Interspecific Eavesdropping on Chemical Communication in the Field between Taif Pomegranate, Aphis punicae, Enemies, and Protectors

doi:10.21203/rs.3.rs-4008859/v1

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Interspecific Eavesdropping on Chemical Communication in the Field between Taif Pomegranate, Aphis punicae, Enemies, and Protectors

https://doi.org/10.21203/rs.3.rs-4008859/v1

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The demand for alternative pesticides for agricultural crops is becoming a public health requirement. In nature, volatile organic compounds (VOCs) play a major role in influencing the qua-trophic relationship between plants, pests, protectors, and predators. By utilizing an ecological approach, these substances can be utilized as a powerful crop biocontrol tool for integrated pest management (IPM) against Aphis punicae, the green pomegranate aphid. This study involved collecting VOCs from aphid-infested pomegranates (AIP) in the presence of protectors and predators in the field for the first time in Taif Governorate to implement IPM. Using headspace open-loop stripping and gas chromatography-mass spectrometry analysis, 30 VOCs were identified from four ecological interactions between AIP, ants, and ladybirds. The VOC profile of AIP indicates that it is experiencing early injury due to low β-farnesene, methyl salicylate, and high caryophyllene. Principal component analysis, cluster analysis, and heatmap were used to identify the key volatile compounds in the field from the four systems studied and demonstrate the variability of the volatile compounds among different insects and host plants. Seven volatile compounds, including 1-methyl-3-tert-butylbenzene, m-ethylcumene, 3-carene, (+)-4-carene, 2,2,4-trimethyl-pentane, and caryophyllene, were detected in the ecological interaction of AIP. The primary ant pheromone compound, 4-heptanone, becomes effective in repelling aphids after 24 hours when ants and ladybirds are present. In 48 hours, a complex pattern was observed consisting of 1-ethyl-3-methylbenzene, 1,3,5-trimethylbenzene, and 1-methyl-1H-imidazole, which was identified as a key factor in repelling aphids. The same ecological system showed that methyl salicylate, sabinene, limonene, pentadecane, and heptadecane were found to attract predators. The composition and quantity of VOCs emitted can cause varying responses. This study serves as a basis for future quantitative studies that can determine the concentration of each volatile compound that is essential for the biological control of A. punicae.

integrated pest management

Punica granatum L.

Aphis punicae

Taif Governorate

multivariate statistical analysis

GC-MS headspace

open-loop stripping

Agroecosystems are constantly changing, which requires significant research and development to maintain the productivity gains already achieved. Research that targets the reduction of crop losses caused by insect pests and pathogens has and will continue to make significant contributions to the necessary increase in yield, productivity, and sustainability (Richard, Qi, & Fitt, 2022). The intention was to develop IPM as a method for controlling insects, by improving specific technologies that deal with insect infestations, which have already played a significant role in reducing crop losses and insecticide use in various countries (Angon et al., 2023). Insecticides fail to eliminate insect pests, leading to a disturbance in the balance between plants, herbivores, their natural enemies, and their associated insects (qua-topic relationship) (Crossley, Snyder, & Hardy, 2021; Sánchez-Bayo, 2021). The use of insecticides excessively in certain crops led to the development of insecticide-resistant insects, the emergence of new pests that were more dangerous than those targeted, the increase in crop losses, and negative environmental impacts (Sharma et al., 2019). IPM has successfully solved these problems by employing a variety of pest monitoring and control strategies to maintain pest populations below levels that can result in economic losses (Angon et al., 2023). The common approach involves integrating genetic defense of host plants with biological control, cultural methods, behavioral techniques, and farmer knowledge (Peterson, Ode, Oliveira-Hofman, & Harwood, 2016). Evolutionary and ecological entomologists are interested in associations that have a purpose, such as plant-herbivore interactions, trophic cascades, and tritrophic relationships (Mooney, Pratt, & Singer, 2012). To understand the evolution of plant-insect relationships, it is vital to comprehend the chemical mechanisms responsible for them, and their effects on populations, communities, and ecosystem levels. (Mbaluto et al., 2020; Trowbridge, 2014). This new science has attracted researchers' attention not only for its fundamental ecological implications, but also for its significance in fields such as biological control (Kansman, Jaramillo, Ali, & Hermann, 2023).

The objective of this research is to explore the quaternary relationship between plants, herbivores, associated insects, and pests in the field. The use of this method significantly contributed to improving yield, productivity, and sustainability. In contrast, interactions are generally more intricate than simple generalized wound responses and are usually influenced by specialized elicitors that are linked to pests or pathogens. Induced responses can affect herbivores either directly or indirectly depending on their mechanism (Heil & Bostock, 2002). For insects, olfaction is crucial in identifying specific behaviors, such as attraction or repulsion toward a food source or sexual partner, fleeing danger, and successfully coping with specific situations (Pellegrino & Nakagawa, 2009). The purpose of chemical compounds that serve as signals between organisms, which are commonly known as semiochemical compounds or chemical mediators. This category contains allelochemicals and pheromones (Dicke & Sabelis, 1988). Allelochemicals are chemicals that aid the exchange of information between different species, which are produced by plants, microorganisms, or animals. These compounds are divided into two subcategories, depending on whether the transmitting or receiving organism is advantageous or disadvantaged by the signal (Kong, Xuan, Khanh, Tran, & Trung, 2019). When the species that produce the compound benefit but the recipient is negatively affected, the compound is referred to as an allomone. When a substance has a positive effect on the recipient species, it is called a kairomone (B. L. Nielsen, O. Rampin, N. Meunier, & V. Bombail, 2015). Pheromones are the second group of semiochemical substances that are chemical messages that cause behavioral reactions or physiological changes in individuals of the same species (Abd El-Ghany, 2019). Insects can use pheromones to respond in different ways, including alarm, defense, aggregation, attraction, recognition of colonies, marking of oviposition sites, mating behavior, recruitment, tracking, and even thermoregulation (Fleischer & Krieger, 2018).

Pomegranate was identified as a significant medicinal plant. It has several pharmaceutical and ornamental uses due to bioactive compounds in its parts (Aboelhana, Ismail, Attia, & Sadik, 2017). The increased attention to this crop must lead to the progressive development of an IPM program against the most insidious pests. Indeed, there are several studies on Taif pomegranate, such as investigating the anti-obesity effects of pomegranate juices from the two Saudi Arabian pomegranates, Taif red and Taif white, in high-fat diets (M. M. Ahmed, Samir el, El-Shehawi, & Alkafafy, 2015). El-Shehawi et al. (2022) focused on determining genetic variations of common pomegranate agriculture in the Taif area. They were also investigated for their protective activity against oxidative stress induced by Acrylamide and its related metabolic changes through its antioxidant and anti-inflammatory properties (El-Shehawi et al., 2022). Al-Barty (2011) carried out a survey and created a list of pests on pomegranate trees in Al-Taif City. During the pre- and post-harvesting periods, this plant is frequently infected with fungal infections, which can cause severe losses to the farming economy (Hassan et al., 2022). Elsayed and Bazaid (2011) observed that pomegranate fruits are attacked by two insects: Ectomyelois ceratoniae and Virachola livia during the period of pomegranate production (May to October) in Taif, Saudi Arabia. The chemical analysis of the pest that infects the pomegranate plant in the Taif governorate has not been carried out. In this study, we are interested in finding an info-chemical method for IPM of the aphid that infects the pomegranate plant, by studying the chemical relationships between the host plant, agricultural pests, natural enemies, and their protectors.

2.1. Plant material and insect

The pomegranate (Punica granatum) those utilized in this study from localities Al-Sir farms (N21.204753,E40.591998) of Taif region. In addition, pomegranate aphid, were identified in our previous study as Aphis punicae (A. punicae) (Aphididae) (Alotaibi et al., 2023). Their natural enemies, i.e. ladybird Coccinella undecimpunctata and protectors, i.e. ant were collected respectively from alfalfa farms and pomegranate farms from the same localities at Taif.

2.2. Rearing of A. punicae natural enemies

The collected ladybird Coccinella undecimpunctata were divided into two groups based on their gender. Each male and female were placed in a box (dimensions)10x10 cm containing cotton moistened with water and honey as well as pollen grains were added to the boxes for providing the food to the insects. The boxes were monitored daily to ensure that eggs were present. The eggs were then picked up and placed in another box and checked daily for hatching and larvae release. Aphids cotton were added to the boxes to feed the larvae. The temperature was set at 28°C and the humidity was between 50–60%.

2. 3 Headspace analysis

The metabolic patterns of pomegranate were characterized at different stages of aphid infection on a farm scale on 22–27 Mai 2021. Thus, (a) the first stage being the infected plant on 22 Mai, (b) second stage: addition of 20 ants on 23–24 Mai, (c) third stage: addition of 10 eleven spot ladybird females on 25–26 Mai, (d) fourth stage: addition of 20 ants and 10 eleven spot ladybird females simultaneously on 27 Mai. Three independent biologic replicates of different steps representing each step were extracted and analyzed.

2.3.1 VOCs Sampling

The collection, separation, identification and quantification steps are critical for VOC analysis. The most widely used method for collecting VOCs, particularly for microorganisms, is closed-loop stripping (CLS) analysis using the reference analysis method of Grob (Badra & Angeli, 2018; Grob, Grob, & Grob, 1975; Kunert, David, Becher, & Boland, 2009), also known as static headspace, and open-loop stripping (OLS) analysis according to the reference analysis method of Hans, also known as dynamic headspace (Borén, Grimvall, & Sävenhed, 1982; Tholl et al., 2006). In this study, VOCs from different steps of AIP were collected using the OLS headspace sampling method as described in (Effah et al., 2020). Pomegranate branches of a suitable size, without visible sign of damage by insects and pathogens, were selected and enclosed in oven cooking bags (KSA).

2.3.2 Opened-Loop Stripping (OLS)

One inlet and one outlet tube were fastened with cable binders at each end of the bag. The portable volatile assay system (PVAS22 pump, VAS Rensselaer NY) was used to circulate carbon-filtered air through the bag. Incoming air was pumped at 1.10 L/min and outgoing air was pulled at 0.90 L/min creating a slight overpressure to avoid contaminants from entering the bag. The VOCs emitted from the all-ecological systems of AIP were trapped in HayeSep Q volatile traps attached to the outlet (pull) tubes. The pump ran for 24 h hours, and then the filters were removed and individually wrapped with labelled pieces of aluminum foil, and stored in a cooler box to prevent contamination and evaporation of the collected volatiles.

2.3.3 Sampling vs treatments and ecological systems of aphid-infested pomegranates

Charcoal and DVB traps were eluted using 60 µL (3 × 20 µL) of ethyl acetate. The samples were analyzed by GC-MS or stored at − 80°C until analysis. The VOCs thus collected were named (PIN24-DVB) for the plant infected with aphids, (PIN48An24-DVB) for plants infected in the presence of ants after 24h. For the second stage, (PIN24An24Lb24-DVB) and (PIN48An48Lb48-DVB) were named respectively for plants infected in the presence of ants and ladybirds after 24 h and 48 h.

2.4 Gas chromatography-mass spectrometry (GC-MS) analysis conditions

Samples were analyzed with a GC (580 Clarus) coupled to an MS (560S Clarus) (Agilent Technologies, Santa Clara, USA). One microliters of each sample were injected into the GC port in split mode. The GC was equipped with a non-polar Elite-5ms Capillary Column (30 m×0.25 mm ID, 0.25 µm film thickness, Agilent Technologies). The GC parameters for the analysis were as follows: The carrier gas was helium at constant flow of 1 mL/min. The injection pre-dwell time was set at 0.1 min (hot needle injection). The injector temperature was 220◦C. An amount of 1 µL was injected into the split mode at a ratio of 1:5. The temperature program of the oven was set at 40◦C for 3 min, and then the temperature was increased at 2.8°C/min to 130°C, followed by an increase of 2.4°C/min to 180°C, where it was ramped up to the final temperature of 250◦C for 6◦C/min. The total GC run time is 67 min. MS parameters were set as follows: Electron energy = 70 eV, trap current emission = 100 µA, source temperature = 150◦C, transfer line temperature = 280◦C. The mass spectrometric detector was operated in the scan mode (m/z 40–450 amu). Data acquisition and analysis were carried out using Turbomass software (Perkin Elmer) and the compounds were identified by comparing their mass spectra with those in the databases NIST 14 (Gaithersburg, MD, USA). NIST library identification was verified by co-injection authentic standards with samples.

2.5 GC–MS data processing for multivariate analysis

All chromatograms were checked with manually and the area of each peak was normalized by the sum of all peak area in sample’s chromatogram. The mean of replicates was used and applied for further statistical analysis.

2.6 Statistical Analysis

For the VOC profiling of the all-ecological systems, 30 VOCs were chosen, based on their consistent occurrence in the samples of at least one species. One replicate of fragilis that did not emit the selected compounds was dropped from analyses. The VOC data were transformed on percentage to achieve normality. The percentage of each major VOC class (aldehydes, alkane, benzoid, ketone, ester, monoterpenes and sesquiterpenes) was calculated by summing up the specific VOC airs for each group, and then dividing by the whole blend (Table S1 and Figs. 1a, and 1b).

Statistical analyses were performed using the SRplot (Scientific and Research plot tool) was a free online platform for data visualization and graphing (version 2023, PMID: 37943830, University of Nebraska-Lincoln, UNITED STATES) (Tang et al., 2023). In case of significant differences. A Principal Component Analysis (PCA) and Cluster, heatmap was also carried out to show the variability of the VOCs between the different insects and host plant.

3.1 Exploitation of chemical communication

In the framework of biocontrol, researches are looking closely at odor. Those emitted by plants first, since they are what enable pest insects to locate them in the landscape and colonize them (Conti et al., 2021; Vargas, Rivera-Pedroza, García, & Jahnke, 2023). Those emitted by insects then, and in particular by females to attract males during reproduction (Lucie Conchou et al., 2019). These odors are called pheromones when they concern exchange between individuals belonging to the same species (Lucie Conchou et al., 2019; B. Nielsen, O. Rampin, N. Meunier, & V. Bombail, 2015). We talk of allelochemical substances to describe the exchanges between individuals of different species, and more specifically of kairomones, when these substances benefit the one who receives the message (B. Nielsen et al., 2015). In recent years, the development of strategies based on semiochemistry has increased considerably, as they are considered effective tools for manipulating the behavior of insects targeting plants with the aim of improving the biological control of crop pests (Conti et al., 2021). However, feeding and/or oviposition by herbivorous insects induces changes in a plant's ecophysiological traits and their emission of VOCs, either as herbivore-induced plant volatiles (HIPVs) or oviposition-induced plant volatiles (OIPVs) (Conti et al., 2021). In this work we interest on the study of the qua-trophic relationship between Punica granatum, A. punicae, natural enemy (ladybird), and pest protector (ant), under field conditions to detect a new way of research for pests protects plants.

3.1.1 The triggering systemic acquired resistance (SAR) in the host plant

The fruit cropping system is localized in the central-western agroclimatic subzone of the Al Baha region and the central and northern areas of the Makkah Region, including the very large Taif governorate. Fruits are grown on more than 35% of the cultivated land. Farms in the Al Baha and Taif regions now see more than 200,000 pomegranate trees produce 30,000 tons of fruit each year (according to the approximate statistics of the Pomegranate Cooperative Society in the Al-Baha and Taif region) (Alghamdi, Aly, & Ibrahim, 2022). The work of identifying aphids associated with plants growing in Taif governorate made it possible to establish a list of the main species encountered during the study period (Alotaibi et al., 2023). Among these aphids, A. punicae Passerini (SUB9563655) is a specialist in Punica granatum. A. punicae is the most important and destructive for the pomegranate tree. This pest could cause heavy yield losses, in some cases reaching 24 tons/ha in the absence of chemical intervention. Plant protection systems based on the intervention threshold can lead to the avoidance of some systematic treatments applied by farmers in the region against these pests.

3.1.2 Open-loop stripping systems-DVB

The effects of cascading in ecological systems that operate across three or more trophic levels can either be based on resources (bottom-up) or natural enemies (top-down). These effects are often considered separately due to their complexity, and their relative strength when acting simultaneously is not known (Senft, Clancy, Weisser, Schnitzler, & Zytynska, 2019). In this work, we studied three field ecological systems based on natural resources (bottom-up), starting with plant-aphid (S1-DVB), plant-aphid-ant (S2-DVB), and plant-aphid-ant-ladybird (S3-DVB) interactions.

During the quadruplet interaction between host plants, pests, protectors, and predators in our setup, herbivores resulted in the appearance volatile lists, which were collected from branches by OLS systems, and identified by GC-MS. Significant differences can be observed in the volatile compounds of field-infected pomegranates with aphids and their predator and protector when analyzed. 30 VOCs were detected in the samples that were trapped from different stages of AIP from all ecologies systems. Plants damaged by A. punicae change their odor emission, as well as the variety of substances classes of VOCs. In most treatments, alkanes, aldehydes, ester, benzene derivatives, and terpenes were the most likely VOCs released from field AIP in the presence or absence of predator or protector. The headspace of AIP (S1-DVB) consisted of benzene derivatives (53.44%), alkanes (43.62%), monoterpenes (5.07%), sesquiterpenes (5.14%), and alcohol (1.94%). In response to adding the ants to the AIP, the headspace profile of the tripartite community (S2-DVB) showed a high level of ketones (75.04%) and alkanes (19.84%) while monoterpenes, sesquiterpenes, and aldehydes disappeared. The profiles of the quadripartite community of AIP that contains ants and ladybirds (S3-DVB) show a decrease of 12.92% in alkanes and an absence of sesquiterpenes (Table S1, Figures 1a and 1b).

Several compounds are present in all samples trapped by DVB and identified by GC-MS. The pomegranate that is damaged by A. punicae (n=15) has the ability to alter its smell. According to the findings, the presence of ants reduces the number of VOCs (n=4), but the presence of ladybirds increases the number of detected VOCs (n=17) as compared to plants affected by A. punicae (Table S1). According to the OLS system, the AIP's qualitative odor composition mainly consists of o-xylene, p-xylene, 1-methyl-3-tert-butylbenzene, m-ethylcumene, 2-methylnaphthalene, 3-carene, (+)-4-carene, benzaldehyde, 2,2,4-trimethyl-pentane, tridecane, caryophyllene, beta-farnesene. Furthermore, the addition of ants changes the profile of the compounds detected. They were essentially formed of o-xylene, p-xylene, 4-heptanone, and unknown alkane-2. After adding ladybird, the number of VOCs detected increased again to 7, and 17 for the OLS system, after 24 and 48 hours of contact. After 48 hours of ecological interaction, OLS discovered new VOCs and the disappearance of others, which had a composition of 99.73% (Table S1, Figures 1a and 1b).

3.2 Analysis of the VOCs profiles from the qua-trophic relationship

The VOC profiles of the three ecologic interactions systems, S1 (two-trophic relationship aphid-plant), S2 (tri-trophic relationship aphid-plant-ant), and S3 (qua-trophic relationship aphid-plant-ant-ladybird), allowed the identification of 30 compounds. Of these 30 compounds, only one p-Xylene was present in all three treatments, while 1-methyl-3-tert-butylbenzene, m-ethylcumene, 3-carene, and (+)-4-carene, alpha-methylbenzeneacetaldehyde, unknown alkane-3, unknown alkane-4, 2,2,4-trimethyl-pentane, caryophyllene, and β -farnesene were only present in the headspace of system S1 (Table S1, Figures 1a and 1b). Ester, ketone, or N-compounds were not present in system S1's headspace, but they were present in other systems (Table S1, Figures 1a and 1b).

The GC/MS analysis showed that the emission of VOCs is highly influenced by ecological interaction. The ecology interaction system (S1-DVB) has ten components, which include 1-methyl-3-tert-butylbenzene, m-ethylcumene, and caryophyllene. In the presence of ants, the major compounds are 4-heptanone and unknown alkane-2. After 24 hours of the presence of ladybird, tricyclene was the main VOC, followed by limonene, and unknown alkane-3 after 48 hours.

3.3 Statistical analysis of fundamental ecological relationships and their impact on the implementation of biological control.

Analysis of HIPVs shows significant differences between the VOCs detected by OLS analysis systems, which were identified by GC-MS. Several compounds were present in all samples that were trapped from different stages of AIP. HIPVs obtained by OLS analysis (n=15), and it shows that β-farnesene was exclusively detected in the HIPVs from AIP. To understand the smell profile of plants attacked by pests and identify behaviorally active components for predators, it's important to use complementary methods, as our results demonstrate. The headspace profiles of AIP and AIP with ants and ladybirds were different due to VOCs. Figure 2c show a heat map graphically displaying results from hierarchical clustering of volatile compounds from AIP without and with ants and ladybirds detected by OLS systems.

This work was conducted to determine the closeness of individual compounds to all ecosystem systems' interactions infested pomegranate with A. punicae, triplet interactions between AIP and ants, and quadruplet interactions between AIP, ants, and ladybirds. Distances between samples and assays were calculated for hierarchical clustering using Pearson's correlation distance. Each volatile compound has a peak area detected by GC-MS, presented on a color scale that illustrates the differences between the replicates of all ecosystem systems interactions. The heat map indicated that the detected compounds and the difference between all ecologies interaction systems are based on the scale of color, and each color corresponds to one detected VOC. The value of the compound is represented by red, orange and dark blue for the maximum (2), average (0) and minimum (–2) (Figure 2c). In addition, principal component analysis (PCA) was performed. The PCA score plot (Figures 2a and 2b) reveals that all OLS analysis ecologic interaction systems are separated. However, the separation shows four different groups based on their profiles of VOCs using significant differences (p < 0.05), relationship between VOCs of AIP and AIP with ants and ladybirds, as shown in Figures 2a and 2b.

To accurately assess the closely connected substances on the topographic plot, all the information provided by the fingerprint analysis technique was used for qualitative characterization. In the fingerprint (Figure 2), each column displays the signal peaks of each sample, and each row displays the same volatile compound for every sample. Additionally, the color of it indicates the amount of volatile compounds, with a brighter color signified a higher content. As illustrated in Figure 2, there was a significant variation in the content of VOCs in the samples of all four groups.

The PCA obtained using the VOCs of the three study systems (Figure 3) showed that all ecology interactions (S1-DVB, S2-DVB, and S3-DVB) are able to form distinct groups on the first plane of the PCA. The specific profiles of each group were observed to have marked chemical variability (Table S2, Figure S1), which allowed for group separation on the PCA. The two dimensions (F1 and F2) of the PCA explain 73.8% of the total data variability (45.9% for the F1 axis and 27.9% for the F2 axis). The first axis, F1, allows for the separation of G-4 rich in 1-ethyl-3-methylbenzene (Bio3), 1,3,5-trimethylbenzene (Bio4), methyl salicylate (Bio10), sabinene (Bio12), limonene (Bio14), unknown alkane-3 (Bio20), pentadecane (Bio24), heptadecane (Bio25), 1-methyl-1H-imidazole (Bio26), and unknown-1 (Bio30), such as PIN48An48Lb48-DVB, located on the right side of the PCA, represented in the positive region of F1. The second dimension of the PCA, F2, allowed to distinguish two groups, G-2 and G-3, which were richer respectively in methyl methanoate (Bio9), unknown alkane-2 (Bio19), and 4-heptanone (Bio8), namely respectively PIN48An24-DVB and PIN24An24Lb24-DVB. The groups that are found in the negative region of F2. However, the first group G-1 was separated by F1 and F2 in the left positive region of F2, which was characterized as being richer in 1-methyl-3-tert-butylbenzene (Bio5), m-ethylcumene (Bio6), 3-carene (Bio11), (+)-4-carene (Bio15), 2,2,4-trimethyl-pentane (Bio18), unknown alkane-6 (Bio22), and caryophyllene (Bio27), namely PIN24-DVB (Figure S1).

4.1 Exploitation of chemical communication

Like any ecological interaction, pest infestation strongly modifies the volatile profiles emitted by host plants and alters the behavioral responses of pests to the volatile compounds emitted. (L. Conchou et al., 2019; Kutty & Mishra, 2023). Studies (Ehrlich & Raven, 1964; Giunti, Palmeri, Algeri, & Campolo, 2018) have validated that the host plant's emission of secondary VOCs during pest attacks plays an essential role in the evolutionary interactions between insects and plants, as their primary aim is to attract or repel insects. Our research indicates that the activity of the ecological interaction systems has an impact on all the volatile profiles of pomegranates infested by aphids (Fig. 1, Table S1, Fig. 2c). This is what other studies have shown that the promotion or reduction of specific VOC emissions has a strong correlation with each pest species (Guo et al., 2023).

4.1.1 Ecological relevance of terpenoid production

Uninfected pomegranate plants contain terpenes, which are the main bioactive compounds in essential oils (Hosseini et al., 2021), perform multiple ecological tasks, including direct and indirect defenses against pathogens and herbivores, the attraction of pollinators, inter-plant communication, and root competition (K. J. Damodaram et al., 2014; Hosseini et al., 2021). Terpenoids exhibit eco-friendly properties, which include insecticidal, herbicidal, fungicidal, and bactericidal effects (Jung, 2014). However, pomegranate has a small amount of monoterpenes. (Maphetu, Unuofin, Masuku, Olisah, & Lebelo, 2022; Marsoul et al., 2020). Specifically, they contain alpha-terpinene, alpha-terpineol, and 3-carene (D. Wang et al., 2018). The AIP's open space supports the findings, concluding that only 5.07% monoterpenes of the 3-Carene and (+)-4-Carene types are present. Studies have confirmed that the smell of pomegranate leaves and floral organs is derived from sesquiterpene hydrocarbons, specifically β-caryophyllene, farnesene and trans-α-bergamotene (Guler & Gül, 2016), and α-humulene is the major compound present in the stem, as well as sesquiterpene found in leaves (Bandeira Reidel, Cioni, & Pistelli, 2018). The VOCs of the uninfected pomegranate has a high concentration of non-terpenes, particularly aldehydes and esters (Bandeira Reidel et al., 2018). The AIP showed a higher concentration of aldehydes (20.47%) and benzenoids (53.44%), while esters were almost absent (Figs. 1a and 1b).

The compounds found in pomegranate are all informative about plant status (K. J. P. Damodaram et al., 2014; Piesik et al., 2013). In this study, AIP's headspace pinpoints β-caryophyllene as the terpenoid with the largest amount. The presence of this sesquiterpene was observed in various phenological stages of pomegranate (Hosseini et al., 2021; Melgarejo et al., 2011). The use of this compound has been documented for its role in various ecological interactions between plants, insects, and other organisms, acting as a pheromone, allomone, or kairomone (Tavera, Dela Cruz, Santos, Amalin, & Janairo, 2023). This VOC is produced by insects, but plants usually do it naturally or after being attacked by herbivores. Numerous studies have revealed that caryophyllene can act as a kairomone for different insect species, including Hemiptera (Aphis fabae Scopoli) (Webster et al., 2008) and (Helopeltis bakeri) (Tavera et al., 2023), Homoptera (aphid Myzus persicae) (Beale et al., 2006), and Hymenoptera (Bruchophagus roddi) (Kamm & Buttery, 1986). Another instance from coffee systems demonstrated that utilizing β-caryophyllene led to a decrease in coffee berry borer (Hypothenemus hampei) infestation by 33–45% (Kansman et al., 2023; Sadeh et al., 2017). Regarding the effects on natural enemies, limonene demonstrated both attraction and repellence depending on its concentration, while β-caryophyllene displayed repellency across a wide range of concentrations (Kansman et al., 2023). This observation did not have a significant impact on all insect pests (Sadeh et al., 2017).

The chemical signal β-farnesene acts as a mediator for interactions between plants, aphids, and natural enemies (B. Wang et al., 2022). This chemical has two origins: it is secreted by the aphid as an alarm pheromone (Vandermoten, Mescher, Francis, Haubruge, & Verheggen, 2012) (no detection was made in the AIP + An + Lb samples) and also produced by the attacked plants as a semi chemical that attracts natural enemies (B. Wang et al., 2022), which accounts for only 0.05% (Table S2). The virtual absence of β-farnesene in treatments S3-24 and S3-48 was proven by VOSTEEN, WEISSER, and KUNERT (2016)., who showed that β-farnesene is emitted in very small quantities by aphids attacked by predators. Our suggestion is that β-farnesene derived from aphids is not a suitable kairomone for most natural enemy species, particularly when they have the ability to use other signals. Our theory is that natural enemies could utilize a modified plant bouquet to determine plants that are infested with aphids by using herbivore-induced plant-derived samples. The aphid attack by ladybirds (S3-24 and S3-48) resulted in the emission of exclusive ladybird VOCs, which can be seen in Table S1. This shows the appearance of tricyclene, limonene, and alkanes (unknown alkane-2, tridecane, pentadecane, and heptadecane) (Fig. 3) specific to the aphid cuticular hydrocarbons, compounds were identified by our first study from the taxonomic data of Taif A. punicae (Alotaibi et al., 2023). The Taif farm has the ability to use this pattern as a bio-insecticide for pest management.

4.1.2 Primary diagnostic for aphid infested pomegranate

In this in-field study, the interaction of aphid-infected pomegranate plants was studied alone and in the presence of ants and ladybirds, where their VOCs were trapped by an OLS system with a DVB trap. Thirty volatile compounds were detected and identified by GC-MS, including esters, N-compounds, and aldehydes. The most abundant VOCs were benzene, alkane, and terpene compounds, specifically emitted by AIP, while ketone was emitted by the aphid-host plant-ant triplet. Their mass spectra were compared with those in the NIST 14 databases and verified by injecting authentic standards with samples (Alotaibi et al., 2023).

The distribution of the chosen target VOCs was greatly affected by the ecological interaction systems during the search. It's interesting to note that the release of terpenes was greatly influenced by the stages of AIP (S1-DVB) and when ladybird entered the system (S3-DVB-24 and S3-DVB-48). This family of VOCs is a response to feeding by herbivorous aphids. Pomegranate plants exposed to volatiles from damaged seedlings release more predator-attracting sesquiterpenes (caryophyllene and β-farnesene) than unaffected plants. Furthermore, these terpenes are capable of repelling aphids at high doses (Warneys et al., 2018). β-farnesene is a constituent of essential oils in several species of pomegranate (Bandeira Reidel et al., 2018; Zhaleh, Mahdizadeh, & Khoshkhoy, 2023). The field has demonstrated that seedlings without herbivores produce sesquiterpene at a low rate (Kivimäenpää et al., 2016), but herbivores chewing can also induce or increase it (Kivimäenpää, Babalola, Joutsensaari, & Holopainen, 2020; Kivimäenpää et al., 2016; Mäntylä, Kleier, Kipper, & Hilker, 2017). According to Kivimäenpää et al. (2020), the high β-farnesene emissions coupled with high methyl salicylate (MeSA) could indicate an infestation of bark-feeding aphids, but it is not the sole cause. They also demonstrate that the MeSA seems to be a more sensitive indicator because it responds to aphid infestation 2–3 weeks earlier than sesquiterpenes (Kivimäenpää et al., 2020).

The headspace of S1-DVB system, show a minor amount of β-farnesene and absence MeSA vis à vis the high amount of caryophyllene in the VOC emission blend of AIP seedlings (Table S1). This result, was proved by (Kivimäenpää et al., 2020) which indicate that β-farnesene and MeSA respond to the aphid attack after 10 days, but not significantly. At the same time, caryophyllene emission increased by a factor of 27 immediately after an aphid infestation (Kivimäenpää et al., 2020). They suggest that the profile containing compounds such as β-farnesene and MeSA has the potential to be used for the diagnosis of aphid infestations in host plants. Our profile indicates that the infestation by aphids (randomly chosen branch) is in its early stages because there is a low amount of β-farnesene, no MeSA, and a high amount of caryophyllene. For successful plant protection in agriculture and horticulture, early diagnosis of plant pathogens and insect pest infestations is crucial (Li et al., 2019).

4.2 How can statistical analysis aid in research for integrated pest management?

The oligophagous insect, A. Punicae, known as the pomegranate aphid, is responsible for attacking pomegranates. It has been discovered that it can colonize and consume freshly cultivated leaves, tender shoots, flowers, flower buds, and fruit that are in development (Pathania, Arora, Pathania, & Kumar, 2019). According to our previous study, A. punicae is a significantly preferred choice over pomegranate in Taif Governorate (Alotaibi et al., 2023). The early diagnosis of plant pathogens and insect pest infestation types was important to focus research on visualizing a pattern of VOCs that can act as a bioinsecticide for integrated pest management. To achieve this, we examined the VOCs profiles of the ecological quadruplet interaction between AIP, predators, and protectors. The ideal pattern for VOCs' profiles was determined through a statistical analysis using a heat map of hierarchical clustering analysis (HCA) and principal component analysis (PCA).

A heat map result from hierarchical clustering analysis (HCA) confirmed that the plant host infested with aphids had distinct VOCs from those that interact with ants and predators (Fig. 2c). The map clearly indicates a good clustering of VOCs depending on the ecological treatments. The three quadruplet interaction systems display the compositional profile of field VOCs detected (Fig. 2c). It's interesting that we observed from a total of 30 VOCs, but there was a higher concentration of pomegranate infested with aphids (15/30) and with ants (4/30). In contrast, adding ladybird after 24 hours (7/30) and after 48 hours (17/30). At the same figure, the results show that PIN48An48Ln48-DVB is alone on the classification tree. This indicates that in this system, the VOCs related to aroma reach their maximum when the host plant interacts with the predator and protector after 48 hours. This system presents an interaction with a cluster formed by PIN48An72-DVB and PIN24An24Ln24-DVB. They are grouped in the same classification branch, so their VOC profiles are not significantly different. On the other hand, PIN24-DVB is not paired with any other system, so the ecological interaction/system of PIN24-DVB is different from the others in terms of its VOC profile (Figs. 1a and 1b). Overall, we found that among the ecological systems tested, those in the presence of ants and ladybird after 24 hours (PIN48-An24-DVB and PIN24An24Lb24-DVB) had a low number of differential VOCs with lower chemical abundance (blue color). PIN24-DVB showed an increased number of differential compounds with a greater chemical abundance (red color) comparing after the adding of ant (PIN48An24-DVB), follow up the ladybird (PIN24An24Lb24-DVB). The addition of ants to ecological systems (host plant-aphid) can lessen the negative effect of inhibiting the movement of mutualist apterous aphids, which is also reduced by ant semiochemicals (Oliver, Mashanova, Leather, Cook, & Jansen, 2007; Thurman, Northfield, & Snyder, 2019). In order to gain advantage, some organisms have the ability to manipulate other organisms' nervous systems or alter their physiology (Oliver et al., 2007). Ants are known to limit alate aphid dispersal by physically removing wings and also through chemical manipulation of the alate developmental pathway (Oliver et al., 2007). The presence of ants in AIP decreases dispersal and increases the local density of aphids, with the benefit of enhancing the availability of honeydew. Our research shows that ant semiochemicals also reduce the walking motion of mutualistic apterous aphids and, consequently, the VOCs emitted in the ecological interaction system.

The quadruplet interaction of aphid-infested pomegranate with the presence of ants and ladybirds after 48 hours indicates an increase in the number of VOCs detected with CLS. At this point, aphid walking dispersal has evolved as a means of natural enemy escape, then ant chemicals may act as a signal indicating protection (Oliver et al., 2007). Therefore, the presence of a high amount of VOCs, which can play several ecological roles, such as helping natural enemies locate prey or hosts, attract or repel other herbivores, and mediate communication between neighboring plants, and within the same plant (Bezerra, Souto, Sant'Ana, & Ambrogi, 2021).

4.3 Quadruplet interaction-derived repellent and attractant substances for use against IPM

PCA statistical analysis divided the headspace profile of study systems into four distinct groups. For the first group G1 of AIP system, only seven volatile compounds were identified, which included 1-methyl-3-tert-butylbenzene, m-ethylcumene, 3-carene, (+)-4-carene, 2,2,4-trimethyl-pentane, unknown alkane-3, and caryophyllene were identified as the key volatile patterns with a marked repellent effect on A. punicae, and which acts directly on the insect.

The addition of ants can affect the interaction between aphids and host plants. Figures 3 and 4 shows that the pattern biomarkers of the G2 contain methyl methanoate and an unknown alkane-1, which is a result of the integration of ants into the AIP system. Methyl methanoate, as proven by many studies, plays a role in both plant protection products and insecticides. (Kaiser, Beckmann, Walter, & Bertau, 2021). Moreover, it is utilized as a fumigant and a larvicide for pest control in agricultural and pest management industries (Haritos, Damcevski, & Dojchinov, 2006; Stejskal, Vendl, Aulicky, & Athanassiou, 2021).

The pattern generated by the presence of a ladybird in the G3 is solely formed by the 4-heptanone biomarker. This biomarker in the headspace of the G3, with its high variability, could be linked to the activities of Azteca ants (Adams, Wells, Yanoviak, Frost, & Fox, 2020; Mayer, de Hoog, Cristescu, Vera, & Prenafeta-Boldú, 2021). Many studies have shown that n-heptanone is an alarm pheromone in ants, including those belonging to the Azteca genus. According to some authors, this VOC can act as an anesthetic for harmful social insects (Mayer et al., 2021; Papachristoforou et al., 2012). Previous studies of volatile emissions from Azteca ant nests have confirmed the importance of 6-methyl-5-hepten-2-one as a defense pheromone, and 4-heptanone is a common alarm pheromone secreted in the mandibular gland of many ant species (Francelino, Mendonça, Do Nascimento, & Sant'ana, 2006; Jardine et al., 2020; Olubajo, Duffield, & Wheeler, 1980). It seems that ladybirds that prey on aphids cause ants to be disturbed by their attacks, and Azteca emit alarm pheromones outside the G3. The observed pattern of ketone is a result of the ants' alarm-defensive system, which can have a repellent effect on ladybirds (Figs. 3 and 4).

The chemical profiles of quadruplet ecological interaction between AIP, ant, and ladybird G4 were well-defined, with the sample scores primarily distributed on the first F1 axis. (45.9% of total variance; Fig. 2). This axis was correlated with the concentration of several relatively predominant compounds, which were exclusive to the interaction between ladybirds and aphids. Among these biomarkers, some benzenoids (1-ethyl-3methylbenene, and 1,3,5-trimethylbenzene), N-compound (1-methyl-1Himidazole) and alkanes (unknwen alkane-2) specific to aphids cuticular, and terpenes (sabinene, limonene) which were identified in our previous study as being pheromones of mechanical induced aphids (Alotaibi et al., 2023). In this complex pattern, there were also methyl salicylate, pentadecane, heptadecane, and sabinene, present, but they played a less prominent role. Previously, Lee (2010) reported that the MeSA immitted by strawberries attract ladybirds to host plant. In a previous study aimed at investigating the response of predatory insects such as adult hoverflies, ladybirds, and green lacewings to MeSA in cranberries (Rodriguez-Saona, Kaplan, Braasch, Chinnasamy, & Williams, 2011; Salamanca, Souza, Kyryczenko-Roth, & Rodriguez-Saona, 2019). Certain alkanes from infested green leaves were categorized as beneficial hydrocarbons due to their different levels of kairomonal activity for carnivorous insects, while pentadecane and heptadecane were categorized as detrimental hydrocarbons (Paul, Singh, & Singh, 2002). Our results also reveal a decreased interest in their biomarkers. The monoterpenes, namely, limonene, and sabinene, not been detected in the headspace of AIP but it was detected after ecologic interaction with the presence of ladybird. Despite the abundant literature, it has been reported that sabinene and limonene may increase in a variety of plants that are infested with aphids, which could be the reason for their repellent effect (Q. Ahmed, Agarwal, Alobaidi, Zhang, & Ren, 2022; Dieudonné et al., 2022). The pattern biomarkers identified in the quadruple ecologic interaction between the AIP, the ant, and the ladybird were the basis for the formation of two groups. The first was derived from a host plant infested with aphids and played a role in attracting ladybirds, while the second was derived from the aphid and was responsible for repelling effect of the natural enemy.

Compared to pesticides, VOCs are sustainable, renewable, and degrade quickly in the environment, making them a viable crop biocontrol tool for integrated pest management. The interaction between plants, pests, protectors, and predators in nature is dominated by VOCs, which can have different responses based on the composition and quantity of compounds released. The purpose of this research was to evaluate the composition and relative content of volatile compounds in the field of a pomegranate plant that was infected with aphids and was situated near natural enemies and their guardians. In total, thirty different volatile compounds were identified from four ecologic interaction systems. According to principal component analysis (PCA), the four systems studied were divided into four groups. In the AIP system, seven volatile compounds were identified as key volatile patterns with a marked repellent effect on the A. punicae pest. The integration of ants into aphid-infested pomegranate reduces the number of VOCs, and as a result, ants affect the physiological transformation of the aphid. Methyl methanoate and unknown alkane-1 obtained patterns can be implicated in both plant protection products and insecticides for pest control in agricultural applications. The system of quadruplet interaction after 24 hours shows seven biomarkers, the most remarkable of which is 4-heptanone, which is a result of the ants' alarm-defensive system. A complex pattern was discovered after 48 hours of ecological interaction that was classified as VOCs created by the plant as an aphid repellant and by the aphid as an alarm-defensive system. To put it simply, these findings provide fresh insight into the identification of a specific bioinsecticide for controlling aphids through biological control. In the future, the volatile patterns obtained can be used as an insecticidal marker against aphids, which could lead to improved pest management. To develop sustainable biocontrol integrated pest management, a qualitative study will be conducted to determine the optimal concentration of VOC mixtures that can repel pests or attract predators.

Author Contributions: Conceptualization: T.A.S., N.H.M., and N.J.O.; methodology: N.H.M., T.A.S., N.J.O., and M.A.M.; data analysis: N.H.M., T.A.S., draft preparation: N.H.M., T.A.S., N.J.O., M.A.M., and E.M.G.; writing—review and editing: N.H.M., T.A.S., N.J.O., M.A.M, and E.M.G; project administration: T.A.S.; funding acquisition: T.A.S.; All authors have read and agreed to the published version of the manuscript.

Data Availability: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments: Authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number (1/441/119).

Funding Statement: The authors would like to acknowledge the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number (1/441/119).

Conflicts of Interest: The authors declare that no competing interest exists.

Abd El-Ghany, N. M. (2019). Semiochemicals for controlling insect pests. Journal of Plant Protection Research, 59(1), 1-11. doi:10.24425/jppr.2019.126036
Aboelhana, E., Ismail, I. A., Attia, A., & Sadik, A. (2017). Genetic studies, in vitro propagation and conservation of some pomegranate cultivars grown in Taif province, Kingdom of Saudi Arabia (Vol. 45).
Adams, R. M. M., Wells, R. L., Yanoviak, S. P., Frost, C. J., & Fox, E. G. P. (2020). Interspecific Eavesdropping on Ant Chemical Communication. Frontiers in Ecology and Evolution, 8. doi:10.3389/fevo.2020.00024
Ahmed, M. M., Samir el, S. A., El-Shehawi, A. M., & Alkafafy, M. E. (2015). Anti-obesity effects of Taif and Egyptian pomegranates: molecular study. Biosci Biotechnol Biochem, 79(4), 598-609. doi:10.1080/09168451.2014.982505
Ahmed, Q., Agarwal, M., Alobaidi, R., Zhang, H., & Ren, Y. (2022). Response of Aphid Parasitoids to Volatile Organic Compounds from Undamaged and Infested Brassica oleracea with Myzus persicae. Molecules, 27(5), 1522. Retrieved from https://www.mdpi.com/1420-3049/27/5/1522
Al-Barty, A. M. F. (2011). Survey and Enumeration of Pests on Pomegranate Tree with Reference to its Parasite in Al-Taif City. Australian Journal of Basic and Applied Sciences, 5, 1086-1093.
Alghamdi, A. G., Aly, A. A., & Ibrahim, H. M. (2022). Effect of Climate Change on the Quality of Soil, Groundwater, and Pomegranate Fruit Production in Al-Baha Region, Saudi Arabia: A Modeling Study Using SALTMED. Sustainability, 14(20), 13275. Retrieved from https://www.mdpi.com/2071-1050/14/20/13275
Alotaibi, N. J., Alsufyani, T., M’sakni, N. H., Almalki, M. A., Alghamdi, E. M., & Spiteller, D. (2023). Rapid Identification of Aphid Species by Headspace GC-MS and Discriminant Analysis. Insects, 14(7), 589. Retrieved from https://www.mdpi.com/2075-4450/14/7/589
Angon, P. B., Mondal, S., Jahan, I., Datto, M., Antu, U. B., Ayshi, F. J., & Islam, M. S. (2023). Integrated Pest Management (IPM) in Agriculture and Its Role in Maintaining Ecological Balance and Biodiversity. Advances in Agriculture, 2023, 5546373. doi:10.1155/2023/5546373
Badra, Z., & Angeli, S. (2018). Characterisation of volatile compounds released by apple trees infested by different aphid species.
Bandeira Reidel, R. V., Cioni, P. L., & Pistelli, L. (2018). Volatiles from different plant parts of Punica granatum grown in Tuscany (Italy). Scientia Horticulturae, 231, 49-55. doi:https://doi.org/10.1016/j.scienta.2017.12.019
Beale, M. H., Birkett, M. A., Bruce, T. J. A., Chamberlain, K., Field, L. M., Huttly, A. K., . . . Zhang, Y. (2006). Aphid alarm pheromone produced by transgenic plants affects aphid and parasitoid behavior. Proc Natl Acad Sci U S A, 103(27), 10509-10513. doi:10.1073/pnas.0603998103
Bezerra, R., Souto, L., Sant'Ana, A., & Ambrogi, B. (2021). Indirect plant defenses: volatile organic compounds and extrafloral nectar. Arthropod-Plant Interactions, 15. doi:10.1007/s11829-021-09837-1
Borén, H., Grimvall, A., & Sävenhed, R. (1982). Modified stripping technique for the analysis of trace organics in water. Journal of Chromatography A, 252, 139-146. doi:https://doi.org/10.1016/S0021-9673(01)88405-3
Conchou, L., Lucas, P., Meslin, C., Proffit, M., Staudt, M., & Renou, M. (2019). Insect Odorscapes: From Plant Volatiles to Natural Olfactory Scenes. Front Physiol, 10, 972. doi:10.3389/fphys.2019.00972
Conchou, L., Lucas, P., Meslin, C., Proffit, M., Staudt, M., & Renou, M. (2019). Insect Odorscapes: From Plant Volatiles to Natural Olfactory Scenes. Frontiers in Physiology, 10. doi:10.3389/fphys.2019.00972
Conti, E., Avila, G., Barratt, B., Cingolani, F., Colazza, S., Guarino, S., . . . Wajnberg, E. (2021). Biological control of invasive stink bugs: review of global state and future prospects. Entomologia Experimentalis et Applicata, 169(1), 28-51. doi:https://doi.org/10.1111/eea.12967
Crossley, M. S., Snyder, W. E., & Hardy, N. B. (2021). Insect-plant relationships predict the speed of insecticide adaptation. Evol Appl, 14(2), 290-296. doi:10.1111/eva.13089
Damodaram, K. J., Kempraj, V., Aurade, R. M., Rajasekhar, S. B., Venkataramanappa, R. K., Nandagopal, B., & Verghese, A. (2014). Centuries of domestication has not impaired oviposition site-selection function in the silkmoth, Bombyx mori. Sci Rep, 4, 7472. doi:10.1038/srep07472
Damodaram, K. J. P., Kempraj, V., Aurade, R. M., Rajasekhar, S. B., Venkataramanappa, R. K., Nandagopal, B., & Verghese, A. (2014). Centuries of domestication has not impaired oviposition site-selection function in the silkmoth, Bombyx mori. Scientific Reports, 4(1), 7472. doi:10.1038/srep07472
Dicke, M., & Sabelis, M. W. (1988). Infochemical Terminology: Based on Cost-Benefit Analysis Rather than Origin of Compounds? Functional Ecology, 2(2), 131-139. doi:10.2307/2389687
Dieudonné, E., Gautier, H., Dardouri, T., Staudt, M., Costagliola, G., & Gomez, L. (2022). Establishing repellent effects of aromatic companion plants on Dysaphis plantaginea, using a new dynamic tubular olfactometer. Entomologia Experimentalis et Applicata, 170(8), 727-743. doi:https://doi.org/10.1111/eea.13194
Effah, E., Barrett, D. P., Peterson, P. G., Godfrey, A. J. R., Potter, M. A., Holopainen, J. K., & Clavijo McCormick, A. (2020). Natural Variation in Volatile Emissions of the Invasive Weed Calluna vulgaris in New Zealand. Plants, 9(2), 283. Retrieved from https://www.mdpi.com/2223-7747/9/2/283
Ehrlich, P. R., & Raven, P. H. (1964). Butterflies and Plants: A Study in Coevolution. Evolution, 18(4), 586-608. doi:10.2307/2406212
El-Shehawi, A. M., Sayed, S., Hassan, M. M., Al-Otaibi, S., Althobaiti, F., Elseehy, M. M., & Soliman, M. (2022). Taify Pomegranate Juice (TPJ) Abrogates Acrylamide-Induced Oxidative Stress Through the Regulation of Antioxidant Activity, Inflammation, and Apoptosis-Associated Genes. Front Vet Sci, 9, 833605. doi:10.3389/fvets.2022.833605
Elsayed, G., & Bazaid, S. (2011). Field investigation of pomegranate fruit worms in Taif and laboratory evaluation of Bacillus thuringiensis against Ectomyelois ceratoniae. Archives of Phytopathology and Plant Protection, 44, 28-36. doi:10.1080/03235400902831075
Fleischer, J., & Krieger, J. (2018). Insect Pheromone Receptors - Key Elements in Sensing Intraspecific Chemical Signals. Front Cell Neurosci, 12, 425. doi:10.3389/fncel.2018.00425
Francelino, M. R., Mendonça, A. L., Do Nascimento, R. R., & Sant'ana, A. E. G. (2006). The Mandibular Gland Secretions of the Leaf-Cutting Ants Atta sexdens sexdens and Atta opaciceps Exhibit Intercaste and Intercolony Variations. Journal of Chemical Ecology, 32(3), 643-656. doi:10.1007/s10886-005-9020-9
Giunti, G., Palmeri, V., Algeri, G. M., & Campolo, O. (2018). VOC emissions influence intra- and interspecific interactions among stored-product Coleoptera in paddy rice. Scientific Reports, 8(1), 2052. doi:10.1038/s41598-018-20420-2
Grob, K., Grob, K., & Grob, G. (1975). Organic substances in potable water and in its precursor: III. The closed-loop stripping procedure compared with rapid liquid extraction. Journal of Chromatography A, 106(2), 299-315. doi:https://doi.org/10.1016/S0021-9673(00)93838-X
Guler, Z., & Gül, E. (2016). Volatile Organic Compounds (Vocs) In The Aril Juices And Seeds From Selected Five Pomegranate ( Punica Granatum L .) Cultivars. International Journal of Food Properties, 20. doi:10.1080/10942912.2016.1155057
Guo, H. G., Miao, S. Z., Ai, P. P., Zhang, M. Z., Yan, Z., & Du, Y. L. (2023). Bioactive volatile compounds from Penicillium digitatum-infected apples: Oviposition attractants for yellow peach moth Conogethes punctiferalis (Lepidoptera: Crambidae). Frontiers in Sustainable Food Systems, 7. doi:10.3389/fsufs.2023.1119717
Haritos, V., Damcevski, K., & Dojchinov, G. (2006). Improved efficacy of ethyl formate toward stored grain insects by combination with CO2 in a ‘dynamic’ application. Pest management science, 62, 325-333. doi:10.1002/ps.1167
Hassan, M. M., Soliman, M. M., Al-Otaibi, S., El-Shehawi, A. M., Taha, E.-K. A., & Sayed, S. (2022). The effectiveness of Xanthium strumamrium L. extract and Trichoderma spp. against pomegranate isolated pathogenic fungi in Taif, Saudi Arabia. Journal of King Saud University - Science, 34(6), 102185. doi:https://doi.org/10.1016/j.jksus.2022.102185
Heil, M., & Bostock, R. M. (2002). Induced systemic resistance (ISR) against pathogens in the context of induced plant defences. Ann Bot, 89(5), 503-512. doi:10.1093/aob/mcf076
Hosseini, S. A., Goldansaz, S. H., Groot, A. T., Menken, S. B. J., Van Der Wielen, F., Wissel, C., . . . Roessingh, P. (2021). Identification of Bioactive Plant Volatiles for the Carob Moth by Means of GC-EAD and GC-Orbitrap MS. Applied Sciences, 11(18), 8603. Retrieved from https://www.mdpi.com/2076-3417/11/18/8603
Jardine, K. J., Piva, L. R. d. O., Rodrigues, T. B., Spanner, G. C., Rodrigues, J. R., Menezes, V. S., . . . Chambers, J. (2020). Volatiles defenses of Amazon <em>Azteca</em> ants (repellent ants). bioRxiv, 2020.2004.2015.043547. doi:10.1101/2020.04.15.043547
Jung, J.-S. (2014). Analysis of Volatile Compounds in the Root Peel, Stem Peel, and Fruit Peel of Pomegranate (Punica granatum) by TD GC/MS. International Journal of Bio-Science and Bio-Technology, 6, 169-182. doi:10.14257/ijbsbt.2014.6.3.20
Kaiser, D., Beckmann, L., Walter, J., & Bertau, M. (2021). Conversion of Green Methanol to Methyl Formate. Catalysts, 11(7), 869. Retrieved from https://www.mdpi.com/2073-4344/11/7/869
Kamm, J. A., & Buttery, R. G. (1986). Ovipositional Behavior of the Alfalfa Seed Chalcid (Hymenoptera: Eurytomidae) in Response to Volatile Components of Alfalfa 1. Environmental Entomology, 15(2), 388-391. doi:10.1093/ee/15.2.388
Kansman, J. T., Jaramillo, J. L., Ali, J. G., & Hermann, S. L. (2023). Chemical ecology in conservation biocontrol: new perspectives for plant protection. Trends in Plant Science, 28(10), 1166-1177. doi:https://doi.org/10.1016/j.tplants.2023.05.001
Kivimäenpää, M., Babalola, A. B., Joutsensaari, J., & Holopainen, J. K. (2020). Methyl Salicylate and Sesquiterpene Emissions Are Indicative for Aphid Infestation on Scots Pine. Forests, 11(5), 573. Retrieved from https://www.mdpi.com/1999-4907/11/5/573
Kivimäenpää, M., Ghimire, R. P., Sutinen, S., Häikiö, E., Kasurinen, A., Holopainen, T., & Holopainen, J. K. (2016). Increases in volatile organic compound emissions of Scots pine in response to elevated ozone and warming are modified by herbivory and soil nitrogen availability. European journal of forest research, 135, 343-360.
Kong, C. H., Xuan, T. D., Khanh, T. D., Tran, H. D., & Trung, N. T. (2019). Allelochemicals and Signaling Chemicals in Plants. Molecules, 24(15). doi:10.3390/molecules24152737
Kunert, M., David, A., Becher, J., & Boland, W. (2009). Volatile Sampling from Biological Sources by the Closed-Loop-Stripping Technique. Cold Spring Harbor protocols, 2009, pdb.prot5233. doi:10.1101/pdb.prot5233
Kutty, N. N., & Mishra, M. (2023). Dynamic distress calls: volatile info chemicals induce and regulate defense responses during herbivory. Front Plant Sci, 14, 1135000. doi:10.3389/fpls.2023.1135000
Lee, J. C. (2010). Effect of Methyl Salicylate-Based Lures on Beneficial and Pest Arthropods in Strawberry. Environmental Entomology, 39(2), 653-660. doi:10.1603/en09279
Li, Z., Paul, R., Ba Tis, T., Saville, A. C., Hansel, J. C., Yu, T., . . . Wei, Q. (2019). Non-invasive plant disease diagnostics enabled by smartphone-based fingerprinting of leaf volatiles. Nature plants, 5(8), 856-866.
Mäntylä, E., Kleier, S., Kipper, S., & Hilker, M. (2017). The attraction of insectivorous tit species to herbivore-damaged Scots pines. Journal of Ornithology, 158, 479-491.
Maphetu, N., Unuofin, J. O., Masuku, N. P., Olisah, C., & Lebelo, S. L. (2022). Medicinal uses, pharmacological activities, phytochemistry, and the molecular mechanisms of Punica granatum L. (pomegranate) plant extracts: A review. Biomedicine & Pharmacotherapy, 153, 113256. doi:https://doi.org/10.1016/j.biopha.2022.113256
Marsoul, A., Ijjaali, M., Elhajjaji, F., Taleb, M., Salim, R., & Boukir, A. (2020). Phytochemical screening, total phenolic and flavonoid methanolic extract of pomegranate bark (Punica granatum L): Evaluation of the inhibitory effect in acidic medium 1 M HCl. Materials Today: Proceedings, 27, 3193-3198. doi:https://doi.org/10.1016/j.matpr.2020.04.202
Mayer, V. E., de Hoog, S., Cristescu, S. M., Vera, L., & Prenafeta-Boldú, F. X. (2021). Volatile Organic Compounds in the Azteca/Cecropia Ant-Plant Symbiosis and the Role of Black Fungi. Journal of Fungi, 7(10), 836. Retrieved from https://www.mdpi.com/2309-608X/7/10/836
Mbaluto, C. M., Ayelo, P. M., Duffy, A. G., Erdei, A. L., Tallon, A. K., Xia, S., . . . Becher, P. G. (2020). Insect chemical ecology: chemically mediated interactions and novel applications in agriculture. Arthropod Plant Interact, 14(6), 671-684. doi:10.1007/s11829-020-09791-4
Melgarejo, P., Calín-Sánchez, Á., Vázquez-Araújo, L., Hernández, F., Martínez, J. J., Legua, P., & Carbonell-Barrachina, Á. A. (2011). Volatile Composition of Pomegranates from 9 Spanish Cultivars Using Headspace Solid Phase Microextraction. Journal of Food Science, 76(1), S114-S120. doi:https://doi.org/10.1111/j.1750-3841.2010.01945.x
Mooney, K. A., Pratt, R. T., & Singer, M. S. (2012). The tri-trophic interactions hypothesis: interactive effects of host plant quality, diet breadth and natural enemies on herbivores. PLoS One, 7(4), e34403. doi:10.1371/journal.pone.0034403
Nielsen, B., Rampin, O., Meunier, N., & Bombail, V. (2015). Behavioral responses to odors from other species: introducing a complementary model of allelochemics involving vertebrates. Frontiers in Neuroscience, 9. doi:10.3389/fnins.2015.00226
Nielsen, B. L., Rampin, O., Meunier, N., & Bombail, V. (2015). Behavioral responses to odors from other species: introducing a complementary model of allelochemics involving vertebrates. Frontiers in Neuroscience, 9. doi:10.3389/fnins.2015.00226
Oliver, T. H., Mashanova, A., Leather, S. R., Cook, J. M., & Jansen, V. A. (2007). Ant semiochemicals limit apterous aphid dispersal. Proc Biol Sci, 274(1629), 3127-3131. doi:10.1098/rspb.2007.1251
Olubajo, O., Duffield, R. M., & Wheeler, J. W. (1980). 4-Heptanone in the Mandibular Gland Secretion of the Nearctic Ant, Zacryptocerus varians (Hymenoptera: Formicidae). Annals of the Entomological Society of America, 73(1), 93-94. doi:10.1093/aesa/73.1.93
Papachristoforou, A., Kagiava, A., Papaefthimiou, C., Termentzi, A., Fokialakis, N., Skaltsounis, A.-L., . . . Theophilidis, G. (2012). The Bite of the Honeybee: 2-Heptanone Secreted from Honeybee Mandibles during a Bite Acts as a Local Anaesthetic in Insects and Mammals. PLOS ONE, 7(10), e47432. doi:10.1371/journal.pone.0047432
Pathania, M., Arora, P. K., Pathania, S., & Kumar, A. (2019). Studies on population dynamics and management of pomegranate aphid, Aphis punicae Passerini (Hemiptera: Aphididae) on pomegranate under semi-arid conditions of South-western Punjab. Scientia Horticulturae, 243, 300-306. doi:https://doi.org/10.1016/j.scienta.2018.07.027
Paul, A., Singh, S., & Singh, A. K. (2002). Kairomonal effect of some saturated hydrocarbons on the egg parasitoids, Trichogramma brasiliensis (Ashmead) and Trichogramma exiguum, Pinto, Platner and Oatman (Hym., Trichogrammatidae). Journal of Applied Entomology - J APPL ENTOMOL, 126, 409-416. doi:10.1046/j.1439-0418.2002.00661.x
Pellegrino, M., & Nakagawa, T. (2009). Smelling the difference: controversial ideas in insect olfaction. J Exp Biol, 212(Pt 13), 1973-1979. doi:10.1242/jeb.023036
Peterson, J. A., Ode, P. J., Oliveira-Hofman, C., & Harwood, J. D. (2016). Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities. Frontiers in Plant Science, 7. doi:10.3389/fpls.2016.01794
Piesik, D., Delaney, K. J., Wenda-Piesik, A., Sendel, S., Tabaka, P., & Buszewski, B. (2013). Meligethes aeneus pollen-feeding suppresses, and oviposition induces, Brassica napus volatiles: beetle attraction/repellence to lilac aldehydes and veratrole. Chemoecology, 23(4), 241-250. doi:10.1007/s00049-013-0138-x
Richard, B., Qi, A., & Fitt, B. D. L. (2022). Control of crop diseases through Integrated Crop Management to deliver climate-smart farming systems for low- and high-input crop production. Plant Pathology, 71(1), 187-206. doi:https://doi.org/10.1111/ppa.13493
Rodriguez-Saona, C., Kaplan, I., Braasch, J., Chinnasamy, D., & Williams, L. (2011). Field responses of predaceous arthropods to methyl salicylate: A meta-analysis and case study in cranberries. Biological Control, 59(2), 294-303. doi:https://doi.org/10.1016/j.biocontrol.2011.06.017
Sadeh, D., Nitzan, N., Shachter, A., Chaimovitsh, D., Dudai, N., & Ghanim, M. (2017). Whitefly attraction to rosemary (Rosmarinus officinialis L.) is associated with volatile composition and quantity. PLoS One, 12(5), e0177483. doi:10.1371/journal.pone.0177483
Salamanca, J., Souza, B., Kyryczenko-Roth, V., & Rodriguez-Saona, C. (2019). Methyl Salicylate Increases Attraction and Function of Beneficial Arthropods in Cranberries. Insects, 10(12), 423. Retrieved from https://www.mdpi.com/2075-4450/10/12/423
Sánchez-Bayo, F. (2021). Indirect Effect of Pesticides on Insects and Other Arthropods. Toxics, 9(8). doi:10.3390/toxics9080177
Senft, M., Clancy, M. V., Weisser, W. W., Schnitzler, J.-P., & Zytynska, S. E. (2019). Additive effects of plant chemotype, mutualistic ants and predators on aphid performance and survival. Functional Ecology, 33(1), 139-151. doi:https://doi.org/10.1111/1365-2435.13227
Sharma, A., Kumar, V., Shahzad, B., Tanveer, M., Sidhu, G. P. S., Handa, N., . . . Thukral, A. K. (2019). Worldwide pesticide usage and its impacts on ecosystem. SN Applied Sciences, 1(11), 1446. doi:10.1007/s42452-019-1485-1
Stejskal, V., Vendl, T., Aulicky, R., & Athanassiou, C. (2021). Synthetic and Natural Insecticides: Gas, Liquid, Gel and Solid Formulations for Stored-Product and Food-Industry Pest Control. Insects, 12(7), 590. Retrieved from https://www.mdpi.com/2075-4450/12/7/590
Tang, D., Chen, M., Huang, X., Zhang, G., Zeng, L., Zhang, G., . . . Wang, Y. (2023). SRplot: A free online platform for data visualization and graphing. PLOS ONE, 18(11), e0294236. doi:10.1371/journal.pone.0294236
Tavera, M. A. A., Dela Cruz, M. C. V., Santos, K. L. T., Amalin, D. M., & Janairo, J. I. B. (2023). β-caryophyllene as an attractant for the cacao mirid bug, Helopeltis bakeri Poppius (Hemiptera: Miridae), and chemodiversity of its host plant volatiles. Frontiers in Agronomy, 5. doi:10.3389/fagro.2023.1178558
Tholl, D., Boland, W., Hansel, A., Loreto, F., Röse, U. S., & Schnitzler, J. P. (2006). Practical approaches to plant volatile analysis. Plant J, 45(4), 540-560. doi:10.1111/j.1365-313X.2005.02612.x
Thurman, J. H., Northfield, T. D., & Snyder, W. E. (2019). Weaver Ants Provide Ecosystem Services to Tropical Tree Crops. Frontiers in Ecology and Evolution, 7. doi:10.3389/fevo.2019.00120
Trowbridge, A. M. (2014). Evolutionary Ecology of Chemically Mediated Plant-Insect Interactions. In R. K. Monson (Ed.), Ecology and the Environment (pp. 1-28). New York, NY: Springer New York.
Vandermoten, S., Mescher, M. C., Francis, F., Haubruge, E., & Verheggen, F. J. (2012). Aphid alarm pheromone: An overview of current knowledge on biosynthesis and functions. Insect Biochemistry and Molecular Biology, 42(3), 155-163. doi:https://doi.org/10.1016/j.ibmb.2011.11.008
Vargas, G., Rivera-Pedroza, L. F., García, L. F., & Jahnke, S. M. (2023). Conservation Biological Control as an Important Tool in the Neotropical Region. Neotropical Entomology, 52(2), 134-151. doi:10.1007/s13744-022-01005-1
VOSTEEN, I., WEISSER, W. W., & KUNERT, G. (2016). Is there any evidence that aphid alarm pheromones work as prey and host finding kairomones for natural enemies? Ecological Entomology, 41(1), 1-12. doi:https://doi.org/10.1111/een.12271
Wang, B., Dong, W., Li, H., D’Onofrio, C., Bai, P., Chen, R., . . . Wang, G. (2022). Molecular basis of (E)-β-farnesene-mediated aphid location in the predator Eupeodes corollae. Current Biology, 32(5), 951-962.e957. doi:https://doi.org/10.1016/j.cub.2021.12.054
Wang, D., Özen, C., Abu-Reidah, I. M., Chigurupati, S., Patra, J. K., Horbanczuk, J. O., . . . Atanasov, A. G. (2018). Vasculoprotective Effects of Pomegranate (Punica granatum L.). Frontiers in Pharmacology, 9. doi:10.3389/fphar.2018.00544
Warneys, R., Gaucher, M., Robert, P., Aligon, S., Anton, S., Aubourg, S., . . . Degrave, A. (2018). Acibenzolar-S-Methyl Reprograms Apple Transcriptome Toward Resistance to Rosy Apple Aphid. Front Plant Sci, 9, 1795. doi:10.3389/fpls.2018.01795
Webster, B., Bruce, T., Dufour, S., Birkemeyer, C., Birkett, M., Hardie, J., & Pickett, J. (2008). Identification of volatile compounds used in host location by the black bean aphid, Aphis fabae. J Chem Ecol, 34(9), 1153-1161. doi:10.1007/s10886-008-9510-7
Zhaleh, H., Mahdizadeh, H., & Khoshkhoy, S. (2023). Punica granatum Seed Essential Oil Suppressed Methadone-Induced Cell Death by Natural Antioxidant Activity. Asian Pac J Cancer Prev, 24(3), 801-810. doi:10.31557/apjcp.2023.24.3.801

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Interspecific Eavesdropping on Chemical Communication in the Field between Taif Pomegranate, Aphis punicae, Enemies, and Protectors

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Plant material and insect

2.2. Rearing of A. punicae natural enemies

2. 3 Headspace analysis

2.3.1 VOCs Sampling

2.3.2 Opened-Loop Stripping (OLS)

2.3.3 Sampling vs treatments and ecological systems of aphid-infested pomegranates

2.4 Gas chromatography-mass spectrometry (GC-MS) analysis conditions

2.5 GC–MS data processing for multivariate analysis

2.6 Statistical Analysis

3. Results

4. Discussions

4.1 Exploitation of chemical communication

4.1.1 Ecological relevance of terpenoid production

4.1.2 Primary diagnostic for aphid infested pomegranate

4.2 How can statistical analysis aid in research for integrated pest management?

4.3 Quadruplet interaction-derived repellent and attractant substances for use against IPM

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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