The lesser mulberry pyralid larvae of different age groups were collected from infested mulberry orchards in Rasht city (37.3846° N, 49.6545° E) Guilan province, Iran. The collected larvae were transferred to the laboratory and were provided with tender mulberry foliage daily. They were cultured in plastic jars (10 × 20 × 5 cm) in an incubator set at 25±1 °C, 60±5% RH and 16L:8D. The adults were maintained in plastic jars (18 × 7 × 5 cm) and were provided with tender mulberry leaves for oviposition and 10% honey solution for feeding soaked in a cotton wool.
Thymus vulgaris essential oil and its constituents
Thyme essential oil was procured from Barij Essence Pharmaceutical Company (Kashan, Iran), thymol (CAS Number: 89-83-8; 95% purity) and carvacrol (CAS Number: 499-75-2; 98% purity) compounds were purchased from Sigma Aldrich (https://www.sigmaaldrich.com).
The components of the essential oil were determined by gas chromatography mass spectrophotometry (GC-MS) on an Agilent GC-7890A using helium as the carrier gas. A progressive temperature from 50 °C to 290 °C (10 ° C per minute) was performed with the energy of 70 eV for every 1 s. The thyme essential oil compounds were identified through retention times (RT) which were then matched with RT of known compounds in the device library (Ebadollahi et al. 2016).
Bioassays were carried out on newly ecdysed third instar larvae. We used thyme oil and two of its components based on availability (i.e. thymol and carvacrol) The primary tests were performed to find effective doses. Finally, five doses of thyme oil (1, 2, 3, 4 and 5 µg per larvae), thymol (12.5, 25, 37.5, 50 and 62.5 µg per larvae) and carvacrol (37.5, 50, 62.5, 75 and 87.5 µg per larvae) were selected. They were diluted in 80% aqueous acetone, with the addition of 0.01% Tween 80 as adjuvant for topical application in order to find the effective doses (1µg/larva) (Rizvi et al. 2018). Each experiment was replicated 4 times with 10 larvae per replicate and controls received the same amount of acetone instead. The number of dead larvae were recorded after 48 hours and LD50, LD30 and LD10 values were estimated using PoloPlus software (2002).
Insecticidal activity of binary mixtures
We followed the method of Kumrungsee et al.(2014) for making mixtures of essential oil components in the order mentioned below:
1. thymol (LD30) + carvacrol (LD30)
2. thymol (LD50) + carvacrol (LD30)
3. carvacrol (LD50) + thymol (LD30)
Each experiment was performed in four replications, and in each replication ten larvae were used. We used the following formula of Kumrungsee et al. (2014);
E= Oa + Ob (1-Oa/100)
In this formula E is the expected mortality, Oa and Ob are the observed mortality of the pure compounds at the specified concentrations. The comparison using the following formula of X2 was performed in order to define the mixtures as antagonistic, additive or synergistic:
The Om= observed mortality of mixture. Thus, when X2 values > 3.84 and < 3.84, are regarded as synergistic or additive, respectively. Where the observed mortality is less than the expected mortalities, it is regarded as antagonistic (Pavela 2015; Hummelbrunner and Isman 2001).
Third instar larvae 48 h after treatment were homogenized in 500 mL of distilled water or buffer related to each test. The homogenates were centrifuged at 4 °C for 10 min. The resulting supernatants were transferred into new micro tubes and frozen at -20 °C until further use.
Esterase assay (ESTs)
In order to measure the activity of estrases, two substrates namely α-naphthyl acetate (α-NA) and β-naphthyl acetate (β-NA) were used. Thus, 15 μL of α-NA or β-NA (10 mM), 50 μL of fast blue RR salt (1 mM) and 20 mL of enzyme solution were released into each well. After 5 min, optical density (OD) was read at 450 nm using a microplate reader (Han et al. 1995).
Glutathione-S-transferase assay (GSTs)
The method of Oppenoorth et al. (1979) was used to measure the glutathione S-transferase activity. Twenty μL of enzyme solution, 30 μL of universal buffer (pH 7), and 20 μL of 20mM CDNB (1-chloro-2,4- dinitrobenzene) and 20 mM DCNB (1,2-dichloro-4-nitrobenzene) were mixed. The activity was read in a microplate reader at 340 nm.
Mixed function oxidase assay
The method used for cytochrome P450 monooxygenase assay was adopted from Martin et al. (2002). A mixture of 80 µL of potassium phosphate buffer (62.5 mM, pH 7.2) and a 20 µL enzyme solution was prepared. To this a solution of 13 mg 3,3',5,5'-Tetramethylbenzidine in 6.5 ml methanol with 19.5 mL of 0.25 M sodium acetate buffer (NaC2H3O2) at pH 5.0 was mixed and ultimately we added 25 µL of H2O2 (3%). After 30 minutes of incubation at 25 ° C, enzyme activity was recorded at 630 nm.
Alanine and aspartate aminotransferase assays (ALT and AST)
In order to measure the activity of alanine and aspartate aminotransferase, Pars Azmun commercial kit was used (http://www.parsazmun.com/). Reagents R1 and R2 are first incubated for 20 minutes at laboratory temperature and then enzyme activity is monitored by adding 20 µL samples. The absorbance was recorded at 340nm (Thomas 1998).
Lactate dehydrogenase assays (LDH)
Activities of lactate dehydrogenase was assayed using a commercial kit by ParsAzmun Co. (http://www.parsazmun.com/). Reagents R1 and R2 are first incubated for 20 minutes at laboratory temperature and then enzyme activity is monitored by adding 20 µL samples. The absorbance was recorded at 340 nm (King 1965).
Acid and alkaline phosphatase assays (ACP and ALP)
The acid and alkaline phosphatases were measured using para-nitrophenol phosphate substrate (Bessey 1946). Twenty µL of enzyme solution was mixed with 40 µL Tris buffer (Tris-HCl, 20 mM, pH 8 for ALP and pH 5 for ACP) and 20 µL substrate (0.02 m, pH 7.2). They were then incubated for 5min and the absorbance was read at 405 nm.
Using bovine serum protein, the protein content of the samples was determined by protein estimation kit (Ziest- Chem Co., Tehran-Iran) based on the method of Lowry et al. (1951). Fifty μL of reagent was mixed with 30 μL of standard (bovine serum protein at a concentration of 50 mg) and 50 μL of reagent with 30 μL of each sample were incubated separately for 15 minutes and the absorbance was read at 545 nm.
Probit analysis using PoloPlus software 2002(Robertson et al. 2007) was used to estimate mortality after 48 h. The collected data (mortality and biochemical) were first normalized using arcsin. The one-way analysis of variance (ANOVA) was done using Minitab software. Tukey’s test was used to compare differences in means at a probability less than 5%.