169 ISJ 15: 169-182, 2018 ISSN 1824-307X RESEARCH REPORT The effects of plant essential oils on the functional response of Habrobracon hebetor Say (Hymenoptera: Braconidae) to its host M Asadi, H Rafiee-Dastjerdi*, G Nouri-Ganbalani, B Naseri, M Hassanpour Department of Plant Protection, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran. Accepted April 20, 2018 Abstract Habrobracon hebetor Say is an important ectoparasitoid wasp that can control Pyralidae and Noctuidae pests in agricultural crops. In this research, the effects of Allium sativum L., Rosmarinus officinalis L., Piper nigrum L., Salvia officinalis L. and Glycyrrhiza glabra L. essential oils were investigated on the functional response of H. hebetor to its host. The GC-MS analysis showed that tetracosamethyl cyclododeca siloxan, alpha-pinene, caryophyllene, beta-thujone and aristolene were major constituents of mentioned essential oils, respectively. In the experiments; the mated females of H. hebetor (under 24 h old) were exposed to sublethal concentrations (LC30) of isolated essential oils for 24 h with fumigant exposure method. In the control, the treatment was performed by using distilled water. Then, six treated wasps were selected randomly to densities of 2, 4, 8, 16, 32 and 64 Ephestia kuehniella Zeller 5th instar larvae for 24 h under 25 ± 1 °C, 60 ± 5% RH and photoperiod of 16: 8 (L: D) h. Eight replicates were conducted for each host density in all treatments. The regression analysis based on Holling model (1959) indicated the functional response type II in the control, P. nigrum, S. officinalis and G. glabra and type III in A. sativum and R. officinalis essential oils. Also, R. officinalis essential oil and the control showed the longest (0.542 h) and shortest (0.411 h) handling times, respectively. The highest (0.047 h-1) and lowest (0.033 h-1) attack rates were also recorded in the control and R. officinalis essential oil, respectively. In addition, R. officinalis and G. glabra essential oils showed the maximum and minimum negative effects on the functional response type and it’s parameters in H. hebetor, respectively. These results indicated that G. glabra essential oil can be recommended with H. hebetor in integrated pest management. Key Words: Ephestia kuehniella; plant essential oils; functional response; Glycyrrhiza glabra; Habrobracon hebetor Introduction Habrobracon hebetor Say is an important ectoparasitoid wasp with special behavioral characteristics (idiobiont and gregarious) that has been applied successfully in many biological control programs in different regions over the world including Iran (Heimpel et al., 1997; Yu et al., 2002; Darwish et al., 2003; Salvador and Consoli, 2008; Abedi et al., 2012; Mahdavi and Saber, 2013). The mass rearing of H. hebetor are performed on the larval stage of flour moth (Ephestia kuehniella Zeller) as laboratory host in different commercial insectariums (Mudd and Corbet, ___________________________________________________________________________ Corresponding author: Hooshang Rafiee-Dastjerdi Department of Plant Protection Faculty of Agriculture and Natural Resources University of Mohaghegh Ardabili, Ardabil, Iran E- mail: hooshangrafiee@gmail.com 1982). This parasitoid wasp till now has been applied under inundative and inoculative release programs against Helicoverpa armigera (Hübner), Sesamia cretica (Lederer) and Ostrinia nubilalis (Hübner) (Hentz et al., 1998; Baker and Fabrick, 2000; Navaei et al., 2002). One of the important behavioral features of natural enemies including parasitoids and predators is their functional responses. Holling (1959, 1961 and 1966) characterized three types of functional responses. The functional response type I has a linear shape (Hassell, 1978). In functional response type II, the numbers of hosts attacked by natural enemies reach to a fix rate. Most of the natural enemies show this type of functional response (Hassell, 1978; Luck, 1985; Yu et al., 2002; Abedi et al., 2012; Mahdavi and Saber, 2013; Jarrahi and Safavi, 2015). The functional response type III also show sigmoid shape (Holling, 1959; Hassell, 1978). mailto:hooshangrafiee@gmail.com 170 Combination of different pest management methods such as biological and chemical control has been recommended in IPM designs all over the world and the negative effects of different compounds on the biocontrol agents must be considered (Abedi et al., 2012). Abramson et al., (2006) studied the effects of citronella and alfazema essential oils on the fennel Aphids, Hyadaphis foeniculi Passerini (Hemiptera: Aphididae) and it’s predator, Cycloneda sanguinea L. (Coleoptera: Coccinellidae) and concluded that citronella essential oil showed the most adverse effects on this predator. In addition, Poderoso et al., (2016) studied the effects of some plant extracts on developmental of the predator Podisus nigrispinus (Hemiptera: Pentatomidae) and concluded that the examined extracts caused relatively high mortality on the adults of this predator and must be used with care, because they can affect the life cycle of this important biocontrol agent. Therefore, natural enemies can be affecting by different botanical compounds that use against the insect pests. Therefore, estimation of the functional response types and their parameters under treatments of botanical compounds including essential oils and extracts are very important factors in IPM programs. To date there have been no research conducted the effects of plant essential oils on E. kuehniella and functional response of H. hebetor; but, the researches about the lethal and sublethal effects of essential oils on this important biocontrol agent are available (Seyyedi et al., 2011; Hashemi et al., 2014; Ahmadpour, 2017). In addition, chemical pesticides can affect host-finding behavior and behavioral responses of H. hebetor. Rafiee- Dastjerdi et al., (2009b) showed that profenofos, thiodicarb, hexaflumuron and spinosad negatively changed the functional response of H. hebetor. Faal-Mohammad Ali et al., (2010) also concluded that chlorpyrifos and fenpropathrin changed the functional response of this parasitoid wasp to it’s host that these negative effects of pesticides can lead to inefficiency of natural enemies and outbreak of plant pests. Therefore, the effects of different compounds must be investigated in assessment of natural enemies for biological control programs. The effects of azadirachtin, cypermethrin, methoxyfenozide and pyridalil also were studied by Abedi et al., (2012); who stated that cypermethrin had the highest negative effects on H. hebetor. Moreover, Mahdavi and Saber (2013) stated that malathion was compatible insecticide on the functional response of H. hebetor compared with diazinon in IPM programs. In addition, Jarrahi and Safavi (2015) concluded that proteus as a new formulated insecticide showed the highest negative effects on this parasitoid wasp compared with entomopathogenic fungus Metarhizium anisopliae Sensu lato and the control under laboratory conditions. Hence, the main objective of the present research was to investigate the effects of above mentioned essential oils isolated from some selective medicinal plants on the functional response of H. hebetor to evaluate the possibility of these botanical compounds to be integrated with this important ectoparasitoid wasp in IPM programs especially for the management of stored products pest. Materials and Methods The present research was carried out during 2016-2017, in the Department of Plant Protection, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran. Rearing of the parasitoid wasp Parent population of Habrobracon hebetor wasps was provided by a private commercial insectarium (Kesht-Gostar Pishgam, Kermanshah Province, Iran), during 2016. Then, the parasitoid wasps were reared under laboratory conditions in growth chamber that was set at 25 ± 1 °C, 60 ± 5% RH and a photoperiod of 16: 8 (L: D) h, on the larvae of flour moth (E. kuehniella) as laboratory host for parasitism activities. Moreover, the honey solution (10%) was applied as food source for feeding of the adult parasitoids (Rafiee-Dastjerdi et al., 2008, 2009b). The ratio of parasitoid to host in our experiments was one female wasp to ten E. kuehniella larvae and the exposure interval of host to the parasitoid wasp was two days. Isolation of essential oils The selected medicinal plants including garlic, Allium sativum; rosemary, Rosmarinus officinalis; black pepper, Piper nigrum; sage, Salvia officinalis and liquorice, Glycyrrhiza glabra that was available in the Iranian flora and contained suitable amount of essential oil were collected from different regions of Islam-Abad Gharb city (34.11° N, 46.53° E) in Kermanshah Province, Iran, during May 2017. The collected plants were dried at room temperature (25 °C) under shade. Then, the parts of noted plants that contained the most insecticidal components including leaves of R. officinalis, S. officinalis and G. glabra, berries of A. sativum and seeds of P. nigrum were milled by electric grinder and 50 g of milled parts were added to 500 ml of distilled water and their essential oils were isolated by clevenger apparatus at 100 °C in 4h time for each plant (Shiva parsia and Valizadegan, 2015). The water of essential oils was removed by sodium sulfate and pure essential oils in special glasses were covered with aluminum coverage and stored in a refrigerator at 4 °C for using in experiments. Chemical analysis of isolated essential oils Chemical components of each essential oil were identified by using gas chromatography-mass spectroscopy (GC-MS/ Company: Agilent, Series: 7890 B, Manufacturer: USA). The comparative and original analyses are two common types of GC-MS analysis. The original analyses that apply about the essential oils and the other volatile compounds measure the peaks in relation to one another. In this method, the tallest peak is assigned 100% of the value and the other peaks being assigned proportionate values. The total mass of the unknown compounds is normally indicated by the parent peak. The value of this parent peak can be 171 used to fit with a chemical formula containing the various elements which are believed to be in the compound (Hites, 2016). Bioassays experiments For investigation the fumigant toxicity of isolated essential oils on the young females of H. hebetor (under 24 h old); different concentrations of essential oils that lead to mortality between 20-80% put on filter paper (2×2 cm) in 60 ml glass Petri dishes as fumigant chambers by using a microaplicator. Distilled water was used in control treatments. Then, 20 adult females H. hebetor were released in each Petri dish without the presence of the host and the Petri dishes immediately were sealed with parafilm to prevent the exit of essential oils. Honey solution (10%) was used for the feeding of wasps on small pieces of paper. Each concentration of the essential oils was bioassayed in four replications and after 24 h of exposure; the number of dead wasps was recorded (Shiva parsia and Valizadegan, 2015). Functional response experiments In the functional response experiments, the LC30 of each essential oil was applied as the low lethal concentration. In first experimental setup, eighty mated females (under 24 h old) of H. hebetor that previously not in the presence of the host were exposed to LC30 of selected essential oils that were put on filter papers (2×2 cm) by using a microaplicator in 10 cm Petri dishes (volume 60 ml) for 24 h. All procedures were performed for the control treatments with distilled water. After 24 h, six treated wasps were selected randomly and transferred separately to the Petri dishes with the different densities (2, 4, 8, 16, 32, and 64) of E. kuehniella larvae (5th instar) and were placed in growth chamber that was set at 25± 1°C, 60± 5% RH and a photoperiod of 16: 8 (L: D) h for 24 h. Ventilation in the Petri dishes were provided with pores in the lids of Petri dishes and honey solution (10%) was supplied as food source for the parasitoids. The functional response experiments were performed in eight replicates in all treatments and the numbers of parasitized host larvae by the parasitoid wasps were recorded after 24 h. Used model The model of Holling (1959) regarding the functional response of different natural enemies was used in this study as explained below: Na = aTtN0(1+aThN0) Na = number of hosts attacked by H. hebetor N0 = different densities of host (2, 4, 8, 16, 32 and 64 5th instar larvae of E. kuehniella) Tt = total time of experiment (was 24 hour) a = attack rate (area of host discoverage) by H. hebetor Th = handling time (time of handling) of H. hebetor to it’s host The other form of this equation is: a= (d+bN0)/(1+CN0) Here, “a” is host density and “b”, “c” and “d” are estimated constants (Hassell et al., 1977; Juliano and Williams, 1987; Juliano, 1993). Statistical analysis The logistic and non-linear regression models were applied to determine the types of functional response and for the estimation of the parasitoid attack rate and handling time under different essential oils treatments and the control, respectively, using SAS V 9.1 software (SAS Institute, 2002). Results Chemical analysis of isolated essential oils The GC-MS analyses results of isolated essential oils are shown in Tables 1 to 5. Eleven major compounds from A. sativum essential oil, fourty-three compounds from S. officinalis and P. nigrum essential oils and fourty-four compounds from S. officinalis and G. glabra essential oils were detected. Tetracosamethyl cyclododeca siloxan (15.82%) from A. sativum, alpha-pinene (9.99%) from R. officinalis, caryophyllene (36.03%) from P. nigrum, beta-thujone (25.63%) from S. officinalis and aristolene (20.14%) from G. glabra were detected as major constituents of each mentioned essential oil. Table 1 Chemical constitutents of Allium sativum L. essential oil Peak Material Retention Time (RT) % of Total 1 5- 2', 6', 6'-Trimethyl-Cyclohexene 36.49 2.16 2 Octasiloxane 36.87 3.64 3 5, 6, 8, 9-Tetramethoxy-2-Methylpep 37.38 5.99 4 N-Methyl-1-Adamantaneacetamide 37.50 7.97 5 Silicone grease, Siliconfett 37.56 8.80 6 1, 3-Xylyl-15-crown-4, 2, 3-Pinan 37.59 8.63 7 4-Methoxy-3-(3-Methoxyphenyl) 37.80 10.20 8 1, 4-Cyclohexadiene,1, 3, 6-tris 37.92 10.57 9 1-Amino-1-ortho-Chlorophenyl 38.02 12.87 10 Anhydro 5-Hydroxy-3-Piperonyl 38.14 13.34 11 Tetracosamethyl cyclododeca siloxan 38.27 15.82 172 Table 2 Chemical constitutents of Rosmarinus officinalis L. essential oil Peak Material Retention Time (RT) % of Total 1 Tricyclene 5.21 0.27 2 Alpha-Pinene 5.45 9.99 3 Camphene 5.70 4.25 4 Bicyclo [3.1.0] Hex-3-en-2-ol 5.79 0.42 5 Bicyclo [3.1.1] Heptane, 6, 6-Dimet 6.21 1.19 6 3-Octanone 6.36 1.31 7 Beta-Myrcene 6.44 1.73 8 (+)-4-Carene 6.94 0.37 9 Benzene,1-Methyl 7.10 0.96 10 dl-Limonene 7.18 2.88 11 1, 8-Cineole 7.26 6.11 12 Gamma-Terpinene 7.76 0.51 13 Alpha-Terpinolene 8.39 0.52 14 Linalool L 8.63 2.17 15 Chrysanthenone 9.29 0.55 16 Bicyclo [2.2.1] Heptan-2-one 9.85 7.78 17 Bicyclo [3.1.1] Heptan-3-one 10.23 0.57 18 Borneol L 10.41 5.02 19 Bicyclo [3.1.1] Heptan-3-one 10.64 1.07 20 3-Cyclohexen-1-ol, 4-Methyl 10.70 1.01 21 Alpha Terpineol 11.10 1.67 22 Estragole 11.29 0.91 23 Bicyclo [3.1.1] Hept-3-en-2-one 11.77 7.24 24 Bicyclo [2.2.1] Heptan-2-ol 14.50 5.71 25 Caryophyllene 19.37 3.81 26 Alpha-Humulene 20.41 0.66 27 Heptasiloxane, Hexadeca Methyl 36.26 4.78 28 3-(4-Chlorophenyl)-4, 6-Dimethoxy 36.35 1.21 29 1, 3-Xylyl-15-crown-4, 2, 3-Pinan 36.38 2.25 30 Cyclononasiloxane, OctadecaMethyl 36.55 1.70 31 Acetamide, 2-(Adamantan-1-yl) 36.68 3.01 32 Bistri, Methylsilyl n-Acetyl Eicos 36.83 5.14 33 1, 1, 1, 5, 7, 7, 7-HeptaMethyl 37.22 0.77 34 6-Phenyl-3, 5-Dithioxo 37.33 1.09 35 Cyclodecasiloxane, EicosaMethyl 37.46 1.50 36 Octadeca Methyl Cyclononasiloxane 37.56 1.26 37 1-Amino-1-Ortho-Chlorophenyl 37.60 0.64 38 Cyclodecasiloxane, Eicosa Methyl 37.69 2.71 39 5, 6, 8, 9-Tethramethoxy-2-Methylpep 37.82 2.25 40 1, 1, 5, 7, 7, 7- Heptamethyl-3 37.89 0.69 41 Cyclononasiloxane, octadecamethyl 38.91 0.41 42 Benzene, 2, 3-dimethyl 38.12 1.10 43 Iron, Monocarbonyl 38.23 0.82 173 Table 3 Chemical constitutents of Piper nigrum L. essential oil Peak Material Retention Time (RT) % of Total 1 Bicyclo [3.1.0] Hexane, 4-methyl 5.28 0.67 2 R-Alpha-Pinene 5.42 3.05 3 Camphene 5.69 0.18 4 Sabinene 6.15 3.96 5 2-Beta-Pinene 6.22 4.34 6 Beta-Myrcene 6.43 0.73 7 1-Phellandrene 6.71 1.34 8 3-Carene 6.84 5.17 9 Alpha Terpinene 6.94 0.16 10 Benzene,1-Methyl 7.09 0.58 11 l-Limonene 7.19 7.02 12 1, 8-Cineole 7.24 0.11 13 1, 4-Cyclohexadiene,1-Methyl 7.75 0.27 14 Bicyclo [3.1.0] Hexan-2-ol 7.93 0.30 15 Alpha-Terpinolene 8.33 0.10 16 (+)-4-Carene 8.39 0.35 17 1, 6-Octadien-3-ol, 3, 7-Dimethyl 8.61 1.07 18 3-Cyclohexen-1-ol, 4-Methyl 10.67 0.64 19 Beta Fenchyl Alchol 11.05 0.21 20 Estragole 11.29 2.72 21 Alpha-Terpinene 16.50 2.41 22 Alpha-Cubebene 16.93 0.29 23 Alpha-Copaene 17.90 3.84 24 Beta Elemene 18.46 1.69 25 Caryophyllene 19.51 36.03 26 Azulene 19.96 0.38 27 Alpha-Humulene 20.43 2.94 28 Trans-beta-Farnesene 20.52 0.29 29 1, 6-Cyclodecadiene 21.25 0.45 30 Beta-Selinene 21.42 2.70 31 Aalpha-Selinene 21.67 2.40 32 Naphthalene 21.82 0.57 33 Cyclohexene, 1-methyl 22.08 5.13 34 1-Naphthalenol 22.26 0.36 35 Delta-Cadinene 22.49 2.52 36 Cadina-1, 4-Diene 22.70 0.21 37 Cyclohexane methanol, 4-Ethenyl 23.17 0.59 38 1, 6, 10-Dodecatrien 23.55 0.58 39 (-)-Caryophyllene Oxide 24.09 1.45 40 Pentalene, Octahydro 25.24 0.79 41 Bicyclo [4.4.0] dec-1-ene 25.56 0.24 42 Copaene 25.67 1.09 43 4H-1, 3, 5-Thiadiazin-4-one 32.52 0.09 174 Table 4 Chemical constitutents of Salvia officinalis L. essential oil Peak Material Retention Time (RT) % of Total 1 Cis-Salvene 4.00 0.15 2 Tricyclo [2.2.1.0 (2, 6)] Heptane 5.20 0.13 3 1S-Alpha-Pinene 5.41 3.73 4 Camphene 5.69 3.36 5 Bicyclo [3.1.1] Heptane 6.20 0.84 6 beta-Myrcene 6.43 0.35 7 Benzene,1-Methyl 7.09 0.82 8 dl-Limonene 7.17 0.80 9 1, 8-Cineole 7.25 10.56 10 1, 6-Octadien-3-ol, 3, 7-dimethyl 8.70 0.27 11 Beta-Thujone 8.90 25.63 12 Thujone 9.11 6.54 13 Bicyclo [2.2.1] heptan-2-one 9.87 16.46 14 Borneol L 10.37 1.69 15 3-Cyclohexen-1-ol, 4-Methyl-1 10.68 0.52 16 P-Menth-1-en-8-ol 11.06 0.23 17 2 (3H)-Furanone 11.27 0.23 18 Benzene, 1-Methoxy-4-(1-Propenyl) 14.42 0.52 19 Phenol, 2-Methyl-5-(1-Methylethyl) 15.06 0.27 20 Caryophyllene 19.34 1.68 21 1H-Cycloprop[e] Azulene 19.96 0.77 22 Alpha-Humulene 20.42 2.81 23 Ledene 21.66 0.47 24 1H-Cycloprop[e] Azulene 23.94 0.52 25 (-)-Caryophyllene Oxide 24.08 0.86 26 Veridiflorol 24.33 4.71 27 1, 2-Dihydropyridine 24.50 0.35 28 12-Oxabicyclo [9.1.0] Dodeca 24.77 1.37 29 trans-Z-alpha-Bisabolene Epoxide 25.34 0.71 30 Cyclopentan,1-Methylen-2-Vinyl 25.97 0.27 31 Bicyclo [6.1.0] Nonane 30.89 0.21 32 Cembrene 31.50 0.13 33 1-Naphthalenepropanol 31.79 4.27 34 5, 7-Dimethoxy-1-Naphthol 31.96 0.16 35 4-Methoxy-3-(3-Methoxyphenyl) 36.83 0.18 36 1, 3-Xylyl-15-crown-4, 2, 3-Pinan 37.17 0.39 37 Methoxy-3-(3-Methoxyphenyl) 37.39 0.82 38 Cyclononasiloxane, OctadecaMethyl 37.45 0.23 39 Etracosamethyl cyclododeca siloxan 37.56 1.16 40 Hexasiloxane, Tetradecamethyl 37.65 0.63 41 Iron, Monocarbonyl 37.00 1.76 42 Silicone Grease, Siliconfett 83.10 0.73 43 1-Naphthalene Ethanol 37.89 1.20 44 5, 6, 8, 9-Tetramethoxy-2-Methylpep 38.04 0.55 175 Table 5 Chemical constitutents of Glycyrrhiza glabra L. essential oil Peak Material Retention Time (RT) % of Total 1 1S-Alpha-Pinene 5.40 0.38 2 Linalool 8.63 1.02 3 Alpha Terpineol 11.07 0.61 4 Lavandulyl Acetate 18.29 6.26 5 Bicyclo [3.1.1] Hept-2-ene 19.23 0.97 6 Caryophyllene 19.36 0.51 7 Endo-2, 6-Dimethyl-6-(4-Methyl) 19.89 1.81 8 1H-Cycloprop Azulene 20.93 1.33 9 Geranyl Propionate 21.14 3.31 10 Benzene,1-(1,5-Dimethyl-4-Hexen) 21.35 0.76 11 Trans-Beta-Farnesene 21.40 1.16 12 3-Buten-2-ol, Benzoate 21.53 0.97 13 Cyclohexene, 1-Methyl-4-(5-Methyl) 22.06 0.42 14 Propanoic Acid, 2-Methyl 22.24 2.19 15 Beta-Sesquiphellandrene 22.49 0.48 16 Butanoic Acid, 3, 7-Dimethyl 23.59 7.34 17 Nerolidol 23.74 4.11 18 3-Hexen-1-ol, Benzoate 23.89 4.37 19 Linalool L 23.93 0.71 20 Caryophyllene Oxide 24.15 1.90 21 3-Cyclohexene-1-Ethanol 24.26 0.61 22 2, 6-Octadien 24.70 8.54 23 (E)-2-Formyl-6-Methyl 24.81 2.68 24 1, 6, 10-Dodecatrien 25.09 2.52 25 Naphthalene 25.34 2.55 26 Aristolene 25.58 20.14 27 Hinesol 25.67 1.90 28 Beta-Eudesmol 25.90 1.73 29 2-Naphthalene methanol 25.96 2.21 30 Beta-Bisabolol 26.33 0.48 31 Alpha-Bisabolol 26.61 0.20 32 Geranyl tiglate 27.04 3.79 33 Acetic acid, 1-Methylcyclopentyl 27.22 0.71 34 Neryl Propionate 28.19 1.67 35 Geranyl Acetate 28.31 0.84 36 Benzyl benzoate 28.48 2.72 37 Neryl 2-Methylpropanoate 29.47 0.76 38 2-Hexadecen 29.82 0.34 39 2-Pentadecanone 29.92 0.58 40 Neophytadiene 30.35 0.37 41 Neryl Acetate 31.01 0.51 42 Hexadecanoic Acid 31.10 0.40 43 Geranyl Benzoate 31.15 2.88 44 Cyclohexene,1-Methyl-5-(1-Methyl) 31.49 0.30 176 Table 6 The acute toxicity of selected essential oils on the adult females of H. hebetor Treatment n Slope ± E LC30 µl/liter air (95% CL) LC50 µl/liter air (95% CL) LC90 µl/liter air (95% CL) χ2 A. sativum 480 1.41±0.20 2.22 5.22 42.05 9.85 (1.14 - 3.31) (3.57 - 6.76) (29.59 - 74.68) R. officinalis 480 2.35±0.30 2.48 4.15 14.51 15.72 (1.60 - 3.28) (3.11 - 5.04) (12.09 - 18.88) P. nigrum 480 1.39±0.20 5.41 12.88 107.33 7.82 (2.84 - 7.93) (9.06 - 16.42) (73.00 - 205.55) S. officinalis 480 1.12±0.15 6.30 18.36 250.47 6.19 (3.21 - 9.62) (12.69 - 24.30) (154.06 - 551.65) G. glabra 480 1.08±0.12 8.72 26.51 401.83 15.22 (4.81 - 13.07) (18.68 - 35.38) (274.39 - 837.72) CL: Confident limit, χ2: Chi-Square value. Table 7 The logistic regression analysis of E. kuehniella larvae parasitized by H. hebetor. Treatments Coefficient Estimate SE χ2 P-value Control P0 (constant) 2.1769 0.6643 10.74 0.0011 P1 (linear) -0.0116 0.0964 0.01 0.9044 P2 (quadratic) -0.0011 0.0036 0.09 0.7603 P3 (cubic) 0.00001 0.00003 0.11 0.7350 A. sativum P0 (constant) 0.0261 0.4235 0.42 0.9508 P1 (linear) 0.0868 0.0653 1.76 0.1841 P2 (quadratic) -0.0033 0.0025 1.81 0.1782 P3 (cubic) 0.00003 0.00002 1.41 0.2348 R. officinalis P0 (constant) 0.1075 0.4190 0.07 0.7975 P1 (linear) 0.0351 0.0640 0.30 0.5837 P2 (quadratic) -0.0012 0.0024 0.26 0.6075 P3 (cubic) 0.00001 0.00002 0.12 0.7306 P. nigrum P0 (constant) 1.5420 0.5193 8.82 0.0030 P1 (linear) -0.0229 0.0762 0.09 0.7635 P2 (quadratic) -0.0007 0.0028 0.07 0.7927 P3 (cubic) 0.00001 0.00003 0.14 0.7096 S. officinalis P0 (constant) 1.6186 0.5090 10.11 0.0015 P1 (linear) -0.0694 0.0744 0.87 0.3509 P2 (quadratic) 0.0016 0.0027 0.32 0.5727 P3 (cubic) -0.00002 0.00003 0.31 0.5764 G. glabra P0 (constant) 2.1836 0.6279 12.09 0.0005 P1 (linear) -0.0469 0.0891 0.28 0.5986 P2 (quadratic) -0.0004 0.0032 0.02 0.8990 P3 (cubic) 0.00001 0.00003 0.08 0.7764 SE: Standard Error, χ2: Chi-square value 177 Fig. 1 Functional response curve of H hebetor previously exposed to LC30 of selected essential oils and the control to different densities of E. keuhniella larvae Bioassay The LC30 and LC50 values for A. sativum, R. officinalis, P. nigrum, S. officinalis and G. glabra essential oils against the females of H. hebetor are shown in Table 6. The adult bioassays indicated that acute toxicity of R. officinalis essential oil on the female wasps of H. hebetor was higher than the others. Also, G. glabra essential oil showed the lowest acute toxicity in this research. Functional response type Logistic regression model with linear and non- linear parameters indicated the functional response types in the control and essential oils treatments (Table 7). According to the results, the functional response type II (P1< 0) were determined in the control and P. nigrum, S. officinalis and G. glabra and type III (P1≥ 0) in A. sativum and R. officinalis essential oils, respectively (Figs 1 and 2). 178 Fig. 2 The percentage curve of parasitized larvae by H. hebetor previously exposed to LC30 of tested essential oils and the control Functional response parameters The estimation results of handling time, attack rate and theoretical maximum attack rate values from treated wasps of H. hebetor are shown in Table 8. Accordingly, the control and R. officinalis essential oil treatments showed the shortest (0.411± 0.028 h) and longest (0.542± 0.058 h) values of handling time, respectively. Also, the highest and lowest attack rate values were recorded in the control (0.047± 0.003 h-1) and R. officinalis essential oil (0.033± 0.003 h-1) treatment, respectively. In addition, the highest value of the theoretical maximum attack rate base on T/Th was obtained in the control (58.35) and the lowest being in R. officinalis essential oil (44.28) treatment; however, the difference between R. officinalis and S. officinalis wasn’t significant. 179 Table 8 Functional response parameters in H. hebetor previously exposed to LC30 of essential oils Treatment Type of functional response Attack rate (h) a ± SE (Lower-Upper) Handling time (h-1) Th ± SE (Lower-Upper) Theoretical maximum attack rate (T/Th) R2 Control II 0.047 ± 0.003 (0.042 - 0.053) 0.411 ± 0.028 (0.355 - 0.469) 58.35 0.93 A. sativum III 0.036 ± 0.003 (0.023 - 0.042) 0.515 ± 0.055 (0.405 - 0.626) 46.57 0.86 R. officinalis III 0.033 ± 0.003 (0.027 - 0.038) 0.542 ± 0.058 (0.425 - 0.659) 44.28 0.85 P. nigrum II 0.039 ± 0.002 (0.034 - 0.043) 0.462 ± 0.036 (0.389 - 0.534) 51.99 0.85 S. officinalis II 0.041 ± 0.004 (0.034 - 0.048) 0.530 ± 0.052 (0.426 - 0.635) 45.27 0.86 G. glabra II 0.042 ± 0.002 (0.037 - 0.047) 0.444 ± 0.031 (0.381 - 0.506) 54.09 0.86 a: Attack rate, Th: Time of handling (Handling time),R 2: Coefficient of specification, SE: Standard error. Discussion Studies about the effects of different plant compounds such as essential oils on the functional response of H. hebetor can be useful tool for forecasting H. hebetor success in IPM programs, especially in the management of stored pests. The essential oils are safe compounds for human and environment programs and many of them showed high toxic effects due to aromatic and biologically active vapours (Yildirim et al., 2011). There is no study about the effects of selected essential oils on the other natural enemies; but, the effects of these essential oils were investigated on different insect pests; such as A. sativum essential oils on Tribolium castaneum (Herbst), R. officinalis essential oil on larvae of Pseudaletia unipuncta (Haworth) and Trichoplusia ni (Hübner), P. nigrum essential oil on rice weevil, Sitophilus oryzae L. and rice moth, Corcyra cephalonica (St.), S. officinalis essential oils on Drosophila melanogaster Meigen and Bactrocera oleae (Rossi) and G. glabra essential oil on potato tuber moth Phthorimaea operculella (Zeller); and showed suitable effects on control of mentioned insects (Yildirim et al., 2011; Yazdgerdian et al., 2015). In addition, there are few investigations about the effects of selected plant essential oils on the important ectoparasitoid wasp, H. hebetor (Seyyedi et al., 2011; Hashemi et al., 2014; Ahmadpour, 2017). In our study, the tested essential oils showed different acute toxicity on the adult females of H. hebetor that are in agreement with the findings reported by Seyyedi et al., (2011), who studied the impacts of isolated essential oil from Ferula gummosa L. on the female wasps of H. hebetor and concluded that mortality of H. hebetor was increased after 24 h of exposure (LC50= 9.16 µl/liter air). Moreover, Hashemi et al., (2014) concluded that Ferula assafoetida L. essential oil had high toxicity on H. hebetor. Tetracosamethyl cyclododeca siloxan, alpha- pinene, caryophyllene, beta-thujone and aristolene as major components in A. sativum, R. officinalis, P. nigrum, S. officinalis and G. glabra essential oil are volatile and aromatic compounds that showed high toxicity against H. hebetor in our research. These compounds contain active molecules that have fumigant, contact, antifeedants and repellent mode of actions and can be considered as efficient insecticides against different insect pests especially in enclosed environments (Yazdgerdian et al., 2015). Accordingly, the sublethal concentrations of different essential oils and the other botanical compounds can have negative effects on natural enemies especially on the functional response parameters (Croft, 1990; Mahdavi and Saber, 2013; Jarrahi and Safavi, 2015). The results of this research showed that the attack rate values in treated wasps of H. hebetor with sublethal concentrations of studied essential oils was lower than control; but, the handling time values were higher than control; this shows that these essential oils have changed the searching behavior and the other parasitism activities of H. hebetor; because, when handling time increase therefore attack rate decrease and this is a negative effect of a compound on a biocontrol agent. There is no research about the effects of plant essential oils on the functional response of H. hebetor; but, the researches on the insecticides effects in this case are available. Mahdavi (2011) studied the effects of abamectin, carbaryl, chlorpyrifos and spinosad and reported functional response type III in the control and all insecticides treatments that his results are in agreement with our results about A. sativum and R. officinalis essential oil. Mahdavi and Saber (2013) also concluded that 180 malathion had lower negative effects on the functional response of H. hebetor compared with diazinon in IPM programs; but, our results indicated that G. glabra essential oil was compatible compound with H. hebetor. Because, this essential oil showed the lowest adverse effects on the functional response type and it’s parameters in this parasitoid wasp. According to the result of Rafiee- Dastjerdi et al., (2013) and Nazeefullah et al., (2014); G. glabra also showed low toxic effects on against potato tuber moth Phthorimaea operculella (Zeller) and Tribolium castaneum (Herbst), respectively. Rafiee-Dastjerdi et al., (2009b) also reported that the functional response of H. hebetor under hexaflumuron, profenofos, spinosad and thiodicarb and control treatments was type II, and their results are in agreement with our results about the control and P. nigrum, S. officinalis and G. glabra essential oils treatments. Faal-Mohammad Ali et al., (2010) stated that the functional response type in H. hebetor under larval and pupal treatments with chlorpyrifos and fenpropathrin and in the control was type III that their results are in disagreement with our results about the control, P. nigrum, S. officinalis and G. glabra due to differences of treatments, growth stage of parasitoid wasps and it’s response type to different densities of host. Moreover, Abedi et al., (2012) studied the sublethal effects of azadirachtin, cypermethrin, methoxyfenozide and pyridalil on the functional response of H. hebetor and concluded that among them based on obtained handling time values, cypermethrin showed the highest adverse effect on the host-finding behavior of this parasitoid wasp; but, in our study R. officinalis essential oil showed the highest effects on this important characteristic of this parasitoid wasp. In addition, Jarrahi and Safavi (2015) concluded that proteus as a new formulated insecticide (based on combination of thiacloprid and deltamethrin) in pupal stage treatment of H. hebetor showed the highest handling time and the lowest attack rate compared with Metarhizium anisopliae and the control; because the results are same, in our study R. officinalis showed the highest handling time and the lowest attack rate values on H. hebetor and therefore is an incompatible essential oil with this parasitoid wasp. The theoretical maximum attack rate also in all examined treatments was different and the highest value of this parameter was recorded for the control that this is in agreement with the results obtained by Rafiee- Dastjerdi et al., (2009b); Abedi et al., (2012) and Mahdavi and Saber, (2013). Functional response studies under laboratory conditions may have low similarity to the results that obtained in the field conditions (Munyaneza and Obrycki, 1997). Houck and Strauss (1985) and Darwish et al., (2003) concluded that laboratory functional response has important role in understanding of the relations between different natural enemies and their hosts in biological control programs. Such studies can provide valuable informations for developers of biological control programs and release of natural enemies in agricultural crops. In conclusion, this research showed that isolated essential oils affected the functional response and quality control of this parasitoid wasp. This study indicated that there isn’t significant difference between G. glabra compared with the control and G. glabra essential oil hadn’t negative effects on the functional response of H. hebetor and it’s parameters including attack rate, handling time and theoretical maximum attack rate. Therefore, G. glabra essential oil can be recommended as a suitable botanical compound in Integration with H. hebetor in IPM programs. In this research, we investigated the effects of selected essential oils against the ectoparasitoid wasp H. hebetor for the first time. This study shows the potential of essential oils as effective and natural compounds on different insects. The authors recommend more researches about the effects of essential oils on the other natural enemies and also application of these compounds for management of insect pests especially in enclosed environments. References Abedi Z, Saber M, Gharekhani G, Mehrvar A, Mahdavi V. Effects of azadirachtin, cypermethrin, methoxyfenozide and pyridalil on functional response of Habrobracon hebetor Say (Hym.: Braconidae). J. Plant Prot. Res. 52(3): 353-358, 2012. Abramson CI, Wanderley PA, Wanderley MJA, Mina AJS, De Souza OB. Effect of Essential Oil from Citronella and Alfazema on Fennel Aphids Hyadaphis foeniculi Passerini (Hemiptera: Aphididae) and its Predator Cycloneda sanguinea L. (Coleoptera: Coccinellidae). Am. J. Environ. Sci. 3(1): 9-10, 2006. Ahmadpour R. The effects of isolated essential oils from four medicinal plants on the ectoparasitoid wasp Habrobracon hebetor Say in laboratory conditions. M.Sc. thesis of Agriculture Entomology. University of Mohaghegh Ardabili, Ardabil, Iran. 75 pp, 2017. Baker JE, Fabrick JA. Host hemolymph proteins and protein digestion in larval Habrobracon hebetor (Hym.: Braconidae). Insect Biochem. Mol. Biol. 30(10): 937-946, 2000. Croft BA. Arthropod Biological Control Agents and Pesticides. Wiley, New York, 1990. Darwish E, El-Shazly M, El-Sherif H. The choice of probing sites by Bracon hebetor Say (Hymenoptera: Braconidae) foraging for Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). J. Stored Prod. Res. 39(3): 265-276, 2003. Faal-Mohammad Ali H, Seraj AA, Talebi-Jahromi Kh, Shishebor P, Mosadegh MS. The effect of sublethal concentration on functional response of Habrobracon hebetor Say (Hymenoptera: Braconidae) in larval and pupal stages. Proceedings of 19th Iranian Plant Protection Congress. 31 July - 3 August, Tehran, Iran, 2010. Hashemi Z, Goldansaz H, Hosseini-Naveh V. Effects of essential oil of Ferula assafoetida L. on biological parameters of the parasitoid wasp Habrobracon hebetor (Hym.: Braconidae) under laboratory conditions. Proceedings of the 21th Iranian plant protection congress. 9-13 September. University of Urmia, Iran, 2014. 181 Hassell MP, Lawton JH, Beddigton JR. Sigmoid functional responses by invertebrate predators and parasitoids. J. Anim. Ecol. 46(1): 249-262, 1977. Hassell MP. The Dynamics of Arthropod Predator Prey Systems. Monographs in Population Biology. Princeton University Press, Princeton, 1978. Heimpel GE, Antolin MF, Franqui RA, Strand MR. Reproductive isolation and genetic variation between two “strains” of Bracon hebetor (Hymenoptera: Braconidae). Biol. Control. 9(3): 149-156, 1997. Hentz MG, Ellsworrth PC, Naranjo SE, Watson TF. Development, longevity and fecundity of Chelonus sp. nr. curvimaculatus (Hymenoptera: Braconidae), an egg-larval parasitoid of pink bollworm (Lepidoptera: Gelechiidae). Environ. Entomol. 27(2): 443-449, 1998. Hites RA. Development of Gas Chromatographic Mass Spectrometry. Analy. Chem. 88(14): 6955-6961, 2016. Holling CS. Some characteristics of simple types of predation and parasitism. Canadian Entomol. 91(7): 385-398, 1959. Holling CS. Principles of insect predation. Annu. Rev. Entomol. 6: 163-183, 1961. Holling CS. The functional response of invertebrate predators to prey density. Mem. Entomol. Soci. Can. 48:1-86, 1966. Houck MA, Strauss RE. The comparative study of functional responses: experimental design and statistical interpretation. Canadian Entomol. 117(5): 617-629, 1985. Jarrahi A, Safavi SA. Effects of pupal treatment with Proteus and Metarhizium anisopliae sensu lato on functional response of Habrobracon hebetor parasitizing Helicoverpa armigera in an enclosed experiment system. Biocontrol Sci. Techn. 26: 206-216, 2015. Juliano SA. Non-linear curve fitting: predation and functional response curve. Design and Analysis of Ecological Experiments (S.M. Cheiner, J. Gurven, eds.), 1993. Juliano SA, Williams FM. A. comparison of methods for estimation the functional response parameters of the random predator equation. J. Anim. Ecol. 56: 641-653, 1987. Luck RF. Principles of Arthropod Predation. Ecological Entomology. John Wiley & Sons, New York, 1985. Mahdavi V. Evaluation of susceptibility of ectoparasitoid Habrobracon hebetor Say (Hymenoptera: Braconidae) to chlorpyrifos, carbaryl, spinosad and abamectin insecticides and entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana in laboratory. M.Sc. thesis, University of Maragheh, Iran 96 pp, 2011. Mahdavi V, Saber M. Functional response of Habrobracon hebetor Say (Hymenoptera: Braconidae) to Mediterranean flour moth (Anagastra Kuehniella Zeller) in response to pesticides. J. Econ. Entomol. 53(4): 399-403, 2013. Mudd A, Corbet SA. Response of the ichneumonid parasite Nemeritis canescens to kairomones from the flour moth, Ephestia kuehniella. J. Chem. Ecol. 8(5): 843-850, 1982. Munyaneza J, Obrycki JJ. Functional response of Coleomeguilla maculata Coleoptera: Coccinellidae) to Colorado potato beetle eggs (Coleoptera: Chrysomelidae). J. Biol. Control. 8(3): 215-224, 1997. Navaei AN, Taghizadeh M, Javanmoghaddam H, Oskoo T, Attaran MR. Efficiency of parasitoid wasps, Trichogramma pintoii and Habrobracon hebetor against Ostrinia nubilalis and Helicoverpa sp. on maize in Moghan. Proceedings of the 15th Iranian Plant Protection Congress. 7-11 September, Razi University of Kermanshah, Iran, 2002. Nazeefullah S, Dastagir G, Ahmad B. Effect of cold water extracts of Acacia modesta Wall. and Glycyrrhiza glabra Linn. on Tribolium castaneum and Lemna minor. Pak. J. Pharm. Sci. 27(2): 217-222, 2014. Oaten A, Murdoch WW. Functional response and stability in predator-prey systems. Am. Nat. 109: 289-298, 1975. Poderoso JCM , Correia-Oliveira ME, Chagas TX, Zanuncio JC, Ribeiro GT. Effects of Plant Extracts on Developmental Stages of the Predator Podisus nigrispinus (Hemiptera: Pentatomidae). Florida Entomol. 99(1): 113- 116, 2016. Rafiee-Dastjerdi H, Hejazi MJ, Nouri-Ganbalani G, Saber M. Toxicity of some biorational and conventional insecticides to cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) and its ectoparasitoid, Habrobracon hebetor (Hymenoptera: Braconidae). J. Entomol. Soc. Iran 28: 27-37, 2008. Rafiee-Dastjerdi H, Hejazi MJ, Nouri-Ganbalani G, Saber M. Effects of some insecticides on functional response of ectoparasitoid, Habrobracon hebetor Say (Hymenoptera: Braconidae. J. Entomol. Soc. Iran 6: 161-166, 2009b. Rafiee-Dastjerdi H, Khorrami F, Razmjou J, Esmaielpour B, Golizadeh A, Hassanpour M. The efficacy of some medicinal plant extracts and essential oils against potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae). J. Cro. Prot. 2(1): 93-99, 2013. Royama TA. Comparative study of models for predation and parasitism. Res. Popul. Ecol. 1: 1-91, 1971. Salvador G, Consoli LF. Changes in the hemolymph and fat body metabolites of Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) parasitized by Cotesia flavipes (Cameron) (Hymenoptera: Braconidae). Biol. Control. 45(1): 103-110, 2008. SAS Institute. The SAS System for Windows. SAS Institute, Cary, NC, 2002. Seyyedi A. Insecticidal effects of Ferula gummosa L. on Ephestia kuehniella Zeller and its parasitoid wasp Habrobracon hebetor Say. M.Sc. thesis of Agriculture Entomology. University of Shahed, Tehran, Iran.100 pp, 2011. Shiva parsia A, Valizadegan O. Fumigant toxicity and repellent effect of three Iranian Eucalyptus species against the lesser grain beetle, 182 Rhyzopertha dominica (F.) (Col.: Bostrychidae). J. Entomol. Zool. Stu. 3(2): 198-202, 2015. Tostowaryk W. The effect of prey defence on the functional response of Podisus modestus (Hemiptera: Pentatomidae) to densities of the sawflies Neodiprion swainei and N. pratti banksianae (Hymenoptera: Neodiprionidae). Canadian Entomol. 104(1): 61-69, 1972. Yazdgerdian AR, Akhtar Y, Isman MB. Insecticidal effects of essential oils against woolly beech aphid, Phyllaphis fagi (Hemiptera: Aphididae) and rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae). J. Entomol. Zool. Stu. 3(3): 265- 271, 2015. Yildirim E, Kordali S, Yazici G. Insecticidal effects of essential oils of eleven plant species from Lamiaceae on Sitophilus granarius (L.) (Coleoptera: Curculionidae). Romani. Biotech. Let. 16(6): 6702-6709, 2011. Yu SH, Roy MI, Na JH, Choi WI. Effect of host density on egg dispersion and the sex ratio of progeny of Bracon hebetor (Hymenoptera: Braconidae). J. Stored Prod. Res. 39(4): 385- 393, 2002.