Insecticidal activity of garlic essential oil and their constituents against the mealworm beetle, Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae)

This study evaluated the insecticidal activity of garlic, Allium sativum Linnaeus (Amaryllidaceae) essential oil and their principal constituents on Tenebrio molitor. Garlic essential oil, diallyl disulfide, and diallyl sulfide oil were used to compare the lethal and repellent effects on larvae, pupae and adults of T. molitor. Six concentrations of garlic essential oil and their principal constituents were topically applied onto larvae, pupae and adults of this insect. Repellent effect and respiration rate of each constituent was evaluated. The chemical composition of garlic essential oil was also determined and primary compounds were dimethyl trisulfide (19.86%), diallyl disulfide (18.62%), diallyl sulfide (12.67%), diallyl tetrasulfide (11.34%), and 3-vinyl-[4H]-1,2-dithiin (10.11%). Garlic essential oil was toxic to T. molitor larva, followed by pupa and adult. In toxic compounds, diallyl disulfide was the most toxic than diallyl sulfide for pupa > larva > adult respectively and showing lethal effects at different time points. Garlic essential oil, diallyl disulfide and diallyl sulfide induced symptoms of intoxication and necrosis in larva, pupa, and adult of T. molitor between 20–40 h after exposure. Garlic essential oil and their compounds caused lethal and sublethal effects on T. molitor and, therefore, have the potential for pest control.

Lethal time of toxic compounds and garlic essential oil in T. molitor. Larvae effects at different time points (Fig. 2). However, LT 50 values from topical application assays showed that diallyl sulfide took longer to kill insects than the diallyl disulfide and garlic essential oil. At a high LC 50 concentration, diallyl sulfide took longer to kill the larvae (t = 1.29, P < 0.001), pupae (t = 4. 23 50 and CL 90 values of garlic essential oil, diallyl disulfide and diallyl sulfide induced symptoms of intoxication in larvae and adults of T. molitor, such as progressive paralysis, reduced food consumption, and regurgitation. Necrosis was observed in larvae, pupae and adults on the area applied, mainly in mouthparts, pronotum, legs, abdomen segments, and anus (Fig. 3).

Discussion
Insecticidal activity of the garlic essential oil and its compounds against the mealworm beetle, T. molitor were determined from the bioassays in the laboratory conditions. Garlic essential oil caused substantial mortality and repellency in larva, pupa, and adult stages. The best results were obtained with concentrations of 16 and 32% in T. molitor as reported for other stored grain pests according to the concentration of these products 11,32 . The susceptibility of stored pest products such as S. oryzae, S. zeamais, Sitotroga cerealella Oliver (Lepidoptera: Gelechiidae), and T. castaneum may vary with the exposure at garlic essential oil applied to the body of these insects or by fumigation 30,33,34 .
Different concentrations of the garlic essential oil showed toxic effects on larva, pupa and adult of T. molitor 48 h after topical application. The dose-response bioassay confirmed toxicity against T. molitor, reaching a 90% mortality rate. Increasing concentrations of garlic essential oil in this insect have shown immediate toxic responses within 12 h of application 29,30,35,36 . Comparing the contact toxicity of garlic essential oil on developmental stages of T. molitor, the larva was significantly more susceptible followed by pupa and adult. The LC 50 and LC 90 of larva (0.77-1.36%), pupa (2.37-4.01%), and adult (2.03-4.73%) indicate that small quantities of the garlic essential oil are toxic in this insect, being more tolerant with age.
The chemical composition of the garlic essential oil revealed 14 compounds detected, 10 identified and quantified in terms of relative percentages. In particular, diallyl sulfide, diallyl disulfide, diallyl tetrasulfide, dimethyl trisulfide, and 3-vinyl-[4H]-1,2-dithiin were the main compounds that were detected in garlic essential oil. The result is in accordance with those of previous reports 30,36,37 . The above compounds are produced as a result of the degradation of allicin under the harsh thermal treatment in the hydrodistillation procedure. Allicin is not a stable compound and readily degrades via several pathways to form the secondary products of various sulfides contributing the characteristic flavour and odour of garlic 38,39 . Various studies indicated that the rapid decomposition of allicin in garlic aqueous extract involved transformation mainly to diallyl sulfides 40,41 . In this study, the main compounds of garlic essential oil are sulfur compounds (thiosulfinates) such as diallyl sulfide, diallyl disulfide, and diallyl tetrasulfide. However, there are considerable variations in the chemical composition of garlic essential oil where 3-vinyl-[4H]-1,2-dithiin is a main compound. In general, the diallyl sulfide, diallyl disulfide, and diallyl tetrasulfide are the most abundant constituents of fresh garlic oil and commercially can be variations in the relative proportions of these compounds [38][39][40][41] .
Diallyl sulfide and diallyl disulfide demonstrated toxic activity on different developmental stages of T. molitor. Diallyl disulfide have stronger contact toxicity in larvae (LC 50 = 57.68 mg mL −1 ), pupae (LC 50 = 55.13 mg mL −1 ), and adult (LC 50 = 81.52 mg mL −1 ), than diallyl sulfide in larvae (LC 50 = 117.1 mg mL −1 ), pupae (LC 50 = 48.86 mg mL −1 ), and adult (LC 50 = 85.97 mg mL −1 ). Garlic essential oil and its constituents, diallyl sulfide and diallyl disulfide have been highly toxic to S. zeamais and T. castaneum 30,35 at different developmental stages as well as other insects 29,36 . Our results showed that T. molitor was more susceptible in the pupal stage followed by larvae and adults exposed to diallyl sulfide and diallyl disulfide. One possible explanation for the developmental stages difference is that efficacy may be affected by the penetration of the garlic compounds into the body and the ability of the insect to metabolize these compounds. The insects exposed to the garlic essential oil and toxic compounds displayed altered locomotion activity, and muscle contractions that were observed in detailed descriptions at high concentrations in LC 50 and LC 90 test. In some individuals, the paralysis was constant with concentrations near the LC 50 without recovery signs. Paralysis and muscle contractions in individuals of T. molitor at LC 50 can be explained by the toxic effect in the nervous system of the same. The rapid toxicity of essential oils and their constituents in insects indicates neurotoxic action as reported to Delia radicum Linnaeus, Musca domestica Linnaeus (Diptera: Muscidae), Cacopsylla chinensis (Yang et Li), and Diaphorina citri Kuwayama (Hemiptera: Psyllidae) with hyperactivity, hyperextension of the legs and abdomen and rapid knock-down effect or immobilization [42][43][44] . Acetylcholinesterase is an enzyme that has been shown to be inhibited by garlic compounds and can act only or in synergism as diallyl disulfide, diallyl trisulfide, and allicin 45,46 . The presence of the diallyl sulfide in garlic compounds may be responsible for the toxic effect in T. molitor and may cause inhibition by cross-linking with essential thiol compounds in enzyme structures, altering the functional shape of the protein and denaturalization 47 .
The toxic compounds of garlic essential oil induced mortality in larva, pupa and adult of T. molitor within a short period of time. The LT 50 of T. molitor larva applied with LC 90 diallyl disulfide and diallyl sulfide was approximately 20 and 36 h, pupae was 21 and 33 h, and adults was 27 and 40 h, respectively. Toxic compounds affect multiple regions of the insect body over a period of time, ranging from one to 20-40 h for death. In this period, the necrotic areas were increasing progressively on the insect body. The comparative effects on T. molitor between garlic essential oil and toxic compounds were observed at various time points. An essential oil of quick action should be preferred for protection of products stored to be able to prevent feeding and avoid or reduce damage by insect pests 11,15,16 .
The repellency test indicated that garlic essential oil and diallyl disulfide has a greater effect on T. molitor behavior than diallyl sulfide had little effect. Odor produced from volatile compounds was repulsive to larvae and adults of T. molitor and was observed during early hours to finished bioassay. Monosulfides, disulfides, and trisulfide breakdown products of the thiosulfinates are volatile compounds and toxic to insect herbivores 34,43,48 . These compounds are garlic majority compounds and toxic to pests of stored products such as S. zeamais and T. castaneum 35 . Garlic essential oil has high percentage of diallyl disulfide as main volatile compound and repellent properties to S. zeamais and T. castaneum 30 . Our results suggest that garlic essential oil, diallyl disulfide, and diallyl sulfide have high activities of behavioral deterrence against T. molitor, as evaluated by the behavioral responses of larvae and adults to different odor sources and the number of insects repelled, indicating their potential to the pest control in stored products.
The garlic essential oil and their toxic constituents compromised the respiration rate of T. molitor up to 3 h after exposure, likely reflecting in behavior response and subsequently, the locomotors activity. Respiratory rate and body mass of insects can represent the sum of the energy demands of the physiological processes of insects that are necessary to produce defense mechanisms against the essential oils and toxic compounds [49][50][51] . Thus, low respiration rate is an indicator of physiological stress, and essential oils can compromise insect respiration by impairing muscle activity, leading to paralysis [49][50][51] . Several studies of plant volatile and their constituents were shown to effectively disrupt the recognition process of the host substrate and influence the walking behavior on insects 52,53 . The absorption of a fumigant by an insect is positively correlated with its respiration rate 54 . In this study, adults of T. molitor have low respiration rate caused by garlic essential oil and their constituents, resulting in physiological costs due to energy reallocation from other basic physiological processes. This favors the use of these toxic compounds by fumigant action and can cause significant negative effects on T. molitor.
This study showed the potential of garlic essential oil and main compounds to control the T. molitor in starches and stored products. In order to prevent or retard the development of insecticide resistance, the toxicity and repellency effects of garlic essential oil and toxic compounds on T. molitor show that can be used individually or mixture to management populations. The lethalities of diallyl sulfide and diallyl disulfide on T. molitor may have advantages by their mode of action on this insect. These findings show that the compounds of garlic essential oil are a potential source of insecticidal compounds and warrants further exploration.

Methods
Insects. Individuals of T. molitor were obtained from the Laboratory of Biological Control of the Institute of Applied Biotechnology to Agriculture (BIOAGRO, Universidade Federal de Viçosa) in Viçosa, Minas Gerais State, Brasil. Adults of T. molitor were kept in plastic trays (60 cm long × 40 cm wide × 12 cm) and maintained at 25 ± 1 °C, 70 ± 10% RH and 12:12 h L:D photoperiod. These adults were fed ad libitum with wheat bran (12% protein, 2% lipids, 75% carbohydrates and 11% mineral/sugar), pieces of sugarcane, Saccharum officinarum (L.) (Poaceae) and chayote Sechium edule (Jacq.) Swartz (Cucurbitaceae). Sheets of paper were placed on the substrate to prevent the stress from insects and facilitate oviposition. Healthy larvae, pupae and adults of T. molitor without amputations, apparent malformations or nutritional deficiencies were used in the bioassays.
Garlic essential oil. The essential oil of garlic, A. sativum used in this study was commercial sample from and 32% (w/v). Aliquots were taken from the stock solution and mixed with acetone in 5 mL glass vials. Different concentrations of the garlic essential oil were applied in 1 uL solution on the thorax of larva, pupa and adult of T. molitor, using a micropipette. For each developmental stage, fifty insects were used per concentration and were placed individually in Petri dishes (Ø 90 mm × 15 mm) with an absorbent paper, fed with chayote and sugarcane ad libitum (larvae and adults) and maintained in the dark. The number of dead insects was in each cage was counted after essential oil exposure for 48 h after application. Rates were calculated with a correction for natural mortality 57 .
Scientific RepoRts | 7:46406 | DOI: 10.1038/srep46406 Identification of garlic essential oil compounds. Quantitative analyses of the garlic essential oil formulation were performed using a gas chromatograph (GC-17A series instrument, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID). The following chromatographic conditions were used: a fused silica capillary column (30 m × 0.22 mm) with a DB-5 bonded phase (0.25 μ m film thickness); carrier gas N 2 at a flow rate of 1.8 mL min −1 ; injector temperature 220 °C; detector temperature 240 °C; column temperature programmed to begin at 40 °C (remaining isothermal for 2 min) and then to increase at 3 °C min −1 to 240 °C (remaining isothermal at 240 °C for 15 min); injection volume 1.0 μ L (1%w/v in CH 2 Cl 2 ); split ratio 1:10; column pressure 115 kPa.
The compounds were identified using a gas chromatograph coupled with a mass detector GC/MS (GCMS-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 increase of 3 °C/min to 300 °C where it was maintained for 25 min. The split mode ratio was 1:10. One microliter of the garlic essential oil containing 1% (w/v in dichloromethane) was injected, helium was used as the 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 μ m; Bellefonte, USA) using the Crossbond ® stationary phase (35% diphenyl-65% dimethyl polysiloxane). Mass spectrometer was programmed to detect masses in the range of 29-450 Da with 70 eV ionization energy. Compounds were identified by comparisons of the mass spectra with those available in the NIST08, NIST11 library and Wiley Spectroteca Data Base (7 th edition), and by the Retention indices.
Toxicity of commercial compounds of garlic essential oil in T. molitor. Diallyl sulfide and diallyl disulfide identified as toxic compounds in garlic essential oil, were obtained commercially. Diallyl sulfide (purity 97.0%) and diallyl disulfide (purity 70%) were purchased from Across Organics (New Jersey, USA). Six different concentrations of the commercial compounds besides the control (acetone) were adjusted in 1 mL of stock solution (treatment and acetone) used to calculate the lethal LC 50 and LC 90 concentrations: 5, 10, 20, 40, 80, and 160 mg mL −1 . For each treatment, aliquots were taken from the stock solution and mixed with acetone in 5 mL glass vials. Different concentrations of the each treatment were applied in 1 uL solution on the body of larva, pupa and adult of T. molitor. Individuals were placed in Petri dishes with wheat bran and sugarcane. A total of 50 larvae, 50 pupae, and 50 adults were used for each concentration and mortality was evaluated for 48 h after application to compared with toxicity garlic essential oil, followed by rate corrections according to the Abbott' formule 57 .
Lethal time of toxic compounds and garlic essential oil in T. molitor. Toxicity of garlic essential oil and commercially obtained diallyl sulfide and diallyl disulfide at the calculated LC 50 and LC 90 concentrations were compared. Distilled water was used as a control. The LC 50 and LC 90 of each compound were applied in larvae, pupae, and adults of T. molitor. Insects were individualized in Petri dishes with wheat bran and sugarcane. A total of 240 larvae, 240 pupae, and 240 adults were used for each treatment, with a total of four replicates. Mortality was recorded every 6 h for 48 h, and estimated lethal time values for 50% mortality (LT 50 ) were compared.
Repellency test. Four Petri dishes (12 × 1.5 cm) were used as an arena, connected to a central board with plastic tubes (diameter 2 cm) at an angle of 45°. The other dishes were distributed around them in equidistant distances and two plates were put together symmetrically opposed. Two hundred and forty individuals (120 larvae and 120 adults) of T. molitor were released in the central board and the control group received sugarcane and chayote. A total of 5 uL of the estimated LC 90 lethal concentration of garlic essential oil and toxic compounds were applied on absorbent filter paper (2 × 2 cm) placed in two opposite plates used as treatment and two opposite ones with 5 uL of distilled water for absorbent filter paper represented the control. Four replicates per treatment and control were evaluated by the number of individuals per plate after 24 hours calculating the repellency index (IR): RI = 2 G/(L + P), where G is the percentage of insects in the treatment and P is the percentage of insects in control. Treatments were classified as neutral if the index was equal to one (1); repellent, higher than one (1), and; attractive lower than one (1).
Respiration rate. Respirometry bioassays were conducted 3 h after the beetles were exposed or not exposed to garlic essential oil and their constituents, as previously detailed. The doses used corresponded to the recorded LC 50 of each toxic compound and control consisted of insect treated with distilled water. Carbon dioxide (CO 2 ) (μ L of CO 2 h −1 /insect) production was measured with a respirometer of the type CO 2 Analyzer TR 3 C (Sable System International, Las Vegas, USA), using methodology adapted from previous studies 49 . Respirometers (25 mL) were used, each one holding 3 adults of T. molitor with mixed sex and connected to a closed system. CO 2 production was measured after the insects were acclimated in the chambers for a period of 12 h at a temperature of 27 ± 2 °C. To quantify the CO 2 produced inside each chamber, compressed oxygen gas (99.99% pure) was passed through the chamber at a flow of 100 mL min −1 for a period of 2 min. This airflow forces all produced CO 2 molecules to pass through an infrared reader coupled to the system, which makes a continuous measurement of the CO 2 produced by the insects and held inside each chamber. After CO 2 measurement, the insects were removed from the chambers and then weighed using an analytical balance (Sartorius BP 210D, Göttingen, Germany). Respiration rate values were not normalized by body mass because this method masks the individual effects of the variables 58 . Six replicates were used for each treatment.
Statistics. Lethal concentration (LC 50 and LC 90 ) and their confidence limits for garlic essential oil and toxic compounds were determined by logistic regression in dose-response assays based on the concentration Probit-mortality 59 using XLSTAT-PRO (v.7.5) program for Windows 60 . Student's t test was used for pairwise comparisons regarding lethal time effects in T. molitor using SAS User software (v. 9.0) for Windows 61 . Repellency data larvae and adults of T. molitor were transformed using formula + . x 0 05 and analyzed by one-way ANOVA. A Tukey's Honestly Significant test (HSD) was also used for comparisons of the means in the bioassays Scientific RepoRts | 7:46406 | DOI: 10.1038/srep46406 at 5% significance level using SAS User software. Respiration rate were subjected to two-way analyses of variance (time × insecticide treatment) and Tukey's HSD test (P < 0.05) when appropriate (PROC GLM). As the time interval was assessed in different insect samples, they are not pseudoreplicates in time and therefore subject to regular two-way analyses of variance instead of repeated measures analyses of variance using SAS User software.