Cannabinoids function in defense against chewing herbivores in Cannabis sativa L.

Abstract

In the decades since the first cannabinoids were identified by scientists, research has focused almost exclusively on the function and capacity of cannabinoids as medicines and intoxicants for humans and other vertebrates. Very little is known about the adaptive value of cannabinoid production, though several hypotheses have been proposed including protection from ultraviolet radiation, pathogens, and herbivores. To test the prediction that genotypes with greater concentrations of cannabinoids will have reduced herbivory, a segregating F2 population of Cannabis sativa was leveraged to conduct lab- and field-based bioassays investigating the function of cannabinoids in mediating interactions with chewing herbivores. In the field, foliar cannabinoid concentration was inversely correlated with chewing herbivore damage. On detached leaves, Trichoplusia ni larvae consumed less leaf area and grew less when feeding on leaves with greater concentrations of cannabinoids. Scanning electron and light microscopy were used to characterize variation in glandular trichome morphology. Cannabinoid-free genotypes had trichomes that appeared collapsed. To isolate cannabinoids from confounding factors, artificial insect diet was amended with cannabinoids in a range of physiologically relevant concentrations. Larvae grew less and had lower rates of survival as cannabinoid concentration increased. These results support the hypothesis that cannabinoids function in defense against chewing herbivores.

Acknowledgements

We are grateful to the research teams from the Smart, Moore, Wang, and Rose labs for their excellent technical assistance on this project, especially Alex Wares, McKenzie Schessl, Natalie McFadden, Joel DeVries, Emily McFadden, Ryan Crawford, Jesse Chavez, Jason Schiller, Wendy Kain, and the farm crews at Cornell AgriTech and the Cornell Agricultural Experiment Station campus area farms. We are also grateful to Dr Kyle Wickings and Dr María Alejandra Gandolfo-Nixon for the use of their lab equipment, Patrick Woods for providing the OLS/TKS gene sequence for the molecular marker design, and to Dr Heather Grab and Dr Brian Nault for advice on the experimental design. This project was funded by grant AC477 from the New York State Department of Agriculture and Markets through Empire State Development.

Author contributions

G.M.S.: Conceptualization, methodology, formal analysis, investigation, visualization, writing – original draft. S.I.S., J.A.T.: Methodology, investigation. M.A.Q., J.L.C.: Investigation. J.K.M., J.N.J., P.W.: Resources. G.P.: Methodology. J.L.H., V.M.M., J.K.C.R., L.B.S.: Supervision, funding acquisition. All authors contributed to writing-review & editing.

Data availability

The datasets generated during and analyzed during the current study are available in the supplementary materials.

Conflict of interest statement

None declared.

References

[1] Theis N, Lerdau M . The evolution of function in plant secondary metabolites. Int J Plant Sci. 2003; 164: S93-102

[2] Wink M . Plant secondary metabolism: diversity, function and its evolution. Nat Prod Commun. 2008; 3: 1205-16

[3] Futuyma DJ, Agrawal AA . Macroevolution and the biological diversity of plants and herbivores. Proc Natl Acad Sci U S A. 2009; 106: 18054-61

[4] Fellermeier M, Eisenreich W, Bacher A, et al. Biosynthesis of cannabinoids. Eur J Biochem. 2001; 268: 1596-604

[5] Dayanandan P, Kaufman PB . Trichomes of Cannabis sativa L. (Cannabaceae). Am J Bot. 1976; 63: 578-91

[6] Turner JC, Hemphill JK, Mahlberg PG . Quantitative determination of cannabinoids in individual glandular trichomes of Cannabis sativa L. (Cannabaceae). Am J Bot. 1978; 65: 1103-6

[7] Livingston SJ, Quilichini TD, Booth JK, et al. Cannabis glandular trichomes alter morphology and metabolite content during flower maturation . Plant J. 2020; 101: 37-56

[8] Gaoni Y, Mechoulam R . Isolation, structure, and partial synthesis of an active constituent of hashish. J Am Chem Soc. 1964; 86: 1646-7

[9] Andre CM, Hausman J-F, Guerriero G . Cannabis sativa: the plant of the thousand and one molecules . Front Plant Sci. 2016; 7: 19

[10] Lange BM, Zager JJ . Comprehensive inventory of cannabinoids in Cannabis sativa L.: can we connect genotype and chemotype? Phytochem Rev. 2022; 21: 1273-313

[11] van Velzen R, Schranz ME . Origin and evolution of the cannabinoid oxidocyclase gene family. Genome Biol Evol. 2021; 13: 1-18

[12] Melzer R, McCabe PF, Schilling S . Evolution, genetics and biochemistry of plant cannabinoid synthesis: a challenge for biotechnology in the years ahead. Curr Opin Biotechnol. 2022; 75: 1-8

[13] Russo EB, Jiang H-E, Li X, et al. Phytochemical and genetic analyses of ancient cannabis from Central Asia. J Exp Bot. 2008; 59: 4171-82

[14] Gülck T, Møller BL . Phytocannabinoids: origins and biosynthesis. Trends Plant Sci. 2020; 25: 985-1004

[15] Berman P, de Haro LA, Jozwiak A, et al. Parallel evolution of cannabinoid biosynthesis. Nat Plants. 2023; 9: 817-31

[16] Gorelick J, Bernstein N . Chemical and physical elicitation for enhanced cannabinoid production. In: Chandra S, Lata H, ElSohly M (eds) Cannabis. Cannabis Sativa L. - Botany and Biotechnology. Springer International Publishing: New York, NY, USA, 2017, 439-56

[17] Tanney CAS, Backer R, Geitmann A, et al. Cannabis glandular trichomes: a cellular metabolite factory. Front Plant Sci. 2021; 12: 1-8

[18] Pate DW . Possible role of ultraviolet radiation in evolution of cannabis chemotypes . Econ Bot. 1983; 37: 396-405

[19] Lydon J, Teramura AH, Coffman CB . UV-B radiation effects on photosynthesis, growth and cannabinoid production of two Cannabis sativa chemotypes . Photochem Photobiol. 1987; 46: 201-6

[20] McPartland JM . Cannabis systematics at the levels of family, genus, and species . Cannabis Cannabinoid Res. 2018; 3: 203-12

[21] Rodriguez-Morrison V, Llewellyn D, Zheng Y . Cannabis inflorescence yield and cannabinoid concentration are not increased with exposure to short-wavelength ultraviolet-B radiation. Front Plant Sci. 2021; 12: 1-21

[22] Llewellyn D, Golem S, Foley E, et al. Indoor grown cannabis yield increased proportionally with light intensity, but ultraviolet radiation did not affect yield or cannabinoid content. Front Plant Sci. 2022; 13: 1-12

[23] Appendino G, Gibbons S, Giana A, et al. Antibacterial cannabinoids from Cannabis sativa: a structure−activity study . J Nat Prod. 2008; 71: 1427-30

[24] Karas JA, Wong LJM, Paulin OKA, et al. The antimicrobial activity of cannabinoids. Antibiotics (Basel). 2020; 9: 1-10

[25] Saleemi MA, Yahaya N, Zain NNM, et al. Antimicrobial and cytotoxic effects of cannabinoids: an updated review with future perspectives and current challenges. Pharmaceuticals. 2022; 15: 1-23

[26] McPartland J . Pathogenicity of Phomopsis ganjae on Cannabis sativa and the fungistatic effect of cannabinoids produced by the host . Mycopathologia. 1984; 87: 149-53

[27] de Meijer EPM, van der Kamp HJ, van Eeuwijk FA . Characterisation of cannabis accessions with regard to cannabinoid content in relation to other plant characters . Euphytica. 1992; 62: 187-200

[28] Felder CC, Glass M . Cannabinoid receptors and their endogenous agonists. Annu Rev Pharmacol Toxicol. 1998; 38: 179-200

[29] McPartland J, Di Marzo V, De Petrocellis L, et al. Cannabinoid receptors are absent in insects. J Comp Neurol. 2001; 436: 423-9

[30] Rothschild M, Fairbairn JW . Ovipositing butterfly (Pieris brassicae L.) distinguishes between aqueous extracts of two strains of Cannabis sativa L. and THC and CBD . Nature. 1980; 286: 56-9

[31] Mantzoukas S, Ntoukas A, Lagogiannis I, et al. Larvicidal action of cannabidiol oil and neem oil against three stored product insect pests: effect on survival time and in progeny. Biology. 2020; 9: 1-13

[32] Park S-H, Staples SK, Gostin EL, et al. Contrasting roles of cannabidiol as an insecticide and rescuing agent for ethanol-induced death in the tobacco hornworm Manduca sexta. Sci Rep. 2019; 9: 10481

[33] He J, Tan AMX, Ng SY, et al. Cannabinoids modulate food preference and consumption in Drosophila melanogaster. Sci Rep. 2021; 11: 4709

[34] Waser P. Effects of THC on brain and social organization of ants. In: Nahas GG, Sutin KM, Harvey D, et al., eds. Marihuana and Medicine. Humana Press: Totowa, NJ, 1999, 155-61

[35] Ona G, Balant M, Bouso JC, et al. The use of Cannabis sativa L. for pest control: from the ethnobotanical knowledge to a systematic review of experimental studies . Cannabis Cannabinoid Res. 2022; 7: 365-87

[36] de Meijer EPM, Bagatta M, Carboni A, et al. The inheritance of chemical phenotype in Cannabis sativa L. Genetics. 2003; 163: 335-46

[37] de Meijer EPM, Hammond KM . The inheritance of chemical phenotype in Cannabis sativa L. (II): cannabigerol predominant plants . Euphytica. 2005; 145: 189-98

[38] de Meijer EPM, Hammond KM, Sutton A . The inheritance of chemical phenotype in Cannabis sativa L. (IV): cannabinoid-free plants . Euphytica. 2009; 168: 95-112

[39] de Meijer EPM, Hammond KM . The inheritance of chemical phenotype in Cannabis sativa L. (V): regulation of the propyl−/pentyl cannabinoid ratio, completion of a genetic model . Euphytica. 2016; 210: 291-307

[40] Stack GM, Toth JA, Carlson CH, et al. Season-long characterization of high-cannabinoid hemp (Cannabis sativa L.) reveals variation in cannabinoid accumulation, flowering time, and disease resistance . Glob Change Biol Bioenergy. 2021; 13: 546-61

[41] Woods P, Campbell BJ, Nicodemus TJ, et al. Quantitative trait loci controlling agronomic and biochemical traits in Cannabis sativa. Genetics. 2021; 219: 1-13

[42] Grassa CJ, Weiblen GD, Wenger JP, et al. A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana . New Phytol. 2021; 230: 1665-79

[43] Weiblen GD, Wenger JP, Craft KJ, et al. Gene duplication and divergence affecting drug content in Cannabis sativa. New Phytol. 2015; 208: 1241-50

[44] Laverty KU, Stout JM, Sullivan MJ, et al. A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci . Genome Res. 2019; 29: 146-56

[45] Onofri C, de Meijer EPM, Mandolino G . Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype . Phytochemistry. 2015; 116: 57-68

[46] Garfinkel AR, Otten M, Crawford S . SNP in potentially defunct tetrahydrocannabinolic acid synthase is a marker for cannabigerolic acid dominance in Cannabis sativa L. Genes. 2021; 12: 228

[47] Koch SD . Insecticidal Property of Cannabidiol: CBD Disrupts Exoskeleton Development of Tobacco Hornworm (Manduca sexta). MS thesis. Colorado State University - Pueblo. 2021, 90 pp.

[48] Abendroth JA, Gondhalekar AD, Scharf ME, et al. Cannabidiol reduces fall armyworm (Spodoptera frugiperda) growth by reducing consumption and altering detoxification and nutritional enzyme activity in a dose-dependent manner . Arthropod Plant Interact. 2023; 17: 195-204

[49] Sutherland DWS, Greene GL. Cultivated and wild host plants. In: Lingren PD, Greene GL, eds. Suppression and Management of Cabbage Looper Populations. Technical Bulletin no. 1684. United States Department of Agriculture-Agricultural Research Service, 1984, 1-13

[50] Kovalchuk I, Pellino M, Rigault P, et al. The genomics of cannabis and its close relatives . Annu Rev Plant Biol. 2020; 71: 713-39

[51] Ren G, Zhang X, Li Y, et al. Large-scale whole-genome resequencing unravels the domestication history of Cannabis sativa. Sci Adv. 2021; 7: 1-12

[52] Bolt MA, Beckerman JL, Couture JJ . Agronomic management of industrial hemp alters foliar traits and herbivore performance. Arthropod Plant Interact. 2021; 15: 137-51

[53] Jones CG, Firn RD . On the evolution of plant secondary chemical diversity. Philos Trans R Soc Lond Ser B Biol Sci. 1991; 333: 273-80

[54] Franco R, Rivas-Santisteban R, Reyes-Resina I, et al. Pharmacological potential of varinic-, minor-, and acidic phytocannabinoids. Pharmacol Res. 2020; 158: 1-15

[55] Glas JJ, Schimmel BCJ, Alba JM, et al. Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores. Int J Mol Sci. 2012; 13: 17077-103

[56] Lange BM, Turner GW . Terpenoid biosynthesis in trichomes-current status and future opportunities. Plant Biotechnol J. 2013; 11: 2-22

[57] Gang DR, Wang J, Dudareva N, et al. An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol. 2001; 125: 539-55

[58] Treutter D . Significance of flavonoids in plant resistance: a review. Environ Chem Lett. 2006; 4: 147-57

[59] Fan P, Leong BJ, Last RL . Tip of the trichome: evolution of acylsugar metabolic diversity in Solanaceae. Curr Opin Plant Biol. 2019; 49: 8-16

[60] Hammond CT, Mahlberg PG . Morphology of glandular hairs of Cannabis sativa from scanning electron microscopy . Am J Bot. 1973; 60: 524-8

[61] Livingston SJ, Rensing KH, Page JE, et al. A polarized supercell produces specialized metabolites in cannabis trichomes. Curr Biol. 2022; 32: 4040-4047.e4

[62] Kang J-H, Shi F, Jones AD, et al. Distortion of trichome morphology by the hairless mutation of tomato affects leaf surface chemistry. J Exp Bot. 2010; 61: 1053-64

[63] Tian D, Tooker J, Peiffer M, et al. Role of trichomes in defense against herbivores: comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum lycopersicum). Planta. 2012; 236: 1053-66

[64] Cranshaw W, Schreiner M, Britt K, et al. Developing insect pest management systems for hemp in the United States: a work in progress. J Integr Pest Manag. 2019; 10: 1-10

[65] Schreiner M. A Survey of the Arthropod Fauna Associated with Hemp (Cannabis sativa L.) Grown in Eastern Colorado. MS thesis, Colorado State University, Fort Collins, CO, 2019, 90 pp.

[66] Serber ZA . Characterizing the Identity and Seasonality of Common Arthropods on CBD Hemp in Northern Indiana. MS thesis, Purdue University, Lafayette, IN 2022:66 pp.

[67] Knight TJ . Insect Seasonality and Guild Composition on Industrial Hemp (Cannabis sativa L.) Grown for CBD in High Tunnels in Alabama. MS thesis, Alabama State University, Montgomery, AL, 2022, 83 pp.

[68] Ali JG, Agrawal AA . Specialist versus generalist insect herbivores and plant defense. Trends Plant Sci. 2012; 17: 293-302

[69] Cosner J . Feeding Behavior and Influence of Hemp Varieties and Fertilization Rates on Damage Caused by Corn Earworm, Helicoverpa zea. MS thesis, University of Tennessee, Knoxville, TN. 2021, 100 pp.

[70] Stack GM, Carlson CH, Toth JA, et al. Correlations among morphological and biochemical traits in high-cannabidiol hemp (Cannabis sativa L.). Plant Direct. 2023; 7: 1-14

[71] Ashman T-L. The role of herbivores in the evolution of separate sexes from hermaphroditism. Ecology. 2002; 83: 1175-84

[72] Keefover-Ring K, Carlson CH, Hyden B, et al. Genetic mapping of sexually dimorphic volatile and non-volatile floral secondary chemistry of a dioecious willow. J Exp Bot. 2022; 73: 6352-66

[73] de Mendiburu F. agricolae: Statistical Procedures for Agricultural Research. R package version 1.3-5. 2021.

[74] Toth JA, Stack GM, Cala AR, et al. Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L. Glob Change Biol Bioenergy. 2020; 12: 213-22

[75] Getman-Pickering ZL, Campbell A, Aflitto N, et al. LeafByte: a mobile application that measures leaf area and herbivory quickly and accurately. Methods Ecol Evol. 2020; 11: 215-21

[76] R Core Team . R: a language and environment for statistical computing. 2022.

[77] Kuznetsova A, Brockhoff PB, Christensen RHB, et al. lmerTest package: tests in linear mixed effects models. J Stat Softw. 2017; 82: 1-26

[78] Lenth R, Singmann H, Love J, et al. emmeans: Estimated marginal means, aka Least-squares means. 2023.

[79] Bates D, Mächler M, Bolker B, et al. Fitting linear mixed-effects models using lme4. J Stat Soft. 2015; 67: 1-48