Articulating Fragrant Agarwood Formation as an Outcome of the Interaction between the Insect Zeuzera conferta and Aquilaria trees – A Review

Agarwood is the resinous infected wood obtained from Aquilaria species, which is a highly priced product in the flavour and fragrance market. Its formation is a complex process of interaction between the plant, insect, and microorganisms. Multiple studies concerning the interaction of microorganisms with the Aquilaria tree have been reported. However, the significant interaction between the insect Zeuzera conferta Walker (Lepidoptera: Cossidae) with Aquilaria has been overlooked, and only exiguous studies have been accomplished. Considering the dearth of available literature on this interesting phenomenon a review has been attempted. The taxonomical and morphological descriptions proffered by researchers and the insect life cycle are discussed. The review lays emphasis on the chemical ecology of the interaction between Z. conferta , Aquilaria and associating microorganisms as a possible continuum operating in the form of complex chemical signalling via release and sensing of Volatile Organic Compounds (VOCs), Herbivore Induced Plant Volatiles (HIPVs) and Microbial Volatile Organic Compounds (MVOCs). The review also scrutinizes the future perspectives of understanding the interaction in devising suitable management strategies to prevent uncontrolled infestation and, simultaneously, develop artificial rearing technology for the insect Z. conferta as a strategy for ensuring sustainable livelihood of farmers dependent on agarwood production .


Introduction
Zeuzera conferta Walker (Lepidoptera: Cossidae) is one of the principal insect pests that has been found to associate with the Aquilaria trees.It is also known as Neurozera conferta Walker (Syazwan et al., 2019).It belongs to the Class Insecta, Order Lepidoptera, and Family Cossidae in a systemic classification.It is widely distributed in Southeast Asian countries, Eastern Himalayas, Sri Lanka, Bangladesh, Taiwan, Andaman & Nicobar Islands and the Philippines.The insect is particularly prevalent in the agarwood plantations of the north-eastern part of India, particularly in the state of Assam, and is known to influence the formation of the rare resinous and fragrant agarwood by infesting Aquilaria trees.In fact, agarwood from Assam is regarded as a high-quality material in the global agarwood market.The borer insect is known locally as "Pukh" in Assamese and "Emphu" in Bodo languages, with both the terms meaning insect, which is used as a general nomenclature by the cultivators, traders, and people conversant with the Aquilaria trees in Assam.The Aquilaria trees, which belong to the Thymelaeaceae family, are commonly known as Agarwood, Eaglewood, or Aloes wood besides various other regional names.Agarwood is the darkcoloured resinous fragrant wood that has a high commercial value.Further, the formation of agarwood involves a complex process of interaction between the plant, insect, and microorganisms.The process of formation of agarwood is a defense response that is connected to this response to injury, created through natural and artificial means.To prevent or to recover from the injury, the Aquilaria trees produce oleoresins at the site of the injury as a product of plant defense response (Zhang et al., 2012).The site of the injury is colonised further by microorganisms leading to the accumulation of the oleoresin which is called agarwood.
So far, a total of 19 insect pests have been recorded to associate with the Aquilaria trees belonging to 16 families and 5 orders of which the preponderance of the sap-sucker is found higher, followed by leaf defoliators and lastly wood borers (Syazwan et al., 2019).However, the wood borer and the leaf defoliator form the major pest of the Aquilaria trees causing a serious damage to the Aquilaria trees (Ong et al., 2014).The larvae of the wood borer Z. conferta Walker (Figure 1) infest the woody stem of the Aquilaria trees and facilitate subsequent microbial infections.The larvae make vertical tunnels inside the trunk of the Aquilaria trees as they feed and move up spreading the microbial infection where the oleoresin accumulates (Kalita et al., 2015).From brown streaks to dark brown and finally to black coloured wood are the changes that occur in the healthy wood where the initial infestations occur.Successively, these lead to stunted and poor development, formation of cankers on the trunk, swelling, symptoms of dieback on the top and outer branches of the trees (Nath and Saikia, 2002).Subsequently, the scenario of a visible wound, stem distortions, decayed branches, uneven and irregular trunk, and odoriferous dispenses evidence of agar formation inside the tree.The incidence of Z. conferta is, however, not observed in all the Aquilaria trees that are grown.Its selectiveness in infesting the Aquilaria trees is interesting, as significant differences are being observed in the infestation process among the Aquilaria trees that are grown separately at a distance of a few meters with one area being completely infested and the other area with none.These variations in the infestation process have led the traders to practice artificial process of injury and induce infections through physical, chemical, and biological means or by their combinations as a method of treatment in the Aquilaria trees where insect incidence is not usually observed.However, the increase in commercial demand and slow natural process of agarwood formation have also pressurized the traders to execute the process of artificial infections (Chippa and Kaushik, 2017).Despite its success in producing agarwood through the application of artificial techniques, the quality of the agarwood remains an issue and is found incommensurate in comparison to naturally occurring ones (Kalita et al., 2015).Moreover, the price of the agarwood, the durability of the fragrance, long shelf life, and the extent of the microbial attack are all found to be higher in agarwood that is induced only after the insect (Z.conferta) infestation (Hoque et al., 2019).
Even with these evidence about the importance of the Z. conferta in the formation of agarwood, studies are yet to elucidate its actual role.There is a possibility of insect-microorganism in relation which it might have a role in the superior quality agarwood formation (Hoque et al., 2019).However, literature is deficient concerning Z. conferta and, therefore, in the present review, efforts have been made to compile the information scattered in diverse domains and bring out the perspectives that require a closer study to improve agarwood production in future.Zeuzera conferta The genus Zeuzera was classified based on the external characters only, for instance, the presence of the crossvein Subcosta (Sc)-Radius Sector (Rs), the shape of the humeral plate, and the length of the anal plate (Sutrisno, 2015).From a total of 52 species across the world, only 5 are reported to occur in the Indian Subcontinent (Arora, 1976).According to the original illustration given by Walker (1856) on the Z. conferta, the female is whitish with black antennae.The thorax possessed two interrupted green stripes and three rows of green spots in the abdomen.Legs are reported to be mostly green and wings with innumerable tiny transverse green or aeneous streaks with green dots down the border.The fore wings are without streaks at the parts of the disk.The length of the body has 13 lines and wings having 28 lines.The species is known from the Sylhet region of Bangladesh and Labuan and Luzon of the Philippines.It is reported to be close to Zeuzera indica Herr.-Sch regarding the origin of the vein in the fore wing and the evenly rounded outer margin in the hind wing.However, phylogenetic studies based on the Cytochrome Oxidase subunit I gene (COI) sequence revealed Z. conferta to be closely related to the Z. lineate (Sutrisno, 2015).The COI gene is regarded as highly conserved and felicitous in identifying a species due to its low variability (generally less than 1-2%).Even for closely associated species, its value is found to be less than 1%.Furthermore, for Lepidoptera the COI gene is one of the most common to be used in inferring the relationship among the closely related species.Yakovlev, (2011) stated the species to be medium in size with males possessing cupshaped antennae and filiform in females (Figure 2a).Dorsally the thorax of the species is white and patternless, with minute black dots on the lateral surface, rounded minute segment on the abdomen, and laterally minute pair of black dots on every segment.Further, elongated forewings, apically acute, white to coffee-coloured, dark bright dots on wings margins, with minute rows of black dots on veins and patternless hindwing with indistinct dark spots on the outer margin characterize the species morphology.The uncus of the male genitalia was long, with middling thickness, with a minute acute apex of the beak in shape.Separately, thick gnathos, a leaf-like smooth valve at margins of middling thickness, and juxta with a welldeveloped lateral process.The saccus was small and semi-circular and aedeagus short, thick, and slightly curved in its proximal third with no cornutes.Nevertheless, the genitalia of the female were not studied.The pupae and adults have been recorded by Ong et al. (2010) concerning Rhizophora apiculata plant.Furthermore, Senthilkumar and Murugesan (2015) reported the male forewings to have black spots being strongest on the vein and opaque white zone, free of black spots, at the end of the cell and in female presence of typical transverse black striae and again black spots free zone at the end of the cell.Adult females are, however, found to be larger than males, possessing long ovipositor at the end of the abdomen, enabling them to position their egg in the bark crevices (Ong et al., 2010) (Figure 2b).However, the latest studies carried out by Borthakur et al. (2021) on the Z. conferta biology, revealed the adult to be of medium in size with a wing expanse of 27 to 35 mm.The arrangement of the forewings was found to be flickering bluish to black in colour with asymmetrical striations or white pellucid background.The hindwings were also reported to be spotty and translucent.The abdomen was brownish in colour and beard black dots which was covered with fur (Figure 2c).
Overall, the genus Zeuzera has been actively studied by Roepke (1955;1957), based on New Guinea and Malayan fauna, and Holloway (1986), based on Bornean fauna.The latter suggested the genus to be similar and well-defined sections of Xyleutes, a genus of moths that belongs to the Cossidae family.Schoorl (1990) also conducted a detailed study on the morphological aspects of the genus Zeuzera.The genus was interpreted based on the presence of crossvein Sc-Rs, humeral plate triangular in shape and anal plate comparatively long to short.Based on his hand cladogram, various characteristics believed to be apomorphies of genus Zeuzera and its relationship within its genus were also presented.However, it was felt that his study needed further assessment due to the growing evolution of understanding in the field of study to testify its validity (Sutrisno, 2015).
The species of the Zeuzera mostly live as a larva in plants (Sutrisno, 2015).Eggs are laid by the female moth chiefly in groups directly in cracks, crevices of the stem and larger branches of the host plant (Moaty et al., 2019).A total of 180-250 eggs are laid by the single female Z. conferta in one batch with the size ranging from 0.2 mm in length to 0.1 mm in breath (Borthakur et al., 2021).The caterpillars emerge out from the egg after its development and are called first instars till it molts.It enters the second instars after the molt and increases in size.Every stage of molting distinguishes another instar.Typically, a caterpillar passes through a total of five instars as it eats and grows, wherein each instar its general appearance changes from one to the next.According to Borthakur et al. (2021), the size of the larvae increased from 0.03 cm to an average of 4.5±0.7 in length and from 0.02 cm to 0.06 cm in breath from first instars to fifth instars.The change was also observed in the colour pattern of the larvae form light reddish pink to light pinkish from first instars to fifth instars.Before entering the stage of the pupation, the matured larvae prepare the pupal tunnel and also the exit hole near the bark surface.The pupa measured 1.9 to 2.5 cm in length and 0.05 cm in breath and weighted 0.46 gm and completed its pupal period within 14-30 days and emerged out as an adult moth.In conclusion, they reported that Z. conferta have two generations in a year that was also earlier reported by Baksha and Islam (1999), with regards to Sonneratia apetala trees in Bangladesh.

Diversity of Hosts
Besides Aquilaria trees, the Z. conferta has a broad range of host of different families such as Sonneratia apetala, S. alba, S. ovate of family Lythraceae, Aegiceras corniculatum of Myrsinaceae, Avicennia lanata, A. marina, A. officinalis of Avicenniaceae, Ochroma lagopus of Bombacaceae, Eucalyptus deglupta of Myrtaceae, Rhizophora apiculata, R. mucronata of Rhizophoracaea, Theobroma cacao of Sterculiaceae, Coffea of Rubiaceae, Erythroxylum L. of Erythroxylaceae, Elettaria cardamomum of Zingiberaceae and Tamarix indica of Tamaricaceae family (Islam, 2004;Yakovlev, 2011).The preferential habitat of the majority of these host trees are coastal mangrove forest, moist, lowland with few from the tropical forest (Ong et al., 2010;Senthilkumar and Murugesan, 2015).The larvae target the stems, trunks, twigs, and shoots of the host plant for the infestation.The association of Z. conferta with S. apetala is extensively studied in Bangladesh where the larva of Z. conferta is also termed as "bee hole borer".The tree S. apetala is largely utilized as a plantation species to construct a shelter belt along with the coastal areas and offshore islands of Bangladesh.The larva is reported to bore in the barks and later make large, profuse, oval, and ramifying tunnels in the stem rendering the tree to wind breakage.Later, the larvae and pupae of the Z. conferta are found to be eaten by woodpeckers such as Dinopium benghalense and Picoides canicapillus and small black ants (Islam, 2004).In Avicennia spp., the infestation by the larvae Z. conferta is reported to occur at an interval of 7 years in natural mixed forest of Brazil (Vannucci, 2002).Few other host plants of the Z. conferta includes Cocoa (Theobroma cacao), Balsa, Coca (Erythroxylum P. Br), and Barringtonia (Arora, 1976;Schoorl, 1990).It is however interesting to note that with regards to these hosts of Z. conferta, only in Aquilaria trees it is reported to have a productive role i.e., in the formation of resinous wood called as agarwood.

Interactions: Insect-plant-microorganism
Plant and its biotic interactions in natural environment have diverse manifestations.In nature, plant interacts with the insect by attracting pollinators for sexual reproduction on one side and, on the other, protecting itself from herbivores, pathogens and even other plants by synthesizing various chemical compounds (Schiestl, 2010).Plants, being sedentary organisms use volatile compounds as a vernacular to communicate and interact with the surrounding environment (Dudareva et al., 2006).Volatile organic compounds (VOCs) are released by the plants constitutively that herbivores utilize for host location (Penaflor and Bento, 2013).
The chemical signals are perceived by the herbivores with the aid of olfactory system and commence the behaviours for communication with the host (Field et al., 2000;Fatouros et al., 2008;Leal, 2013).The attraction of the insect towards the host plant is due to the volatile phytochemicals, which are perceived by specialized chemoreceptor neurons on the antenna (Loon, 1996).Herbivores are recognized by the plants through damage-associated molecular patterns (DAMPs) and herbivore-associated molecular patterns (HAMPs), also called as elicitors that include extracellular protein fragments, nucleotides, peptides, glucose oxidase, fatty acid-amino acid conjugates (FACs), β-glucosidase, inceptins, and caeliferins (Giron et al., 2018;Hogenhout and Bos, 2011).The herbivore attack on the host plant releases a large diversity and a greater amount of VOCs, called as Herbivore Induced Plant Volatiles (HIPV) (Paré and Tumlinson, 1999;Howe and Jander, 2008;Penaflor and Bento, 2013).HIPVs are important olfactory cues produced by the plants under herbivores attack, in a manner that they reveal indirect information about the presence of the herbivores (Aartsma et al., 2019).Parasitoids, specifically carnivorous insects use HIPVs in locating and regulating the herbivores as their natural enemies (Forbes et al., 2018;Kessler and Heil, 2011) resulting in tritrophic interaction.Terpenoids, aromatics, green leaf volatiles (GLVs-C6 aldehydes, alcohols, and their esters), and amino acid volatile derivatives are some of the volatiles emitted by herbivore-damaged plants (Dudareva et al., 2006).The recognition of the DAMP and HAMP by the plants activates the diverse defense mechanism in the plants aiming to reduce the damage caused by the herbivorous insect (Giron et al., 2018).
Plants respond to the herbivores attack by two mechanisms known as Direct and Indirect defense.Direct defense includes all plant traits enhancing the resistivity of the plant and, thereby, changing the insect's behaviour or physiology.Indirect defense, on the other hand, includes all the plant traits but does not have a direct effect on attacking herbivores but it can attract the natural enemies of the herbivores that can be any carnivorous insects (Aljibory and Chen, 2018).The metabolites of lipoxygenase (LOX) pathway, the shikimic acid pathway, and product of the terpenoid pathway are the prevalent volatile signals involved in direct and indirect defense (Pichersky and Gershenzon, 2002).However, some herbivores have developed resistivity to such a response and alter plant metabolism by injecting effectors into the host plant and repress the plant defense system (Hogenhout and Bos, 2011;Kaloshian and Walling, 2016;Giron et al., 2018).Secondary metabolites of the plant also act as the defense system towards the insect herbivory and also several classes of secondary products are produced through infection, wounding, or herbivory.Insect, however, becomes immune or develops an adaptation mechanism to such a defense mechanism of plants due to feeding on or infecting a particular plant (Bennett and Wallsgrove, 1994).Throughout the phase of feeding or during egg deposition, the herbivores alter the phenotype of the plants through changes in the production of central and specialized metabolites, morphological traits, and architecture (Dicke and Baldwin, 2010;Hilker and Meiners, 2010;Howe and Jander, 2008;Mithofer and Boland, 2012).
The oviposition of the insect has also been found as a threat to the plants.Similar to herbivory induced volatiles, the oviposition by the herbivores also activates the release of oviposition induced volatiles (Hilker et al., 2002;Fatouros et al., 2005b;Salerno et al., 2013).The female wound the trees prior to the egg deposition and the elicitors which is procured from the secretion attaching eggs to plants, forge contact with the inner plant tissues through this wound inflicted (Hilker et al., 2005).The female has the aptitude to acknowledge the finest plant or host quality for the fine growth of larvae.The mechanism of specific site preference for oviposition by the female is also a master plan to procure defense against predation on premature stages of development.The oviposition is a critical step, peculiarly in Lepidoptera, owing to the relative immobility of the hatching larvae and thus depending on the judicious choice of food plant by the adult female (Fenny et al., 1983;Renwick, 1989).Various events leading to oviposition follows a sequence of searching, orientation, encounter, landing, surface evaluation, and acceptance (Renwick and Chew, 1994).All these stages of the sequence depend on the sensory cues of the insect; however, definitive experiments of the sequential mechanism are difficult to perform (Morris and Kareiva, 1991).After the insect gets descend on a plant, it determines its site suitability for oviposition through the physical and chemical contact perception on the various surface of plants.Tarsi, antennae, proboscis, and ovipositor of lepidopterans are the sensory receptors involved.The Central Nervous System (CNS) acts as the final processing of information provided by the various sensory inputs received by the insect acceptance or rejection of a site for oviposition (Renwick and Chew, 1994).
The microorganisms also interact with the insects through the production of microbial volatile organic compounds (MVOC).The MVOCs have been found to closely associate with the behaviour of the insects (Davis et al., 2013).Insects are sensitive to odours and highly responsive to microbial volatile emissions (Ezenwa et al., 2012;Price et al., 2011).MVOCs have ecological functions such as some MVOCs that can attract or repel insects, stimulate oviposition, inhibit the growth of microorganisms competing the associate insects, mimic plant hormones or induce defense resistance (Davis et al., 2011;Ryu et al., 2003Ryu et al., , 2004)).The microbial associates are also responsible for the important physiological functions of the insects (Haine et al., 2008;Rozen et al., 2008) and the interaction of the insect-microbes might even play a significant role in quorum sensing (Lowry et al., 2008;Ma et al., 2012;Tomberlin et al., 2012a).Symbiotic microorganisms associated with the insect also play a role in finding a suitable host and food resources for the insect (Davis et al., 2013).The female moth perceives the volatile compounds through their olfactory system from the surroundings.(fg) Using volatile compounds the moth seeks for the host location and undergoes different series of events preceding the oviposition, checking the suitability for oviposition at the favourable site in the stem.On finding the suitable site the female moth lay eggs and completes its life cycle.The hatched larvae then commence the activity of the infestation by forming tunnels in the stem.(h) The tree releases HIPVs (Herbivore Induced Plant Volatiles) after the infestation to attract the natural enemies (e.g., carnivorous insects) of the larvae and establish a mutualistic relationship with the tree.(i-j) Microbial infection occurs along the tunnels created by the larvae, where the accumulation of resin occurs.The gut microbes of the Z. conferta also act as a source of microbial infection.(k) The accumulated resins across the tunnels are later called as agarwood.(l) The larvae emerge out as an adult moth from the exit hole after completing its life cycle.
Till date there is no scientific evidence and no experimental studies have been executed to explain about the mechanism of interaction between the Aquilaria tree and Z. conferta.Moreover, meagre studies have been accomplished on the biology of Z. conferta.Based on the analogous strategy of insect-plant interaction, therefore, the insect Z. conferta, and Aquilaria tree might also trail a similar pattern of interaction (Figure 3a-l).The Aquilaria tree (Labelled a) might produce aroma related volatiles such as Volatile Organic Compounds (VOCs) to attract the herbivore, Z. conferta (c) as the plant volatiles are the significant molecules in the insect-host recognition (Qiao et al., 2012).The adult moth, Z. conferta then possibly detect the signals of the various volatiles released by the Aquilaria trees through their olfactory system and receives through the chemoreceptors (e).As VOCs possess the potential to trigger the behaviour of the insects, it might stir up the adult Z. conferta to look for the host plant for the oviposition.On finding the host plant, the adult female moth then undertakes various sequence of events preceding the oviposition in order to confirm the suitability of the oviposition at a favourable site in the host plant (f).On obtaining the suitable site the female moth lay eggs and completes its life cycle (g).The larvae which emerged from the eggs then initiates the phase of infestation in the Aquilaria trees by chewing the wood.Throughout the period of infestation, the larvae might inject various elicitors into the woody stem of the host plant.The host plant then activates its diverse defense mechanism to combat the herbivory by synthesizing disparate secondary metabolites and also releasing various HIPVs.The HIPVs are released by the Aquilaria trees to attract the natural enemies of the Z. conferta and accordingly maintain a mutualistic relationship with the trees (h).Hitherto, however there has been no literature available with reference to the natural enemies of the Z. conferta.The injection of the elicitors represses the defense mechanism of the Aquilaria trees and the larvae of Z. conferta might survive the phytochemicals released by the Aquilaria trees.The microorganisms present in the tree might also emit the Microbial Volatile Organic Compounds (MOVCs) triggering the behaviour of the insect Z. conferta (d).Fungi of the genera Fusarium and Lasiodiplodia have been reported to produce two compounds, δ-lactones and mullein that releases an odour having the potential to act as an insect attractant (Nago and Matsumoto, 1994).The interesting finding is that both these fungi are associated with the Aquilaria trees (Mohamad et al. 2010;Chippa and Kaushik, 2017).Therefore, such microorganisms might also be releasing the volatile compounds and alluring the insect Z. conferta towards the Aquilaria trees.In a chemometric evaluation of interaction study carried out by Sen et al. (2017), between the agarwood and fungus Fusarium, revealed the appearance of ecologically important semio-chemicals (e.g., Pheromones).Semio-chemicals are the organic compounds that have the potential to stimulate the activity of organisms used by insects and other animals for the purpose of biological communications.The role of semio-chemicals, however, in the fungal colonization in Aquilaria trees through the insect Z. conferta intervention needs further investigation.Consequently, after the infestation of the larvae in the Aquilaria tree, spiral, oval, or ring-shaped injury or wound (Figure 4a) is developed serving as the gateway for initiation of microbial infection (Kalita et al., 2015) and the tree response to it by the formation of resinous wood called agarwood along the zone of infestation as a mechanism of defense reaction (Figure 4c) (j-k).
The size of the tunnel increases as the larvae grow and also the metamorphosis from larva to pupa takes place inside the tree.The pupa partially moves out from the exit hole before finally attaining its maturity as a moth (Ong et al., 2010).The larvae then complete its life cycle and emerges out as adult moths through the exit hole leaving the exuvia intact (l).However, the release of the HIPVs by the Aquilaria trees to attract the natural enemies of the Z. conferta and to prey on it might debarred it from completing its life cycle.
Research carried out on the antennal and behaviour response of the Heortia vitessoides, one of the major leaf defoliator pest of the Aquilaria tree also showed that the female moth was attracted to the volatiles of the green leaves possessing the compounds such as nonanal, decanal, hexanal, and (Z)-3-hexenylacetate rather than the dry leaves, forming the vital constituent for the minimal attraction of the insect (Qiao et al., 2012;Syazwan et al., 2019).Finally, as the Aquilaria tree matures, it closes its exterior entry and exit hole leaving a distinct lesion (Figure 4b).
The microorganisms present in the gut of the Z. conferta might also have an indispensable role in infecting the Aquilaria trees as they are assigned with various significant roles in their host metabolism, physiology, growth, reproduction (Breznak, 1982;Chen and Purcell, 1997;Lemke et al., 2003).The larvae might act as vectors and release potential microbes to induce the infection through the excretion process during the tunnelling pursuit.Study on the diversity of the microorganisms associated with the gut of the larvae and comparative analysis with the microbes associated with Aquilaria trees and in resinous agarwood might help us understand and correlate the mechanism of insect-plant and microorganism's interactions.Analysis on the potential role of the endophytic microbes associated with the Aquilaria trees in producing odorous compounds will help us in a deeper understanding of its capability of alluring the Z. conferta towards Aquilaria trees.Furthermore, the study of the difference between the insect-infested and non-insect infested Aquilaria trees will also contribute to a broader way of understanding of the mechanism of insect infestation, as Z. conferta is not found to invade all the Aquilaria trees in the same environment.

Present and Future Perspectives
Zeuzera confertaa pest?
The association of the Z. conferta with the Aquilaria trees can be referred to as "necessary evil".The infestation by the borer in the young Aquilaria plants causes a major threat to its survival as the activity of tunnelling by the borer damages the tissues of the plants.However, its desideratum cannot be ruled out, as agarwood of good grades is obtained only when the trees are infested by it (Hoque et al., 2019).The damage caused by the Z. conferta in Aquilaria trees is still considered as moderate level apart from the other 19 pests that are found to associate with the trees (Syazwan et al., 2019).The people familiar with the Aquilaria habitats and plantations are very much cognizant of the necessity of its association with the Aquilaria trees.
Even people in the locality who are not directly associated with agarwood farming are also familiar with its essentiality.Yet, the scientific knowledge of different mechanisms of formation of agarwood is unknown to them.
Despite having a prominent role in assisting fine agarwood formation, it remains as a major drawback for most of the Aquilaria cultivators owing to the damage it causes by infesting in the young Aquilaria plants leading to the stunted growth and sometimes death of the plants.The term pest is reckoned for an insect or a microbe in the agricultural practices if it interrupts the progression or the development of the plant (Syazwan et al., 2019).The commencement of the infestation of the pest Z. conferta is observed in the plants attaining 5 years, and maximum in the trees of age group 8-16 years and moderately above 16 years (Kalita et al., 2015).The cultivators have, therefore, taken quite a few measures to protect the Aquilaria plants from infestation by the Z. conferta.Some of the measures are (i) spraying of the insecticides on the surface of the tree, (ii) spraying of the insecticides directly on the holes made by the borer on trees, (iii) killing of the insect directly when found, and (iv) killing the female moth before oviposition.The measures taken are performed seasonally based on the availability of the insects.The event of killing is intermittent as it is performed only when the insects are found.However, the spraying of the insecticides is performed when the rate of an infestation is found to be higher based on the observation.Few other management practices that have hitherto been executed are trimming and removal of the infected branches, shutting off the hole made by the insect with plasticine (liquid-based pesticides), and application of the granule-systemic based pesticides (Syazwan et al., 2019).Pheromones, mass trapping, mating disruption, entomopathogenic fungi, and nematodes are other control measures that have been successful in controlling the lepidopteran pest (Ibrahim et al., 2019;Ong et al., 2010).The practise of spraying insecticides is carried out when the trees are young and are heavily infested by Z. conferta.However, when the trees attain its maturity, the incidence of the Z. conferta is not much of a concern and the practice of spraying insecticides lessens as the tree becomes less vulnerable to breakage and damage.The insecticides used and their effectiveness in controlling the incidence of Z. conferta is needed to be analysed in detail.However, the application of the pesticides in controlling the incidence of the pest has become increasingly knotty due to concern about human health hazards, environment and pest resistance (Atreya et al., 2012).Furthermore, the obscure habitat of the larvae inside the trees and prolonged ovipositional period makes the chemical treatment less successful (Shamseldean et al., 2009).

Frass
The frass of the Aquilaria trees acts as an indicator of the incidence of the Z. conferta (Figure 5).Frasses are the excrement or the excreted pellets which are released by the larvae after feeding on their food source.
In wood borers the frass from the tunnelling activity is expelled out from the entry and exit hole on the ground (Ong et al., 2010).The expelled frass helps the seeker, seeking the activity of Z. conferta in the Aquilaria trees for confirmation about the infestation just by scrutinizing the presence or absence of the frass on the ground surrounding the Aquilaria trees.The frass varies in shape and colour with some spherical or oval in shape and white in colour and some wet sticky powdered with brownish appearance.In assessing between the old and the new infestations by the Z. conferta, frass plays a prominent role through its morphological appearance.Dry frass and dimmed coloured (Figure 5a) indicate old infestation, whereas wet and bright coloured indicates the new ones (Figure 5b).According to the cultivators, the trees which expels brownish coloured frass after the insect infestation produces a better grade of agarwood (Figure 5c).Ants and spiders are later reported to occupy the abandoned stem to take refuge (Ong et al., 2010).
According to Reynold and Hunter (2004), frass also provides important source of nutrients in the soil system increasing the diversity of the soil invertebrates.The deposition of the frass in the forest floor increased the nitrogen content excessively.Collembola, fungal-feeding nematodes, bacterial-feeding nematodes, and prostigmatid mites are some of the soil invertebrates that have significantly increased in the Southern Appalachians due to the depositions of the frass in the forest floor (Reynolds et al., 2003).Careful investigations on frass can lead to future diagnostic mechanisms on staging insect infestations leading to agarwood resin formation.Biochemical, microbiological and image analysis of frass appear to be suitable initial analytical candidates in this regard.

Artificial Rearing
The incidence of Z. conferta is not observed in all the Aquilaria trees that are cultivated.The incidence or the prevalence of the pest attack in the Aquilaria trees is mostly observed in monocultures (Ong et al., 2014).The practise of growing as monocultures by the farmers into small or a large-scale came into existence due to the overexploitation outside of their natural habitat (Irianto et al., 2011).However, there are some regions where the incidence of the insect is not observed despite being grown as monocultures or mix cultures.Owing to those innumerable artificial techniques are applied for production of agarwood, as naturally the formation of agarwood very much relies on the insect and microorganism's interactions (Syazwan et al., 2019).Despite its success in production of agarwood through artificial means, quality has always been a matter of concern for the cultivators as the superior grades are only harvested from the insect infected ones (Kalita et al., 2015;Hoque et al., 2019).The technique of artificially rearing the insect might be a key alternative to overcome this hurdle.The artificially reared Z. conferta larvae can be introduced in the Aquilaria trees where the incidence is not generally observed.Consequently, the larvae might select suitable site in the tree introduced and initiate its tunnelling activity and execute its role in insect-plant and microorganism's interaction leading to the agarwood formation.The latest study carried out by Borthakur et al. (2021) on the life cycle of the Z. conferta bestows hope of artificially rearing the insect and making it available in close proximity.This technique can be an alternative source of livelihood and also helps in catalysing the production of quality agarwood, generating more advantage to the cultivators.Moore and Navon (1966) were the first researchers to develop artificial medium for the wood borers, specifically for the leopard moth, Zeuzera pyrina L. The artificial media of their preparation comprised of a basal medium of three variants, composed of full fat soya meal (30.0 g), sucrose (48.0 g), Brewer's yeast (24.0 g), agar-agar flakes (24.0 g), nipagin (1.5 g), acetic acid 20% v/v (30.0 ml); sodium ascorbate 10% w/v (30.0 ml), pear bark homogenate (10 g/ 70 ml H2O), and distilled water.The media was successful in raising the successive generation of the leopard moth considerably within a short duration of 3-4 months than in nature where it takes a year.However, the artificially bred larvae were found glabrous, and they differed in colour as compared to those developed in woods.Moreover, apart from the laboratory conditions the rearing can also be tried in its natural state by providing the cambium portion of the Aquilaria stem as the larvae depends on it as food source (Borthakur et al., 2021).Therefore, the introduction of the larval stage of the Z. conferta at the right age of the Aquilaria plant might be a solution to the problem of lack of insect incidence and also might be less susceptible to breakage and death as matured trees are less vulnerable to pest (Ong et al., 2014).

Conclusion
Agarwood resin formation which is a unique phenomenon is still not clearly understood by science.The study of the insect should secure equal eminence with other areas of research associated with Aquilaria trees.The superior quality of agarwood garnered after the insect infestation makes the need to study the insect Z. conferta highly significant.Since the accumulation of the oleoresin is observed along the tunnels generated by the insect, there is also a possibility that the microbes existing in the gut of the larvae might as well initiate the microbial infection through the excrement of the larvae.The possibility of the involvement of chemical signalling in the ecology of the insect and its interaction with the plants and microorganisms further strengthens the argument that Z. conferta plays a pivotal role in the famous agarwood aroma development.Future studies are likely to unravel the intricacies of this involvement.The phrase "necessary evil" parallels in describing the Z. conferta, as the infestation is a critical necessity to fine agarwood formation but also remains a major threat for the plantation when infestation occurs at early age.The selectiveness in infesting a particular Aquilaria tree, environmental conditions, and soil quality of the regions where the insect infestation is observed could be interesting themes for future research.Possibly the insights from the future research on Z. conferta and the plant-insect-microbe continuum can help understand the aroma of agarwood better.

Figure 1 :
Figure 1: Larva of Zeuzera conferta Walker: The process of extracting larvae from the Aquilaria trees firstly involves selecting the trees with the help of frass.The texture of the frass is checked to differentiate between the old and the new infestations.Dimmed coloured and dry frass indicates the old infestations whereas brightly coloured and wet with moisture intact indicates the new ones.The newly infected trees are selected and cut off and are brought into the agarwood processing centres.The stems are first cut into sections horizontally with the help of the chainsaw, few centimeters below and above the infestation point.The later are split into many symmetrical parts from top to the bottom edge vertically, thoroughly until the larvae comes out.[Source: Field trip, 2021]

Figure 3 :
Figure 3: Interactions of Zeuzera conferta Walker linked in agarwood formation: (a) Aquilaria tree (b) Insect mediated agarwood formation in the Aquilaria tree through series of interactions between insect (Z.conferta)plant (Aquilaria) and microorganisms.(c) The Aquilaria tree releases VOCs (Volatile Organic Compounds) (d) Microorganisms from the host tree also releases MOVCs (Microbial Organic Volatiles Compounds).(e)The female moth perceives the volatile compounds through their olfactory system from the surroundings.(fg) Using volatile compounds the moth seeks for the host location and undergoes different series of events preceding the oviposition, checking the suitability for oviposition at the favourable site in the stem.On finding the suitable site the female moth lay eggs and completes its life cycle.The hatched larvae then commence the activity of the infestation by forming tunnels in the stem.(h) The tree releases HIPVs (Herbivore Induced Plant Volatiles) after the infestation to attract the natural enemies (e.g., carnivorous insects) of the larvae and establish a mutualistic relationship with the tree.(i-j) Microbial infection occurs along the tunnels created by the larvae, where the accumulation of resin occurs.The gut microbes of the Z. conferta also act as a source of microbial infection.(k) The accumulated resins across the tunnels are later called as agarwood.(l) The larvae emerge out as an adult moth from the exit hole after completing its life cycle.

Figure 4 :
Figure 4: Resin formation at the site of infestation: (a) Spherical wound created by Zeuzera conferta infestation.(b) Closure of the wound as the tree attains maturity.(c) Formation of agarwood along the tunnels created by Zeuzera conferta inside the stem of the Aquilaria tree.[Source: Field trip, 2021]