Assessing the adaptive role of cannabidiol (CBD) in Cannabis sativa defense against cannabis aphids

Abstract

Cannabis sativa is known for having unique specialized or secondary metabolites, cannabinoids that are derived from an extension of the terpene pathway in the Cannabis lineage and includes more than 100 other similar metabolites. Despite the assumption that cannabinoids evolved as novel herbivory defense adaptations, there is limited research addressing the role of cannabinoids in C. sativa responses to insect herbivores. Here we investigated the role of cannabidiol (CBD), the predominant cannabinoid in hemp, in plant defense against cannabis aphid (Phorodon cannabis), one of the most damaging pests of hemp. We hypothesize that insect feeding may induce changes in cannabinoids as an adaptive strategy for defense. We found that mean fecundity, net reproductive rate (R₀) and adult longevity of cannabis aphids was reduced on the high cannabinoid cultivar compared to the low- cannabinoid cultivar in whole plant assays. In contrast, supplementation of CBD in artificial feeding assays increased aphid fecundity from day 1 to day 3. Additionally, aphid feeding did not impact cannabinoid levels in leaf tissues with the exception of Δ⁹-tetrahydrocannabinol (THC). This suggests that other cannabinoids and/or metabolites such as terpenes are causing the observed decrease in aphid performance in the whole plant assays. In addition to cannabinoids, C. sativa also possesses a range of defense mechanisms via phytohormone signaling pathways that are well described in other plant species. Indeed, cannabis aphid feeding significantly increased levels of the major phytohormones, salicylic acid, jasmonic acid, and abscisic acid, which are known to be involved in plant defense responses against aphid species. These results highlight the interplay between cannabinoid synthesis and phytohormone pathways and necessitate further investigation into this complex interaction.

Keywords: Cannabis sativa; cannabinoids; cannabis aphids; insect performance; phytohormones.

Figure 1

CBD has a positive effect on cannabis aphid fecundity in artificial feeding assays. Cannabis aphids were maintained on artificial diet supplemented with either DMSO or DMSO + 1 mM CBD. A cohort of ten 8-day old adult aphids were transferred to artificial diet and allowed to feed for 4 days and the number of (A) nymphs and (B) surviving adults remaining each day was monitored (n = 14, n =12 for 1 mM CBD). Analysis with GLM followed by one-way ANOVA with Tukey-HSD post-hoc tests were performed for each day. Different lowercase letters indicate significant differences.

Figure 2

Cannabis aphids have delayed preference for low-cannabinoid cultivar. Fifty alatae cannabis aphids were placed in a Petri dish and introduced to a 12” cube cage to choose between three-week old high-cannabinoid (Unicorn) or low-cannabinoid (Elite) on either side of the cage. The number of alatae on either plant was documented at the indicated timepoints. Values are the mean ± SE of n=18. Analysis with both Friedman (full model) and Kruskal-Wallis (individual time points) tests were performed. Asterisks indicate significance **P < 0.01.

Figure 3

Cannabis aphid infestation induces plant defense responses. Ten adult cannabis aphids were clip-caged on a 3-week-old cannabis single leaf. After 48 hours aphids were removed and leaves were collected and relative expression of phytohormone marker genes for salicylic acid (PR1), jasmonic acid (HEL), abscisic acid (PP2C-6), and CBD (CBDAS) were measured with RT-qPCR. Error bars represent SE of the mean of four biological replicates with two technical replicates each. Statistical analysis was performed using a t-test. Asterisks indicate significance, *P < 0.05 (t-test).

Figure 4

Cannabis aphid feeding impacts phytohormone levels. Eight-week-old hemp plants were infested with 10 mixed life stages of aphids and cannabinoid levels were analyzed 20 days post-infestation. The cannabinoids include (A) salicylic acid (SA), (B) jasmonic acid (JA), and (C) abscisic acid (ABA) production in infested and uninfested leaves for each cultivar. Statistical analysis was performed using a t-test. Asterisks indicate significance *P < 0.05, **P < 0.01, ***P < 0.001.