In Cannabis, two phytochemicals, THC and CBD, are abundantly produced in the glandular trichomes, and are expected to play pivotal defensive roles. Indeed, these cannabinoids are known to confer resistance against a variety of bacteria27. Also, several anecdotes show that high THC and CBD producing Cannabis varieties are more protective against microbial and insect pathogens. A recent study suggests the possible use of the crop-residue of fiber hemp containing CBD as an insecticide5. Despite the escalating pharmaceutical uses of CBD, the intrinsic questions of why Cannabis plants produce the secondary metabolites, how they exploit the molecules for their survival, and what evolutionary benefits they provide still remain unanswered.

To investigate which constituents of hemp confer the insecticidal function, two hemp varieties containing different levels of CBD at 3.2% and 0.8% in leaf tissues were used for the two-way feeding choice study. The test reveals that 3rd instar M. sexta larvae showed clear preferences on the Cannabis leaf tissues containing 0.8% CBD over the 3.2% CBD tissues (Fig. 1A,B). When neonates were tested, they were unable to ingest any of the hemp tissues. Also, another feeding study shows that the high dosage of CBD (>1 mM) significantly inhibits the growth, mortality, and development of the insects, while the low dose (<1 mM) had no effect (Fig. 1C,D,E). Based on our data, we could not determine if the detrimental mechanism was due to neurotoxic effect or biochemical effect that CBD has on M. sexta’s digestive system. However, the feeding assay clearly demonstrated the preventive role of the CBD against pest insects. Indeed, numerous plants have been shown to exploit secondary metabolites (e.g., terpenes and phenolics) to protect plants from predators and microbial pathogens28. Additionally, it has been reported that high cannabinoid producing hemp varieties are more pest-resistant6 and also hypothesized that the cannabinoid level can be mediated by herbivory. It can be postulated that when CBD is highly accumulated in flowers, it is beneficial for the plants’ survival and reproduction. Despite the potential of CBD as a potent insecticide, in all likelihood, CBD use may not be feasible currently due to the high production and extraction costs. However, crude CBD-containing hemp extract of mature flowers may be used as a repellent in other crop species, as well as a companion crop5,6.

The latter feeding assay used MCT oil as a CBD solvent agent. To ensure that the inhibitory effects were not caused by the vehicle, another solvent agent, ethanol (200 proof) was used for dissolving CBD, and the feeding assay was repeated. Unexpectedly, greater than 1% EtOH in the diet appeared to be lethal for the caterpillars within 3 days. However, the larvae reared on the EtOH-containing AD with CBD display significantly lower mortalities. The CBD supplementation effectively rescued the insects from the EtOH stress, increasing the survival rate by up to 40% (Figs 1E and 4C).

To confirm the preventive role of CBD against EtOH-stress, another EtOH feeding study was implemented in a controlled-environment. The study confirmed that M. sexta larval growth and development were negatively correlated to the increase of EtOH in the diet. The chronic exposure to >1% EtOH significantly reduced the length, body weight, and survival rate. While Drosophila showed an increased mobility in the lower EtOH level29, the caterpillars used in this study did not show any increase in their mobility within sample subset of less than 1% EtOH. Additionally, the high dose of EtOH (>2%) significantly disrupted the food searching behavior (Fig. 3B).

Invertebrates have been used as a model system to investigate alcoholism and its social and behavioral effects due to the simplicity in anatomy, their sophisticated genetics, and molecular similarities to higher animals30. A series of EtOH feeding studies in honeybees demonstrated the negative influence in social communication and behaviors, a decrease in locomotion, and significant impairment of Pavlovian conditioning of proboscis extension31. Furthermore, a similar study shows that excessive ethanol intoxication by solutions containing ≥5% ethanol causes significant ethanol-induced stress in brain tissue that impairs honeybee behavior and associative learning32. Another EtOH study performed in Drosophila reveals the increase of locomotion at the low level of EtOH vapor, while higher doses cause reduced movement, loss of posture control, and immobility30.

Notably, M. sexta represents a lower EtOH tolerance when compared to mice that were able to survive at 36% EtOH ingestion33. The higher mortality is likely due to less alcohol-dehydrogenase (ADH) enzymes in insects than mammals that contain at least six medium-chain ADH classes (class I-VI)34. Drosophila produces only class III ADH enzyme, and the transcriptomic analysis performed on the M. sexta male moth antenna also revealed the presence of transcript encoding a class III-like ADH enzyme35. In addition, it is assumed that the respiratory system of M. sexta being mediated via spiracles caused the high casualty rate. The spiracles are external tracheal apertures located on each segment of the body for gas exchange, which may escalate the alcohol absorption into the insect system via the vapor.

The detoxifying mechanism of CBD against EtOH is still unknown. However, it is assumed that CBD effectively lowers the EtOH-induced oxidative stress, resulting in the increased growth, food consumption, mobility, and survival rate. Interestingly, CBD functions differently in the absence or presence of EtOH in the diet. In the absence of EtOH, >2 mM CBD became lethal to the insects (Fig. 1C,D,E). In contrast, the lethal doses show rescuing effects in the diets containing high EtOH concentrations (2%, 5%, and 10%). Our findings suggest that <1 mM CBD has no adverse effects on the insects’ growth, but a high dose of CBD (>2 mM CBD) can be inhibitory.

The chemical analysis indicates that CBD accumulation in the body of M. sexta was positively correlated to the amounts added to the diets (Fig. 4D). CBD was not detected from the control media (AD) itself and the larvae fed on AD. Also, CBD was not detected from the larvae reared on +10 uM CBD because it was under the detection limit. However, the larvae reared on AD + 1% EtOH + 100 uM CBD accumulates 0.0003% in the insect body, 0.0007% and 0.001% on +1 mM and +2 mM CBD diets, respectively. Only trace amounts were metabolized since the majority of CBD seemed to be excreted (Fig. 5C).

The electrophysiology data clearly show that CBD affects the signal transduction the ganglion axons, responded differently when treated EtOH and/or CBD. In Fig. 5C, two electric response peaks were observed, possibly the rapid first peak may have been caused by the myelinated axon and the delayed second peak by an unmyelinated axon. Based on the appearance of the first peak, EtOH caused a more rapid response compared to the larvae reared on AD and CBD-treated AD. Notably, the larvae treated with 1 mM CBD had delayed electric responses, but the magnitude of slope was much larger. Figure 5D presents the slope at different electric stimuli, revealing a significantly larger response in ganglia of CBD-treated larvae at all stimulus intensity after first stimulus at zero mA.

Cannabinoids, whether endogenous or exogenous, are potent regulators of neurotransmission36. THC and CBD bind to two distinct endocannabinoid receptors, CB1 and CB2, which belong the G-protein coupled receptor superfamily37. THC shows high affinity to CB1/CB2 receptors, which are primarily located in the brain and nervous system37,38,39. CBD has low affinity to both receptors, which is mostly found in peripheral organs and immune tissues37,40. Although CB1/2 receptors have been found in the nematode Panagrellus redivivus and several other invertebrates41, cannabinoid receptors have not been identified in insects to date42. Despite the absence of cannabinoid receptors in M. sexta, differential signal responses in the larval ganglia were evident between larvae reared on AD, AD + 1% EtOH, and AD + 1% EtOH + 1 mM CBD (Fig. 5C,D). The electrophysiology results suggest that the differential electric responses in the central nervous system (CNS) might have been caused by ‘non-CB receptors’ that are known to interact with cannabinoids in some species43. The non-CB receptors present in insects include 5-hydroxytryptamine (serotonin) receptors (5HT1A/2 A), transient receptor potential vanilloid cation channel receptors (TRPV), and nicotinic acetylcholine receptors (α7nACh) that are involved in a number of neurological and psychiatric responses44. In mammals, CBD has been found to act on these receptors. However, the binding affinity of CBD varies from species to species43,45.

The central nervous system is essential for signal transduction to modulate a variety of physiological responses including modulation of spiracles that are essential for respiration, gas and water exchange. This study’s findings demonstrate CBD’s role in modifying signal conductivity of the CNS. This modification may be the cause of the observed physiological changes, i.e., increased food searching activity, which would increase diet consumption, thus causing the differences in the length and weight observed in these caterpillars. All of these positive activities increased the survival rate of the EtOH-intoxicated M. sexta.

Here we report dual, contrasting roles of CBD in M. sexta larvae. The naturally occurring CBD in Cannabis primarily acts as a feeding deterrent against pests. Consequently, cannabidiol ingested effectively inhibits the larval growth and development, resulting in high mortality. Our results support a long-held belief for the defensive function of cannabinoids and would suggest the potential use of CBD-rich hemp extract as a repellent and/or as a companion crop. Secondly, lethal amounts of CBD function differently in the presence of EtOH stress, becoming protective. The CBD-administered larvae outperformed the larvae fed on the EtOH-only diet, significantly increasing survival rate by 40%. The electrophysiology results suggest that CBD affects the signal transduction of ventral ganglia in the CNS. This modification was beneficial to their growth and survival under chronic EtOH exposure. These findings could be a step forward to understanding the production of CBD as a part of defense mechanism in Cannabis, as well as the use of CBD as a non-traditional therapeutic treatment for alcohol abuse, addiction, and intoxication.