Induced volatiles in the interaction between soybean (Glycine max) and the Mexican soybean weevil (Rhyssomatus nigerrimus)

The present study analyzed the volatile compounds emitted by Glycine max (cv. FT-Cristalina-RCH) soybean plants: healthy plants and plants damaged mechanically or by the Mexican soybean weevil Rhyssomatus nigerrimus. The SPME method was used to compare the volatile profile of soybean plants in four different conditions. The volatile profile of G. max plants infested by R. nigerrimus was qualitatively and quantitatively different from that of healthy and mechanically damaged plants. Emission of 59 compounds was detected in the four treatments. Of these compounds, 19 were identified by comparison of the Kovats index, mass spectrum and retention times with those of synthetic standards. An increase in concentration of the volatiles (Z)-3-hexenyl acetate and the compound 1-octen-3-ol was observed when the soybean plants were mechanically damaged. The compounds mostly produced by the soybean plant during infestation by male and female R. nigerrimus were 1-octen-3-ol, 6-methyl-5-hepten-2-one, (E)-β-ocimene, salicylaldehyde, unknown 10, linalool, methyl salicylate, (Z)-8-dodecenyl acetate (ester 5), ketone 2 and geranyl acetone. Behavioral effects of the identified compounds during the insect-plant interaction and their conspecifics are discussed.


Introduction
Plants produce and emit volatile compounds (VC) that are released by flowers, fruits and vegetative tissues (Marín and Céspedes, 2007). Generally, these substances are made of terpenes derived from fatty acids and aromatic compounds. Plant volatiles act as signals for other organisms and for the plant itself. They can also be exported and modify the surroundings of the plant species that produce them, its neighbors and its enemies (Rostás and Eggert, 2008). The main functions of VC in plants are to attract pollinators, seed dispersers, and natural enemies of herbivores; to be intraspecific and interspecific messengers; and to defend the plants by repelling insects or detaining colonization of pathogenic bacteria and fungi (Byers et al., 2014). VC can also be used by herbivores as cues to direct their flight to host plants (Visser, 1986).
VC are classified into two types according to the way in which they are released: constitutive and induced. Constitutive VC are those inherent to the plant, while induced VC are emitted by plants in response to a stimulus caused by mechanical damage or damage by herbivore insects to plant tissues (Sánchez and Délano, 2003). When the latter type of damage occurs, the emitted compounds are called herbivore-induced plant volatiles (HIPV) (Vivanco et al., 2005;Rostás and Eggert, 2008).
The volatiles induced by herbivores are emitted at the damaged site as well as in undamaged tissues (War et al. 2011). The plant uses these metabolites as chemical defense; outstanding of these metabolites are toxins, repellents, anti-feeding compounds and digestibility reducers (Moraes et al., 2000). Recent studies indicate that compounds released by a plant vary with species, plant variety and herbivore species (Michereff et al., 2011).
This study focuses on volatiles induced by herbivores that attack soybean Glycine max L. (Fabales: Fabaceae), which is cultivated worldwide and is economically important for production of oil and biodiesel (FAO, 2017). In the state of Chiapas, Mexico, soybean cultivation has extended over an area of 130 ha with a production of 273 t of soybeans and yield of 2.11 t/ha (SIAP, 2017).
In the Mexican state of Tamaulipas, San Luis Potosí, Veracruz and Chiapas, the major economically important pest of soybeans is the Mexican soybean weevil Rhyssomatus nigerrimus Fahraeus 1837 (Coleoptera: Curculionidae) (Terán-Vargas and López-Guillén, 2014). Cortés (2016) reported that this insect is attracted by volatiles emitted by the soybean plant. However, differences in the VC profile have been observed between healthy plants and plants attacked by the weevil (Cruz-López, unpublished data).
Although the volatile profile of soybean plants damaged by other pests is known, there are no reports regarding the curculionid R. nigerrimus. Therefore, the objective of this study was to identify the volatile compounds produced in leaves and pods of the soybean plant when it is attacked by R. nigerrimus, an insect considered a key pest of soybean crops in the Soconusco region, Chiapas. The knowledge generated in this study will provide useful information for future biological tests on attraction between plant and insect, which will eventually favor development of attractants for control and monitoring of the soybean weevil.

Biological material
R. nigerrimus adults were collected manually from soybean crops in the Ejido el Manzano in the municipality of Tapachula, Chiapas, every week for four months. These specimens were stored in plastic recipients covered with organdy fabric for later transfer to the chemical ecology insectary of El Colegio de la Frontera Sur (ECOSUR). In the laboratory, the collected insects were separated by sex using a stereoscopic microscope following the method proposed by López-Guillén et al. (2016). Female and male R. nigerrimus were kept separately in plastic containers and fed sweet potato slices (Ipomoea batatas L.). The insects were kept in the ECOSUR insectary at a temperature of 25±2 ºC and relative humidity (RH) of 70±5% with a 12:12 h light:dark photoperiod.
The soybean plants (cv. FT-Cristalina-RCH) used in the experiments were planted in pots containing Miracle-Gro  substrate and watered daily. The experiment was conducted in a room with controlled climate (25±2 °C, 60±10% RH). The pods used in the experiments were obtained from the soybean plants grown to the R6 state.

Sampling of volatiles in the laboratory
Soybean plant emitted compounds were collected by solid phase microextraction (SPME). Four treatments were set up. The first treatment consisted of introducing 30 adult R. nigerrimus females or males into a 125 mL Erlenmeyer flask with a soybean plant. In the second treatment 30 adult R. nigerrimus males were introduced into the flask without soybean plant. The third treatment a flask held a soybean plant with mechanical damage in the trifolio with a 1 mm diameter, 250 mm long entomological needle. The fourth treatment a control flask held a healthy soybean plant. Soybean pods emitted compounds were collected by SPME. Four treatments were set up. In the first treatment 30 adult females or males of R. nigerrimus were added into a 10 mL vial containing 12 grams of soybean pods, in the second treatment 30 adult males of R. nigerrimus were placed in the vial without pods, the third treatment consisted of a vial containing 12 grams of pods mechanically damaged with an entomological needle of 1 mm in diameter and a length of 250 mm, and the fourth treatment was a vial containing 12 grams of healthy soybean pods used as a control. Six repetitions were performed for each treatment.
The entrance of the flask or vial was covered with aluminum foil and the SPME device containing the 65 µm polydimethylsiloxane-divinylbenzene (PDMS/DVB) (Supelco  , Toluca, Mexico) fiber was introduced. The fiber had been previously heated to 250 ºC for 5 min in the gas chromatograph (GC) to prevent possible contamination by volatiles from the previous sample absorbed in the fiber. The PDMS/DVB fiber was left for 24 h to absorb the volatiles, after which time it was removed. The volatile capture room was at 25±2 °C and 50 to 60% RH. Illumination was provided by four 39 W fluorescent lamps located 3 m above the volatile collection devices.

Chemical analysis
Desorption of the captured volatile compounds were desorbed inside the injection port of the gas chromatograph for 1 min at 250 ºC. The volatile compounds captured by SPME were identified chemically in a gas chromatograph (Varian CP/3800) coupled to a mass spectrometer (Varian Saturn 2200), using a cast non-polar SPB-1 capillary column 30 m long, 0.25 mm interior diameter (Supelco  , Toluca, Mexico). Analysis was performed with a temperature ramp beginning with an initial temperature of 50 ºC (for 2 min), increasing 15 ºC every min until reaching 280 ºC held for 10 min. Helium was the carrier gas and injector temperature was 250 ºC. Ionization was carried out by electron impact at 70 eV. The compounds were identified by comparing the retention index, mass spectra and retention times with those of synthetic standards. Other compounds were tentatively identified based on comparison with spectra from the National Institute of Standards and Technology (NIST) library, version 2.0.

Statistical analysis
Data were analyzed using the classification type random forest multivariate technique (Liaw & Wiener, 2002) with R software (R Development Core Team) to determine associations among the compositions of induced volatiles and to compare the treatments applied to soybean plants and pods.

Results
SPME analysis of the samples by gas chromatography-mass spectrometry shows that the volatile compounds emitted by soybean plant and pods include aromatics, esters, terpenes, ketones, alcohols and aldehydes (Tables 1 and 2).

Discussion
This study revealed that the volatile profile of healthy soybean plants differs from the volatile profiles of plants damaged mechanically and by R. nigerrimus, both qualitatively and quantitatively. Of the 19 identified compounds, 12 had not been reported in previous studies on volatiles emitted by soybean (Liu et al., 1989;Boué et al., 2003;Zhu and Park, 2005;Cai et al., 2015;Cortés, 2016).
Regarding quantitative differences, the results of this study demonstrated that four of the compounds produced by plants infested by females ((E) β-ocimene, linalool, methyl salicylate and α-copaene) were produced in greater proportion than by plants in the other treatments and the control. Recent     studies report that the reason that volatiles such as linalool and methyl salicylate in plants increase is because they are HIPVs produced in response to herbivory (War et al., 2011;García, 2017). Emission of these compounds has also been detected in Capsicum spp. and Camellia sinensis, host plants of chili and tea weevils, respectively. Studies on these insect-plant interactions have demonstrated that induced volatiles, such as benzylic alcohol, (Z)-3-hexenal, myrcene, benzaldehyde and γ-terpinene, attract conspecific weevils of both sexes, while linalool and (Z)-3-hexenyl acetate attracts only females, and (E)-β-ocimene attracts only males (Muñiz-Merino et al., 2014;Sun et al., 2010). This suggests that the soybean weevil can respond similarly to these volatiles. It has been reported that (E)-β-ocimene is a volatile commonly released by leaves and flowers of diverse plant species, inducing plant defense response and establishing tritrophic interactions as well as promoting mating and oviposition of insect pests (Dicke and Baldwin, 2010;Farré-Armengol et al., 2017;Tang et al., 2016).
Under the condition of infestation by females and males of the black soybean weevil, salicylaldehyde and  2-octanone were detected. These compounds had not been previously reported in soybean plants (Liu et al., 1989). Possibly, salicylaldehyde was detected because it is a precursor of methyl salicylate (Asghari & Mosaeybi, 2009), which was also detected in the insect-plant treatments. Also, 2-octanone is a volatile compound that is not produced by plants; it is possibly produced by the weevils while they feed as a signal for other insect species and/or their conspecifics (Meiners et al., 2003). The presence of methyl salicylate and (Z)-3-hexenyl acetate has been reported in the soybean varieties London and Davis (Boué et al., 2003;Moraes et al., 2008). Dong et al. (2011) report that Camellia sinensis plants emit numerous volatile compounds, such as (Z)-3-hexen-1-ol, linalool, α-farnesene, benzylic nitryl, indol, nerolidol, and ocimene, in high concentrations as a defense response to herbivore attack. They also underline that, for identification of induced volatile compounds, not only is it necessary to consider qualitative differences but also quantitative differences compared with the control without damage since, depending on the concentration in which these metabolites are found, they will cause an effect on the behavior of conspecifics and of parasitoids.
The study conducted by Moraes et al. (2008) indicates that (Z)-3-hexenyl acetate is produced when vegetative tissue is damaged by herbivory. Some compounds produced by soybeans are considered components of plant defense against herbivores are (Z)-3-hexenyl acetate, farnesene, caryophyllene and humulene (Cai et al., 2015). In our study, we observed that the soybean plant (cv FT-Cristalina-RCH) emitted higher concentrations of (Z)-3-hexenyl acetate when damaged mechanically than when damaged by insects ( Figure 2). This difference in emission of volatiles depends on both the physiological state of the soybean plant and the variety of soybean when it is attacked by an insect pest (Liu et al., 1989;Rostás and Eggert, 2008).
However, this study should be complemented with additional experiments to determine the total compounds present in this variety of soybean as well as their effect on the insect pest and on natural enemies.