Bioactivity of the Cymbopogon citratus (Poaceae) essential oil and its terpenoid constituents on the predatory bug, Podisus nigrispinus (Heteroptera: Pentatomidae)

Podisus nigrispinus Dallas (Heteroptera: Pentatomidae), released in biological control programs, is a predator of Lepidopteran and Coleopteran species. Lemongrass essential oil and its constituents can be toxic to this natural enemy. The major constituents of lemongrass essential oil are neral (31.5%), citral (26.1%), and geranyl acetate (2.27%). Six concentrations of lemongrass essential oil and of its citral and geranyl acetate constituents were applied to the thorax of P. nigrispinus nymphs and adults. The walking and respiratory behavior of the P. nigrispinus third-instar nymphs, treated with citral and geranyl acetate at the LD50 and LD90 doses, were analyzed with video and respirometer. The lemongrass essential oil toxicity increased from first- to fifth-instar P. nigrispinus nymphs. The P. nigrispinus respiration rates (μL de CO2 h−1/insect) with citral and geranyl acetate in the LD50 and LD90 differed. Nymphs exposed to the lemongrass essential oil and its constituents on treated surfaces presented irritability or were repelled. Podisus nigrispinus adults were tolerant to the lemongrass essential oil and its constituents, geranyl acetate and citral. The altered respiratory activity with geranyl acetate and the fact that they were irritated and repelled by citral suggest caution with regard to the use of the lemongrass essential oil and its constituents in integrated pest management incorporating this predator, in order to avoid diminishing its efficiency against the pests.

Predatory insects play an important role in insect communities, and are used in biological control to reduce herbivorous arthropod populations 1,2 . The predatory bug, Podisus nigrispinus Dallas (Heteroptera: Pentatomidae) can control Lepidopteran and Coleopteran pest species which prey on agricultural crops and forest plantations in the Americas [3][4][5] . The biology and ecology of P. nigrispinus, including its development, morphology 6 , predator-prey interaction 4 , and feeding strategies such as extraoral digestion 1 have been studied. This insect is reared in the laboratory and released in biological control programs in cotton 7 , soybean 4 , and tomato 8 crops.
Synthetic insecticides may induce resistance in insects 9 , cause toxic reactions in mammals 10 and other non-target organisms such as parasitoids, pollinators, and predators [11][12][13] , and may also leave residues 14 and cause environmental pollution 15 . Exposure to insecticide causes adverse effects on the development, longevity and fecundity, and may alter behavior related to mobility and feeding [16][17][18] . The search for safer insecticides for human health and the environment has resulted in the development of specific compounds for pests which are selective 1 19,20 . In this sense, effective use of P. nigrispinus in integrated pest management (IPM) programs depends on the compatibility of the predator with the other control methods being employed 21 .
Plant essential oils represent an alternative for pest control with low pollution and quick degradation in the environment, making them suitable for managing insects even in organic farming [22][23][24] . Plant essential oils are volatile substances, mainly composite mixtures of terpenoids which are used for their aromatic qualities. In plants, terpenoids are products of secondary metabolism and are found in glandular hairs or secretory cavities of the plant cell wall in bark, flowers, fruits, leaves, roots and stems 25 . Essential oils and their constituents cause lethal and sublethal effects on insects, such as biocide activity, infertility, irritability, phagoinhibition and repellency 23,26,27 . Essential plant oils can control pests 22,23,26,27 .
Lemongrass, Cymbopogon citratus (DC. Stapf.), a plant native to India and Sri Lanka 28 , has antifungal 29 , anti-inflammatory 30 and anti-protozoa 31 properties. Predatory insects have a tolerance in relation to essential oils, which emphasizes the importance of the potential success of these natural enemies in IPM programs 24 . Essential oils have been shown to possess toxic effects against lepidopteran pests such as Euprosterna elaeasa Dyar (Limacodidae) 32 , Spodoptera exigua Hübner 33 and Trichoplusia ni Hübner (Noctuidae) 34 , and these insects are natural prey of the P. nigrispinus in Brazilian agricultural crops. However, the lethal and sublethal effects caused by essential oils have also been demostrated on this predatory bug 35 . Podisus nigrispinus is a predator of defoliating pests in different crop systems, but the action of essential oils as insecticide on this natural enemy of those pests needs further studies in order to avoid harming this natural ally.
The objective of this study was to evaluate the lethal and sublethal effects of lemongrass essential oil and its terpenoid constituents (geranyl acetate and citral) on P. nigrispinus.

Results
Lemongrass essential oil toxicity test. Lethal doses of the lemongrass oil increased from first to fifth instars with LD 50 of 1.08 to 139.30 μg/insect −1 and LD 90 of 2.02 to 192.05 μg/insect −1 . The LD 50 and LD 90 of the lemongrass for third instar P. nigrispinus nymphs was 21.58 and 28.35 μg/insect −1 , respectively. Mortality was always <1% in the control ( Table 1).

Discussion
The increase in the lethal doses (LD 50 and LD 90 ) of the lemongrass essential oil from first to fifth instars of P. nigrispinus suggests that this predator progressively developed a tolerance as it matured. The toxicity of this essential oil is similar to that reported in other studies for this insect 36 and for other predators 37,38 , showing a relatively favorable safety profile for P. nigrispinus, whether through direct use of the oil or by the individual components of the oil serving as precursors for the synthesis of active ingredients of new selective insecticides. Insects may present selectivity mechanisms such as reduction of insecticide penetration through the cuticle, or  www.nature.com/scientificreports www.nature.com/scientificreports/ site insensitivity and/or detoxification or metabolization of the insecticide by enzymes 39 to reduce the effect on acetylcholinesterase 40 or inhibition of octopamine receptors 41 . Comparing the contact toxicity of lemongrass essential oil on developmental of P. nigrispinus nymphs showed that the first and second instar were more susceptible followed by the third, fourth and fifth instars; this indicates that high quantities of the lemongrass essential oil are toxic in the early stages of this insect, and that they become more tolerant with age.
The chemical composition of lemongrass essential oil revealed 13 constituents, identified and quantified. Neral, citral, nonan-4-ol, camphene, 6-metil-hept-5-en-2-one, and citronelal were the main compounds that were detected, according to previous reports on terpenoids obtained from lemongrass essential oil [42][43][44] . However, variations in the abundance of the constituents was observed, including geranial as its main compound [42][43][44] , depending on the extracted organ, plant age, geographical area of the collection and extraction method 45,46 . Terpenoids are frequently found in plants, where they play numerous vital roles in plant physiology as well as important functions in all cellular membranes 47 . Also, the defensive role in plants containing simple terpenoids has been demonstrated, as well as more complex compounds 48 . In this study, terpenoids are the most abundant constituents of lemongrass essential oil, but the relative proportions of the constituents with insecticide potential 45 can vary.
The low toxicity of citral or geranyl acetate for P. nigrispinus may be related to the cuticle of this insect, which acts as a barrier, as reported for Bombyx mori Linnaeus (Lepidoptera: Bombycidae) exposed to deltamethrin 49 . Cuticular lipids prevent the desiccation and penetration of xenobiotics into insects 50 , as well as promoting thickening and cuticle composition that can delay the penetration of insecticidal molecules into the body of the insect 51 , thus reducing the essential oil effect post-application due to rapid degradation or evaporation in the environment 52 . The lack of detoxifying enzymatic activity (inhibitors of cytochrome P450s, esterases or glutathione S-transferases) 53 was observed in Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) treated with citral, which penetrated through the cuticular layer 54 . Toxic constituents can affect multiple regions of the insect body, causing necrotic areas which increase progressively throughout the entire insect body 26,55 . One possible explanation for the low toxicity caused in P. nigrispinus is that there may be differences in the penetration rate of the lemongrass constituents into the body, coupled with the ability of this insect to rapidly detoxify.
The terpenoid constituents of lemongrass essential oil had a negative effect on the P. nigrispinus respiration rate. The reduction of the respiratory rate of third-instar P. nigrispinus nymphs after exposure to citral and geranyl acetate may be due to muscle paralysis, disruption of oxidative phosphorylation processes and dysregulation of the breathing activities 18,22,26,56 . In this study, P. nigrispinus nymphs exposed to the terpenoid constituents of the essential oil developed low respiration rates, which further unbalanced the organism physiology, as described for Sitophilus granarius Linnaeus (Coleoptera: Curculionidae) 57 and Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) 55 .
The short distance traveled in the arenas towards the opposite side from the geranyl acetate by P. nigrispinus nymphs suggest repellent activity. Various insect pests show altered behavioral responses when exposed to lemongrass essential oil constituents, as reported for Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) 58 , Culex quinquefasciatus Say (Diptera: Culicidae) 59 , and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) 60 , influencing the olfactory orientation and insect walking behavior. Insects can identify the presence of compounds, as reported for Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), after the activation of olfactory receptors in the presence of geranyl acetate 61 . The results indicate that P. nigrispinus exhibits behavioral avoidance by means of repellence to geranyl acetate, minimizing contact with insecticide-contaminated surfaces. In contrast, nymphs of P. nigrispinus, exposed in arenas with citral, presented irritability and decreased resting periods, which may be related to intoxication in the octapaminergic system, causing hyperactivity or hyperextension in the legs and abdomen 62 . Essential oils have caused sublethal effects such as increased heart rate, changes in the cAMP level in the nervous system and decreased binding to octopamine receptors, as decribed for Periplaneta americana Linnaeus (Blattodea: Blattidae) 62 .
Podisus nigrispinus tolerates lemongrass essential oil and its constituents, but geranyl acetate repelled this predator and citral caused irritability. This suggests caution in the use of lemongrass essential oil and these constituents in integrated pest management involving P. nigrispinus. This study may support future research with Cymbopogon citratus and its constituents in the search for bioinsecticides, based on nanoscience, against pests but without effect on this predator.

Methods
Insect mass rearing. Nymphs and adults of P. nigrispinus were obtained from the mass rearing of the 'Laboratorio de Controle Biológico' (LCBI) of the 'Universidade Federal de Viçosa' (UFV) in Viçosa, Minas Gerais state, Brazil. This predatory bug is reared at room temperature at 25 ± 1 °C, 70 ± 10% RH, and 12 h photophase. Podisus nigrispinus eggs were placed in Petri dishes (12 × 1.5 cm) with cotton soaked with water. Nymphs and adults of this insect were monitored in cubic wooden cages (30 × 30 × 30 cm) covered with nylon. These nymphs and adults were fed ad libitum with Tenebrio molitor L. (Coleoptera: Tenebrionidae) pupae and received Eucalyptus sp. (Myrtaceae) leaves and water 2 . essential oil toxicity test. The lemongrass essential oil was acquired from the 'Destilaria Bauru Ltda. ' company (Catanduva, São Paulo, Brazil), extracted by hydrodistillation on an industrial scale 63 . Lemongrass essential oil was diluted in 1 mL of acetone to obtain a stock solution. Six different doses of lemongrass were prepared and used to assess the insecticide toxicity and determine relevant toxicological endpoints; a dilution series of doses (8.1, 16.2, 31.2, 62.5, 125, and 250 µg/insect −1 ) was used to determine dose-mortality relationship and lethal dose (LD 50 and LD 90 ). Acetone was used as a control. Each solution (1 μL) was applied to the thorax of first-, second-, third-, fourth-and fifth-instar nymphs using a micropipette. For each nymph instar, fiften nymphs were tested, placed individually in Petri dishes with one T. molitor pupa per day and cotton soaked with water. The number of dead nymphs in each Petri dish was counted after 36 h. www.nature.com/scientificreports www.nature.com/scientificreports/ Identification of the lemongrass essential oil constituents. Quantitative analyses of lemongrass essential oil were performed in triplicate using a gas chromatograph (GC-17A, Shimadzu, Kyoto, Japan) equipped with flame ionization detector (FID). Chromatographic conditions were: a fused silica capillary column (30 m × 0.22 mm) with a DB-5 bonded phase (0.25 μm film thickness); carrier gas N 2 at a flow rate of 1.8 mL min −1 ; injector temperature of 220 °C; detector temperature of 240 °C; column temperature programmed to begin at 40 °C (remaining isothermal for 2 min) and increase at 3 °C min −1 to 240 °C (remaining isothermal at 240 °C for 15 min); 1 μL injection volume (1% w/v in dichloromethane); 1:10 split ratio and 115 kPa column pressure.
Constituents were identified using a gas chromatograph coupled with a mass detector GC/MS (CGMS-QP 5050 A; Shimadzu, Kyoto, Japan). The injector and detector temperatures were 220 °C and 300 °C, respectively. The initial column temperature was 40 °C for 3 min, with a programmed temperature increasing of 3 °C/min to 300 °C, where it was maintained for 25 min. The split mode ratio was 1:10. One microliter of lemongrass essential oil containing 1% (w/v in dichloromethane) was injected and helium used as carrier gas with a flow rate constant of 1.8 mL −1 on the Rtx ® -5MS capillary column (30 m, 0.25 mm × 0.25 μm, Bellefonte, USA) using Crossbond ® stationary phase (35% diphenyl, 65% dimethyl polysiloxane). The Mass Spectrometer was programmed to detect masses in the range of 29-450 DA with 70 eV ionization energy. Constituents were identified by comparisions of the mass spectra with those available from the National Institute of Standards and Technology (NIST08, NIST11) libraries, the Wiley Spectroteca database (7 th edition), and by the retention indices. toxicity of lemongrass constituents. Geranyl acetate (97.0% purity) and citral (95.0% purity), identified as constituents of the lemongrass essential oil, were obtained from Sigma Aldrich (Darmstadt, Germany). The efficacy of these constituents was determined by their lethal doses (LD 50 and LD 90 ) in the laboratory. Six different doses of each constituent were prepared and used to assess the insecticide toxicity and determine relevant toxicological endpoints; a dilution series of doses (8.1, 16.2, 31.2, 62.5, 125, and 250 µg/insect −1 ) was used to determine dose-mortality relationship and lethal dose. Acetone was used as a control. Each solution (1 μL) was applied to the thorax of third-instar nymphs using a micropipette. Fiften nymphs were tested, placed individually in Petri dishes with one T. molitor pupa per day, and cotton soaked with water. The number of dead nymphs in each Petri dish was counted after 36 h.
testing the respiratory rate. Respiration rate bioassays were conducted for 3 h after P. nigrispinus nymphs were exposed to geranyl acetate or and citral (LD 50 and LD 90 levels). Insects treated with distilled water were used as control. Carbon dioxide (CO 2 ) production (μL of CO 2 h −1 /insect) was measured with a TR3C CO 2 Analyzer (Sable System International, Las Vegas, USA) according to methods adapted from previous studies 18,22,55 . A third-instar nymph of P. nigrispinus was placed in each respirometry chamber (25 mL) connected to a closed system. After insect acclimation, CO 2 production was measured for 12 h at 27 ± 2 °C. Subsequently, compressed oxygen gas (99.99% pure) was introduced into the chamber at 100 mL min −1 for 2 min. The gas flow forces the CO 2 through an infrared reader, which continuously measures the CO 2 contained inside the chamber. Before and after the experiment, P. nigrispinus nymphs were weighed on an analytical balance (Sartorius BP 210D, Göttingen, Germany). Ten replicates were used for each insecticide treatment and control following a completely randomized design. testing locomotion behavior. Nymphs of P. nigrispinus were placed in a Petri dish (90 mm diameter × 15 mm high) lined with filter paper (Whatman no. 1). Then the inner walls of the Petri dish were covered with polytetrafluoroethylene (Dupont ® , Barueri, SP, Brazil) to prevent insect escape. Behavioral locomotor response bioassays were conducted in arenas half-treated with 250 µL of geranyl acetate or citral; dishes treated with acetone only were used as control. One P. nigirispinus nymph was released at the center of the arena treated with geranyl acetate or citral (on filter paper) and kept in the Petri dish for 10 min. Forty-eight third-instar P. nigrispinus nymphs were used for each lethal dose (16 per each treatment: control, geranyl acetate or citral), following a completely randomized design. For each insect, walking activity within the arena was recorded using a digital camcorder (XL1 3CCD NTSC, Canon, Lake Success, NY, USA) equipped with a 16 × video lens (Zoom XL 5.5-88 mm, Canon, Lake Success, NY, USA). A video tracking system (ViewPoint LifeSciences, Montreal, Quebec, Canada) was used to analyze the videos and measure the distances that the insects walked and the time spent resting on each half of the arena. Insects that spent less than 1 s on the half of the arena treated with the essential oil or constituent were considered repelled, whereas those that remained less than 50% of the time on the insecticide-treated surface were considered to have been irritated 26,55,57 . statistical analysis. Dose-mortality data were subjected to Probit analysis, generating a dose-mortality curve 64 . Respiration rates were subjected to two-way ANOVA and Tukey's HSD test (P < 0.05). Locomotor behavior response data were analyzed by one-way ANOVA, and a Tukey Honestly Significant Difference (HSD) test was also used for comparison of means at the 5% significance level. Toxicity, respiration rate, and locomotor behavior response data were analyzed using SAS for Windows v. 9.0 65 .