The Role of Green Gram Plant Volatile Blends in the Behavior of Arctiid Moth, Spilosoma Obliqua

doi:10.21203/rs.3.rs-1761169/v1

Larvae of Spilosoma obliqua Walker (Lepidoptera: Arctiidae) feed on the leaves and flowers of green gram [Vigna radiata (L.) Wilczek] and reduce seed yield. Therefore, it is interesting to observe whether undamaged (UD) and insect-damaged (ID, plants after feeding by S. obliqua larvae) plants cause attraction of conspecific adult moths. Females showed attraction towards volatile blends of UD, ID and mechanically-damaged (MD) plants of three green gram cultivars [PDM 54 (PDM), Pusa Baisakhi (PUSA) and Samrat (SAM)] in Y-tube olfactometer bioassays. Females were more attracted towards volatile blends of ID plants of a particular cultivar than UD plants of the same cultivar. Total amount of volatiles was higher in ID plants than UD plants. Some herbivore-induced plant volatiles − 3Z-hexenal, 1-hexanol, 3Z-hexenyl acetate, 2-octanol and ocimene were attractive to the insect. Females were attracted towards three synthetic blends (µg in 25 µl CH₂Cl₂) comprised − 2.97 µg 3Z-hexenal, 4.92 µg 1-hexanol, 5.56 µg benzaldehyde, 9.76 µg 3Z-hexenyl acetate, 7.27 µg 2-octanol, 7.97 µg ocimene and 5.71 µg acetophenone resembling PDM, 4.68 µg 3Z-hexenal, 8.38 µg benzaldehyde, 7.66 µg 3Z-hexenyl acetate, 5.96 µg ocimene and 7.60 µg acetophenone resembling PUSA, and 3.39 µg 3Z-hexenal, 6.29 µg benzaldehyde, 8.74 µg 3Z-hexenyl acetate, 6.17 µg ocimene and 3.69 µg acetophenone resembling SAM in olfactometer and wind tunnel bioassays. Females could not distinguish between these three synthetic blends in olfactometer and wind tunnel bioassays, suggesting that these three synthetic blends could be used as lures in developing baited traps in integrated pest management strategies.

Green gram

Spilosoma obliqua

Volatiles

Olfactometer bioassay

Wind tunnel bioassay

Green gram [Vigna radiata (L.) Wilczek] (Fabaceae), commonly known as mung bean, is one of the most important pulse crops in India. This leguminous crop is thought to be originated from India, and is grown as a fallow crop after cultivation of rice in India. Cultivation of green gram enhances soil nitrogen content, which is helpful for cultivation of other crop plants. Asian countries (India, Bangladesh, Korea, Pakistan, China, Japan, Sri Lanka, Indonesia, Philippines, Burma and Thailand) are the main producers of this pulse crop (Gurung et al. 2020; Singh and Sehgal 1992). Now, this pulse crop is also grown in America and Africa (Anonymous 2020). At present, approximately 70% of this pulse crop is contributed by India (Anonymous 2020). The cooked green gram seeds are valuable sources of proteins, carbohydrates, manganese, magnesium, phosphorous, iron, copper, potassium, zinc, and vitamins B1, B2, B3, B5, B6 and B9. The green gram seeds are also consumed as sprouts. The sprouting green gram seeds had higher level of protein, calcium, iron and zinc content as well as lower level of antinutrients such as total phenols, flavonoids and tannins (Oghbaei and Prakash 2017). But, attacks by various insect pests associated with green gram plants cause a reduction in pulse yield.

The polyphagous insect pest, Spilosoma obliqua Walker (Lepidoptera: Arctiidae), commonly known as Bihar hairy caterpillar, is of oriental origin and known to cause serious damage to various crops of agricultural importance (Gurung et al. 2020) such as cotton, cauliflower, cabbage, broccoli, mulberry, castor, broad bean (Gurung et al. 2020), jute (Selvaraj et al. 2015), black gram, cowpea (Haribhai 2015), soybean (Chaudhary 2009), sesame (Biswas 2006), sunflower (Varatharajan et al. 1998), groundnut and brinjal (Edde 2021). The insect has been recorded to feed on 126 plant species, which belongs to ca. 24 plant families (Gurung et al. 2020; Singh et al. 1999). This insect is also a serious pest of green gram plant in India as the larvae of this insect pest feed on the leaves and flowers of green gram, and reduces yield of this pulse crop (Mobarak et al. 2020a). Females of this insect lay eggs on the underside of leaves, and the emerged first instars consume green and soft tissues of the leaves behind the veins, while fourth to sixth instars are the most damaging as they defoliate the whole plant. Nineteen to 20 days are required to complete the larval development of S. obliqua on green gram leaves, and 8–9 days are required to complete pupation, while newly emerged adults (males and females) live for 4–5 days (Mobarak et al. 2020a).

It has been demonstrated that long-chain alkanes and free fatty acids present in leaf and flower surface waxes served as short-range attractants, and stimulate females of S. obliqua to lay eggs on the green gram plants (Mobarak et al. 2020b and 2021). However, volatile organic compounds (VOCs) emitting from green gram plants causing attraction of adult S. obliqua have not been investigated. So, it is necessary to find out the composition of VOCs emitted by green gram plants that causes attraction of S. obliqua. Several reports indicate that damage by an insect on its host plants results in the changes of total amounts of VOCs compared to undamaged plants, and some novel herbivore-induced plant volatiles (HIPVs) that are emitted by insect-damaged plants (Das et al. 2021; Karmakar et al. 2018; Malik et al. 2016; Mitra et al. 2021). It is also proved that the composition of VOCs varies between undamaged plants and mechanically-damaged plants as mechanical damage causes the fastest changes in the production of VOCs (Piesik et al. 2010; Portillo-Estrada et al. 2015, 2021). Further, it also helps us to compare the composition of VOCs between insect-damaged plants and mechanically-damaged plants. Therefore, it is of substantial interest to monitor whether there are changes in the composition of VOCs in volatile blends emitted by mechanically-damaged green gram plants compared to volatile blends emitted by undamaged and insect-damaged green gram plants.

The aims of the current study were to (i) assess the behavioral responses of S. obliqua towards volatile blends emitted from undamaged, insect-damaged and mechanically damaged plants of three green gram cultivars (PDM 54, Pusa Baisakhi and Samrat, as these three cultivars are currently grown in West Bengal, India) and determine whether volatile blends from these treatments were attractive to S. obliqua, (ii) identify and quantify the VOCs emitted by undamaged, insect-damaged and mechanically-damaged plants of these cultivars by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID) analyses, respectively, to compare the VOC profiles among these cultivars, and (iii) evaluate the role of individual synthetic VOCs and synthetic blends in the insect responses to determine whether synthetic blends could act as olfactory cues to attract S. obliqua, which can be used as lures in developing baited traps as a part of an integrated pest management (IPM) strategy to control this insect pest without using synthetic insecticides.

Plants

Seeds of three green gram cultivars (PDM 54, Pusa Baisakhi and Samrat, hereafter these three cultivars will be expressed as PDM, PUSA and SAM, respectively) were germinated on moistened filter papers. Sufficient amounts of soil were collected from the field of Crop Research Farm (CRF), The University of Burdwan (23°16′ N, 87°54′ E), West Bengal, India and sterilised in an autoclave at 121°C for 30 min. Each seed with cotyledon was sown in an earthen pot containing ca. 1500 cm³ of sterilised soil, and grown under natural photoperiod of 13:11 L/D at 30–35°C during July – September, 2020. The whole plant with the pot was covered with fine mesh nylon net (180 cm height, 90 cm diameter) to avoid insect attack and unintentional infection. Plants (2–3-wks-old) of each green gram cultivar with a height of ca. 20–25 cm and 10 fully developed leaves were used for the experiments.

Insects

Sixth instars of S. obliqua were collected from jute plants adjoining to the fields of CRF of this University. Larvae were fed on castor plants in an environmental chamber at 27 ± 2°C, 65 ± 10% relative humidity (r. h.) and a photoperiod of 12L:12D. Emerged adults were kept on castor leaves as females could lay eggs and F1 eggs were collected. The emerged larvae of S. obliqua were also fed on castor leaves. Second generation newly emerged 4th instars of S. obliqua were placed on the plants of each green gram cultivar for insect feeding damage treatments. Newly emerged males and females (males were identified by bipectinate antenna, while females were identified by serrated antenna) were paired, and mating occurred between four and six hours of emergence. Second generation females (1–2 days old) were used for olfactometer and wind tunnel bioassays. Larvae were not reared on green gram leaves as insects might be habituated to the volatile blends of green gram plants, which might cause a bias of S. obliqua females towards volatile blends of green gram plants in bioassays.

Plant volatile blend collection

Undamaged (UD) plants of three green gram cultivars (PDM, PUSA and SAM) were used for collection of volatile blends. For insect damage (ID) treatments, a group of five newly emerged 4th instars of S. obliqua had been continuously fed for 12 hr (on the plants of PDM, PUSA and SAM treatments hereafter referred as ID PDM, ID PUSA and ID SAM, respectively). Volatile blends were collected from ID plants just after feeding by 4th instars. For the treatment of mechanical damage (MD), 10 leaves of a plant were wounded twice with hole punches (each hole 0.5 cm), and volatile blends were collected without delay after wounding. An experimental plant was positioned in a closed glass dome (4 L) with Teflon base having a small opening for passing the stem of plant (Das et al. 2021) (Supplementary Fig. S1). After placing each plant in the closed glass dome, sterilized non-absorbent cotton was encircled in the stem of an experimental plant to avoid damage in the stem by the Teflon base. Five experimental plants of each treatment were used to collect volatile blends for 4 hours between 9 am and 1 pm, and a fraction of volatile blends was applied to identify and quantify the VOCs while remaining fractions were used for olfactory bioassays. For olfactory bioassays, volatile blends were collected from 33 UD plants of each green gram cultivar, 82 plants of ID PDM, 75 plants each of ID PUSA and ID SAM, and 33 MD plants of each green gram cultivar over 4 hours between 9 am and 1 pm. Charcoal-filtered air was propelled (4 L min^-1) into the top of the closed glass dome and pulled (2 L min^-1) through four volatile collector traps (150 mm long × 5 mm o.d.). Each trap comprised 80 mg HayeSep Q (80–100 mesh) as adsorbent, and four volatile collector traps were put into four side sampling ports round about the base of closed glass dome.

To elute volatile blends from the adsorbent of an experimental plant, HayeSep Q from four volatile collector traps were amalgamated and 600 µl methylene chloride were applied. The collected volatile blends in methylene chloride were concentrated to 100 µl through a gentle nitrogen flow. A fraction (75 µl) was employed for olfactory bioassays, and the rest (25 µl) was applied for identification and quantification of VOCs through GC-MS and GC-FID, respectively (the volatile blends collected from five experimental plants of each treatment). For olfactory bioassays, an aliquot of 25 µl (equivalent to volatile blends emitted by a plant during 1 hr) was applied to Whatman No. 41 filter paper (1 cm²).

Olfactometer bioassays

We performed olfactory bioassays of S. obliqua females using a glass Y-tube olfactometer (each arm 15 cm, radius of each arm 4 cm and 45° Y angle) (Supplementary Fig. S2). The stem of the olfactometer was attached with a porous glass vial (4 cm radius × 7 cm long) (as the air can flow through the olfactometer). Each arm of the olfactometer was connected with an adapter attached with a glass vial (4 cm radius × 4 cm long), which contained a piece (1 cm²) of filter paper (Whatman No. 41) loaded with 25 µl volatile blends (VOCs collected from variously treated experimental plants or individual synthetic compounds or synthetic blends) and other glass vial contained a filter paper of the same size loaded with 25 µl control solvent (methylene chloride), and a female was placed in the stem of the olfactometer. Charcoal-filtered air was passed through each arm of the Y-tube at 125 ml min^-1. Teflon tubes were used to connect between different parts of the olfactometer set-up.

Behavioral responses of S. obliqua females (1–2 days old) using Y-tube olfactometer were conducted in the laboratory between 16.30 and 20.30 hr at 27 ± 2 ºC, 70 ± 5% r. h. and a light intensity of 150 lux. Females did not show bias towards the control solvent (methylene chloride) in preliminary olfactory bioassays. The choice of a female was recorded as positive response when the female reached the end of the odor-loaded arm of the Y-tube, while a negative response was recorded when the female moved towards the solvent-loaded arm. Behavioral response was recorded as no choice when a female was in the stem of the Y-tube for 3 min (Karmakar et al. 2020). A female was tested once and it was discarded from bioassays. Responses of S. obliqua females towards one volatile blend sample were finished when 60 adult females had responded. After testing five insects, the filter papers with either volatile blend sample or the control solvent were changed and the Y-tube was rotated 180° to keep away from directional effects. Further after testing five insects, the Y-tube olfactometer set-up was thoroughly washed with petroleum ether followed by acetone and dried in a hot-air oven at 50°C for 1 hr. Teflon tubes were cleaned by 70% ethanol two times and dried at 27 ± 2 ºC for 1 hr after testing five insects.

Behavioral responses of S. obliqua towards natural volatile blends in olfactometer bioassays

Responses of unmated females or gravid females or males towards volatile blends of UD plants of each green gram cultivar (PDM or PUSA or SAM) were tested against the control solvent [methylene chloride (CH₂Cl₂)] to observe whether there were differences in the responses of unmated females, gravid females and males towards natural volatile blends of UD plants of each green gram cultivar compared to the control solvent.

Bioassays using unmated females towards volatile blends emitted by differently treated plants (UD, ID and MD) of three green gram cultivars (PDM, PUSA and SAM) were performed in a variety of combinations (Table 1).

Table 1

Bioassays conducted in Y-tube olfactometer using *Spilosoma obliqua* females towards natural volatile blends emitted from plants of three green gram cultivars [PDM 54 (PDM), Pusa Baisakhi (PUSA) and Samrat (SAM)] that have received specific treatments: undamaged (UD), insect-damaged (ID) and mechanically-damaged (MD) plants (volatile blends emitted by plants during 1 hr)
Bioassays	T1	T2
Bioassay 1	Volatile blends from differently	Control solvent
	treated green gram plants	(CH₂Cl₂)
	UD PDM
	UD PUSA
	UD SAM
	ID PDM
	ID PUSA
	ID SAM
	MD PDM
	MD PUSA
	MD SAM
Bioassay 2	UD plants	UD plants
	PDM	PUSA
	PDM	SAM
	PUSA	SAM
Bioassay 3	ID plants	ID plants
	PDM	PUSA
	PDM	SAM
	PUSA	SAM
Bioassay 4 & 5	ID or MD plants	UD plants
	PDM	PDM
	PDM	PUSA
	PDM	SAM
	PUSA	PDM
	PUSA	PUSA
	PUSA	SAM
	SAM	PDM
	SAM	PUSA
	SAM	SAM
Bioassay 6	ID plants	MD plants
	PDM	PDM
	PDM	PUSA
	PDM	SAM
	PUSA	PDM
	PUSA	PUSA
	PUSA	SAM
	SAM	PDM
	SAM	PUSA
	SAM	SAM

Volatile blend analysis

Identification of collected volatile blends from five separate experimental plants of each treatment were carried out through a Perkin-Elmer Clarus 690 coupled to a Mass selective detector fitted with a HP-5 capillary column (Agilent; Palo Alto, CA, USA; length: 30 m × 0.32 mm × 0.25-µm film thickness). The oven temperature program was held at 50°C for 3 min then programmed at 3.8°C/min to 240°C and finally held for 5 min. Carrier gas was helium (1 ml/min), and sample (1 µl) was injected with a split ratio of 1:5 (Koner et al. 2022). The MS parameters were 250°C at the interface, ionization energy 70 eV and scan speed 5 scans/sec. The identities of VOCs were confirmed by comparing the diagnostic ions (NIST 2014 library) including GC retention times with those of relevant authentic synthetic compounds.

For quantification of VOCs through GC-FID, nonyl acetate was incorporated as internal standard (IS) at 1 µg µl^− 1 in each sample. Analysis of volatile blends were carried out by a Techcomp GC (Em Macau, Rua De Pequim, Nos. 202A-246, Centro Financeiro F7, Hong Kong) model 7900 attached with a HP-5 capillary column and a flame ionization detector, which was performed at the same temperature conditions mentioned for GC-MS analysis. Nitrogen was carrier gas (18 ml/min), and 1 µl sample injected with a split ratio of 1:5. Temperature of the injector port was 280°C. In addition, straight-chain alkanes from C7 to C28 were injected in this equipment at the same temperature programming. Retention time was applied to compute the Kovats retention indices (for temperature-programming) according to van Den Dool and Kratz (1963).

Synthetic compounds

HayeSep Q (80–100 mesh) was purchased from Sigma Aldrich, Germany. All synthetic compounds were purchased from Sigma Aldrich, Germany (Supplementary Table S1).

Behavioral responses of S. obliqua females towards individual synthetic compounds and synthetic blends in olfactometer bioassays

Responses of females towards individual synthetic compounds or complete synthetic blends resembling amounts emitted by UD, ID and MD plants of each green gram cultivar (µg/4 hr) were dissolved in 100 µl CH₂Cl₂, and 25 µl of this odor (volatile blends emitted by plants during 1 hr) were tested against the control solvent (25 µl) to observe the role of individual synthetic compounds in S. obliqua (Table 2, Supplementary Table S2). In addition, responses of S. obliqua towards synthetic blends (consisting of those individual synthetic compounds to which S. obliqua showed responses or attraction) (25 µl volatile odor) resembling amounts emitted by UD, ID and MD plants of each green gram cultivar (volatile blends emitted by plants during 1 hr) vs. the control solvent (25 µl) were conducted (Table 2, Supplementary Table S3).

Table 2

Bioassays conducted in Y-tube olfactometer using *Spilosoma obliqua* females towards complete synthetic blends or individual synthetic compounds or certain synthetic blends resembling natural volatile blends emitted from plants of three green gram cultivars [PDM 54 (PDM) Pusa Baisakhi (PUSA) and Samrat (SAM)] that have received specific treatments: undamaged (UD), insect-damaged (ID) and mechanically-damaged (MD) plants (µg in 25 µl CH₂Cl₂)
Bioassays	T1	T2
Bioassay 1	Complete synthetic blends from	Control solvent
	differently treated plants	(CH₂Cl₂)
	UD PDM
	UD PUSA
	UD SAM
	ID PDM
	ID PUSA
	ID SAM
	MD PDM
	MD PUSA
	MD SAM
Bioassay 2a	Individual synthetic compounds resembling
	amounts of individual VOCs present in plants
	of differently treated each green gram cultivar
Bioassay 2b	Blends comprised of those compounds on
	which insect showed responses resembling
	amounts present in plants of differently
	treated each green gram cultivar
Bioassay 2c	Blends comprised of those compounds on
	which insect showed attraction resembling
	amounts present in plants of differently
	treated each green gram cultivar
Bioassay 3a	Natural volatile blends from	Individual synthetic compounds or certain
	ID PDM plants	synthetic blends resembling amounts
		present in volatile blends of ID PDM plants
Bioassay 3b	Natural volatile blends from	Individual synthetic compounds or certain
	ID PUSA plants	synthetic blends resembling amounts
		present in volatile blends of ID PUSA plants
Bioassay 3c	Natural volatile blends from	Individual synthetic compounds or certain
	ID SAM plants	synthetic blends resembling amounts
		present in volatile blends of ID SAM plants
Bioassay 4a	Certain synthetic blends resembling	Certain synthetic blends resembling
	amounts present in ID PDM plants*	amounts present in ID PUSA plants**
Bioassay 4b	Certain synthetic blends resembling	Certain synthetic blends resembling
	amounts present in ID PDM plants*	amounts present in ID SAM plants***
Bioassay 4c	Certain synthetic blends resembling	Certain synthetic blends resembling
	amounts present in ID PUSA plants**	amounts present in ID SAM plants***
*: A synthetic blend 2.97 µg 3Z-hexenal, 4.92 µg 1-hexanol, 5.56 µg benzaldehyde, 9.76 µg 3Z-hexenyl acetate, 7.27 µg 2-octanol, 7.97 µg ocimene and 5.71 µg acetophenone (PDM blend 7)
**: A synthetic blend 4.68 µg 3Z-hexenal, 8.38 µg benzaldehyde, 7.66 µg 3Z-hexenyl acetate, 5.96 µg ocimene and 7.60 µg acetophenone (PUSA blend 5)
***: A synthetic blend 3.39 µg 3Z-hexenal, 6.29 µg benzaldehyde, 8.74 µg 3Z-hexenyl acetate, 6.17 µg ocimene and 3.69 µg acetophenone (SAM blend 5)

Responses of females towards natural volatile blends from ID plants of each green gram cultivar (volatile blends usually released by plants during 1 hr) were tested against individual synthetic compounds or certain synthetic blends [PDM blend 7–2.97 µg 3Z-hexenal, 4.92 µg 1-hexanol, 5.56 µg benzaldehyde, 9.76 µg 3Z-hexenyl acetate, 7.27 µg 2-octanol, 7.97 µg ocimene and 5.71 µg acetophenone, PUSA blend 5–4.68 µg 3Z-hexenal, 8.38 µg benzaldehyde, 7.66 µg 3Z-hexenyl acetate, 5.96 µg ocimene and 7.60 µg acetophenone, and SAM blend 5–3.39 µg 3Z-hexenal, 6.29 µg benzaldehyde, 8.74 µg 3Z-hexenyl acetate, 6.17 µg ocimene and 3.69 µg acetophenone] resembling amounts emitted by ID plants of the same green gram cultivar (µg in 25 µl CH₂Cl₂) (Table 2, Supplementary Table S4).

Responses of females towards certain synthetic blends resembling natural volatile blends of ID plants of each green gram cultivar were tested against certain synthetic blends resembling natural volatile blends of the other green gram cultivars (µg in 25 µl CH₂Cl₂) (Table 2).

Dose response bioassays of S. obliqua towards individual synthetic compounds were conducted to find out the lowest and highest doses where the female started responding and showed the highest attraction. Dose response bioassays of females towards nine individual compounds were tested at different doses against the control solvent (3Z-hexenal or acetophenone or decanal: 2.50, 5 and 10 µg in 25 µl CH₂Cl₂; hexanal or 1-hexanol or ocimene: 4, 8 and 16 µg in 25 µl CH₂Cl₂; benzaldehyde: 3.50, 7 and 14 µg in 25 µl CH₂Cl₂; 3Z-hexenyl acetate: 6, 12 and 24 µg in 25 µl CH₂Cl₂ and 2-octanol: 6.50, 13 and 26 µg in 25 µl CH₂Cl₂) as the insects were attracted towards nine individual compounds.

Behavioral responses of S. obliqua females towards certain synthetic blends in wind tunnel bioassays

The wind tunnel comprised a plexiglass observation chamber (120 × 30 × 30 cm) with attachments for regulating the speed, filtering, channeling and expelling the air. Charcoal-filtered air was passed through the chamber at 0.20 m s^− 1 with air temperature 27 ± 1°C and 70 ± 5% r. h. The sides and floor of the chamber were covered with black paper to insulate insects from external visual stimuli in the chamber. A plexiglass platform 15 cm above the floor of the chamber was used for release of the test insect, whereas a plexiglass platform 3 cm above the floor of the chamber was used for the attachment of filter papers. A piece (1 cm²) of filter paper (Whatman no. 41) was fixed on a plexiglass stick (4 mm diameter × 2 cm high), and the stick was inserted into a rubber stopper (2.5 cm diameter × 1 cm high). Filter papers were placed centrally in inverted direction 30 cm from the upwind end on the floor of the observation chamber and the test insect was released 90 cm from the upwind end. All tests with females were carried out between 16.30 and 20.30 hr. Each female was introduced into the wind tunnel, observed for 5 min for a given treatment and then recaptured. A female was tested once and it was discarded from bioassays. After testing every five insects, the chamber and release platform were cleaned with deionised water and the chamber was ventilated for 15 min before next bioassays. The target positions of filter papers with volatile cues were changed after each replicate to avoid positional bias in two-choice test. Each filter paper with volatile blends was replaced by a new one after testing five insects. A total of 50 insects (each replicate five insects) were tested in each experiment excluding the number of females that did not respond. The responses of the female S. obliqua were carried out by the following treatments:

Condition 1: In no-choice assay, certain synthetic blend (25 µl of PDM blend 7 or PUSA blend 5 or SAM blend 5) resembling each green gram cultivar (equivalent to volatile blends released by each green gram cultivar during 1 hr, that elicited highest attraction to the insect in Y-tube olfactometer bioassays) was applied on the centre of each filter paper.

Condition 2: In two-choice test, two filter papers were placed with a gap of 5 cm in between them at the centre of the observation chamber. The two-choice test was carried out in the following combinations: PDM blend 7 vs. PUSA blend 5 or SAM blend 5, and PUSA blend 5 vs. SAM blend 5.

Statistical analyses

The data recorded as total amounts of VOCs, and amounts of individual VOCs from UD, ID and MD plants of three green gram cultivars were log (x + 1) transformed before performing statistical analyses. The distribution pattern of the data was normal as indicated by Kolmogorov-Smirnov tests, and Levene’s test for homogeneity of variance demonstrated that the data were also homogeneous with respect to treatments (except those individual compounds which are not recorded in all treatments) using SPSS software (version 25.0). Discriminant function analysis (DA) was conducted using different treatments of three green gram cultivars – UD plants (UD PDM, UD PUSA and UD SAM), ID plants (ID PDM, ID PUSA and ID SAM) and MD plants (MD PDM, MD PUSA and MD SAM) as response variables against the VOCs recorded as explanatory variables to substantiate the differences among green gram cultivars in the context of VOC bouquets using XLSTAT software (Zar 1999). Data obtained on the behavioral responses of S. obliqua towards VOCs were analysed assuming the null hypothesis that the possibility of scores for the test sample(s) or the control solvent is equivalent to 50%, i.e., Chi-square analysis (H₀: P = 50%) (Koner et al. 2022). Insects that remained in the stem of olfactometer were not used for the analysis. Data recorded on the responses of S. obliqua in wind tunnel bioassay experiments were subjected to t-test (Rojas and Wyatt 1999; Karmakar et al. 2016).

Behavioral responses of adult S. obliqua towards natural volatile blends

Males or unmated females or mated females were similarly attracted towards volatile blends of UD plants of each green gram cultivar compared to the control solvent (UD PDM: χ² = 9.60, df = 1, P = 0.0019; UD PUSA: χ² = 8.07, df = 1, P = 0.0045 and UD SAM: χ² = 6.67, df = 1, P = 0.0098). So, unmated females were used in further olfactory bioassays.

Females showed attraction towards volatile blends of UD, ID and MD plants of each green gram cultivar compared to the control solvent (Fig. 1). Females did not differentiate between volatile blends of UD plants of a particular green gram cultivar and volatile blends of the other cultivar (Table 3). Females were attracted towards volatile blends of ID plants of each green gram cultivar when tested against volatile blends of UD plants of the same cultivar (Table 3). Females showed attraction towards volatile blends of ID PDM when tested against volatile blends of UD PUSA or UD SAM, while females displayed attraction towards volatile blends of ID PUSA against volatile blends of UD SAM (Table 3). Females failed to distinguish between volatile blends of ID plants of these green gram cultivars when tested against each other (Table 3). Females could not distinguish between volatile blends of MD plants of a green gram cultivar and volatile blends of UD plants of the same or other cultivar (Table 3). Females did not differentiate between volatile blends of ID plants of a green gram cultivar and volatile blends of MD plants of the same or other cultivar (Table 3).

Table 3

Behavioral responses of *Spilosoma obliqua* females towards natural volatile blends emitted by undamaged (UD), insect-damaged (ID) and mechanically-damaged (MD) plants of three green gram cultivars [PDM 54 (PDM), Pusa Baisakhi (PUSA) and Samrat (SAM)] were tested against each other. Each test was carried out with at least 60 responding females. Non-responders were other than 60 responding females
Comparison		Insects responded		Non-	χ2	P
T1	T2	T1	T2	responders	(df = 1)	values
UD plants	UD plants
PDM	PUSA	31	29	5	0.07	0.7963
PDM	SAM	32	28	6	0.27	0.6056
PUSA	SAM	31	29	6	0.07	0.7963
ID plants	UD plants
PDM	PDM	38	22	4	4.27	0.0389
PDM	PUSA	39	21	4	5.40	0.0201
PDM	SAM	39	21	5	5.40	0.0201
PUSA	PDM	37	23	6	3.27	0.0707
PUSA	PUSA	38	22	5	4.27	0.0389
PUSA	SAM	38	22	4	4.27	0.0389
SAM	PDM	35	25	6	1.67	0.1967
SAM	PUSA	36	24	6	2.40	0.1213
SAM	SAM	38	22	5	4.27	0.0389
ID plants	ID plants
PDM	PUSA	31	29	6	0.07	0.7963
PDM	SAM	32	28	5	0.27	0.6056
PUSA	SAM	31	29	6	0.07	0.7963
MD plants	UD plants
PDM	PDM	34	26	5	1.07	0.3017
PDM	PUSA	34	26	6	1.07	0.3017
PDM	SAM	35	25	6	1.67	0.1967
PUSA	PDM	32	28	5	0.27	0.6056
PUSA	PUSA	33	27	6	0.60	0.4386
PUSA	SAM	34	26	5	1.07	0.3017
SAM	PDM	31	29	6	0.07	0.7963
SAM	PUSA	32	28	5	0.27	0.6056
SAM	SAM	33	27	6	0.60	0.4386
ID plants	MD plants
PDM	PDM	34	26	4	1.07	0.3017
PDM	PUSA	35	25	4	1.67	0.1967
PDM	SAM	36	24	5	2.40	0.1213
PUSA	PDM	33	27	4	0.60	0.4386
PUSA	PUSA	34	26	5	1.07	0.3017
PUSA	SAM	35	25	5	1.67	0.1967
SAM	PDM	32	28	5	0.27	0.6056
SAM	PUSA	33	27	4	0.60	0.4386
SAM	SAM	34	26	4	1.07	0.3017

Identification And Quantification Of Vocs

The DA elucidated that first two factors F1 and F2 expressed more than 97% of the variation, and the biplot represented the spatial direction of response variables, UD PDM, UD PUSA, UD SAM, ID PDM, ID PUSA, ID SAM, MD PDM, MD PUSA and MD SAM against the eight extracted factors (Fig. 2). As indicated through the Fisher’s distance, plants are significantly different in the context of VOC bouquet (Fig. 2, Supplementary Table S5). The eigen values and canonical correlations specify > 99% of variation being illustrated by three axes (Supplementary Table S5).

The total amounts of VOCs were higher in ID or MD plants of each green gram cultivar compared to UD plants of the same cultivar (Table 4). There were no significant differences in the total amounts of VOCs among three cultivars in the context of UD or ID or MD treatments.

Table 4

Composition (GC-FID analysis) of volatile organic compounds (VOCs) (N = 5, Mean ± SE) emitted by undamaged, insect-damaged (plants after 12 hr of continuous feeding by fourth instar *Spilosoma obliqua*) and mechanically-damaged plants of three green gram cultivars, PDM 54 (PDM), Pusa Baisakhi (PUSA) and Samrat (SAM) (µg/4 hr)
Compounds	Undamaged plants			Insect-damaged plants			Mechanically-damaged plants			Retention
	PDM	PUSA	SAM	PDM	PUSA	SAM	PDM	PUSA	SAM	index
2E-Penten-1-ol	0.41 ± 0.03^a	0.47 ± 0.04^a	0.74 ± 0.06^b	2.68 ± 0.19^c	23.46 ± 1.46^d	4.09 ± 0.29^e	4.65 ± 0.33^e	1.43 ± 0.09^f	19.06 ± 1.12^d	760.62
3Z-Hexenal	-	-	-	11.88 ± 0.62^a	18.73 ± 1.05^b	13.57 ± 0.78^c	6.78 ± 0.39^d	5.79 ± 0.37^d	4.19 ± 0.33^e	773.30
Hexanal	2.51 ± 0.16^a	23.08 ± 1.65^b	3.48 ± 0.22^a	10.09 ± 0.62^c	12.63 ± 0.80^c	-	17.73 ± 1.07^d	36.70 ± 2.07^e	10.43 ± 0.67^c	800.97
3-Hexanol	0.95 ± 0.07^a	1.02 ± 0.09^a	1.25 ± 0.11^ac	1.68 ± 0.09^bc	41.11 ± 2.19^d	42.81 ± 2.45^d	15.97 ± 0.97^e	44.90 ± 2.71^d	30.67 ± 1.68^f	817.24
3Z-Hexen-1-ol	-	-	-	5.22 ± 0.31^a	10.58 ± 0.71^b	6.79 ± 0.45^cd	6.31 ± 0.44^ad	-	23.74 ± 1.33^e	834.74
2Z-Hexen-1-ol	0.72 ± 0.06^a	0.80 ± 0.06^a	1.05 ± 0.08^a	17.90 ± 1.15^b	7.33 ± 0.47^c	1.89 ± 0.14^d	2.31 ± 0.16^d	25.38 ± 1.53^e	8.36 ± 0.58^c	858.37
1-Hexanol	-	-	-	19.68 ± 1.10^a	-	-	28.98 ± 1.65^b	-	-	868.01
Heptanal	2.10 ± 0.14^a	1.44 ± 0.10^b	2.39 ± 0.17^a	1.41 ± 0.09^b	1.41 ± 0.10^b	2.31 ± 0.18^a	1.44 ± 0.11^b	8.41 ± 0.54^c	11.76 ± 0.71^d	893.78
Nonane	0.42 ± 0.03^a	0.48 ± 0.04^ab	0.75 ± 0.05^b	29.02 ± 1.55^c	8.27 ± 0.48^d	31.85 ± 1.87^c	-	-	-	915.52
1-Heptanol	-	-	-	17.79 ± 1.05^a	3.16 ± 0.20^b	11.51 ± 0.71^c	7.24 ± 0.50^d	8.86 ± 0.54^d	8.76 ± 0.60^d	950.48
Benzaldehyde	10.30 ± 0.68^a	4.34 ± 0.29^b	14.82 ± 0.87^c	22.26 ± 1.29^d	33.51 ± 1.77^e	25.14 ± 1.44^d	20.29 ± 1.20^d	20.22 ± 1.20^d	-	974.59
Myrcene	16.73 ± 1.02^a	10.87 ± 0.72^b	14.82 ± 0.94^a	13.93 ± 0.93^a	2.74 ± 0.18^c	6.35 ± 0.43^d	-	-	-	1000.17
3Z-Hexenyl acetate	-	-	-	39.03 ± 2.19^a	30.62 ± 1.65^b	34.94 ± 1.91^ab	-	-	-	1006.57
2-Octanol	-	-	-	29.08 ± 1.59^a	-	-	35.61 ± 2.12^b	-	-	1015.90
Benzyl alcohol	4.10 ± 0.27^a	2.67 ± 0.19^b	8.52 ± 0.57^c	7.97 ± 0.52^c	5.47 ± 0.41^ae	2.85 ± 0.21^b	12.61 ± 0.84^d	7.78 ± 0.47^c	7.14 ± 0.56^ce	1033.06
Ocimene	-	-	-	31.88 ± 1.86^a	23.86 ± 1.30^b	24.70 ± 1.36^b	23.28 ± 1.42^b	15.80 ± 0.86^c	-	1048.31
1,3-Diethyl benzene	-	-	-	3.18 ± 0.25^a	1.67 ± 0.09^b	15.80 ± 1.02^c	9.00 ± 0.64^d	22.33 ± 1.29^e	8.98 ± 0.58^d	1052.58
Acetophenone	9.21 ± 0.56^a	2.22 ± 0.17^b	-	22.86 ± 1.25^c	30.41 ± 1.69^d	14.74 ± 0.91^eg	16.20 ± 1.04^eg	11.24 ± 0.66^f	13.16 ± 0.82^fg	1056.85
Linalool	8.89 ± 0.60^a	2.03 ± 0.13^b	-	2.65 ± 0.19^b	13.56 ± 0.74^c	22.01 ± 1.40^de	10.85 ± 0.69^a	25.69 ± 1.63^d	19.27 ± 1.11^e	1079.83
Undecane	2.86 ± 0.20^a	0.68 ± 0.05^b	3.19 ± 0.18^ad	0.94 ± 0.07^b	0.87 ± 0.06^b	1.77 ± 0.15^c	0.74 ± 0.05^b	0.86 ± 0.06^b	4.08 ± 0.31^d	1098.93
Limonene oxide	3.17 ± 0.23^a	15.47 ± 0.93^b	1.55 ± 0.10^c	5.71 ± 0.36^d	6.03 ± 0.35^d	-	5.27 ± 0.44^d	-	-	1127.62
1-Nonanol	1.21 ± 0.09^a	0.84 ± 0.06^a	10.48 ± 0.70^b	25.31 ± 1.55^c	1.01 ± 0.07^a	-	26.37 ± 1.55^c	-	5.35 ± 0.42^d	1163.58
Decanal	6.05 ± 0.39^a	-	2.85 ± 0.16^b	7.93 ± 0.55^c	2.66 ± 0.18^b	8.95 ± 0.60^c	-	13.62 ± 0.81^d	15.91 ± 0.97^d	1206.11
Cuminaldehyde	0.91 ± 0.07^a	1.00 ± 0.07^a	1.24 ± 0.09^ad	2.14 ± 0.21^b	1.94 ± 0.13^b	3.59 ± 0.28^c	1.68 ± 0.13^bd	2.03 ± 0.11^b	4.81 ± 0.37^e	1218.60
Nerol	0.76 ± 0.06^a	17.71 ± 1.08^b	1.09 ± 0.08^a	7.19 ± 0.54^c	20.75 ± 1.24^b	2.58 ± 0.18^d	20.61 ± 1.32^b	1.75 ± 0.10^e	4.65 ± 0.37^f	1249.37
Geraniol	14.59 ± 0.85^a	-	-	13.10 ± 0.84^a	14.55 ± 0.80^a	2.52 ± 0.17^b	-	-	-	1267.04
1-Undecanol	1.64 ± 0.13^a	1.78 ± 0.11^a	1.96 ± 0.13^a	0.81 ± 0.05^b	0.76 ± 0.06^b	1.63 ± 0.12^a	0.65 ± 0.05^b	0.75 ± 0.04^b	3.96 ± 0.27^c	1372.60
β-Caryophyllene	1.33 ± 0.09^a	9.72 ± 0.64^b	9.41 ± 0.62^b	1.06 ± 0.07^a	8.01 ± 0.57^b	8.30 ± 0.53^b	1.19 ± 0.08^a	1.02 ± 0.06^a	4.46 ± 0.36^c	1415.18
Farnesene	-	-	-	1.08 ± 0.07^a	1.05 ± 0.09^a	1.91 ± 0.14^b	0.91 ± 0.09^a	1.03 ± 0.07^a	4.20 ± 0.33^c	1489.88
Nerolidol	-	0.43 ± 0.04^a	0.70 ± 0.05^bc	0.59 ± 0.05^ac	0.50 ± 0.04^ac	1.37 ± 0.11^d	0.42 ± 0.04^a	0.46 ± 0.03^ac	3.85 ± 0.31^e	1526.10
1-Hexadecene	2.02 ± 0.13^a	9.35 ± 0.65^b	9.09 ± 0.60^b	1.44 ± 0.12^a	1.36 ± 0.11^a	2.21 ± 0.14^c	1.16 ± 0.09^a	1.36 ± 0.10^a	4.37 ± 0.31^d	1594.28
Methyl jasmonate	8.31 ± 0.53^a	-	7.28 ± 0.46^a	5.71 ± 0.40^b	5.84 ± 0.38^b	3.67 ± 0.28^c	5.00 ± 0.36^d	6.23 ± 0.34^e	7.32 ± 0.48^e	1617.79
1-Hexadecanol	16.28 ± 0.95^a	1.33 ± 0.08^b	4.41 ± 0.26^c	1.24 ± 0.08^b	1.41 ± 0.10^b	2.47 ± 0.18^d	1.32 ± 0.10^b	1.11 ± 0.07^b	4.21 ± 0.24^c	1902.58
1-Heptadecanol	-	-	-	1.43 ± 0.12^a	1.20 ± 0.08^a	2.07 ± 0.14^b	1.02 ± 0.08^a	1.22 ± 0.08^a	3.26 ± 0.22^c	1951.15
Geranyl linalool	1.89 ± 0.12^ac	2.19 ± 0.13^a	2.21 ± 0.14^a	1.68 ± 0.14^ac	1.63 ± 0.10^ac	9.25 ± 0.54^b	1.40 ± 0.10^c	1.68 ± 0.11^ac	4.58 ± 0.33^d	2033.83
Total	117.36 ± 7.46^a	109.90 ± 7.34^a	103.25 ± 6.64^a	367.57 ± 21.87^b	338.09 ± 19.65^bc	313.66 ± 19.08^bcd	287.00 ± 18.04^bcd	267.66 ± 15.92^cd	240.51 ± 15.36^d
Within the rows means followed by different letters are significantly different (P < 0.05) by Tukey test.

The volatile profile of UD PDM, UD PUSA and UD SAM comprised 24, 22 and 22 compounds with 79%, 86% and 86% of the compounds shared among UD plants of these cultivars, respectively (Table 4, Supplementary Fig. S3). In ID PDM, ID PUSA and ID SAM, 35, 33 and 30 compounds were detected, demonstrating 86%, 91% and 100% of the compounds shared among ID plants of these cultivars, respectively (Table 4, Supplementary Fig. S3). Thirty, 26 and 26 VOCs were identified in volatile blends of MD PDM, MD PUSA and MD SAM, respectively, indicating 77%, 88% and 88% of the compounds shared among MD plants of these cultivars, respectively (Table 4, Supplementary Fig. S3). Myrcene and 1-hexadecanol were predominant in UD PDM. Hexanal was predominant in UD PUSA. In UD SAM, both benzaldehyde and myrcene were predominant. 3Z-Hexenyl acetate predominated in ID PDM, while 3-hexanol was predominat in ID PUSA, MD PUSA and MD SAM. 2-Octanol was predominant in MD PDM.

Behavioral responses of adult S. obliqua females towards individual synthetic compounds and synthetic blends

Females showed attraction towards complete synthetic blends resembling natural volatile blends of UD, ID and MD plants of each green gram cultivar (PDM, PUSA and SAM) compared to the control solvent (Supplementary Table S6, Tables 5 and 6).

Table 5

Behavioral responses of *Spilosoma obliqua* females towards complete synthetic blends, individual synthetic compounds and blends equivalent to those emitted by insect-damaged (ID) plants of each green gram cultivar [PDM 54 (PDM), Pusa Baisakhi (PUSA) and Samrat (SAM)] (T1) vs. the control solvent (T2: CH₂Cl₂). Each test was carried out with at least 60 responding females. Non-responders were other than 60 responding females
	Insects responded		Non-	χ²	P
	T1	T2	responders	(df = 1)	values
Synthetic VOCs at similar amounts emitted by ID PDM (µg in 25 µl CH₂Cl₂)
Complete synthetic blend	47	13	1	19.27	0.0001
a. 3Z-Hexenal (2.97)	38	22	3	4.27	0.0389
b. Hexanal (2.52)	33	27	2	0.60	0.4386
d. 1-Hexanol (4.92)	38	22	3	4.27	0.0389
e. Benzaldehyde (5.56)	39	21	3	5.40	0.0201
f. Myrcene (3.48)	34	26	4	1.07	0.3017
g. 3Z-Hexenyl acetate (9.76)	41	19	2	8.07	0.0045
h. 2-Octanol (7.27)	38	22	2	4.27	0.0389
i. Benzyl alcohol (1.99)	34	26	4	1.07	0.3017
j. Ocimene (7.97)	40	20	3	6.67	0.0098
k. Acetophenone (5.71)	39	21	3	5.40	0.0201
m. Decanal (1.98)	35	25	4	1.67	0.1967
n. Geraniol (3.28)	36	24	4	2.40	0.1213
a + b + d + e + f + g + h + i + j + k + m + n (PDM blend 12)	47	13	2	19.27	0.0001
a + d + e + g + h + j + k (PDM blend 7)	46	14	1	17.07	0.0001
Synthetic VOCs at similar amounts emitted by ID PUSA (µg in 25 µl CH₂Cl₂)
Complete synthetic blend	46	14	2	17.07	0.0001
a. 3Z-Hexenal (4.68)	39	21	3	5.40	0.0201
b. Hexanal (3.16)	35	25	3	1.67	0.1967
c. 3Z-Hexen-1-ol (2.64)	34	26	3	1.07	0.3017
e. Benzaldehyde (8.38)	41	19	2	8.07	0.0045
g. 3Z-Hexenyl acetate (7.66)	39	21	4	5.40	0.0201
i. Benzyl alcohol (1.37)	32	28	4	0.27	0.6056
j. Ocimene (5.96)	38	22	2	4.27	0.0389
k. Acetophenone (7.60)	40	20	2	6.67	0.0098
l. Linalool (3.39)	33	27	4	0.60	0.4386
n. Geraniol (3.64)	37	23	4	3.27	0.0707
a + b + c + e + g + i + j + k + l + n (PUSA blend 10)	46	14	1	17.07	0.0001
a + e + g + j + k (PUSA blend 5)	45	15	2	15.00	0.0001
Synthetic VOCs at similar amounts emitted by ID SAM (µg in 25 µl CH₂Cl₂)
Complete synthetic blend	45	15	2	15.00	0.0001
a. 3Z-Hexenal (3.39)	38	22	3	4.27	0.0389
c. 3Z-Hexen-1-ol (1.70)	33	27	5	0.60	0.4386
e. Benzaldehyde (6.29)	40	20	3	6.67	0.0098
f. Myrcene (1.59)	32	28	4	0.27	0.6056
g. 3Z-Hexenyl acetate (8.74)	40	20	2	6.67	0.0098
i. Benzyl alcohol (0.71)	32	28	4	0.27	0.6056
j. Ocimene (6.17)	39	21	3	5.40	0.0201
k. Acetophenone (3.69)	38	22	3	4.27	0.0389
l. Linalool (5.50)	37	23	4	3.27	0.0707
m. Decanal (2.24)	36	24	5	2.40	0.1213
a + c + e + f + g + i + j + k + l + m (SAM blend 10)	45	15	1	15.00	0.0001
a + e + g + j + k (SAM blend 5)	44	16	2	13.07	0.0003

Table 6

Behavioral responses of *Spilosoma obliqua* females towards complete synthetic blends, individual synthetic compounds and blends equivalent to those emitted by mechanically-damaged (MD) plants of each green gram cultivar [PDM 54 (PDM), Pusa Baisakhi (PUSA) and Samrat (SAM)] (T1) vs. the control solvent (T2: CH₂Cl₂). Each test was carried out with at least 60 responding females. Non-responders were other than 60 responding females
	Insects responded		Non-	χ²	P
	T1	T2	responders	(df = 1)	values
Synthetic VOCs at similar amounts emitted by MD PDM (µg in 25 µl CH₂Cl₂)
Complete synthetic blend	44	16	2	13.07	0.0003
a. 3Z-Hexenal (1.70)	35	25	2	1.67	0.1967
b. Hexanal (4.43)	37	23	4	3.27	0.0707
c. 3Z-Hexen-1-ol (1.58)	33	27	5	0.60	0.4386
d. 1-Hexanol (7.24)	39	21	3	5.40	0.0201
e. Benzaldehyde (5.07)	38	22	3	4.27	0.0389
h. 2-Octanol (8.90)	40	20	2	6.67	0.0098
i. Benzyl alcohol (3.15)	35	25	2	1.67	0.1967
j. Ocimene (5.82)	38	22	3	4.27	0.0389
k. Acetophenone (4.05)	38	22	3	4.27	0.0389
l. Linalool (2.71)	32	28	4	0.27	0.6056
a + b + c + d + e + h + i + j + k + l	44	16	1	13.07	0.0003
d + e + h + j + k	43	17	2	11.27	0.0008
Synthetic VOCs at similar amounts emitted by MD PUSA (µg in 25 µl CH₂Cl₂)
Complete synthetic blend	44	16	2	13.07	0.0003
a. 3Z-Hexenal (1.45)	35	25	3	1.67	0.1967
b. Hexanal (9.18)	39	21	3	5.40	0.0201
e. Benzaldehyde (5.05)	38	22	3	4.27	0.0389
i. Benzyl alcohol (1.95)	34	26	5	1.07	0.3017
j. Ocimene (3.95)	36	24	3	2.40	0.1213
k. Acetophenone (2.81)	35	25	3	1.67	0.1967
l. Linalool (6.42)	38	22	3	4.27	0.0389
m. Decanal (3.41)	38	22	3	4.27	0.0389
a + b + e + i + j + k + l + m	44	16	2	13.07	0.0003
b + e + l + m	41	19	2	8.07	0.0045
Synthetic VOCs at similar amounts emitted by MD SAM (µg in 25 µl CH₂Cl₂)
Complete synthetic blend	42	18	2	9.60	0.0019
a. 3Z-Hexenal (1.05)	32	28	3	0.27	0.6056
b. Hexanal (2.61)	34	26	3	1.07	0.3017
c. 3Z-Hexen-1-ol (5.94)	39	21	5	5.40	0.0201
i. Benzyl alcohol (1.78)	33	27	4	0.60	0.4386
k. Acetophenone (3.29)	37	23	4	3.27	0.0707
l. Linalool (4.82)	35	25	5	1.67	0.1967
m. Decanal (3.98)	38	22	3	4.27	0.0389
a + b + c + i + k + l + m	42	18	2	9.60	0.0019
c + m	40	20	2	6.67	0.0098

Females showed responses towards seven individual synthetic compounds (benzaldehyde, myrcene, benzyl alcohol, acetophenone, linalool, decanal and geraniol) resembling amounts present in volatile blends of UD PDM in comparison to the control solvent, and females were attracted towards a synthetic blend of these seven compounds compared to the control solvent (Supplementary Table S6). Females demonstrated responses towards five individual synthetic compounds (hexanal, benzaldehyde, myrcene, benzyl alcohol and acetophenone) resembling amounts present in volatile blends of UD PUSA in comparison to the control solvent, and females were attracted towards a synthetic blend of these five compounds in comparison to the control solvent (Supplementary Table S6). Females displayed responses towards four individual synthetic compounds (hexanal, benzaldehyde, myrcene and benzyl alcohol) resembling amounts present in volatile blends of UD SAM in comparison to the control solvent, and females showed attraction towards a synthetic blend of these four compounds compared to the control solvent (Supplementary Table S6).

Females showed responses towards 12 individual compounds (3Z-hexenal, hexanal, 1-hexanol, benzaldehyde, myrcene, 3Z-hexenyl acetate, 2-octanol, benzyl alcohol, ocimene, acetophenone, decanal and geraniol) resembling amounts present in volatile blends of ID PDM in comparison to the control solvent, and females were attracted towards a synthetic blend of these 12 compounds (PDM blend 12) (Table 5). Females were attractive towards seven individual compounds – 3Z-hexenal, 1-hexanol, benzaldehyde, 3Z-hexenyl acetate, 2-octanol, ocimene and acetophenone or a synthetic blend of these seven compounds (PDM blend 7) compared to the control solvent (Table 5).

Females demonstrated responses towards 10 individual compounds (3Z-hexenal, hexanal, 3Z-hexen-1-ol, benzaldehyde, 3Z-hexenyl acetate, benzyl alcohol, ocimene, acetophenone, linalool and geraniol) resembling amounts present in ID PUSA compared to the control solvent, while a synthetic blend of these 10 compounds (PUSA blend 10) attracted the females (Table 5). Females displayed responses towards 10 individual compounds (3Z-hexenal, 3Z-hexen-1-ol, benzaldehyde, myrcene, 3Z-hexenyl acetate, benzyl alcohol, ocimene, acetophenone, linalool and decanal) resembling amounts present in ID SAM compared to the control solvent, while a synthetic blend of these 10 compounds attracted the females (SAM blend 10) (Table 5). Females were attracted towards five individual compounds – 3Z-hexenal, benzaldehyde, 3Z-hexenyl acetate, ocimene and acetophenone or a synthetic blend of these five compounds (PUSA blend 5 or SAM blend 5) resembling amounts present in ID PUSA or ID SAM plants in comparison to the control solvent (Table 5).

Females exhibited responses towards 10 individual compounds (3Z-hexenal, hexanal, 3Z-hexen-1-ol, 1-hexanol, benzaldehyde, 2-octanol, benzyl alcohol, ocimene, acetophenone and linalool) resembling amounts present in MD PDM plants compared to the control solvent, while a synthetic blend of these 10 compounds attracted the females (Table 6). Females revealed attraction towards five individual compounds – 1-hexanol, benzaldehyde, 2-octanol, ocimene and acetophenone or a synthetic blend of these five compounds compared to the control solvent (Table 6).

Females demonstrated responses towards eight individual compounds (3Z-hexenal, hexanal, benzaldehyde, benzyl alcohol, ocimene, acetophenone, linalool and decanal) resembling amounts present in MD PUSA compared to the control solvent, while a synthetic blend of these eight compounds attracted the female moths (Table 6). The insect showed attraction towards four individual compounds – hexanal, benzaldehyde, linalool and decanal or a synthetic blend of these four compounds compared to the control solvent (Table 6).

Females exhibited responses towards seven individual compounds (3Z-hexenal, hexanal, 3Z-hexen-1-ol, benzyl alcohol, acetophenone, linalool and decanal) resembling amounts present in MD SAM compared to the control solvent, while a synthetic blend of these seven compounds elicited attraction of the females (Table 6). The insects showed attraction towards two individual compounds – 3Z-hexen-1-ol and decanal or a synthetic blend of these two compounds compared to the control solvent (Table 6).

Females did not differentiate between natural volatile blends released by ID PDM plants and PDM blend 12 or PDM blend 7 (Table 7). Females did not discriminate between volatile blends emitted by ID PUSA plants and PUSA blend 10 or PUSA blend 5 (Table 7). They also did not discriminate between volatile blends emitted by ID SAM plants and SAM blend 10 or SAM blend 5 (Table 7).

Table 7

Behavioral responses of *Spilosoma obliqua* females towards natural volatile blends emitted by insect-damaged (ID) plants of a particular green gram cultivar [PDM 54 (PDM) or Pusa Baisakhi (PUSA) or Samrat (SAM)] vs. individual synthetic compounds or synthetic blends at similar amounts usually emitted by ID plants of the same cultivar. Each test was carried out with at least 60 females. Non-responders were other than 60 responding females
Comparison		Insects responded		Non-	χ²	P
T1	T2	T1	T2	responders	(df = 1)	values
Natural volatile blends emitted by	Synthetic VOCs and blends at similar amounts
ID PDM plants (µg in 25 µl CH₂Cl₂)	emitted by ID PDM plants (µg in 25 µl CH₂Cl₂)
	a. 3Z-Hexenal (2.97)	41	19	2	8.07	0.0045
	b. Hexanal (2.52)	46	14	2	17.07	0.0001
	d. 1-Hexanol (4.92)	41	19	2	8.07	0.0045
	e. Benzaldehyde (5.56)	40	20	3	6.67	0.0098
	f. Myrcene (3.48)	45	15	2	15.00	0.0001
	g. 3Z-Hexenyl acetate (9.76)	38	22	3	4.27	0.0389
	h. 2-Octanol (7.27)	41	19	2	8.07	0.0045
	i. Benzyl alcohol (1.99)	45	15	2	15.00	0.0001
	j. Ocimene (7.97)	39	21	3	5.40	0.0201
	k. Acetophenone (5.71)	40	20	2	6.67	0.0098
	m. Decanal (1.98)	44	16	2	13.07	0.0003
	n. Geraniol (3.28)	43	17	2	11.27	0.0008
	a + b + d + e + f + g + h + i + j + k + m + n (PDM blend 12)	32	28	3	0.27	0.6056
	a + d + e + g + h + j + k (PDM blend 7)	33	27	4	0.60	0.4386
Natural volatile blends emitted by	Synthetic VOCs and blends at similar amounts
ID PUSA plants (µg in 25 µl CH₂Cl₂)	emitted by ID PUSA plants (µg in 25 µl CH₂Cl₂)
	a. 3Z-Hexenal (4.68)	39	21	3	5.40	0.0201
	b. Hexanal (3.16)	43	17	2	11.27	0.0008
	c. 3Z-Hexen-1-ol (2.64)	44	16	2	13.07	0.0003
	e. Benzaldehyde (8.38)	37	23	4	3.27	0.0707
	g. 3Z-Hexenyl acetate (7.66)	39	21	3	5.40	0.0201
	i. Benzyl alcohol (1.37)	46	14	2	17.07	0.0001
	j. Ocimene (5.96)	40	20	2	6.67	0.0098
	k. Acetophenone (7.60)	38	22	3	4.27	0.0389
	l. Linalool (3.39)	45	15	2	15.00	0.0001
	n. Geraniol (3.64)	40	20	2	6.67	0.0098
	a + b + c + e + g + i + j + k + l + n (PUSA blend 10)	32	28	3	0.27	0.6056
	a + e + g + j + k (PUSA blend 5)	33	27	4	0.60	0.4386
Natural volatile blends emitted by	Synthetic VOCs and blends at similar amounts
ID SAM plants (µg in 25 µl CH₂Cl₂)	emitted by ID SAM plants (µg in 25 µl CH₂Cl₂)
	a. 3Z-Hexenal (3.39)	39	21	3	5.40	0.0201
	c. 3Z-Hexen-1-ol (1.70)	44	16	2	13.07	0.0003
	e. Benzaldehyde (6.29)	37	23	3	3.27	0.0707
	f. Myrcene (1.59)	45	15	2	15.00	0.0001
	g. 3Z-Hexenyl acetate (8.74)	37	23	4	3.27	0.0707
	i. Benzyl alcohol (0.71)	45	15	2	15.00	0.0001
	j. Ocimene (6.17)	38	22	3	4.27	0.0389
	k. Acetophenone (3.69)	39	21	3	5.40	0.0201
	l. Linalool (5.50)	40	20	2	6.67	0.0098
	m. Decanal (2.24)	41	19	2	8.07	0.0045
	a + c + e + f + g + i + j + k + l + m (SAM blend 10)	32	28	3	0.27	0.6056
	a + e + g + j + k (SAM blend 5)	33	27	4	0.60	0.4386

Females did not distinguish between PDM blend 7 and PUSA blend 5 or SAM blend 5 (Fig. 3). Females also did not distinguish between PUSA blend 5 and SAM blend 5 (Fig. 3).

In dose response bioassays, females started to show attraction towards 3Z-hexenal or acetophenone or decanal at 5 µg in 25 µl CH₂Cl₂ and showed the highest attraction at 10 µg in 25 µl CH₂Cl₂ (Table 8). Females showed some attraction towards hexanal or 1-hexanol or ocimene at 8 µg in 25 µl CH₂Cl₂ and the highest attraction at 16 µg in 25 µl CH₂Cl₂ (Table 8). Females displayed some attraction towards benzaldehyde at 7 µg in 25 µl CH₂Cl₂ and the highest attraction at 14 µg in 25 µl CH₂Cl₂ (Table 8). Females exhibited some attraction towards 3Z-hexenyl acetate at 12 µg in 25 µl CH₂Cl₂ and the highest attraction at 24 µg in 25 µl CH₂Cl₂ (Table 8). Females demonstrated some attraction towards 2-octanol at 13 µg in 25 µl CH₂Cl₂ and the highest attraction at 26 µg in 25 µl CH₂Cl₂ (Table 8).

Table 8

Dose responses of *Spilosoma obliqua* females towards individual synthetic compounds (T1) vs. the control solvent (T2: CH₂Cl₂). Each test was carried out with at least 60 females. Non-responders were other than 60 responding females
Synthetic	Concentration	Insects responded		Non	χ2	P
compounds	( µg in 25 µl CH₂Cl₂)	T1	T2	responders	(df = 1)	values
3Z-Hexenal	2.5	36	24	5	2.40	0.1213
	5	39	21	3	5.40	0.0201
	10	45	15	1	15.00	0.0001
Hexanal	4	36	24	4	2.40	0.1213
	8	39	21	2	5.40	0.0201
	16	45	15	1	15.00	0.0001
1-Hexanol	4	36	24	5	2.40	0.1213
	8	40	20	2	6.67	0.0098
	16	46	14	1	17.07	0.0001
Benzaldehyde	3.5	37	23	4	3.27	0.0707
	7	40	20	3	6.67	0.0098
	14	45	15	1	15.00	0.0001
3Z-Hexenyl acetate	6	37	23	5	3.27	0.0707
	12	43	17	3	11.27	0.0008
	24	47	13	1	19.27	0.0001
2-Octanol	6.5	36	24	4	2.40	0.1213
	13	43	17	1	11.27	0.0008
	26	47	13	1	19.27	0.0001
Ocimene	4	36	24	4	2.40	0.1213
	8	40	20	2	6.67	0.0098
	16	45	15	1	15.00	0.0001
Acetophenone	2.5	35	25	5	1.67	0.1967
	5	39	21	4	5.40	0.0201
	10	44	16	1	13.07	0.0003
Decanal	2.5	36	24	5	2.40	0.1213
	5	40	20	2	6.67	0.0098
	10	46	14	1	17.07	0.0001

Wind tunnel bioassays

In no-choice tests, females landed significantly on filter papers with synthetic blends comprised PDM blend 7 (t = 7.407, df = 18, P < 0.0001) or PUSA blend 5 (t = 4.950, df = 18, P = 0.0001) or SAM blend 5 (t = 2.424, df = 18, P = 0.0261).

In two-choice tests, females did not show preference on landing on the filter papers when compared between PDM blend 7 and PUSA blend 5 (t = 0.640, df = 18, P = 0.531), PDM blend 7 and SAM blend 5 (t = 1.701, df = 18, P = 0.106), and PUSA blend 5 and SAM blend 5 (t = 1.325, df = 18, P = 0.202).

The present study focuses on olfactory cues as insects can locate host plants from long-range using volatile blends from plants as opposed to visual cues which are usually involved at short-range of the host plants. The findings of this current research were focussed on how active volatile components of three green gram cultivars could act as cues to attract adults of S. obliqua, which could facilitate to realize the processes fundamental in the interactions through chemical point of view among volatile blends of differently treated green gram cultivars and the insect pest. In this study, acetophenone and linalool were present in volatile blends of UD PDM and UD PUSA; whereas decanal and methyl jasmonate were present in UD PDM and UD SAM. Geraniol was present in volatile blends of UD PDM; whereas nerolidol was detected in volatile blends of UD PUSA and UD SAM. Therefore, our findings support the hypothesis that the VOC profiles differ within cultivars of a same plant species (Avellaneda et al. 2021; Mitra et al. 2021). The insect showed responses to some compounds resembling amounts present in undamaged plants (benzaldehyde, myrcene and benzyl alcohol – common among three cultivars, acetophenone – common in PDM and PUSA, hexanal – common in PUSA and SAM, and linalool, decanal and geraniol – specific in PDM), suggesting the attractions of insects are dependent on the key compounds as well as their amounts present in volatile blends (Karmakar et al. 2020; Mitra et al. 2021).

The composition and emission of VOCs from insect-damaged plants are influenced by the nature of insect feeding damage (Lin et al. 2016; Rodriguez-Saona et al. 2009). Eight compounds (3Z-hexenal, 3Z-hexen-1-ol, 1-heptanol, 3Z-hexenyl acetate, ocimene, 1,3-diethyl benzene, farnesene and 1-heptadecanol) were specific in volatile blends of insect-damaged plants of three green gram cultivars compared to volatile blends of undamaged plants of these green gram cultivars, supporting the hypothesis that insect feeding causes qualitative and quantitative changes in VOCs among volatile blends of insect-damaged plants compared to volatile blends of undamaged plants (Das et al. 2021; Koner et al. 2022; Mitra et al. 2020). However, four herbivore-induced plant volatiles (HIPVs) – 1-heptanol, 1,3-diethyl benzene, farnesene and 1-heptadecanol present in volatile blends of these green gram cultivars did not elicit response in the females of S. obliqua. Two HIPVs (1-hexanol and 2-octanol) specific in volatile blends of ID PDM compared to volatile blends of UD PDM elicited attraction of the insect pest. The above observations suggest the hypothesis that feeding by an insect herbivore on different cultivars of a plant species influences production of different VOCs (Mitra et al. 2021).

Here, nonane and myrcene were detected in volatile blends of UD and ID plants of three green gram cultivars but not in volatile blends of MD plants of these cultivars. Two compounds (3Z-hexenyl acetate and geraniol) were recorded in volatile blends of ID plants of three green gram cultivars but not in volatile blends of MD plants of these cultivars. There were also qualitative variations in individual VOCs emitted by MD plants of three green gram cultivars compared to VOCs present in ID plants of these cultivars such as hexanal was not detected in volatile blends of ID SAM plants. Five compounds (1-hexanol, benzaldehyde, 2-octanol, ocimene and acetophenone) were responsible for attraction of S. obliqua towards volatile blends of MD PDM; whereas four compounds (hexanal, benzaldehyde, linalool and decanal) were responsible for attraction of the insect towards volatile blends of MD PUSA. Two compounds (3Z-hexen-1-ol and decanal) caused attraction of female moths towards volatile blends of MD SAM. Therefore, our findings support the hypothesis that HIPVs differ qualitatively as well as quantitatively from the volatile blends of mechanically-damaged plants (Das et al. 2021; Koner et al. 2022; Mitra et al. 2017; Piesik et al. 2010;). It is in agreement with the idea that the composition of VOCs differs between plants after insect-damage and mechanical-damage due to the metabolic effects of oral discharges by the insect herbivore on the insect-damaged plants (Holopainen and Gershenzon 2010; Portillo-Estrada et al. 2021).

HIPVs attract herbivore enemies of the host plant (McCormick et al. 2012; Qiao et al. 2018; Rodriguez-Saona et al. 2020). In the current study, 3Z-hexenal, 3Z-hexenyl acetate and ocimene, which were specific in volatile blends of ID plants of three green gram cultivars compared to volatile blends of UD plants of these cultivars, attracted females of S. obliqua. 1-Hexanol and 2-octanol specific in volatile blends of ID PDM compared to other two ID green gram cultivars induced attraction of S. obliqua. Except for UD SAM, where acetophenone was not detected, increased emissions of benzaldehyde and acetophenone from ID plants compared UD plants caused attraction of S. obliqua towards volatile blends. Therefore, these findings suggest that HIPVs not only assist predators and parasitoids to locate the insect-herbivore, but also assist conspecific adults to further attract the host plant (El-Sayed et al. 2016; Guo and Wang 2019). Several studies demonstrated that caterpillar-induced plant volatiles attract conspecific-adults in nature such as feeding by the larvae of the light brown apple moth, Epiphyas postvittana (Walker) on apple seedlings elicited more attraction of the adult moth on the insect-damaged apple seedlings compared to undamaged plants (El-Sayed et al. 2016), and attack by the larvae of the cabbage moth, Mamestra brassicae (Linnaeus) on cabbage plants resulted in more attraction of the conspecific adult moths compared to undamaged plants (Rojas 1999). Caterpillars of Spodoptera littoralis (Boisduval) fed on cotton plants resulted in preference of conspecific female moths to oviposit on insect-damaged plants compared to undamaged plants (Anderson and Alborn 1999). Further, females of diamondback moth, Plutella xylostella (Linnaeus) showed preference to oviposit on conspecific larvae-damaged plants compared to undamaged plants (Shiojiri and Takabayashi 2003). Thus, herbivory by the larvae of S. obliqua on green gram plants in crop fields could result in conspecific females laying more eggs on green gram plants, which could lead to further damage by the larvae on green gram plants.

The current study reveals that plants of each green gram cultivar damaged by the larvae of S. obliqua emit a unique volatile blend that causes attraction of conspecific adult moths, and these synthetic blends could offer valuable insights for the development of eco-friendly baited traps in integrated pest management (IPM) strategies of S. obliqua. Additional work is necessary to determine whether females oviposit or males mate on the conspecific larvae-damaged green gram plants including attraction of natural enemies of S. obliqua, which would help in better understanding of the counteracting factors affecting herbivore attack.

Funding: The financial assistance from the Govt. of India as Council of Scientific and Industrial Research (CSIR) Senior Research Fellowship [File No: 09/025(0258)/2018-EMR-I] to Syed Husne Mobarak is gratefully acknowledged.

Conflicts of interest/Competing interests: The authors declare that they have no conflict of interest/competing interests.

Availability of data material (data transparency): The authors confirm that the data supporting the findings of this study are available within the article and its supplementary material.

Code availability: Not applicable

Author contributions: AMB and SHM conceived and designed the study. SHM, AK and RD conducted experimental work, collected and analysed data. AMB drafted the manuscript. AMB supervised SHM, AK and RD. All authors critically revised the draft and approved the final manuscript for publication.

Ethics approval: Not applicable

Consent to participate: Not applicable

Consent for publication: All authors agreed to submission of final manuscript.

Acknowledgments: We are thankful to Dr. Rahul Joshi, Scientist, Zoological Survey of India, Gangetic Plains Regional Centre, Bihar for identifying the insect. We thank Department of Science and Technology Promotion of University Research and Scientific Excellence (DST-PURSE) of Phase-II for providing necessary instrumental facilities.

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Supplementarymaterial.doc

The Role of Green Gram Plant Volatile Blends in the Behavior of Arctiid Moth, Spilosoma Obliqua

Status:

Version 1

Abstract

Figures

Introduction

Methods And Materials

Plants

Insects

Plant volatile blend collection

Olfactometer bioassays

Volatile blend analysis

Synthetic compounds

Statistical analyses

Results

Identification And Quantification Of Vocs

Wind tunnel bioassays

Discussion

Declarations

References

Supplementary Files

Status:

Version 1