Lethal Effect and Behavioral Responses of Leaf ...

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Acefylline. 2.60 ± 0.02. 2.06 ± 0.008. 1506 α-Bulnesene. 13.75 ± 0.013. 11.75 ± 0.006. 1568. Longicanfenilona. 0.96 ± 0.003. 1.01 ± 0.005. 1589. Caryophyllene ...
Neotrop Entomol https://doi.org/10.1007/s13744-018-0615-6

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Lethal Effect and Behavioral Responses of Leaf-Cutting Ants to Essential Oil of Pogostemon cablin (Lamiaceae) and Its Nanoformulation AG ROCHA1, BMS OLIVEIRA1, CR MELO1, TS SAMPAIO 2, AF BLANK 1,3, AD LIMA2, RS NUNES2, APA ARAÚJO4, PF CRISTALDO1,5, L BACCI1,3 1

Programa de Pós Graduação em Agricultura e Biodiversidade (PPGAGRI), Univ Federal de Sergipe, São Cristóvão, SE, Brasil Rede Nordeste de Biotecnologia (RENORBIO), Univ Federal de Sergipe, São Cristóvão, SE, Brasil 3 Depto de Engenharia Agronômica, Univ Federal de Sergipe, São Cristóvão, SE, Brasil 4 Depto de Ecologia, Univ Federal de Sergipe, São Cristóvão, SE, Brasil 5 Depto de Agronomia/Entomologia, Univ Federal Rural de Pernambuco, Recife, PE, Brasil 2

Keywords Bioinsecticides, patchouli, Formicidae, formicidal, insecticidal plant Correspondence L Bacci, Programa de Pós Graduação em Agricultura e Biodiversidade (PPGAGRI), Univ Federal de Sergipe, São Cristóvão, SE, Brasil; [email protected] Edited by Gabriel Manrique – Univ de Buenos Aires Received 4 April 2018 and accepted 7 June 2018 * Sociedade Entomológica do Brasil 2018

Abstract Leaf-cutting ants belonging to the genus Atta (Formicidae: Myrmicinae) are important pests in agricultural and forest environments. In the present study, we evaluated the formicidal activity of the essential oil of Pogostemon cablin and its nanoformulation on the leaf-cutting ants: Atta opaciceps (Borgmeier, 1939), Atta sexdens (Linnaeus, 1758), and Atta sexdens rubropilosa Forel, 1908. The nanoformulation was developed by magnetic stirring using polyoxyethylene (36%), pure ethanol (36%), essential oil of P. cablin (18%), and water (10%). Bioassays of acute toxicity by fumigation and behavioral bioassays in treated arenas, with and without choice, were performed. The essential oil of P. cablin and its nanoformulation demonstrated efficient insecticidal activity and irritability to ant species. The concentration required to kill 50% of workers varied from 1.06 to 2.10 μL L−1, with a mean time to death of less than or equal to 42 h. The essential oil of P. cablin and its nanoformulation reduced the displacement and velocity speed of the workers of A. opaciceps and A. sexdens rubropilosa in totally treated arenas. In the bioassays with choices, the three species of ants walked less and at a greater speed on the treated side of arena. This work demonstrates the potential of the essential oil of P. cablin and its nanoformulation to the generation of new formicidal products.

Introduction Leaf-cutting ants of the genus Atta are dominant in tropical ecosystems, where they play an important ecological role as ecosystem engineer species (Vasconcelos & Cherrett 1997, Sternberg et al 2007). However, such species can become pests in agricultural and forest environments (Della Lucia 2011, Urbas et al 2007), due to the behavior of cutting leaves from plants to maintain the

symbiotic fungi colony in their nests. In savannas and Neotropical forests, for example, these insects can remove up to 17% of leaf production (Cherrett 1989, Costa et al 2008, Herz et al 2007). In addition to direct losses due to death or reduced plant growth, ants increase the susceptibility of plants to be attacked by other insects and pathogens (Cantarelli et al 2008, Zanetti et al 2014). In Brazil, the control of leaf-cutting ants is mainly performed through the use of organic synthetic insecticides, although

Rocha et al

there is a pressure from the certification agencies to prohibit the use of these products (e.g., non-governmental organization: Forest Stewardship Council 2010). Despite being polyphagous, leaf-cutting ants are selective foragers (Blanton & Ewel 1985, Coley & Barone 1996, Farji-Brener 2001), mainly in relation to the chemical composition of plants—avoiding antifungal compounds, repellents, and terpenoids (Chen et al 1983, Howard 1985, Hubbell et al 1983, Hubbell et al 1984, Wiemer & Ales 1981). This natural bioactivity of secondary metabolism of compounds of plants has been extensively studied focusing on the pest control (Bakkali et al 2008, Knaak & Fiuza 2010). Among these compounds, the essential oils (EOs) of aromatic plants—composed by a complex mixture of compounds in different concentrations—stand out due to their toxicity to herbivores, besides their rapid degradation and low toxicity to mammals (Koul et al 2008, Regnault-Roger et al 2012, Vojoudi et al 2014). Studies have demonstrated the insecticidal potential of EO of patchouli Pogostemon cablin Benth (Lamiaceae) against a range of insects (Lima et al 2013, Park & Park 2012, Pavela 2008, Swamy & Sinniah 2015, van Beek & Joulain 2018), including urban ants (Albuquerque et al 2013). Since EOs from plants exhibit high volatility, their use under natural conditions requires improved technologies. The encapsulation of EOs in nanoparticles can allow the slow and gradual release of these products resulting in a delay in their degradation (Kah & Hofmann 2014), consequently increasing their potential of use for pest control. In this study, we evaluated the formicidal activity of P. cablin and its nanoformulation on leaf-cutting ant species: Atta opaciceps (Borgmeier, 1939), Atta sexdens (Linnaeus, 1758), and Atta sexdens rubropilosa Forel, 1908 (Formicidae: Myrmicinae). The bioassays consisted in evaluating the toxicity, repellency, and walking behavior of ant individuals submitted to the action of compounds.

for approximately 140 min after the onset of boiling (Ehlert et al 2006)The analyses of the EO components were performed using a GC/MS/FID (GCMSQP2010 Ultra, Shimadzu Corporation, Kyoto, Japan) equipped with an autosampler AOC-20i (Shimadzu). Separations were accomplished using an Rtx®-5MS Restek fused silica capillary column (5%-diphenyl-95%-dimethyl polysiloxane) of 30 m × 0.25 mm i.d., 0.25 mm film thickness, at a constant helium flow rate of 1.0 mL min−1. The injection temperature was 280°C and 1.0 μL (10 mg mL−1) of sample was injected, with a slip ratio of 1:30. The oven temperature was programmed from 50°C (isothermal during 1.5 min), with an increase of 4°C min −1 –200°C, then 10°C min −1 –300°C, ending with a 5-min isothermal at 300°C. For GC/MS, molecules were ionized by ionization of electrons with energy of 70 eV. The fragments were analyzed by a quadrupole system programmed to filter fragments/ions with m/z in the order of 40–500 Da and detected by an electron multiplier. The data processing was performed with CG-MS software Postrun Analysis (Labsolutions Shimadzu). The ionization process for CG/ FID was performed by the flame from hydrogen gases 5.0 (30 mL min−1) and synthetic air (300 mL min−1). The quantification of each constituent and the electronic current generated were amplified and processed using CGMS software Postrun Analysis. The identification of constituents from EO was performed by comparison of retention index in the literature (Adams 2007). Retention indices were obtained using equation proposed by van Den Dool and Kratz (1963) in relation to a series of n-alkanes (nC8–nC31). Three libraries (WILEY8, NIST107, and NIST21) from GC-MS were also used to compare acquired mass spectra using a similarity index of 80%.

Material and Methods

The nanoformulation of EO of P. cablin was prepared by magnetic stirring using polyoxyethylene (20) sorbitan (Tween 80) (36%), ethanol (Synth, Diadema, Brazil; 99.8% of purity) (36%), EO of P. cablin (18%) as oil phase, and water (10%) as aqueous phase. The nanoformulation was maintained at room temperature for 24 h to allow its complete homogenization (El Maghraby et al 2008). The active principles contained in the EO of P. cablin present in the nanoformulation were isolated by hydrodistillation in a Clevenger-type apparatus. One milliliter of the nanoformulation was added in 2 L of water in a volumetric flask (3 L) over a boiling period of 140 min. The EO extracted was kept in amber vial (5 mL) and maintained at − 20°C. Identification and quantification of compounds were performed as described above.

Essential oil of Pogostemon cablin: obtaining and chemical analyzes Plants of P. cablin [POG (002) access] were obtained from the Germplasm Active Bank of the Universidade Federal de Sergipe (UFS), located at the experimental station “Campus Rural da UFS,” São Cristóvão, Sergipe, Brazil (11°00′ S, 37°12′ W). The average annual temperature and precipitation of the region are 27°C and 1590 mm, respectively. Leaves of the plant were collected in January 2015, during the plant development phase and dried in an oven (Marconi MA 037) at 40 ± 1°C for 4 days. The EO of the leaves of P. cablin was extracted by hydrodistillation in a Clevenger-type apparatus

Nanoformulation: obtaining, isolation, quantification of compounds, and characterization

Effect of Essential Oil and Nanoformulation on Ants

Nanoformulation: physical characterization and polarized light microscopy The nanoformulation was physically characterized in relation to micro- and macroscopic aspects: (i) polydispersity—index indicating the variation of the particle size from the mean, and (ii) microparticle diameter. The polydispersity index and particle size were determined by photon correlation spectrometry, after dilution of the sample with ultra-pure water in the ratio of 1:100 ( v :v ) i n Z e t a s i z e r N a n o s e r i e s N o n o - Z s ( M a l v e r n Instruments, Worcestershire, RU). The analyses were performed at 25°C, in triplicate. The identification of isotropy in the obtained system was performed by polarized light microscopy. For each replicate (N = 4), one drop of the formulation was transferred to a glass side, covered by cover slip. After 5 days, the samples were analyzed under Olympus BX-51 microscope equipped with Color Evolution LC (PL-A662) digital camera and PexelLINK Image Analyzer software. Collection and maintenance of leaf-cutting ants Adult workers of A. opaciceps, A. sexdens, and A. sexdens rubropilosa were collected directly from the nest, at the campus of Universidade Federal de Sergipe (UFS), SãoCristóvão-SE. Nest fragments with insects were maintained in plastic container (50 cm diameter × 20 cm height) under ambient (25–27°C, 60 ± 5% RH) conditions for 24 h before the bioassays were performed. The identification of ant species was conducted in comparison with samples from the Agricultural Entomology Lab. - UFS, where samples were deposited. Bioassays For all bioassays, the treatments used were the solution of the EO of P. cablin, its nanoformulation, and the control (acetone solvent: Panreac, UV-IR, HPLC-GPC PAI-ACS, 99.9% purity). Preliminary tests have shown that acetone does not alter the survival of ants. The bioassays were conducted at the Agricultural Entomology Laboratory of UFS, São Cristóvão-SE, Brazil. During bioassays, no water and food were offered to the ant workers. Acute toxicity: lethal concentration and lethal time Toxicity bioassays were performed by fumigation exposure. For this, seven workers were kept in a glass pot (280 mL) covered with filter paper moistened with 0.5 mL of distillated water. The experiment was conducted in a completely randomized design, with four (lethal concentration) and eight (lethal time) repetitions per treatment, for each ant species.

To determine the lethal concentrations (LC50 and LC90), concentration-response curves were performed with six to 13 concentrations starting with a 5% standard solution of the treatments. The standard solution was obtained by mixing 80 μL of the EO of P. cablin or the nanoformulation in 1520 μL of solvent (acetone). The solutions were applied to a dispenser (1 cm2 filter paper—Unifil, cod. 501.009) using a 10-μL Hamilton® microsyringe. The dispenser was fixed internally on the lid of the pot, out of reach of the ants. The pots were hermetically sealed and kept in an incubator chamber (B.O.D.—25 ± 1°C, 70 ± 5% RH and photoperiod of 12 h). Mortality evaluations were performed 48 h after insect exposure to treatments. The LC90 of each treatment was used to determine the survival curves and lethal times to kill 50% of the populations (LT 50 ), according to the procedure described above. Mortality evaluations were performed for 60 h every 2 h. Behavioral bioassays The walking behaviors of ant species were analyzed by choice (half-treated arena) and no-choice (fully treated arena) tests. In both experiments, the experimental unit consisted of an arena (glass Petri dish: 9 cm of diameter and 1.5 cm height) covered with a filter paper (Unifil, cod. 501.009). After application of the treatments (acetone [control], EO of P. cablin [0.1%], and its nanoformulation [0.1%]), the filter papers were placed in the exhaust hood for 5 min for solvent evaporation. The EO of P. cablin and its nanoformulation were diluted in acetone at concentration of 0.1%. In the center of each arena, an adult female worker was inserted. The experimental design was completely randomized with 30 arenas for each species and treatment, totaling 450 arenas in both bioassays. For the bioassay of no-choice, 0.4 mL of the solutions of each treatment was added to the filter paper. The parameters evaluated were the following: the average displacement (cm) and the walking speed (cm s−1). For the bioassay of choice, the filter paper discs were separated into two halves (treated and untreated) and fixed with double-face tape at the bottom of the arenas (N = 180). Each half was treated separately with 0.2 mL solution of the treatments and fixed to the arena after evaporation of the solvent. In the untreated half, 0.2 mL of the solvent (acetone) was applied. The parameters evaluated were as follows: the average displacement (cm), mean distance to the opposite area (cm), walking speed (cm s−1), time (s) (< 1 s) for first contact in the treated area (repellency–avoidance without contact), and time (s) spent in each area (irritability–avoidance after contact) (Cordeiro et al 2010). The movements of insects were video-recorded for 10 min without interruption using EthoVision® XT software version

Rocha et al

8.5 (Noldus Integration System, Sterling, VA) and Panasonic SD5 SuperDynamics camera (model WV-CP504), equipped with a Spacecom 1/3” 3–8-mm F1.2 lens. The videos were analyzed in a Studio 9 software (Pinnacle Systems, Mountain View, CA). Statistical analysis To determine the lethal concentrations, Probit analyses were performed to determine the concentration-mortality curves of EO of P. cablin and its nanoformulation for each species of ant after 48 h of exposure. From these curves, the lethal concentrations (LC50 and LC90) with their respective confidence intervals were obtained at 95% probability in the SAS software (PROC PROBIT SAS 2001). The LCs were compared by the criterion of not overlapping the confidence intervals (CI95) with the origin of the interval. Survival analysis (Kaplan-Meier) was performed (LIFETEST, SAS Institute 2001) to estimate the time spent for mortality of 50% of the populations (LT50). Multivariate analysis of variance (PROC GLM with MANOVA, SAS) was performed to evaluate whether there is variation of the ants’ walking behavior submitted to the different treatments. Univariate analyses of variance were performed individually for the significant parameters. The comparison of means was performed by the Fisher LSD test (PROC GLM; LSD; SAS).

required to provoke 50% of mortality on workers after 48 h of exposure varied from 1.30 to 1.45 μL L−1 for P. cablin EO and from 1.06 to 2.10 μL L−1 for the nanoformulation (Table 2). Nanoformulation was 1.3 times more toxic to A. opaciceps workers than the EO of P. cablin. For A. sexdens rubropilosa, EO was 1.6 times more toxic than the nanoformulation. However, no significant differences between EO and nanoformulation were observed for A. sexdens. Nanoformulation potentiated the toxicity of P. cablin EO. The EO present in the nanoformulation (18%) was 7.1, 5.4, and 3.4 times more toxic to workers of A. opaciceps, A. sexdens, and A. sexdens rubropilosa, respectively (Table 2).

Lethal time

Results

The survival of the workers of the ant species exposed to LC90 of EO of P. cablin and its nanoformulation was significantly reduced over time (log-rank test: χ2 = 388.3, d.f. = 8, p < 0.001; Table 3). The treated ants presented tremors, paralysis, and folding of the legs. In general, the EO of P. cablin and its nanoformulation acted quickly causing mortality in 50% of ant populations in less than or equal to 42 h (Table 3). The EO of P. cablin acted more quickly on A. opaciceps and A. sexdens compared to its nanoformulation. The opposite was observed for A. sexdens rubropilosa, which showed the lowest LT50 (28.6 h) when exposed to nanoformulation.

Characterization of EO of P. cablin and the prototype of nanoformulation

Walking behavior in arenas with no chance of choice

A total of 14 compounds (95.77% of total composition) were identified in the EO of P. cablin, which consisted of nonoxygenated (45.28%) and oxygenated sesquiterpenes (50.49%) (Table 1). Patchoulol (44.30%) was the major compound, followed by α-bulnesene (13.75%), α-guaiene (10.87%), and seychellene (7.97%) (Table 1). The chemical composition of the nanoformulation showed a variation of 10.5% in relation to EO. Exceptions were observed for α-humulene (0.45%) that was not detected in the nanoformulation and for β-patchoulene, which showed an increment of 146% in the nanoformulation (Table 1). The nanoformulation of EO presented droplets with an average size of 42.39 mm, polydispersity index of 0.768, and pH of 6.13. Lethal concentration The EO of P. cablin and its nanoformulation showed insecticidal activity by fumigation on workers of A. opaciceps, A. sexdens, and A. sexdens rubropilosa. The concentration

The walking behavior in arenas totally treated varied among ant species (Wilks’ lambda = 0.503, F4,520 = 53.26, p < 0.001), treatments (Wilks’ lambda = 0.758, F4,520 = 19.27, p < 0.001), and with the interaction between ant species × treatment (Wilks’ lambda = 0.855, F4,520 = 5.30, p < 0.001). The total displacement and walking speed were significantly affected by ant species (F2,261 = 81.67, p < 0.001 and F2,261 = 69.20, p < 0.001, respectively), treatments (F2,261 = 15.94, p < 0.001 and F2,261 = 37.81, p < 0.001, respectively), and by the interaction between ant species × treatment (F4,261 = 5.72, p < 0.001 and F4,261 = 10.16, p < 0.001, respectively). Representative trails of walking behavior of ant species in arenas totally treated are shown in Fig 1. The workers of A. sexdens and A. sexdens rubropilosa walking at higher velocity were compared to the workers of A. opaciceps in the control treatment (Fig 2a). The displacement and walking speed of workers of A. opaciceps and A. sexdens rubropilosa were reduced in the arenas totally treated when compared to control. The behavior of workers of A. sexdens did not change (Fig 2a, b).

Effect of Essential Oil and Nanoformulation on Ants Table 1 Chemical composition of the essential oil of Pogostemon cablin and its nanoformulation containing 18% of the essential oil of Pogostemon cablin.

RIa

Concentration (%)b

Compound

Essential oil of P. cablin

Nanoformulation (18%)

β-Patchoulene β-Elemene

1.81 ± 0.002 0.79 ± 0.008

4.44 ± 0.007 0.82 ± 0.002

1413

Cycloseychellene

0.60 ± 0.002

0.49 ± 0.005

1420 1436

β-Caryophyllene α-Guaiene

1.85 ± 0.008 10.87 ± 0.05

1.66 ± 0.003 9.05 ± 0.004

1449 1455

Seychellene α-humulene

7.97 ± 0.006 0.45 ± 0.005

7.91 ± 0.007 –

1461

allo-Aromadendrene

4.58 ± 0.011

4.18 ± 0.007

1497

Acefylline

2.60 ± 0.02

2.06 ± 0.008

1506

α-Bulnesene

13.75 ± 0.013

11.75 ± 0.006

1568

Longicanfenilona

0.96 ± 0.003

1.01 ± 0.005

1589

Caryophyllene oxide

2.50 ± 0.005

2.46 ± 0.006

1662 1678

Pogostol Patchoulol

2.73 ± 0.012 44.30 ± 0.02

2.66 ± 0.02 47.30 ± 0.005

Non-oxygenated sesquiterpenes (%) Oxygenated sesquiterpenes (%)

45.28 50.49

42.35 53.43

Total detected (%)

95.77

95.78

1382 1387

a

Retention index calculated using the equation by Van den Dool and Kratz (1963) in relation to a homologous series of n-alkanes (nC9-nC18).

b

Values (± SEM) of the content of the compounds obtained from three different measurements.

Walking behavior in arenas with choice Significant differences were observed in the walking behavior of the workers of A. opaciceps (Wilks’ lambda = 0.669, F5,114 = 11.30, p < 0.001), A. sexdens (Wilks’ lambda = 0.282, F5,114 = 58.15, p < 0.001), and A. sexdens rubropilosa (Wilks’ lambda = 0.428, F5,114 = 30.43, p < 0.001) between treated and untreated arenas (Figs 3 and 4). The total displacement and walking speed were significantly different among workers of A. opaciceps (F1,116 = 21.73, p < 0.001 and F1,116 = 6.13, p = 0.015, respectively), A. sexdens (F 1,116 = 138.78, p < 0.001 and F 1,116 = 4.56; p = 0.035, Table 2 Toxicity of the essential oil of Pogostemon cablin and its nanoformulation (18% of essential oil of Pogostemon cablin) on workers of Atta opaciceps, Atta sexdens, and Atta sexdens rubropilosa after 48 h of exposure by fumigation.

Treatment

respectively), and A. sexdens rubropilosa (F1;116 = 59.79, p < 0.001 and F1,116 = 18.41, p < 0.001, respectively). For A. sexdens rubropilosa, the displacement and walking speed were significantly affected by treatments (F1,116 = 11.36, p < 0.001 and F1,116 = 5.73, p = 0.018, respectively); however, these parameters were not altered for A. opaciceps (F1,116 = 0.03, p = 0.869 and F1,116 = 2.82, p = 0.096) and A. sexdens (F1,116 = 0.11, p = 0.729 and F1,116 = 0.80, p = 0.288). The total displacement and walking speed were not affected by the interaction between area × treatments, for A. opaciceps (F1,116 = 3.33, p = 0.071 and F1,116 = 2.97, p = 0.088), A. sexdens (F 1,116 = 0.12, p = 0.729 and F 1,116 = 1.14, p = 0.288), and

No. of insects

LC50 (CI 95%) (μL L−1)

LC90 (CI 95%) (μL L−1)

Slope

χ2

p value

305 450 253

1.36 (1.25–1.47) 1.45 (1.40–1.49) 1.30 (1.15–1.45)

2.54 (2.28–2.91) 1.78 (1.70–1.88) 2.91 (2.57–3.40)

4.70 14.32 3.66

1.28 1.16 1.74

0.53 0.56 0.58

250 250 378

1.06 (0.94–1.17) 1.48 (1.31–1.66) 2.10 (2.01–2.20)

2.43 (2.14–2.86) 4.05 (3.43–5.06) 3.11 (2.87–3.50)

3.53 2.93 7.55

2.90 4.11 3.43

0.23 0.12 0.17

Essential oil of P. cablin A. opaciceps A. sexdens A. sexdens rubropilosa Nanoformulation (18%) A. opaciceps A. sexdens A. sexdens rubropilosa

μL L−1 , microliters of treatments (EO of P. cablin or nanoformulation) per liter of air (glass vial of 0.28 L)

Rocha et al Table 3 Lethal time (h) (confidential intervals 95%) of control, essential oil of Pogostemon cablin, and its nanoformulation (18% of essential oil of Pogostemon cablin) on workers of Atta opaciceps, Atta sexdens, and Atta sexdens rubropilosa. Treatment

Control

Essential oil of P. cablin

A. opaciceps

56.67 (54.88–58.47)

30.46 (26.79–34.13)

39.82 (35.28–44.36)

A. sexdens A. sexdens rubropilosa

55.12 (53.24–57.00) 55.89 (53.96–57.81)

34.14 (30.19–38.08) 40.17 (36.27–44.07)

42.00 (39.00–44.99) 28.60 (23.89–33.31)

A. sexdens rubropilosa (F1,116 = 0.03; p = 0.864 and F1,116 = 0.49, p = 0.487). The mean displacement walked to the opposite area of the arena was similar between the treated and untreated areas for A. opaciceps (areas: F1,116 = 0.04, p = 0.848; treatment: F1,116 = 0.01, p = 0.930; and interaction area × treatment: F1,116 = 0.32, p = 0.572), A. sexdens (areas: F1,116 = 1.26, p = 0.264; treatment: F1,116 = 1.67, p = 0.199; and interaction area × treatment: F1,116 = 0.39, p = 0.532), and A. sexdens rubropilosa (areas: F1,116 = 0.85, p = 0.358; treatment: F1,116 = 0.11, p = 0.740; and interaction area × treatment: F1,116 = 0.06, p = 0.813). The time for first contact (repellency) between the treated and untreated areas did not differ for any of the ant species: A. opaciceps (areas: F1,116 = 1.55, p = 0.216; treatment: F1,116 = 1.44, p = 0.232; and interaction area × treatment: F1,116 = 0.16, p = 0.686), A. sexdens (areas: F1,116 = 2.12, p = 0.148, treatment: F 1,116 = 0.24, p = 0.624; and

Fig 1 Most representative trails of displacement of workers of Atta opaciceps, Atta sexdens, and Atta sexdens rubropilosa during 10 min in arenas totally treated with acetone (control), essential oil of Pogostemon cablin, and its nanoformulation (18%).

Nanoformulation (18%)

interaction area × treatment: F1,116 = 0.42, p = 0.518), and A. sexdens rubropilosa (areas: F1,116 = 3.01, p = 0.084; treatment: F1,116 = 0.43, p = 0.514; and interaction area × treatment: F1,116 = 0.06, p = 0.801). Most representative trails of walking behavior in the three studied ant species in arenas half-treated are shown in Fig 3. The ant species walked less and at a higher velocity in the treated area (Fig 4a). For A. sexdens rubropilosa, this effect was more pronounced for the EO of P. cablin than for nanoformulation. Significant differences were observed in the time spent in each area of arena (treated or untreated) for A. opaciceps (F 1,116 = 27.67, p < 0.001), A. sexdens (F 1,116 = 276.60, p < 0.001), and A. sexdens rubropilosa (F1,116 = 146.29, p < 0.001) (Fig 5). There was no influence of the interaction between area × treatment for A. opaciceps (F1,116 = 6.81, p = 0.010), A. sexdens (F1,116 = 0.18, p = 0.671), and A. sexdens

Effect of Essential Oil and Nanoformulation on Ants

(a)

Displacement (cm)

3000

2000

Control EO of P. cablin Nanoformulaon

Aa

Aa

a a

b

b

b

b

Ba b

b

1000

Fig 2 Displacement (a) and walking speed (b) (± standard error) of workers of Atta opaciceps, Atta sexdens, and Atta sexdens rubropilosa exposed by contact in arenas totally treated with essential oil of Pogostemon cablin (0.1%) and its nanoformulation (18%). Histograms with the same capital letter, for comparison among species in the control treatment, and lowercase, for comparison among treatments, do not differ among themselves by Fisher’s LSD test at p < 0.05.

Fig 3 Most representative trails of displacement of workers of Atta opaciceps, Atta sexdens, and Atta sexdens rubropilosa during 10 min in arenas with half-treated with essential oil of Pogostemon cablin or its nanoformulation (18%) (right side). On the left side, only acetone was applied.

(b)

8

Velocity (cm s-1)

0

6

Aa Ba

a

a

Ca 4

b

b

2

0 A. opaciceps

A. sexdens

Species

A. sexdens rubropilosa

Rocha et al EO of P. cablin

EO of P. cablin + nanoformulaon

(a)

1000

a Untreated area Treated area

Displacement (cm)

Nanoformulaon

aA

750

aA

500

bA a

b bB b

250

0

(b)

Fig 4 Displacement (a) and walking speed (b) (± standard error) of workers of Atta opaciceps, Atta sexdens, and Atta sexdens rubropilosa exposed by contact in arenas with halftreated with essential oil of Pogostemon cablin or its nanoformulation (18%) in the CL90 of the toxicity bioassays. Histograms with the same capital letter, for comparison among treatments in the same area (Atta sexdens rubropilosa), and lowercase, for comparison between treated and untreated areas, do not differ among themselves by Fisher’s LSD test at p < 0.05.

a b

Velocity (cm s-1)

2,5

bA bB

2,0 a

1,5 b

1,0 0,5 0,0

A. opaciceps

A. sexdens

A. sexdens rubropilosa

Species

rubropilosa (F1,116 = 2.99, p = 0.087). The time spent in each area of arena was similar among treatments for A. opaciceps

(F1,116 = 0.01, p = 0.970), A. sexdens (F1,116 = 0.01, p = 0.944), and A. sexdens rubropilosa (F1,116 = 0.01, p = 0.958).

* n.s. Fig 5 Irritability (%) (± standard error) of workers of Atta opaciceps, Atta sexdens, and Atta sexdens rubropilosa exposed by contact in arenas with halftreated with essential oil of Pogostemon cablin (0.1%) and its nanoformulation (18%). * indicates significant differences between areas by Fisher’s LSD test at p < 0.05. n.s. not significant.

aA

aA

A. opaciceps

Nanoformulaon

EO of P. cablin + nanoformulaon

A. sexdens rubropilosa

*

80

EO of P. cablin

A. sexdens

*

100

A. opaciceps

60

40

20

Time in untreated area (%)

0

20

40

60

80

Time in treated area (%)

100

Effect of Essential Oil and Nanoformulation on Ants

The species A. sexdens and A. sexdens rubropilosa remained most of the time on the side not treated with the EO of P. cablin and its nanoformulation (Fig 5). However, A. opaciceps remained less time on the side treated with EO of P. cablin and showed no differences between the untreated side and the side treated with the nanoformulation (Fig 5).

Discussion The essential oil of P. cablin and its nanoformulation showed lethal and sublethal effects on leaf-cutting ants of the genus Atta, being potential formicides for the management of these pests. The insecticidal activity of EO of patchouli on urban ants has been already demonstrated (Albuquerque et al 2013); however, this is the first study to show the potentiation of nanoformulation in the toxicity of EO on ants. The main compounds found in the EO of P. cablin showed similar values obtained in other studies (Albuquerque et al 2013, Liu et al 2015). On the other hand, the EO of P. cablin present in the nanoformulation showed small variations in its chemical composition. The increase of β-patchoulene content in the nanoformulation may have been caused by oxidation of its precursor compounds, which are present in smaller proportions in the EO. Since α-humulene originates from a common intermediate compound of patchoulou, its non-detection in the nanoformulation may be due to its oxidation in the presence of water (Chen et al 2014, Deguerry et al 2006), which resulted in increased patchoulol. The same may occur with other compounds which had their reduced concentrations in the nanoformulation (seychellene, α-guaiene, and αbulnesene) and which also have the same patchoulol intermediates (Chen et al 2014, Deguerry et al 2006). The rapid mortality and symptoms (tremors, paralysis, and folding of the legs) showed by the different species of ants when in contact with the EO of P. cablin and its nanoformulation suggest that these compounds present neurotoxic activity, acting directly on target sites in the nervous system of these insects (Zhu et al 2003). In fact, the mechanisms of insecticidal bioactivity of sesquiterpenes present in the EOs include the following: interference in calcium channels modulated by gamma-aminobutyric acid (GABA) (Isman 2006); action in vulnerable sites such as cytochrome P450 (Bacci et al 2007); inhibition of acetylcholinesterase (López & Pascual-Villalobos 2010); and interference in the action of octopamine (neuromodulator found in invertebrates) (Rattan 2010). Although the nanoformulation presents only 18% of the EO of P. cablin, the nanoformulation potentiated the effect of EO. The reduced particle size of the nanoformulation increases its contact surface enhancing absorption and interaction with biological tissues. In addition, the particles have greater mobility and are released gradually resulting in increased systemic activity (Liu et al 2008, Nel et al 2006, 2009, Oliveira et al 2014, Weiss et al 2009, Yang et al

2009). The A. sexdens rubropilosa was the species more tolerant to nanoformulation, which may be associated with a higher efficiency of their detoxification enzymes or a lower sensitivity of the target site (Bacci et al 2007, 2009). Sublethal effects of EO of P. cablin and its nanoformulation were observed even at low concentration (0.1%). In fact, when individuals had a choice, individuals of the three ant species preferred the untreated side of arena (greater displacement and longer time spent) and increased their walking velocity in the treated area to avoid the compounds. Thus, these results show that although the treatments were not repellent to the ants, they caused irritability, since the individuals remained most of the time on the untreated area of arena, avoiding the compounds when they had a choice. Many active ingredients used to control ants have been banned or restricted because they are classified as persistent organic pollutants. In the recent years, the use of products based on EO has been considered a viable and safe solution in relation to environmental impact, human health, and the absence of deleterious effects on non-target organisms. A viable alternative for the use of products based in EO is the nanoformulation, which allows a greater stability of the compounds. Thus, the nanoformulation of the EO of P. cablin can be considered as a potential alternative, since it allows a slower and continuous release of the active compounds directly in the nest. Such compounds could compromise the viability of the colonies by immediate action on workers or even by promoting change in the collective behavior of the workers (Casida & Durkin 2013, Gilbert & Gill 2010, Guedes et al 2016). Studies indicate that the control of leaf-cutting ants can occur both via toxicity of colony individuals and the symbiotic fungus (Castaño-Quintana et al 2013, Ribeiro et al 2008). Since EOs from plants have shown fungicidal activity, it can be hypothesized that EO of P. cablin also influences the reduction of leaf-cutting ants via effect on fungal mortality. In conclusion, the lethal and sublethal effects of the EO of P. cablin and its nanoformulation highlight the potential of these compounds as a formicide. Author Contribution AGR, APAA, and LB conceived the study. AGR, BMSO, and CRM performed experiments and analyzed the data. TSS, AFB, ADL, and RSN performed the chemical analyses. AGR, APAA, PFC, and LB wrote the paper. All authors read and approved the manuscript. Funding InformationThis study was funded by the Brazilian National Council for Scientific and Technological Development (CNPq), the Foundation for Research Support and Technological Innovation of the State of Sergipe (FAPITEC/SE), and the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES).

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