2.1 Plant materials and preparation
Leaves and barks from 14-year-old plants of C. loureirii were collected in Maoming County, one of the major cinnamon-producing areas of Guangdong Province in South China, in May 2019. Samples were washed with tap water and left to shade-dry. Then, the dried samples were ground into powders (60 mesh) and stored in a desiccator for further analysis.
2.2 Essential oil extraction
The volatile oils of the leaves and bark were obtained by hydrodistillation in accordance with the method in the Pharmacopoeia of the People’s Republic of China (China Pharmacopoeia Committee 2020) with minor modifications. The powder of the sample (15 g) was weighed into a 1000 mL distillation flask and placed in a sealed vessel along with a mixture of distilled water and zeolites (w/v = 1: 20, 1: 15, and 1: 10) and distilled after soaking for 1, 1.5, or 2 h. Afterwards, the distillate was transferred into a 750 mL separating funnel. Methylene dichloride (40 mL) was used to extract the volatile compounds from the aqueous layer three times. The combined organic layer was transferred into a 250 mL conical flask, dehydrated over anhydrous sodium sulfate for 30 min, and filtered through mid-speed filter paper. After most of the distillate was evaporated over a vapor bath in a fume hood, the concentrated solution was transferred into a dried brown sample bottle and placed at room temperature until the methylene chloride was completely evaporated. The obtained yellow volatile oil was weighed and stored at 4°C until further use. The yield of the essential oil was determined using the following equation:
Three repeats were conducted for each sample, and the average essential oil content was calculated.
2.3 Origin and rearing of RIFA micrergates and macrergates
Red imported fire ants were obtained from the suburbs of Zengcheng County of Guangzhou city and stored in the laboratory for bioassays in plastic containers at 25 ± 2°C and 60%–80% elative humidity. A test tube (25 mm × 200 mm) partially filled with 10% sugar water or tap water and plugged with cotton to reduce the evaporation of water was used as the water source. A Petri dish (8.5 cm × 1.5 cm) containing the larvae of Tenebrio molitor (Coleoptera Tenebrionidae) and ham was used as a food source. Red imported fire ants were kept in a dry indoor environment at 25 ± 2°C during the bioassay experiment. Tenebrio molitor was purchased from Guangzhou Insect Market.
2.4 GC–MS analysis
GC–MS analysis was performed on a 7890A gas chromatograph (Agilent, American) interfaced with a 5975C Plus mass spectrometer (Agilent, American). A fused silica capillary Agilent Technology HP-5ms (5% phenyl methyl siloxane) column (Rxi-5MS, 30.0 m×0.25 mm×0.25 μm, film thickness = 0.1 m) was used for separation. The injector temperature was 260°C. The initial temperature was 100°C, and the temperature was gradually increased to 150°C at a rate of 4°C/min and held for 3 min at 150°C. Then, the temperature was gradually increased to 180°C at a rate of 4°C/min. Finally, the temperature was gradually increased to 220°C. The linear velocity of the helium carrier gas was 1mL/min at a split ratio of 1:10. The injection volume was 1 μL.
EI was used as the ion source, and the ion source temperature was 200°C. The sector mass analyzer was set to scan from 45 to 450 amu (m/z). The ionization energy was 70 eV. The interface temperature was 250°C.
The identification of the components was performed by linear retention indices which were calculated using the generalized equation of Van Den Dool and Kratz (1963), with literature data, and by matching their recorded mass spectra with the standard mass spectra from the National Institute of Standards and Technology (NIST 14) library data provided by 5977A HP-MSD (Agilent, American) ChemStation (Adams 2007). The GC-MS peak area normalization method was used to calculate the relative percentage content of each component in the leaf and bark oils of C. loureirii. Three replicates were performed for each sample.
2.5 Quantitative analysis of trans-cinnamaldehyde and cinnamyl acetate in samples by GC
The standard solutions were prepared by mixing 3 mg/mL cinnamyl acetate with 10 mg/mL trans-cinnamaldehyde and diluted to different concentrations (0.13, 0.26, 0.52, 0.75, 0.98, 1.24, and 1.5 mg/mL) with dichloromethane and were kept at 4°C until further use. The samples were subjected to three parallel determinations to analyze their trans-cinnamaldehyde and cinnamyl acetate contents.
A fused silica capillary Agilent Technology HP-5ms (5% phenyl methyl siloxane) column (Rxi-5MS, 30.0 m × 0.25 mm × 0.25 μm, film thickness = 0.1 m) was used for separation. The injector temperature was set to 200°C. The detector temperature was set to 300°C. The initial column temperature was maintained at 50°C for 2 min, and the temperature was gradually increased to 300°C at a rate of 15°C/min and was then held for 10 min. The linear velocity of the nitrogen carrier gas was 2.57 mL/min at a split ratio of 1:40.
2.6 Physiological indices of RIFAs
The biological fumigation activities of plant essential oils and main compounds were evaluated against RIFAs according to the method of Seo et al. (2009), with slight modifications. Cinnamon essential oils were placed into 200 µL capped centrifuge tubes with 10 small holes in the cap. Then, the centrifuge tubes were placed in a cup precoated with polytetrafluoroethylene (PTFE) emulsion and dried for 24 h. Twenty medium-sized RIFA workers were transferred into each cup (body length was 3.0-4.3 mm, head width was 0.7-1.0 mm), which was then sealed with an airtight lid. The test was carried out at a temperature of 25 ± 1°C and a humidity of 80%.
Experiment 1: Essential oils at concentrations of 320, 160, and 80 µg/cm3 were tested. The cumulative mortality caused by essential oil at a concentration of 320 µg/cm3 was determined every 10 min for 1 h. At other concentrations, cumulative mortality was measured every 2 h for a period of 12 h.
Experiment 2: Trans-cinnamaldehyde at concentrations of 5, 50, and 100 µg/cm3 and cinnamyl acetate at 2.5, 25, and 50 µg/cm3 were tested, and cumulative mortality by fumigation was measured every 2 h. Mortality was measured continuously for 20 h. The mixtures of trans-cinnamaldehyde and cinnamyl acetate (v/v = 2:1) at concentrations of 7.5, 75, and 150 µg/cm3 were tested for cumulative mortality every 2 h for a period of 20 h.
Experiment 3: Mixtures of trans-cinnamaldehyde and cinnamyl acetate in different ratios (v/v = 1:2, 2:1, and 4:1) at a concentration of 75 µg/cm3 were tested for cumulative mortality by fumigation every 2 h for a period of 20 h.
Experiment 4: Quantitative cinnamyl acetate was added to the essential oil of Cinnamomum cassia bark to obtain a 2:1 (v/v) mixture of trans-cinnamaldehyde and cinnamyl acetate, and cumulative mortality by fumigation was measured every 2 h. The rates were measured continuously for 12 h.
Taking an empty centrifuge tube as a control. According to the provisions of the pesticide control institute of the Ministry of Agriculture on the efficacy test methods and evaluation standards of health insecticides, only the mortality and knockdown ability observed in the control group must be lower than 20%, all experiments were considered valid. All treatments were repeated three times. The following formula was used to calculate mortality:
2.6.2 Grasping ability
The experimental procedure was the same as that described in section 2.6.1. The RIFA workers were transferred into a disposable plastic cup, covered with white paper, and the disposable cup was gently turned upside-down and allowed to stand for 3 s. Then, the cup was gently turned back, and the number of RIFAs that fell down into the bottom of the cup was recorded. Taking an empty centrifuge tube as a control and assuming that the mortality and knockdown ability observed in the control group must be lower than 20%, all experiments were considered valid. All treatments were repeated three times. The following formula was used to calculate the grasping ability:
2.6.3 Knockdown ability
The experimental operation was the same as that described in Section 2.6.1. The knockdown ability of RIFA workers was observed at the corresponding time point. The total number of ants being knocked down was the sum of the dead and ant workers who had lost the ability to walk. Taking an empty centrifuge tube as a control and assuming that the mortality and knockdown ability observed in the control group must be lower than 20%, all experiments were considered valid. All treatments were repeated three times. The following formula was used to calculate the knockdown ability:
2.6.4 Scanning electron microscopy (SEM) observation of the antennae of RIFA treated with cinnamon essential oils
Fumigation was performed with essential oil, trans-cinnamaldehyde, cinnamyl acetate, and a mixture of trans-cinnamaldehyde and cinnamyl acetate (v/v = 2:1) at concentrations of 320, 100, 50, and 150 µg/cm3, respectively. The worker ants were treated for 24 h and then stored. The test used ant workers without fumigation as a control group.
Sample preparation method for SEM: Under an anatomical lens, the antennae and heads of the test ants were cut off with a blade. The surface attachments were washed with 70% ethanol, and the samples were fixed with glutaraldehyde for 24 h. After dehydration with 70%, 80%, 90%, and 100% ethanol gradients, they were glued onto the SEM sample stage with conductive glue, and after spraying with gold, the antennae of RIFAs were observed and photographed under SEM (Hitachi S-3400N-Ⅱ, Japan).
2.6.5 GC-EAG analysis of the main components in essential oils
Medium-sized RIFA workers were chosen, and the antennae were cut off quickly with a scalpel. The cut end was connected to a reference electrode filled with Ringer’s saline and placed on the electrode. The tip of the antenna was connected to the recording electrode. The electrode was connected to the signal amplifier through a silver chloride-plated silver wire, and the signal was monitored by the probe, and the data were recorded and analyzed by GC-EAG (Agilent 7890B-5977B, SYNTECH IDAC-2).
GC-EAG analysis was conducted with an HP-SMS capillary column with an injection volume of 1 µL and no split injection. The inlet temperature was 230°C, the initial temperature of the program was 50°C, and the temperature was gradually increased to 100°C at a rate of 5°C/min. Then, the temperature was gradually increased to 240°C at a rate of 10°C/min and held for 4 min. The total time was 20 min. The detector was a flame ionization detector (FID) with a temperature of 250°C.
2.6.6 EAG analysis of single and mixed components of trans-cinnamaldehyde and cinnamyl acetate on the antenna electroantennogram (EAG) responses of RIFA
The antenna EAG responses of RIFA to trans-cinnamaldehyde and cinnamyl acetate were measured by the method described by Yan et al. (2012), with slight modifications. First, the heads of medium-sized RIFA workers were completely cut off. Second, a small portion at the end of the antenna was cut off. Finally, the antenna was cut off from the head. The antenna was connected to a PRG-2 electrode painted with conductive glue, and the EAG baseline in the EAG IDAC2 record was measured. The continuous humidified airflow was 500 mL/min, the stimulation airflow was 450 mL/min, the stimulation airflow duration was 0.3 s, the signal drift was automatically controlled, and the maximum recording time was 5 s. The measurement was repeated 10 times, and a new worker antenna was applied in each group. The sample volume was 1 μL each time (at concentrations of 1, 10, 100, and 1000 μg/mL), which was dripped onto a 5 mm × 30 mm filter paper strip. Each repetition interval was 1 min, and n-hexane was used as the control.
The fumigation effects of different concentrations of C. loureirii leaf and bark essential oils, trans-cinnamaldehyde, cinnamyl acetate, and the mixture on the mortalities, grasping abilities, and knockdown abilities of ant workers were compared. The variance analysis (ANOVA) of the data was performed by the Tukey method (P<0.05).