The mulberry pyralid moth, Glyphodes pyloalis (Lepidoptera: Crambidae)(Walker 1859), is considered a key pest of mulberry trees, which are exclusively used for silkworm rearing. Considering that silkworm cultivation is dependent on mulberry leaves, it is inevitable to choose a suitable control method against mulberry pyralid moths. In recent years, research about pheromones in insects and how they affect the Intraspecies and interspecific relationships has received much attention. These studies have included the identification of pheromone compounds and their biosynthesis, recognition of pheromone receptors in insects, as well as behaviors caused by receiving pheromones in insects, in order to be used in pest control programs. Considering that sex pheromones are non-toxic and specific compounds, the present study was conducted to identify pheromone compounds in G. pyloalis. Three compounds, including hexadecanoic acid methyl ester (H), (Z)-9-octadecenoic acid methyl ester (S), and octadecanoic acid methyl ester (O), were identified and introduced as pheromone compounds in G. pyloalis. Examining the results of the wind tunnel showed that these compounds in the ratio of 8H:4S:1O show the best attraction in the male population. However, this ratio of compounds, when used in pheromone traps in a mulberry orchard, was found to be less effective than control traps (non-mated females). It seems that this decrease in performance happened due to the rapid decomposition of the compounds in the environment. According to the results of the present study, it can be said that identifying the sex pheromone of G. pyloalis can lead to the development of efficient strategies for monitoring and controlling the population of this pest.

Insect rearing

In order to form a colony, the larvae of the mulberry snout moth were collected from infested mulberry orchards in the vicinity of Rasht (37°17'02.3"N 49°35'17.8" E) around the second half of June 2020-2022. and were transferred to the Department of Plant Protection, University of Guilan. Larvae were placed in transparent plastic containers with dimensions of 18×15×7 cm, and fresh mulberry (Shin Ichinoise) leaves were provided to them daily until pupation. The rearing was carried out in a BOD incubator with a temperature set at 25 ± 1 ºC, relative humidity of 60± 5% and a photo period of 16:8 (dark: light) hours (Khosravi & Jalali Sendi, 2010). Upon adult emergence, cotton soaked in a 10% honey solution was provided for feeding.

Extraction of the sex pheromone from female glands

Our choice to extract pheromones from the sex pheromone-producing gland was due to the initial focus on identifying precursor compounds or compounds that are produced directly in the secretory gland. This step can provide accurate initial information on potentially active compounds.

We are fully aware that the final composition of the naturally released sex pheromone may differ from that found in the gland, as changes may occur along the release pathway (such as chemical transformation, combination with other substances, or selective evaporation) (Lin et al., 2018). However, our goal at this stage is to precisely identify the compounds found in the gland, which are known to be the main source of the pheromone. On the other hand, the method implemented in this research has been used in various sources to extract sex pheromones from Lepidoptera (Brakefield et al., 2009)

After the emergence of female adults, the pheromone glands in the eighth and ninth abdominal rings of 2-5 days old female moths were collected (Fig. 1). For this purpose, they were first kept on ice for 1 minute to make them immobile, and then the ovipositor was removed by gently pressing the end of the abdomen, and then separated from the body using a scalpel (disinfected with 70% alcohol). Thirty adult females were used in this experiment. Then, 50 µL of hexane was added to each gland inside a microtube. After 60 minutes of storing the samples at room temperature, the tissues were removed from the tube, and the prepared extract was stored in a freezer at -20 ºC until chemical analysis. (Honda et al., 1990).

Gas chromatograph-mass spectrometry (GC-MS) analysis

The chemical analysis of the extracted pheromone was performed by gas chromatography-mass spectrometer (Agilent Technologies 7890B-Agilent Technologies 5977A). GC-MS, equipped with a 30-mm-long, 0.25-mm-width, and 0.25-μm-internal thickness HP-5MS (5%-phenyl-methylpolysiloxane) capillary column. Helium gas (99.999% purity) was applied at a 3 mL/min flow rate, and the column temperature first started at 50 ◦C and then reached 280 ◦C at a rate of 5 ◦C /min.  In the next step, 2 μL of pheromone solution was injected. The spectra were captured with the ionization energy of 70 eV in the electron impact mode (Ebadollahi et al., 2016). Detected compounds were compared and interpreted with sex pheromone compounds of other insects using the http://www.pherobase.net website.

Chemicals

Hexadecanoic acid methyl ester (purity 95%) (CAS Number: 112-39-0), (Z)-9-Octadecenoic acid methyl ester (purity 95%) (CAS Number: 112-62-9), and Octadecanoic acid methyl ester (purity 99%) (CAS Number: 112-61-8) were purchased from Sigma Aldrich (https://www.sigmaaldrich.com). Then, different concentrations of compounds were evaluated individually and in combinations. After evaluation in laboratory conditions, the desired concentration was evaluated in the field.

Examining the effectiveness of the identified compounds in laboratory conditions

Evaluation of the effectiveness rate of unmated male insects to sex pheromone in the wind tunnel (Specifications length of 1.2 meters, width and height 30 cm) was done (Fig. 2). The wind tunnel was placed in a controlled room with 25 ± 1 ºC and a relative humidity of 60 ± 1 percent. This tunnel was made of Plexiglass and two air regulators (one for air flow and the other for air suction and exit). Various concentrations of pheromone compounds were evaluated individually and in triple mixtures. Each standard mixture was dissolved in n-hexane, and 500 µL of each mixture was loaded into an Eppendorf tube sealed with cotton wool. For control, the same amount of n-hexane was added to the vial without pheromone. Then, individual males were released in the wind tunnel at a distance of 100 cm from the odor source. Male insects were exposed to a wind flow speed of 0.3 m/s and a light intensity of 10 lux. The response time of male insects in the wind tunnel included moving against the wind direction and up to 40 cm away from the release site, flying up to 10 cm away from the synthetic pheromone source, sitting on the pheromone source, and trying to mate for several minutes. Each male insect was used only once in an experiment. When no reaction was shown by the male insect, it was considered non-responsive (Miller & Roelofs, 1978). This experiment was repeated in three replicates, each replicate with 5 individual insects in the wind tunnel for each combination.

Investigating the efficiency of identified compounds in field conditions (Mulberry orchards assay)

In order to increase the performance and gradual release of pheromone compounds, 500 µL hexane and 500 mg clinoptilolite zeolite were mixed together. This mixture was placed in the environment for 30 minutes until the hexane evaporated and reached the initial weight. Then three-component mixture of Hexadecanoic acid methyl ester, Octadecanoic acid methyl ester, and (Z)-9-Octadecenoic acid methyl ester compounds was added to the vial containing clinoptilolite zeolite at a ratio of 8:4:1.

To investigate the effectiveness of pheromone in capturing mulberry pyralid moths, a completely randomized design experiment was conducted with 3 treatments, including the combination of prepared pheromone, control (trap containing hexane), and natural pheromone (live adult female) in four replications in the Iranian Silkworm Research Center (Pasikhan-Rasht; 37°16'51.5"N 49°27'18.7 "E). A yellow delta trap was used in this research. Traps were installed at a height of 1.5 meters from the ground. The distance between the treatments in the rows was 2.5 meters. The traps were visited at intervals of one week. In order to randomize the traps, using a table of random numbers, the order of installing the traps in each specific block was determined first, and then the installation of the traps was done. After a week had passed again in the same way, the order of the traps was determined, and the installation process was carried out.

Statistical analysis

In order to evaluate the pheromone compounds in the wind tunnel, an experiment was conducted as a randomized complete block design with 3 replications, and each replication with 5 unmated male insects. Alsototo investigate the efficiency of pheromone traps, an experiment was conducted as a randomized complete block design with 4 replications. Each replication included 3 treatments (virgin female, pheromone trap, and n-Hexane). The number of insects hunted was recorded weekly for 4 weeks. SAS version 9.1 software was used for data analysis. The number of captured insects was log-transformed to normalize the data. One-way analysis of variance (ANOVA) was used to compare the mean number of insects caught in different pheromone treatments. Tukey's test was used to compare the mean data (p<0.05).