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New Zealand Journal of Crop and Horticultural Science

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Nematicidal effects of hemp (Cannabis sativa) may not be mediated by cannabinoid receptors J. M. Mcpartland & M. Glass To cite this article: J. M. Mcpartland & M. Glass (2001) Nematicidal effects of hemp (Cannabis sativa) may not be mediated by cannabinoid receptors, New Zealand Journal of Crop and Horticultural Science, 29:4, 301-307, DOI: 10.1080/01140671.2001.9514191 To link to this article: http://dx.doi.org/10.1080/01140671.2001.9514191

Published online: 22 Mar 2010.

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Nematicidal effects of hemp (Cannabis sativa) may not be mediated by cannabinoid receptors

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J. M. MCPARTLAND Faculty of Health & Environmental Sciences UNITEC Private Bag 92 025, Auckland New Zealand M. GLASS Department of Pharmacology University of Auckland Private Bag 92 019, Auckland New Zealand Abstract Few nematodes infest the roots of hemp (Cannabis sativa L.) plants, and hemp plant extracts have been utilised as botanical nematicides. The responsible constituent may be Δ9-tetrahydrocannabinol (Δ9-THC). In humans, Δ9-THC exerts its effects via a family of G protein-coupled receptors, known as cannabinoid (CB) receptors. CB receptors are phylogenetically ancient, and occur in many vertebrates and invertebrates. We therefore searched for evidence of CB receptors in nematodes. All nematode cDNA sequences at GenBank, including the entire genome of Caenorhabditis elegans, were screened for homologs of human CB receptors using BLAST 2.0 as a sequence alignment search engine. We also searched for homologs of fatty acid amide hydrolase (FAAH), the enzyme in vertebrates that metabolises the endogenous ligands of CB receptors. Several C. elegans gene products with low homology to CB receptors and FAAH were identified. Close examination of these sequences revealed crippling substitutions at critical amino acid residues. These results suggest the genes for CB receptors are absent in C. elegans, and the nematicidal activities of Δ9-THC and Cannabis are not mediated through CB receptors.

H01015 Received 18 April 2001; accepted 20 September 2001

Keywords nematodes; Caenorhabditis elegans; hemp; cannabis; integrated pest management; cannabinoid receptors; fatty acid amide hydrolase; sequence homology; amino acid

INTRODUCTION Hemp, Cannabis sativa L., is rarely infested by nematodes (McPartland et al. 2000). This makes hemp attractive to farmers using Integrated Pest Management (IPM), as a rotation crop with nematode-susceptible plants. About 4000 species of nematodes are plant parasites, and they damage crops around the world (Weischer & Brown 2000). The damage they do tends to be under appreciated, because most nematodes attack roots, causing unseen, underground damage. Although nematodes are very small, simple organisms they can occur in high numbers and it is their accumulated impact that causes damage. Their nervous system is so simple it can be described at the level of individual cells: Caenorhabditis elegans, for instance, has exactly 302 neurons. A complete wiring diagram of its nervous system has been compiled. The entire DNA sequence needed to build C. elegans has been described—19 000 genes, a 97-megabase genomic sequence (Bargmann 1998). In New Zealand, the most economically damaging crop nematodes are Globodera pallida and G. rostochiensis, which threaten potato yields and export status (Mercer 1994; Bulman & Marshall 1997). Hemp has been rotated with potatoes to suppress G. rostochiensis (Kir'yanova & Krall 1980). In New Zealand pastures, the most important nematodes of white clover are Heterodera trifolii, Ditylenchus dipsaci (Mercer 1994), and four Meloidogyne species (Mercer & Miller 1997). Hemp rotations suppress soil populations of//, glycines (Scheifele 1998) and M. chitwoodi (Kok et al. 1994). Some hemp cultivars are resistant to M. hapla (Meijer 1993) and other Meloidoygne species (Mateeva 1995). Soil mixed with 3% w/w dried Cannabis seed cake suppressed M. incognita (Goswami & Vijayalakshmi

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1986). Aqueous leaf extracts have been shown to kill plant-pathogenic nematodes, including M. incognita (Vijayalakshmi et al. 1979), M. javanica (Bajpai & Sharma 1992), H. cajani (Mojumder et al. 1989), and Hoplolaimus indicus, Rotylenchulus reniformis, and Tylenchorynchus brassicae (Haseeb et al. 1978). Cannabis and cannabis extracts have been used as vermifuges for domestic animals (Kabelik et al. 1960). Tn human medicine, physicians treated "worms" with juice extracted from crashed hemp seed (Parkinson 1640; Culpepper 1814). This practice dates to ancient Roman medicine (Pliny 1950 reprint). In medieval Arabic medicine, crushed leaves and seeds were ingested for treatment of Ascaris infection (Lozano 2001). The Cannabis constituents responsible for these nematicidal effects have not been ascertained. Other nematicidal plants express high levels of terpenoids, phenolics, and fatty acid derivatives with anti-nematode activity (Chitwood 1992). Cannabis uniquely biosynthesises the cannabinoids, a small family of C2i terpenophenolic compounds, exemplified by A9tetrahydrocannabinol (A9-THC). A9-THC is toxic to the flatworm Dugesia tigrina (Platyhelminthes: Tricladia), with an LD50 = 6 x 10~7M (Lenicque et al. 1972). Another cannabinoid, cannabiorcichromemic acid, is toxic to the nematode Heterorhabditis sp., with an LD50 = 20 mg litre"1 (Quaghebeur et al. 1994). A9-THC and cannabiorcichromemic acid may exert their effects on nematodes via cannabinoid (CB) receptors. CB receptors are G protein-coupled receptors (GPCRs) expressed on cell membranes. In humans two genes that encode CB receptors have been cloned: CB 1 predominates in cells of the central nervous system, but can also be detected in the pituitary, heart, lung, prostate, uterus, testis, ovary, and elsewhere. CB2 is largely restricted to cells of immune function, such as leukocytes, tonsils, spleen, thymus, and bone marrow, but also occurs in the pancreas, uterus, lung, and elsewhere (Felder & Glass 1998). CB receptors appear to be phylogenetically ancient, and occur in many mammals, reptiles, fish, molluscs, sea urchins, leeches, and even Hydra (Salzet et al. 2000). The presence of CB receptors in nematodes has not been investigated. Hundreds of other GPCRs have been identified in nematodes; the ligands signaling these receptors are mostly unknown (Bargmann 1998). To determine whether the nematicidal effects of A9-THC and Cannabis are CB receptor-mediated we searched for genetic evidence of CB receptors

in nematodes. The present study screened for homologs of human CB receptors in all nematode cDNA sequences deposited at GenBank, including the entire genome of C. elegans. We also searched for homologs of fatty acid amide hydrolase (FAAH), the enzyme in vertebrates that metabolises the endogenous ligands of CB receptors (Cravatt et al. 1996).

MATERIALS AND METHODS The genome of C. elegans is available on a computerised database, GenBank (National Center for Biotechnology Information, http:// www.ncbi.nlm.nih.gov). These sequences were compared with the deduced amino acid sequences of human CB1, human CB2, and human FAAH (GenBank accession numbers g.i. 4502927, g.i. 4502929, and g.i. 6225310 respectively). All other nematode cDNA sequences deposited at GenBank were also screened (these included Meloidogyne, Heterodera, Pratylenchus, and Ditylenchus species). Sequences were aligned using gapped BLAST (Basic Local Alignment Search Tool) version 2.0 (Altschul et al. 1997). BLAST 2.0 uses the SEG program as a default filter to eliminate low-complexity regions within sequences (i.e., amino acid repeats), because low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment (Wootton & Federhen 1996). The SEG filter, however, can confound BLAST queries with sequences that have low-complexity regions within their functional motifs, such as the PXX(3 repeat in CB1 (Reggio et al. 2000) and the GGSSGGEGALI catalytic core in FAAH (Matias et al. 2001). To overcome this potential problem, BLAST searches were done twice, once with the SEG filter on, and once with the filter off. Homologs identified by BLAST can be distinguished as orthologs (gene products found in different organisms, derived by descent from a common ancestor) or paralogs (gene products found in one given organism, derived by a gene duplication event, exampled by CB1 and CB2). Sequences identified by BLAST were considered orthologs if they had greater sequence identity to human CB genes than to any other sequences in that given organism (Tatusov et al. 2000). Homologies were calculated as percent identity (identical amino acid residues), aligned over a designated number of amino acid residues. Homology is considered significant in the presence of >30% identity for >80%

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McPartland & Glass—Nematodes and CB receptors Fig. 1 Comparison of human cannabinoid (CB) receptors CB1, CB2, rat CB 1, and nematode sequences, aligned in two transmembrane regions. A, Transmembrane helix 3, from positions 3.25 to HUMAN CB1 3.52. B, Transmembrane helix 5, RHESUS CB1 from positions 5.39 to 5.65. Amino RAT CB1 acid residues differing from the human CB 1 sequence are printed FINCH CB1 NEWT CB1 in reverse (white on black). Residues known to confer CB F I S H CB1B receptor specificity are boxed. Sin- LEECH CB1 gle-letter abbreviations for amino acid residues are as follows: A, C.e.CEUl5167 Ala; C, Cys; D, Asp; E, Glu; F, C.e.Fl5A8.5 Phe; G, Gly; H, His; I, He; K, Lys; C.e.C02H7.2 L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

B

303 333 33333333333333333333333 « • • - 2 3 3 3333333 4444444444555 2222 567 901 34567890123456789012 FLPK JGG V rASFTASVGSLFLTAIDRYI FLJ K uGG V rASFTASVGSLFLTAIDRYI FLE K LGG V rASFTASVGSLFLTAIDRYI FLE K LJGG V rASFTASVGSLFLTAIDRYI FLE K LGG V rASFTASVGSLFLTAIDRYI FLE K LEG V PASFTASVGSLFLTAIDRYa FLE K LGG V rASg0ASVGSHBLTAIDRYJ3 g 0 H B

5555555555 5555555555555555 4444444444 5555555555666666 0123456789 0123456789012345 LMFWIGVTSV L LLFIVYAYMYILWKA LMFWIGVTSV LLLFIVYAYMYILWKA YLMFWIGVTSV L LLFIVYAYMYILWKA XLMFWIGVTSE LLLFIVYAYMYILWKA LMFWIGVTSE L L J L F I V Y A Y M Y I L W K A

HUMAN CB1 RHESUS CB1 RAT C B 1 FINCH C B 1 NEWT C B l F I S H CB1B C.e.CEU15l67 C.e.Fl5A8.5 C.e.C02H7.2 Y »'AWiarJvi31SE»gJLMiiare»ri51Vfevi.i i i l t c M

of amino acid residues (Rubin et al. 2000). For a significant comparison, the putative CB 1 receptors in other organisms were also BLASTed, with the following GenBank accession (g.i.) numbers: rhesus monkey, 9664881; rat, 111475; finch, 8575561; newt, 8575561; puffer fish, 1545940; and leech (sequence not deposited at GenBank, obtained from Stefano et al. 1997). Sequences with the closest identity to human CB receptors were subsequently analysed for functional motifs in their transmembrane regions, using the numbering scheme of Ballesteros & Weinstein (1995).

SEG filter off. The next greatest fit, C. elegans C02H7.2 (g.i. 1208820), exhibited 23% identity with 61 % of CB 1. Another noteworthy sequence, C. elegans CEU15167 (g.i. number 2317845), exhibited 26% identity with 61% of the CBl sequence; this sequence, however, has been identified as a serotonin receptor. No sequences with significant homology to the CB2 receptor were identified. Close examination of the C. elegans sequences revealed crippling substitutions at significant amino acid residues, discussed below. A non-redundant search of all other nematode sequences deposited at GenBank identified only gene products with much lower identity than the aforementioned C. elegans RESULTS sequences (e.g., Heterodera schachtii, g.i. The C. elegans sequence with greatest BLAST 12585495). alignment, F15A8.5 (g.i. 7499160), exhibited 24% BLASTing with human FAAH identified three identity with 64% of the CB 1 sequence (aligned C. elegans gene products, B0218.1 (g.i. 1326392), with Cb 1 amino acid residues 109 to 411), with the B0218.2 (g.i. 1326390), and F58H7.2 (g.i.

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Table 1 Homologues of human CB1 (cannabinoid) receptors, receptors, with % identity calculated with BLAST 2.0 algorithm. Species

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Monkey (Macaca mulatto) Rat (Rattus norvegicus) Finch (Taeniopygia guttata) Newt (Taricha granulosa) Puffer fish (Fugu rubripes) Leech (Hirudo medicinalis)

% identity with human CB1 gene sequence 100% of 472 amino acids 97% of 473 amino acids 91% of 473 amino acids 83% of 473 amino acids 72% of 468 amino acids 58% of 153 amino acids

2854176), that shared significant identity with human FAAH, exhibiting a mean of 36% identity over 92% of their sequences.

DISCUSSION BLAST did not reveal any nematode gene products with significant identity to human CB receptors. The best C. elegans alignments did not meet the "30% identity for 80% length" cut-off values established by Rubin et al. (2000). In comparison, BLAST identified gene products in other animals with high sequence identity to human CB1 receptors (Table 1). The percentage sequence identity in the other animals varied, due to accumulated mutations. For example, the CB 1 gene from the rhesus monkey was 100% identical to human CB1, whereas the partial CB1 gene cloned from the leech shared only 58% identity with human CB 1. Their percent identity is proportional to the evolutionary distances between them. The primordial ancestors of humans and leeches diverged at least 600 million years ago, so CB genes in the two species had over half a billion years to accumulate differences. In contrast, the CB genes in humans and monkeys had only 10 million years to accumulate differences. Sequence identity is especially important in receptor transmembrane regions. In these locations, the rhesus and rat sequences were 100% identical to human CB1. The finch, newt, and leech sequences exhibited a modest scatter of substituted amino acid residues, whereas the "best fit" C. elegans sequences were rife with substitutions (Fig. 1).

Not to be dissuaded by arbitrary cut-off values, we analysed the C. elegans sequences for functional motifs in their transmembrane regions, using the numbering scheme of Ballesteros & Weinstein (1995). For example, in transmembrane Helix 3, Iysinel92 (position 3.28) is important for CB1 binding affinity (Song & Bonner 1996), and a basic residue at that position appears critical (Chin et al. 1998). None of the C. elegans sequences have a lysine or a basic amino acid residue at that position (Fig. 1A). One turn up at position 3.32, CB1 receptors uniquely have a valine residue (Song et al. 1999). None of the identified C. elegans sequences have a valine residue at 3.32; two of them substitute an aspartate residue, which is characteristic of biogenic amine receptors (Song et al. 1999). In transmembrane Helix 5, tyrosine 275 (position 5.39) is critical for CB receptor binding affinity (Abood et al. 1998); all the nematode sequences have crippling substitutions at this position (Fig. IB). CB receptors uniquely have a leucine residue at 5.50 (Mountjoy et al. 1992), which is lacking in the nematode sequences. In conclusion, the best fit nematode sequences identified by BLAST were not functional CB receptors. The GenBank database incorrectly identifies C. elegans C02H7.2 as a punitive CB receptor, predicted by GENEFINDER (Wilson et al. 1994). GENEFINDER is an algorithm utilised by GenBank to recognise and annotate sequences that resembles known protein-coding genes, but the algorithm does not accurately map functional motifs in transmembrane regions (Bird et al. 1999). The lack of genes encoding CB receptors in C. elegans is remarkable, because its genome encodes c. 1100 GPCRs— c. 5% of C. elegans genes are dedicated to GPCRs (Bargmann 1998). The lack of CB receptors is equally exceptional when one considers the genes are conserved in nearly all animals, including mammals, reptiles, fish, leeches, molluscs, sea urchins, and even the most primitive animal with a nervous system, Hydra vulgaris (Salzet et al. 2000). Equally remarkable is our finding of three C. elegans genes with sequences suggesting they are orthologs of human FAAH. FAAH is a hydrolytic enzyme found in the large "amidase signature family" (Patricelli & Cravatt 2000), which includes amidases from species as incongruent as the yeast Saccharomyces cerevisiae and the bacterium Rhodococcus rhodochrous. Therefore, it is possible that the three genes identified in C. elegans express a non-FAAH amidase, which does not specifically

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McPartland & Glass—Nematodes and CB receptors recognise the endocannabinoids. An example of such an enzyme has been recently reported in the leech (Matias et al. 2001). Indeed, the C. elegans sequences have crippling substitutions in one or two regions shown to be critical for FAAH enzymatic activity. The FAAH catalytic core (GGSSGGEGALI, amino acid residues 215-225) has mutated to GGSSGGESALI in C. elegans B0218.2 and to GGSSGGEAALV in C. elegans F58H7.2. Arreaza & Deutsch (1999) identified a proline-rich domain PPLPFR (amino acid residues 310-315) essential for FAAH function; it has mutated to PPVHWN in B0218.1, to PPVTWN in B0218.2, and PPVKFQ in F58H7.2. Thus the three C. elegans genes identified by BLAST may not express FAAH, but an amidase related to FAAH, such as asparagine amidase (Tsuji et al. 1999). This study examined the complete genome of C. elegans (Nematoda: Rhabditida) because no complete genome is available for a plant-parasitic nematode (Nematoda: Tylenchida). However, the predictive power of C. elegans has been demonstrated in many studies (Bird et al. 1999), and it would be unusual if CB receptors were cloned from another nematode. Evidence suggests the phylum Nematoda displays considerable synteny (conservation of gene order); most genes of other nematodes deposited at GenBank have homologs in the C. elegans genome (Bird et al. 1999). Although Cannabis and cannabis extracts act as nematicides, these effects do not appear to be mediated by CB receptors. These findings are corroborated by the work of Lenicque et al. (1972), who demonstrated that the toxicity of A9-THC against Dugesia tigrina was nearly equalled by that of cannabidiol (LD50 = 13 x 10~7M), a cannabinoid with little affinity for CB receptors (Felder & Glass 1998), suggestive of a non-receptor-mediated mechanism. The effect of cannabinoids on nematodes has not yet been elucidated; analogues of these compounds may prove to be potent nematicidal agents. Although hemp cultivation in New Zealand has been proscribed since 1927, the cultivation of trial plots was recently approved, with a first planting scheduled for this year (Dearnaley 2001 (http:// www.nzherald. co. nz/story display. cfm?the section = n e w s & t h e s u b s e c t i o n = &storyID =179848)). In addition to its other uses, the crop could serve in rotation with nematode-susceptible plants, such as potatoes, maize, peas, grains, and pasture.

305 NOTE ADDED IN PROOF While this manuscript was being reviewed, a paper was published by Elphick & Egertova (2001), which tested for the presence of CB receptors in C. elegans, using a simpler methodology (p-BLAST search without SEG filter manipulation, and no functional analysis of significant residues in transmembrane regions). Although these authors reported slightly different results, they came to the same conclusion, i.e., that nematodes do not express CB receptors.

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