Mulberry & silkworm: The chemistry behind a remarkable plant–insect relationship

Introduction

The mulberry silkworm, Bombyx mori, has a remarkable relationship with its food plant, mulberry (Morus spp.). Unlike Eri, Muga and Tasar silkworms that can feed on a wide range of host plants, the mulberry silkworm shows a strong preference for mulberry leaves. This specialized feeding behaviour is one of the most fascinating examples of plant-insect interaction (Kang et al., 2019; Lu et al., 2021) [1,2] . Two step verification process is there before host plant selection, In the first screening step of detection of edible leaves, B. mori larvae demonstrate chemotaxis behaviour by using olfaction (Iizuka et al., 2024) [3] . Once they reach their target leaves, a biting action is initiated, and swallowing factor such as cellulose, phosphate, and silica in leaves aids the larvae in the continuous consumption of food.

Mulberry: More than just a food plant

For a silkworm, a mulberry leaf is much more than a source of nutrients. It is a complex chemical package containing carbohydrates, proteins, amino acids, minerals, vitamins, sterols and secondary metabolites, which collectively influence feeding, growth and development. Some of these compounds act as feeding stimulants, while others may serve as chemical cues for host recognition. The silkworm therefore evaluates the leaf through a sophisticated chemosensory system rather than simply responding to its appearance. In B. mori, compounds such as chlorogenic acid, isoquercetin and β-sitosterol present in mulberry are involved in the recognition and stimulation of feeding (Lu et al., 2021; Sato, 2024) [2,4] . The larva possesses specialized chemosensory structures, particularly the maxillary palps and other mouthpart and body sensilla, containing gustatory receptors that help distinguish suitable food from unsuitable plants. Recent studies describe this as a two-step authentication process: first, the maxillary palps contact the leaf surface and detect trace amounts of mulberry-specific compounds such as chlorogenic acid, isoquercetin and β-sitosterol; this is followed by a test bite, during which sugars and other compounds released from the leaf tissue provide additional information about its suitability. Receptors such as BmGr 6 , BmGr 9 and BmGr 63 have been shown to participate in the recognition of feeding-promoting compounds, demonstrating how chemical signals from mulberry are translated into the behavioural response of biting and sustained feeding (Kang et al., 2019; Yang et al., 2022; Sato, 2024) [1,4] .

The invisible signals released by mulberry

Plants continuously release a variety of volatile organic compounds (VOCs) into their surroundings. These airborne chemical signals play an important role in plant-insect communication and can influence insect behaviour. For Bombyx mori, such signals provide information about the presence and possible location of a suitable food plant. The larva detects these volatile cues through its olfactory system and moves towards the source, a directed response commonly described as chemotaxis. This initial olfactory recognition represents the first stage in the silkworm's interaction with its host plant and helps it distinguish potential food sources before physical contact with the leaf (Tanaka et al., 2009; Lu et al., 2021) [5,2] . Once the larva reaches the mulberry leaf, the feeding decision involves a second level of chemical evaluation. Non-volatile compounds present on the leaf surface and within the leaf tissue are detected through specialized gustatory structures associated with the mouthparts. These chemical cues help the larva determine whether the leaf is suitable for feeding and can stimulate biting and continued ingestion. In addition to chemical cues, the physical characteristics of the leaf and its nutritional composition influence feeding and subsequent utilization of the food. Thus, the interaction between B. mori and mulberry involves a coordinated sequence of olfactory attraction, gustatory recognition, biting, ingestion and nutritional utilization, demonstrating the remarkable precision with which the silkworm identifies and consumes its preferred food plant (Kang et al., 2019; Lu et al., 2021) [1,2] . . From a scientific perspective, these compounds should not be considered as independent or exclusive determinants of mulberry attraction. Under natural conditions, the silkworm encounters a mixture of volatile compounds, and its response depends on the composition and concentration of this chemical blend. Subsequent studies have demonstrated that B. mori possesses highly specialized olfactory receptors capable of detecting particular mulberry volatiles, providing a molecular basis for chemotactic behaviour (Tanaka et al., 2009; Tanaka et al., 2023) [5] . Thus, the combined action of mulberry volatiles can be viewed as an initial chemical "address" that helps the silkworm locate its preferred host plant. Once the larva reaches the leaf, the feeding decision shifts from airborne olfactory cues to contact-based gustatory and nutritional cues that regulate biting and continued consumption (Lu et al., 2021) [2] .

Three phases of silkworm feeding 1. Attracting factors -the pre-bite phase

Biting factors -the feeding initiation phase

β-Sitosterol: A major plant sterol that contributes significantly to the stimulation of biting and feeding behaviour in silkworm larvae.

Flavonoids: Isoquercitrin and Morin

Once the silkworm larva reaches the mulberry leaf, non-volatile chemical constituents present on the leaf surface and within the leaf tissue provide the next level of information for host recognition. Early feeding studies identified β-sitosterol and flavonoids such as isoquercitrin as important biting stimulants, while morin was also reported to possess biting-stimulating activity (Nayar et al., 1962; Hamamura et al., 1962) [8,7] . β-Sitosterol isolated from mulberry was subsequently shown experimentally to stimulate feeding in B. mori, although the study also demonstrated that sterols have both nutritional and phagostimulatory roles (Ito et al., 1964) [9] . These findings established that the larva does not begin sustained feeding simply because it has reached a green leaf; rather, specific chemical constituents are detected by its sensory system and contribute to the initiation of biting. More recent research has provided a molecular explanation for these observations. Ueno et al. (2020) demonstrated that the maxillary palps of B. mori function as highly sensitive chemosensory organs during the first stage of host acceptance. Neurons in these palps can detect extremely small quantities of chlorogenic acid, quercetin glycosides and β-sitosterol on the leaf surface; detection of this combination promotes a test bite. After the test bite, chemicals released from the leaf tissue, particularly sugars, are evaluated by sensory neurons associated with the mouthparts, allowing the larva to decide whether to proceed to persistent feeding. Thus, the earlier concept of "biting stimulants" has now been refined into a two-stage host-acceptance mechanism, in which surface chemical cues trigger test biting and tissue-derived nutritional cues help sustain continuous feeding (Ueno et al., 2020; Sato, 2024) [4,10] . [11] demonstrated that sucrose applied to the mouthparts of Bombyx mori larvae directly triggered swallowing movements, and higher sucrose concentrations increased the rate of ingestion and strengthened the rhythmic contractions of the cibarial pump. Earlier feeding experiments also showed that sucrose was one of the strongest feeding stimulants among the sugars and related compounds tested (Ito, 1960). These findings indicate that soluble nutrients do not merely provide nutritional value after ingestion; they can also act as sensory signals that promote the transition from biting to ingestion. Myo-inositol provides another important example of a nutrient-associated feeding signal. Electrophysiological studies identified specific sensory neurons and the gustatory receptor BmGr 10 that respond to myo-inositol, demonstrating that the silkworm possesses a dedicated sensory mechanism for detecting this compound (Koh et al., 2016) [12] . Furthermore, Sasaki et al. (2013) [13] showed that a combination of myo-inositol and sucrose enhanced feeding activity and modified both chewing and swallowing motor patterns, suggesting that these compounds can influence the neural circuits controlling ingestion. A recent review also confirms that myo-inositol functions as a feedinginducing factor in silkworm larvae (Sato, 2024) [4] . The roles of cellulose, silica and phosphate/mineral components should be described more cautiously. Earlier nutritional studies reported that cellulose could enhance feeding when incorporated into artificial diets, suggesting that physical or nutritional properties of the diet can influence feeding behaviour (Ito, 1960). However, current molecular studies of B. mori feeding focus much more strongly on soluble sugars, myo-inositol and other gustatory stimuli and on the neural mechanisms controlling chewing and swallowing. Therefore, rather than stating that cellulose, silica and potassium phosphate are established swallowing stimulants, it is scientifically safer to describe them as dietary or physical factors that may contribute to the suitability and continued ingestion of plant material, while giving stronger emphasis to sucrose and myo-inositol as experimentally demonstrated feeding/swallowing-related signals

Swallowing factors -the continuous consumption phase

Potent attractant

The chemical invitation: cis-jasmone

Volatiles emitted by green leaves trigger chemotaxis behaviour in the insects, in silkworm Bombyx mori chemotaxis behaviour is majorly driven by potent attractant named as cis-jasmone. Cis-jasmone is a derivative of the plant hormone jasmonic acid and is synthesized as a metabolite from linolenic acid via the octadecanoid pathway. Researchers analysed volatile compounds emitted from mulberry leaves and tested their ability to attract silkworm larvae. Among the compounds examined, cis-jasmone showed particularly strong attraction. Remarkably, behavioural experiments demonstrated a response to extremely small quantities: the threshold amount required to elicit attraction from a distance of 20 cm was approximately 0.3 pg for cisjasmone, 3 pg for trans-jasmone, 30 pg for dihydrojasmone and 300 pg for 2-amylcyclopentenone. Thus, cis-jasmone showed the highest potency among the compounds tested, with attraction occurring at an extremely small quantity. These results demonstrate the exceptional sensitivity of the silkworm olfactory system and highlight the importance of volatile chemical cues in host-plant localization before physical contact with the leaf (Tanaka et al., 2009) [5] .

The receptor: BmOr-56

The silkworm possesses a specialized odorant receptor called BmOr-56, which is remarkably sensitive to cis-jasmone, allowing the insect to detect even tiny amounts of this volatile chemical released mulberry leaves. Studies examining olfactory receptor genes expressed in larval olfactory organs identified BmOr-56 as highly responsive to cis-jasmone (Tanaka et al., 2009; Morinaga et al., 2023) [5,14] . Functional experiments showed that the receptor has a strong sensitivity to this compound, and the receptor response corresponded closely with the behavioural attraction observed in silkworm larvae. Thus, BmOr-56 can be viewed as an important molecular component of the silkworm's cis-jasmone detection system. Scientists generated silkworms in which BmOr-56 was deleted using gene-editing technology. These mutant silkworms lost the attractive behavioural response to cis-jasmone, providing direct genetic evidence that BmOr-56 plays a critical role in this specific chemoattraction. Among a total of 66 Ors identified in the B. mori genome, we found that 23 were expressed in the olfactory organs during larval stages.

Potent gustatory stimulant

Finding a plant is only the first step. The silkworm must also decide whether the leaf is suitable for feeding. This is where gustatory receptors become important. Chemical compounds present in mulberry leaves interact with sensory receptors associated with feeding behaviour. Some compounds act as phagostimulants, encouraging the larva to bite and continue feeding. Major plant sterols present in mulberry is β-sitosterol which act as a Gustatory stimulant. The feeding response is determined by a combination of compounds rather than by one chemical alone (Nayar & Fraenkel, 1962; Ueno et al., 2020; Sato, 2024) [8,4,10] . Instead, it is the result of a complex chemical profile containing several compounds that together make mulberry highly acceptable to B. mori. Mulberry leaves provide proteins and amino acids needed for tissue development and enzyme synthesis, carbohydrates that provide energy, lipids and sterols required for physiological processes, and minerals and other micronutrients required for normal development.

The genetic switch: GR66

Perhaps the most striking evidence for the genetic control of mulberry preference came from studies of the gustatory receptor gene GR66. In 2019, Zhang et al., used CRISPR/Cas9-mediated gene editing to dis-rupt GR 66 in Bombyx mori. GR 66 encodes a putative bitter gustatory receptor. The result was remarkable. Silkworms strongly preferred mulberry and generally rejected several other plant materials. However, silkworms carrying mutations in GR 66 lost much of their normal mulberry specificity and began feeding on a surprisingly broad range of materials, including apple, pear, soybean, corn and other plants. This provided powerful experimental evidence that a specific gustatory receptor can have a major influence on host-plant preference. The chemosensory neurons in the maxillary palp (MP) of the larvae detect mulberry secondary metabolites, chlorogenic acid (CGA), and isoquercetin (ISQ), with ultrahigh sensitivity, for host plant recognition and feeding initiation. BmGr 6 is regarded as a putative sugar receptor and BmGr 9 was reported as a fructose receptor, we investigated their response to major sugars present in the mulberry leaves (Dfructose, D-glucose, sucrose, and myo-inositol). results indicate that the two Grs are highly tuned to CGA and ISQ, whereas BmGr 9 also exhibits a high affinity with D-fructose.

The fascinating defence system of mulberry

Mulberry provides food to the silkworm, but at the same time, the plant has evolved its own defence mechanisms against herbivores. Plants cannot run away from insects that feed on them. Instead, they defend themselves through physical barriers and chemical compounds known as secondary metabolites. Mulberry is particularly interesting because it contains 1-deoxynojirimycin (DNJ), a naturally occurring iminosugar (azasugar) characteristic of Morus species. DNJ closely resembles the structure of glucose and can therefore interact with carbohydrate-digesting enzymes. Studies have demonstrated that DNJ is a potent inhibitor of α-glucosidases, enzymes involved in the breakdown of dietary carbohydrates into absorbable sugars. This biochemical property can interfere with carbohydrate digestion and has made DNJ one of the best-studied bioactive compounds in mulberry (Wang et al., 2021; Liu et al., 2015) [16,15] . Interestingly, DNJ may also have an ecological role beyond its biochemical activity. Comparative studies of Morus alba have reported that DNJ possesses antifeedant activity against some insects, suggesting that it may contribute to the plant's chemical defence. However, this effect is not universal, and B. mori is highly specialized for feeding on mulberry. Thus, rather than describing DNJ simply as a "defence compound," it is more accurate to view it as one component of the complex chemical environment of mulberry that can influence herbivore physiology and feeding interactions (Yagi et al., 1976, Liu et al., 2015) [17] .

Conclusion: A remarkable chemical partnership

The relationship between Bombyx mori and mulberry is a beautiful example of nature's chemical engineering. Mulberry provides nutrients and chemical cues that help the silkworm recognize and utilize its host plant. At the same time, the plant produces defensive compounds such as DNJ, while the silkworm has evolved physiological mechanisms that allow it to thrive on its specialized food plant.

Volatile compounds such as cis-jasmone demonstrate how plants can communicate chemically with insects, while compounds such as βsitosterol, Morin and other leaf constituents contribute to the complex nutritional and physiological environment experienced by the insect. Ultimately, the silkworm's preference for mulberry is the outcome of sensory recognition, feeding stimulation, nutritional suitability and long-term evolutionary adaptation.

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