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.

The mealworm beetle, Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) is a pest of stored products such as starches, food for cats and dogs, and pasta. This insect may also infest broken grains of Zea mays (L.) (Poales: Poaceae), Triticum aestivum (L.) (Poales: Poaceae) and Glycine max (L.) (Fabales: Fabaceae)123. The presence of T. molitor in stored grain and bran can contaminate food with fragments of the body, faeces and indirectly by saprophytic microorganisms causing loss of food quality456. T. molitor causes losses up to 15% of grains and flour production in worldwide789.

T. molitor is controlled primarily with chemical insecticides, but this method has restrictions against stored product insects10, due to residual toxicity and insect resistance11, especially in countries with extensive cereal production for export and domestic consumption1012. Chemical control of this insect can be achieved by methyl bromide and phosphine treatment; however, fumigants cannot kill the eggs of storage pests and several issues have been discussed in the employment of insecticides, such as residue, environment impact and toxicity to humans1012. Economic, social and environmental concerns have caused a gradual change to reduce chemical control in starches and stored products111213. More selective and biodegradable products, including “green pesticides”, can reduce the use of synthetic chemicals in warehouses1415.

Plant essential oils have favorable ecotoxicological properties (low toxicity to humans, further degradation, and lower environmental impact), making them suitable to managing insects in organic farming1617. These oils are plants secondary metabolites and include alkaloids, amides, chalcones, flavones, kawapirones, lignans, neolignans or phenols which are important in insect-plant relationships141518. In this sense, essential oils represent an alternative for pest control as repellents, deterrent of oviposition and feeding, growth regulators, and toxicity to insects with low pollution and quick degradation in the environmental1617. Various studies have focused on the possibility of using plant essential oils for application to stored grain to control insect pests192021.

Garlic, Allium sativum Linnaeus (Amaryllidaceae), is a native of temperate western Asia and has been used throughout the world as a food spice and medicine22. Antimicrobial23, cardiovascular24, anticancer25, hypo- and hyper-glycaemic, and other beneficial properties of garlic have been reported26. In different studies, garlic essential oil was demonstrated to possess insecticidal activity against Blattella germanica Linnaeus (Blattodea: Blatellidae)27, Lycoriella ingénue Dufour (Diptera: Sciaridae)28, Reticulitermes speratus Kolbe (Isoptera: Rhinotermitidae)29, and several grain storage insects as Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), Sitophilus oryzae Linnaeus, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), and Tribolium castaneum Herbst (Coleoptera: Curculionidae)3031. There are a variety of insecticides that have toxicological properties, deterrents, and repellents used for the control of T. molitor; however, essential oil of garlic could be an alternative for the control in stored products. Identification of toxic compounds of garlic is important in understanding toxicity as it relates to pest control. In this study, we hypothesized that garlic essential oil and their constituents have insecticidal activity in T. molitor.

We examined the insecticidal activity of garlic essential oil as well as compounds identified on T. molitor, explain in various experiments: (i) garlic oil composition, (ii) toxicity test, (iii) lethal time test, (iv) repellency index and, (v) respiration rate, in order to contribute for the development of new strategies for controlling this insect pest affecting an important source of food.

Results

Susceptibility of T. molitor exposed to garlic essential oil

Mortality of T. molitor was obtained with 16 and 32% (w/v) of the garlic essential oil and three different lethal concentration levels (LC50 and LC90) were estimated by Probit (X, P < 0.0001) (Table 1). The LC50 and LC90 values indicated that garlic essential oil was the most toxic to T. molitor larvae (X = 260.1, df = 5), followed by pupae (Χ = 149.5, df = 5) and adults (Χ = 46.6, df = 5). Mortality was always <1% in the control.

Table 1

Lethal concentrations of the garlic essential oil against different developmental stages of Tenebrio molitor after 48 hours exposure.

1IS	2LC	3VE	4IC	5X2
Larva	LC50	0.771	0.668–0.871	260.15
	LC90	1.365	1.236–1.544
Pupa	LC50	2.371	2.171–2.615	149.51
	LC90	4.016	3.676–4.465
Adult	LC50	2.032	1.751–2.326	46.65
	LC90	4.736	4.215–5.465

1IS, insect stage; 2LC50 and 99, lethal concentration causing 50, 90 and 99% mortality; 3EV, estimated value; 4CI, confidential interval; 5X, chi-squared value for the lethal concentrations and fiducial limits based on a log scale with significance level at P < 0.001.

Garlic essential oil composition

A total of 14 compounds from the garlic essential oil were obtained, 10 compounds were identified and 4 unknown which accounted for 97.54% of the total composition (Fig. 1, Table 2, Supplementary Fig. 1). The primary compounds of the garlic essential oil were dimethyl trisulfide (19.86%), diallyl disulfide (18.62%), diallyl sulfide (12.67%), diallyl tetrasulfide (11.34%), and 3-vinyl-[4 H]-1,2-dithiin (10.11%), followed by diallyl trisulfide (5.74%), allyl trisulfide (4.41%), 1,4-dimethyl tetrasulfide (4.06%), allyl disulfide (3.95%), methyl allyl disulfide (3.87%), and methyl allyl trisulfide (3.76%).

Figure 1

Gas chromatogram profiles of peak retention of compounds of the garlic essential oil: Diallyl sulfide (1), methyl allyl disulfide (2), dimethyl trisulfide (3), diallyl disulfide (4), diallyl tetrasulfide (5), unknown (6), methyl allyl trisulfide (7), 3-vinyl-[4H]-1,2-dithiin (8), allyl trisulfide (9), unknown (10), 1,4-dimethyl tetrasulfide (11), diallyl trisulfide (12), unknown (13), and unknown (14).

Table 2

Chemical composition of the garlic essential oil.

Peak number	Compound	Formula	MM	RI	Ri	Rt	m/z
1	Diallyl sulfide	C6H10S	114	17.70	849	5.650	114.05
2	Methyl allyl disulfide	C4 H8 S2	120	4.60	911	7.850	122.00
3	Dimethyl trisulfide	C2H6S3	126	38.40	972	9.950	127.90
4	Diallyl disulfide	C6H10S2	146	5.40	1099	14.90	145.95
5	Diallyl tetrasulfide	C6H10S4	210	16.10	1601	15.56	147.95
6	Unknown	C6H10S2	146	14.9	1099	16.11	145.95
7	Methyl allyl trisulfide	C4 H8 S3	152	5.10	1128	17.42	110.90
8	3-vinyl-[4H]-1,2-dithiin	C6 H8 S2	144	26.40	1134	21.18	157.85
9	Allyl trisulfide	C6H10S3	178	4.90	1350	25.03	114.05
10	Unknown	C4 H8 S2	120	8.60	911	28.45	183.85
11	1,4-Dimethyl tetrasulfide	C6H10S4	210	6.60	1601	34.83	209.85
12	Diallyl trisulfide	C6H10S3	178	11.80	1350	43.26	113.05
13	Unknown	C6H10S3	178	5.40	1350	44.85	146.95
14	Unknown	C6H10S3	178	13.40	1350	49.91	186.95

MM - Molecular mass, RI - Relative intensity, Ri - Retention indices, Rt - Retention time, m/z - Molecular weight.

Toxicity assessment of compounds

The toxicity of commercially obtained diallyl sulfide and diallyl disulfide in T. molitor were estimated by Probit (X, P < 0.0001) and evaluated at different concentrations (Table 3). Toxicity was higher with diallyl disulfide, while diallyl sulfide was lower. Dose-response bioassays showed optimal results with diallyl disulfide by pupae (Χ = 46.45, df = 5) with a LC50 = 55.13 mg mL−1 and LC90 = 109.1 mg mL−1, followed larvae (Χ = 76.64, df = 5) with a LC50 = 57.68 mg mL−1 and LC90 = 154.3 mg mL−1, and adults (Χ = 31.32, df = 5) with a LC50 = 81.52 mg mL−1 and LC90 = 168.1 mg mL−1. The LC50 and LC90 values indicated that diallyl sulfide was toxic to pupae (X = 67.68, df = 5) with a LC50 = 48.86 mg mL−1 and LC90 = 210.5 mg mL−1, followed by larvae (Χ = 7.43, df = 5) with a LC50 = 117.1 mg mL−1 and LC90 = 222.8 mg mL−1, and adults (Χ = 50.83, df = 5) with a LC50 = 85.97 mg mL−1 and LC90 = 222.8 mg mL−1. Mortality was always <1% in the control.

Table 3

Lethal concentrations of the diallyl sulfide and diallyl disulfide on different developmental stages of Tenebrio molitor after 48 hours exposure.

Compounds	1IS	2LC	3VE mg mL−1	4IC mg mL−1	5X2
Diallyl sulfide	Larva	LC50	117.1	104.9–132.4	7.43
		LC90	222.8	198.1–256.9
	Pupa	LC50	48.86	36.71–61.85	67.68
		LC90	210.5	176.1–265.8
	Adult	LC50	85.97	77.84–95.51	50.83
		LC90	247.5	207.3–311.7
Diallyl disulfide	Larva	LC50	57.68	44.88–66.37	76.64
		LC90	154.3	136.9–177.8
	Pupa	LC50	55.13	49.78–61.31	46.45
		LC90	109.1	98.77–122.3
	Adult	LC50	81.52	57.96–91.25	31.32
		LC90	168.1	152.1–188.7

1IS, insect stage; 2LC50 and 99, lethal concentration causing 50, 90 and 99% mortality; 3EV, estimated value; 4CI, confidential interval; 5X, chi-squared value for the lethal concentrations and fiducial limits based on a log scale with significance level at P < 0.001.

Lethal time of toxic compounds and garlic essential oil in T. molitor

Larvae, pupae, and adults of T. molitor applied with LC50 and LC90 concentrations of garlic essential oil vs toxic compounds showed lethal effects at different time points (Fig. 2). However, LT50 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 LC50 concentration, diallyl sulfide took longer to kill the larvae (t = 1.29, P < 0.001), pupae (t = 4.23, P < 0.001), and adults (t = 2.16, P < 0.001) with LT50 values of 45.9 ± 0.52 h, 40.1 ± 0.38 h, and 54.2 ± 0.85 h, respectively. Diallyl disulfide took less time to kill the larvae (t = 3.24, P < 0.001), pupae (t = 4.16, P < 0.001), and adults (t = 2.12, P < 0.001) with LT50 values of 40.7 ± 0.14 h, 43.9 ± 0.52 h, and 49.7 ± 0.91 h, respectively. At a high LC90 concentration, diallyl sulfide took longer to kill the larvae (t = 4.45, P < 0.001), pupae (t = 4.51, P < 0.001), and adults (t = 3.96, P < 0.001) with LT50 values of 36.8 ± 0.85 h, 33.3 ± 0.43 h, and 40.3 ± 0.27 h, respectively. Diallyl disulfide took less time to kill the larvae (t = 4.31, P < 0.001), pupae (t = 4.31, P < 0.001), and adults (t = 4.08, P < 0.001) with LT50 values of 20.3 ± 0.45 h, 21.4 ± 0.16 h, and 27.7 ± 0.75 h, respectively.

Figure 2

Survivorship of Tenebrio molitor after 48 h topical applied with a LC50 and LC90 garlic essential oil vs toxic compounds: larva (A,D,G,J), pupa (B,E,H,K), and adult (C,F,I,L) (control insects were applied with water). Control (⦁), garlic essential oil (◻), diallyl disulfide and diallyl sulfide (▴).

The CL50 and CL90 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).

Figure 3

Time-course of garlic essential oil and toxic compounds on larva, pupa and adult of Tenebrio molitor after application to level LC90.

Control (A,E,I) and sequential necrosis effects at 12 h (B,F,J), 24 h (C,G,K), and 48 h (D,H,L). Necrosis point (arrows).

Repellency test

The larvae of T. molitor repellency index by garlic essential oil and compounds with concentrations estimated for the LC90 values differ between them (F1,17 = 7.61, P < 0.05) (Fig. 4A). The essential oil of garlic was the most repellent (RI = 1.11 ± 0.05), followed by that of the diallyl disulfide (RI = 1.07 ± 0.04), and diallyl sulfide (RI = 0.96 ± 0.03). The repellency index for adults of T. molitor differed with the concentration of the garlic essential oil and compounds, the estimated LC90 values (F1,17 = 6.81, P < 0.05) (Fig. 4B). Diallyl disulfide (RI = 1.11 ± 0.03) and garlic essential oil (RI = 1.07 ± 0.07) were the most repellent followed by diallyl sulfide (RI = 0.89 ± 0.03).

Figure 4

Repellency of Tenebrio molitor by the garlic essential oils and toxic compounds to level LC90 application on larvae (A) and adult (B). Treatments (Means ± SD) differ at P < 0.05 (Tukey’s mean separation test).

Respiration rate

The respiration rate (μL of CO2 h−1/insect) of T. molitor was significantly different for garlic essential oil and compounds with concentrations estimated for the LC90 values in larva (F3,71 = 6.84, P < 0.001), pupa (F3,71 = 44.35, P < 0.001), and adult (F3,71 = 10.45, P < 0.001). Respiration rate observed between 1 and 3 h were different in larva (F2,71 = 4.95, P < 0.001), pupa (F2,71 = 12.75, P < 0.001), and adult (F2,71 = 58.23, P < 0.001). The interaction treatments and time in was different in larva (F3,71 = 10.45, P < 0.001), but was not different in pupa (F3,71 = 0.15, P = 0.932) and adult (F3,71 = 3.19, P = 0.041). In general, garlic essential oil, diallyl disulfide, and diallyl sulfide reduced the respiration rate of T. molitor at 1 and 3 h after exposure (Fig. 5).

Figure 5

Respiration rate (Mean ± SE) of Tenebrio molitor after exposure to garlic essential oils and toxic compounds to level LC90 application on larvae (A), pupa (B), and adult (C). Treatments (Means ± SD) differ at P < 0.05 (Tukey’s mean separation test).

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 products1132. 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 fumigation303334.

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 application29303536. 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 LC50 and LC90 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 reports303637. 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 garlic3839. Various studies indicated that the rapid decomposition of allicin in garlic aqueous extract involved transformation mainly to diallyl sulfides4041. 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 compounds38394041.

Diallyl sulfide and diallyl disulfide demonstrated toxic activity on different developmental stages of T. molitor. Diallyl disulfide have stronger contact toxicity in larvae (LC50 = 57.68 mg mL−1), pupae (LC50 = 55.13 mg mL−1), and adult (LC50 = 81.52 mg mL−1), than diallyl sulfide in larvae (LC50 = 117.1 mg mL−1), pupae (LC50 = 48.86 mg mL−1), and adult (LC50 = 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. castaneum3035 at different developmental stages as well as other insects2936. 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 LC50 and LC90 test. In some individuals, the paralysis was constant with concentrations near the LC50 without recovery signs. Paralysis and muscle contractions in individuals of T. molitor at LC50 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 immobilization424344. 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 allicin4546. 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 denaturalization47.

The toxic compounds of garlic essential oil induced mortality in larva, pupa and adult of T. molitor within a short period of time. The LT50 of T. molitor larva applied with LC90 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 pests111516.

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 herbivores344348. These compounds are garlic majority compounds and toxic to pests of stored products such as S. zeamais and T. castaneum35. Garlic essential oil has high percentage of diallyl disulfide as main volatile compound and repellent properties to S. zeamais and T. castaneum30. 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 compounds495051. Thus, low respiration rate is an indicator of physiological stress, and essential oils can compromise insect respiration by impairing muscle activity, leading to paralysis495051. 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 insects5253. The absorption of a fumigant by an insect is positively correlated with its respiration rate54. 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 Ferquima Industry & Commerce Ltda. (Vargem Grande Paulista, São Paulo State, Brasil), produced in industrial scale by hydrodistillation drag of water vapor55. Ferquima Industry & Commerce Ltda is accredited by the Ministry of Agriculture, Livestock and Supply of Brazil and by international organizations according to ISO Guide56. This provides Brazilian producers with licenses, and ensures unrestricted access to major world organic product markets.

Mortality test

Garlic essential oil efficacy was determined by calculating the lethal concentrations (LC50, and LC90) values under laboratory conditions and conducted in triplicate. Six concentrations of garlic essential oil besides the control (acetone) were adjusted in 1 mL of stock solution (essential oil and acetone): 1, 2, 4, 8, 16 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 mortality57.

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 μm film thickness); carrier gas N2 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 μL (1%w/v in CH2Cl2); 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 μm; 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 (7th 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 LC50 and LC90 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’ formule57.

Toxicity of garlic essential oil and commercially obtained diallyl sulfide and diallyl disulfide at the calculated LC50 and LC90 concentrations were compared. Distilled water was used as a control. The LC50 and LC90 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 (LT50) were compared.

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 LC90 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).

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 LC50 of each toxic compound and control consisted of insect treated with distilled water. Carbon dioxide (CO2) (μL of CO2 h−1/insect) production was measured with a respirometer of the type CO2 Analyzer TR3C (Sable System International, Las Vegas, USA), using methodology adapted from previous studies49. Respirometers (25 mL) were used, each one holding 3 adults of T. molitor with mixed sex and connected to a closed system. CO2 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 CO2 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 CO2 molecules to pass through an infrared reader coupled to the system, which makes a continuous measurement of the CO2 produced by the insects and held inside each chamber. After CO2 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 variables58. Six replicates were used for each treatment.

Statistics

Lethal concentration (LC50 and LC90) 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-mortality59 using XLSTAT-PRO (v.7.5) program for Windows60. Student’s t test was used for pairwise comparisons regarding lethal time effects in T. molitor using SAS User software (v. 9.0) for Windows61. Repellency data larvae and adults of T. molitor were transformed using formula and analyzed by one-way ANOVA. A Tukey’s Honestly Significant test (HSD) was also used for comparisons of the means in the bioassays 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.

Additional Information

How to cite this article: Plata-Rueda, A. et al. Insecticidal activity of garlic essential oil and their constituents against the mealworm beetle, Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae). Sci. Rep. 7, 46406; doi: 10.1038/srep46406 (2017).

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