 instar of Aedes aegypti revealed LC50 0.9887 and 0.4653 ppm in 24 and 48 hours (CG71 methanolic extract), LC50 0.7834 ppm in 48 hours (CG71 ethyl acetate), LC50 0.7834 and 1.8149 ppm (UNI 40 for ethyl acetate and methanolic extracts, respectively) in 48 hours. These findings highlight the potential of B. bassiana metabolites for controlling the vector of Dengue and Zika diseases.

Recently, new analogues of beauvericin were described, including beauvericin A, B, 24 D, E and F. 25 Bassianolide is another cyclooctadepsipeptide metabolite produced B. bassiana and Lecanicillium lecanii. 9It is toxic to insects and probably also acts as an ionophore, such as other cyclodepsipeptides. 15The structural diversity of metabolites from entomopathogenic fungi, associated with diverse biological activities, stimulates the research by on bioactive fungal extracts for controlling insect pests and disease vectors.

Dengue is a viral infection transmitted to humans by
Aedes aegypti mosquitoes that became a major concern of the public health sector. 26There were 96 million apparent dengue infections globally in 2010, of which the Americas contributed 14% (13 million infections) of worldwide and over half developed in Brazil and Mexico. 27Due to no effective antiviral agents and licensed vaccine for infection, the decrease in transmission depends only on vector control by elimination of artificial and disposable water inundated larvae breeding sites and application of insecticides. 27,28Furthermore, studies reveal resistance in the vector population to various substances, in Brazil there is an irregular distribution of insecticide resistance. 28urthermore, Aedes mosquitoes also transmit Zika virus; attracting worldwide attention in 2016, when the vector caused widespread epidemic cases in Brazil. 29This disease has been recognized in Brazil since late 2014, but in 2015 coincided with an increase in the number of cases of microcephaly.The more affected Brazilian states were Bahia, Paraíba, and Pernambuco, where in the first trimester of pregnancy coincided with reports of cases of febrile and allergy compatible with Zika virus disease, suggesting an association between Zika virus infection during early pregnancy and the occurrence of microcephaly. 30onsequently, there is an urgent need to develop new methods for A. aegypti mosquitoes infections control.So, fungal extracts are an alternative because they constitute a rich source of bioactive metabolites.
The paper reports the activity of two strains of Beauveria bassiana extracts on mortality of Aedes aegypti larvae and identification of beauvericins and bassianolide using ultra-high pressure liquid chromatography-high resolution mass spectrometry in tandem mode (UHPLC-HRMS/MS).Additionally, the fragmentation data of beauvericin E and A of F is provided.

Morphological characterization
The strains were preliminary identified as Beauveria bassiana based on the macro and micromorphological characteristics of colonies, conidia and conidiophores after cultivation for 7 days at room temperature on malt extract agar (Himedia, Mumbai, India). 32

DNA sequencing and phylogenetic analyses
Strains CG 71 and UNI 40 were cultivated on malt extract agar for five days at 24 °C.The mycelium was scrapped from the colonies, macerated under liquid nitrogen, and subjected to genomic DNA extraction with the Wizard Genomic Deoxyribonucleic Acid (DNA) Purification Kit (Promega, Madison, USA).A fragment of the second largest subunit of the ribonucleic acid (RNA) polymerase gene (RPB2) was amplified from the extracted DNA using the primers 5F and 7cR, and the polymerase chain reaction (PCR) conditions of Liu et al. 33 The purified PCR products were sent for DNA sequencing by a commercial service.
Consensus sequences were assembled using SeqAssem ver.07/2008 (SequentiX -Digital DNA Processing, Germany).Blast searches against the GenBank database suggested that both strains belonged to Beauveria bassiana.DNA sequences of RPB2 from reference strains of Beauveria bassiana and related taxa 34 were used to compose a multiple sequence alignment using multiple sequence comparison by log-expectation (MUSCLE), as implemented by Mega 6 software. 35Maximum likelihood (ML) phylogenetic analysis was conducted using Mega 6. 1000 bootstrap pseudo-replicates were employed for estimating node support.The general time reversible model with gamma distribution of rates across sites (GTR+G) model of evolution, estimated using JModeltest, 36 was used in the ML analysis.Isaria farinosa (ARSEF 4029) was used as the outgroup.

Culture of B. bassiana, extraction and chromatographic fractionation
The B. bassiana CG71 and UNI 40 strains were cultivated on potato dextrose agar (PDA) at 26 °C for 7 days, and pre-inoculated in 60 mL of supplemented liquid medium (ML) divided into 4 Erlenmeyer flasks (50 mL) containing 30.0 g glucose; 20.0 g malt extract; 2.0 g bactopeptone; 1.0 g yeast extract; 0.5 g KCl; Vol. 28, No. 6, 2017 0.5 g MgSO 4 .7H 2 O; 0.5 g KH 2 PO 4 ; 1 L water, as reported by Bunyapaiboonsri et al. 37 The flasks were incubated on a rotary shaker (200 rpm) for 8 days.These seed cultures (240 mL of each strain) were used to inoculate 11 liters ML (55 Erlenmeyer flasks of 1 L with 200 mL of medium for each strain).After incubation at room temperature for 20 days in static mode, the mycelium was separated by reduced pressure filtration and the liquid phase was submitted to liquid-liquid fractionation with ethyl acetate (EtOAc).After lyophilization, the mycelial masses were combined and extracted totally with methanol (MeOH).The organic solvents were removed by vacuum distillation at 55 and 60 °C using a rotary evaporator.

Cyclodepsipeptides identification
The UHPLC-HRMS/MS analyzes were performed on an Accela High Speed LC coupled to a LTQ Orbitrap Velos FT-MS (ThermoFisher Scientific, San Jose, CA, USA), equipped with an electrospray source (HESI-II) and operated at a resolution of 60,000 FWHM, with positive ionization and 25 eV at HRMS/MS mode.The chromatographic system was fitted with an RP Luna C 18 column (150 × 4.6 mm, 5 μm, Phenomenex) applying as mobile phase H 2 O/CH 3 OH (30:70 → 0:100, v/v, both buffered with 0.1% of formic acid) in 15 min at a flow rate of 0.4 mL min -1 .The software Thermo Xcalibur 2.1 (ThermoFisher Scientific) was used to control the full system.The sample injection (auto injector) volume was 0.5 μL.

Bioassay to control Aedes aegypti larvae Rockefeller strain
The bioassay was performed at the Laboratory of Biocontrol of Arthropods (LABIART), Veterinary Institute, Department of Animal Parasitology, Universidade Federal Rural do Rio de Janeiro (UFRRJ).The extracts tested were CG71a, CG71m, UNI40a and UNI40m.
Aedes aegypti of the Rockefeller strain were maintained in colonies at 27 ± 3 °C, and 80% humidity on a 12-h light/ dark cycle.Four replications of ten (10) third instar larvae (L-3) of Aedes aegypti susceptible in the insecticide were tested in five concentrations: 25, 50 100, 150, and 200 μL of each extract dissolved in dimethyl sulfoxide 10% (DMSO).
Two control groups were tested, one containing the solvent used in extracts and the other containing only pure water.The analyzes of the results of larval mortality were assessed at 15 minutes and at successive counts of 15, 30 minutes, 1, 2, 3 and 24 and 48 hours.The lethal concentrations LC 50 and their respective confidence intervals were calculated through Probit analyses, using the R software (R Core Team).

Cyclodepsipeptides fragmentation
Mass spectrometry has played essential role in the structure determination of cyclodepsipeptides originated from diverse fungal species, grains and foods.The fragmentation is a main step in sequencing and primary stage usually is bond rupture in the ester or amide group. 38nterpretation and elucidation of the amino acid sequence using collision-induced dissociation (CID) depends essentially on the sequence of specific ions present (a n , b n , c n and x n , y n , z n ).The fragment ions produced in this process can be divided in two series. 39eauvericin is a cyclohexadepsipeptide isolated in 1969 from B. bassiana, derived from two dipeptidol monomers alternately, D-hydroxyisovalerate (D-HiV) and N-methyl-L-phenylalanine (N-Me-Phe), while bassianolide isolated in 1977 from B. bassiana and Verticillium lecanii is a cyclooctadepsipeptide also formed by two dipeptidol monomers units of D-hydroxyisovalerate (D-HiV) and N-methyl-L-leucine (N-Me-Leu). 18he cyclodepsipeptides beauvericin, beauvericin A or F, beauvericin E and bassianolide were detected in two strains of B. bassiana (CG71 and UNI 40) extracts and fractions semi-purified according to the observed accurate masses for protonated molecule and the corresponding cationized molecules, displayed at their full scan spectra (Figure 2) and selected ion chromatogram (Figure 3).][42][43][44] The electrospray ionization tandem mass spectra (ESI-MS/MS) and fragmentation mechanism proposal for beauvericin A or F are depicted in Figures 4 and 5, respectively.The N-methyl-L-phenylalanine (−161 Da) loss gave rise to the fragment ion m/z 637.3486.While the peaks m/z 555.3066 and 294.1701 are detected due to the isopropylideneketene losses (82 Da), the ion m/z 376.2121 occurs through the rupture of the amide bond.It is worth to highlight that this new fragmentation proposal is fundamental for the confirmation of the beauvericin A or F presence in the organics extracts of entomopathogenic fungi.
The inspection of the MS/MS data showed that the peaks at m/z 523.2809 in beauvericin and m/z 682.4630 in bassianolide spectra represent the loss of an 2-hydroxy-3-methyl-1-buten-1-one molecule (100 Da), the identical D-HiV monomer (Figures 6 and 7).Additionally, the peaks at m/z 623.3333 (Figure 6) and 782.5159 (Figure 7) show the loss of N-methyl-L-phenylalanine (−161 Da) and N-methyl-L-leucine (−127 Da), respectively.Similar fragments containing phenylalanine residues (m/z 262) are described for beauvericin and E, which then lost H 2 O to give m/z 244 (Table 1).
Figure 8 shows fragmentation mechanism proposal for beauvericin E (precursor ion m/z 736.4722).Differently from the mechanism of the other cyclodepsipeptides produced by the fungus, the MS/MS data from beauvericin E showed

Phylogenetic analyses
Strains CG 71 and UNI 40 were grouped in a wellsupported clade with reference strains Beauveria bassiana in the phylogenetic analyzes conducted with partial RPB2 DNA sequences (Figure 9), thus confirming their identity.

Larvicidal activity
Table 2 shows larvicidal activity of the B. bassiana CG71 and UNI40 extracts against 3 rd instar of Aedes aegypti.The methanolic extract of strain CG71 revealed the highest activity with LC 50 values of 0.9887 and 0.4653 ppm after 24 and 48 hours, respectively.The ethyl acetate extract of CG71 displayed LC 50 of 29.1777 and 1.2309 ppm after 24 and 48 hours, respectively.
The UNI 40 strain showed LC 50 values of 0.7834 and 1.8149 ppm for the ethyl acetate and methanolic extracts after 48 hours of incubation, respectively.These extracts showed no larvicidal activity in the first 24 hours of incubation.Besides the cyclodepsipeptides detected in both CG 71 and UNI 40 extracts, other peptides with peaks at m/z above 1000 were detected in the ethyl acetate and methanolic extracts of UNI 40.
New studies using fungal organic extracts aim to identify new bioactive secondary metabolites, such as Fusarium sp.extract with trypanocidal activity, which revealed beauvericin as the responsible for the toxicity of Fusarium sp. to Trypanosoma cruzi. 45auvericin has been reported to be toxic on Aedes aegypti larvae with LC 50 26 ppm, 24 and 10 and 20 μg mL -1 showed 39 and 86% of mortality in 48 hours, respectively. 46The presence of beauvericin, beauvericin A or F, beauvericin E and bassianolide in the extracts of two B. bassiana strains may explain the excellent larvicidal activity observed.

Conclusions
The cyclodepsipeptides beauvericin, beauvericin A or F, beauvericin E and bassianolide were identified in the B. bassiana CG71 and UNI 40 extracts.The methanolic extract of CG71, ethyl acetate and methanolic extracts of UNI40 showed larvicidal activity against 3 rd instar of Aedes aegypti with LC 50 values of 0.4653, 0.7834 and 1.8149 ppm after 48 hours of incubation, respectively.These results suggest    that cyclodepsipeptides are probably the active principles responsible for the larvicidal action.Therefore, further studies must be undertaken in order to isolate and confirm the compounds responsible for this activity.Therefore, they may be considered as potential insecticidal components in the formulations for the Dengue and Zika vector.The liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodology used proved to be an excellent tool for the identification of cyclodepsipeptides in fungal extracts, and contributed with new fragmentation studies for beauvericin A or F and E.

Figure 1 .
Figure 1.Structures of compounds 1-4 identified from the extracts of B. bassiana.

Figure 2 .
Figure 2. Full scan spectra of (A) beauvericin and (B) beauvericin A or F, produced by B. bassiana CG 71.Data acquired at ESI.

Figure 9 .
Figure 9. Maximum likelihood phylogenetic tree of partial RPB2 sequences of CG 71 and UNI 40, and reference strains of Beauveria bassiana and related species.Bootstrap values equal or higher than 70% are showed by the nodes.Isaria farinosa (ARSEF 4029) was used as the outgroup.T identifies ex-type strains.

Table 1 .
Characterization (mass accuracy) of named ions cyclodepsipeptides and MS/MS parameters of Beauveria bassiana extracts