ArticlePDF Available


Two cannabinoid receptors, CB1 and CB2, are expressed in mammals, birds, reptiles, and fish. The presence of cannabinoid receptors in invertebrates has been controversial, due to conflicting evidence. We conducted a systematic review of the literature, using expanded search parameters. Evidence presented in the literature varied in validity, ranging from crude in vivo behavioural assays to robust in silico ortholog discovery. No research existed for several clades of invertebrates; we therefore tested for cannabinoid receptors in seven representative species, using tritiated ligand binding assays with [3H]CP55,940 displaced by the CB1-selective antagonist SR141716A. Specific binding of [3H]CP55,940 was found in neural membranes of Ciona intestinalis (Deuterstoma, a positive control), Lumbricusterrestris (Lophotrochozoa), and three ecdysozoans: Peripatoides novae-zealandiae (Onychophora), Jasus edwardi (Crustacea) and Panagrellus redivivus (Nematoda); the potency of displacement by SR141716A was comparable to measurements on rat cerebellum. No specific binding was observed in Actinothoe albocincta (Cnidaria) or Tethya aurantium (Porifera). The phylogenetic distribution of cannabinoid receptors may address taxonomic questions; previous studies suggested that the loss of CB1 was a synapomorphy shared by ecdysozoans. Our discovery of cannabinoid receptors in some nematodes, onychophorans, and crustaceans does not contradict the Ecdysozoa hypothesis, but gives it no support. We hypothesize that cannabinoid receptors evolved in the last common ancestor of bilaterians, with secondary loss occurring in insects and other clades. Conflicting data regarding Cnidarians precludes hypotheses regarding the last common ancestor of eumetazoans. No cannabinoid receptors are expressed in sponges, which probably diverged before the origin of the eumetazoan ancestor.
Cannabinoid receptors in invertebrates
*GW Pharmaceuticals, Ltd., Salisbury, Wiltshire, UK
Department of Pharmacology, University of Auckland, Auckland, New Zealand
àLandcare Research NZ Ltd, Auckland, New Zealand
§Cawthron Institute, East Nelson, New Zealand
Two cannabinoid receptors have been described to date,
CB1 and CB2. They are G-protein-coupled receptors
(GPCRs) named after their exogenous ligand, D
hydrocannabinol (THC) (Mechoulam et al., 1998). CB1 is
primarily expressed in the central nervous system,
whereas CB2 occurs in leukocytes and immune tissues
(Felder & Glass, 1998). Orthologs of cannabinoid
receptors are expressed in mammals, birds, reptiles,
amphibians, and fish (McPartland, 2004). Within the
vertebrate lineage, cannabinoid receptors track a ‘classic’
evolutionary pattern, with gene duplication in teleost
fish followed by paralog divergence (Yamaguchi et al.,
1996). The evolution of cannabinoid receptors in inver-
tebrates has been disputed, due in part to conflicting
in vivo,in vitro, and in silico evidence. The validity of this
evidence is hierarchical, as indicated by Elphick &
Egertova (2001), and delineated below:
Level 0: in vivo studies, behavioural changes evoked by
cannabis extracts;
Level I: in vivo studies, behavioural changes evoked by
specific cannabinoid ligands;
Correspondence: J. M. McPartland, 53 Washington Street Ext., Middlebury,
VT 05753, USA.
Tel.: +1 802 388 8303; fax: +1 802 399 8304; e-mail:,
G-protein coupled receptor;
Two cannabinoid receptors, CB1 and CB2, are expressed in mammals, birds,
reptiles, and fish. The presence of cannabinoid receptors in invertebrates has
been controversial, due to conflicting evidence. We conducted a systematic
review of the literature, using expanded search parameters. Evidence
presented in the literature varied in validity, ranging from crude in vivo
behavioural assays to robust in silico ortholog discovery. No research existed for
several clades of invertebrates; we therefore tested for cannabinoid receptors
in seven representative species, using tritiated ligand binding assays with
H]CP55,940 displaced by the CB1-selective antagonist SR141716A. Specific
binding of [
H]CP55,940 was found in neural membranes of Ciona intestinalis
(Deuterstoma, a positive control), Lumbricus terrestris (Lophotrochozoa), and
three ecdysozoans: Peripatoides novae-zealandiae (Onychophora), Jasus edwardi
(Crustacea) and Panagrellus redivivus (Nematoda); the potency of displacement
by SR141716A was comparable to measurements on rat cerebellum. No
specific binding was observed in Actinothoe albocincta (Cnidaria) or Tethya
aurantium (Porifera). The phylogenetic distribution of cannabinoid receptors
may address taxonomic questions; previous studies suggested that the loss of
CB1 was a synapomorphy shared by ecdysozoans. Our discovery of cannabi-
noid receptors in some nematodes, onychophorans, and crustaceans does not
contradict the Ecdysozoa hypothesis, but gives it no support. We hypothesize
that cannabinoid receptors evolved in the last common ancestor of bilaterians,
with secondary loss occurring in insects and other clades. Conflicting data
regarding Cnidarians precludes hypotheses regarding the last common
ancestor of eumetazoans. No cannabinoid receptors are expressed in sponges,
which probably diverged before the origin of the eumetazoan ancestor.
doi: 10.1111/j.1420-9101.2005.01028.x
Level II: in vitro studies, effects of cannabinoid ligands
upon signal transduction effectors, or immunohisto-
chemical studies using tagged antibodies raised against
Level III: in vitro studies, tritiated ligand binding assays, or
PCR cloning techniques;
Level IV: in silico studies, ortholog identification in
whole-genome sequences.
In vivo studies provide the lowest levels of evidence.
For example, Parkinson (1640), described a Cannabis
extract exerting in vivo behavioural effects, ‘poured
into the holes of earthwormes, will draw them forth, and
fishermen have used this feate to get wormes to baite
their hookes’. This evidence is nearly anecdotal, given
the polypharmaceutical content of crude Cannabis. The
behavioural effects elicited by Cannabis could have been
caused by an entourage of compounds acting at a variety
of targets. The next level of evidence (Level I) appraises
the behavioural effects of specific cannabinoid receptor
agonists and antagonists. For example, Buttarelli et al.
(2002) exposed the planaria worm Dugesia gonocephala to
WIN55212-2, which elicited dose-dependent changes in
motor behaviour, and this effect was attenuated by the
CB1 antagonist SR141716A.
In vitro assays offer more precise levels of evidence, such
as the Level-II study of THC’s effects at the neuromuscular
junction of the lobster Homarus americanus (Turkanis &
Karler, 1988). This study provided indirect proof of
cannabinoid receptors, because THC could have affected
the neuromuscular junction via other lobster protein
targets or via membrane disrupting effects. Immunohis-
tochemical assays that use tagged antibodies raised against
mammalian CB1 also represent Level-II evidence, suscep-
tible to Type-I error (false positives due to cross reactions
with other proteins). On the other hand, detection of
specific binding sites with [
H]CP55,940 and other tritiated
cannabinoids represents Level-III evidence, especially
when the potency of binding displacement is compared
to mammalian receptors (Elphick & Egertova, 2001).
Cloning of CB1 genes by reverse-transcription polymerase
chain reaction (RT-PCR) technology is Level III, possibly
prone to Type-I error: a cannabinoid receptor gene cloned
from the leech Hirudo medicinalis (Stefano et al., 1997b)
was contested as an artifact of PCR contamination
(Elphick, 1998). Similarly, a cannabinoid gene cloned
from the fruit fly Drosophila melanogaster (Abbott, 1990)
has been refuted by a tritiated ligand binding study
(McPartland et al., 2001) and in silico studies (Elphick &
Egertova, 2001, McPartland et al., 2001).
Whole genome in silico studies provide a rigorous level
of evidence, Level IV. Thanks to high-throughput
sequencing and advances in computational biology, the
entire genomes of many organisms have been sequenced
and deposited in internet-accessible databases. Using this
approach, no orthologs of CB1 or CB2 were found in the
genome of the fruit fly D. melanogaster (Elphick &
Egertova, 2001; McPartland et al., 2001) and the
nematode Caenorhabditis elegans (Elphick & Egertova,
2001; McPartland et al., 2001). Unfortunately the in silico
approach poorly assesses invertebrates, because only four
invertebrate genomes have been sequenced, namely sea
squirt, Ciona intestinalis (a deuterostome), and three
protostomes: fruit fly, D. melanogaster; mosquito, Anoph-
eles gambiae; and nematode, C. elegans. To wit, all three
protostomes are members of the Ecdysozoa clade, a
recently described clade of invertebrates (Aguinaldo
et al., 1997). No genomes have been sequenced from
the other major protostome clade (the Lophotrochozoa),
or from basal animals, such as cnidarians and sponges.
Evidence suggests cannabinoid receptors evolved in basal
animals, based on a Level-III study of the cnidarian Hydra
vulgaris (De Petrocellis et al., 1999). Yet the receptors
evidently lack expression in D. melanogaster and C. elegans,
as aforementioned. This led McPartland et al. (2001) to
hypothesize that cannabinoid receptors evolved in early
metazoans, at least 525 million years ago (MYA), but
were secondarily lost in the Ecdysozoa.
The purpose of this study was three-fold: (1) conduct a
systematic review of the literature concerning cannabi-
noid receptors in invertebrates; (2) utilize this literature
database to identify subgroups of invertebrates that have
not been studied; (3) search for cannabinoid receptors in
organisms representative of subgroups lacking data.
Rather than using RT-PCR technology, which has been
problematic when cloning cannabinoid receptors, we
utilized tritiated ligand binding assays, coupled with
comparisons to mammalian receptors. Tritiated ligand
binding results were then scrutinized within the context
of evidence assembled in the systematic review, weighed
with respect to levels of evidence.
Materials and methods
Systematic review
MEDLINE (1966-June 2005) and AGRICOLA (1990-June
2005) were searched using MeSH keywords alone or in
various Boolean combinations: invertebrate, cannabi-
noids, cannabinoid receptors, THC, cannabis, marijuana.
All reports were scanned for supporting citations; antece-
dent sources were retrieved, except for doctoral disserta-
tions. Unindexed conference proceedings and textbooks
were scanned by hand. Data validity was assessed by
source (peer-reviewed journal article vs. chapter in edited
book vs. conference proceeding abstract), experimental
methodology (segregated by levels of evidence, Table 1)
and the frequency of independent observations.
Tissue sampling
Based on the literature review, seven groups were
selected for experimentation: a deuterostome (positive
control), lophotrochozoan, three sub-clades within the
Ecdysozoa. a cnidarian, and a sponge. The UNITEC
Cannabinoid receptors in invertebrates 367
institutional review board approved the study protocol
prior to the experiment. Seven species were assayed:
(1) Sea squirt, C. intestinalis (Chordata-Urochordata),
30 individuals collected from infralittoral mussel beds in
the Marlborough Sounds, and couriered to Auckland in
sea water on ice. Harvested tissues included neural
ganglia and neural gland, testis, intestine (which may
include peyer’s patch-like lymphoid tissues), and hemo-
lymph (cells that exhibit immune functions) mixed with
heart tissue. (2) Earthworm, Lumbricus terrestris (Lophot-
rochozoa-Annelida-Oligochaeta), about 100 individuals
were purchased from Biosuppliers, Ltd, commercial
insectary, Auckland. The cephalic end was opened at
the prostomium and cerebral ganglia removed from
around the anterior pharynx. (3) Velvet worm, Peripato-
ides novae-zealandiae (Ecdysozoa-Onychophora-Peripati-
dae), 25 individuals were collected from a protected
location near Auckland (under permit by Landcare
Research, NZ Ltd). The cephalic end was decapitated
and adhesive secretions from head glands removed. (4)
Rock lobster, Jasus edwardi (Ecdysozoa-Arthropoda-
Crustacea), one individual, sourced from Sea World,
Auckland. Brain tissue (subesophageal ganglion) was
dissected from the dorsal aspect of the head and thorax.
Identity of the ganglion was confirmed by tracing the
optic nerves caudally. (5) Beer mat nematode, Panagrellus
redivivus (Ecdysozoa-Nematoda-Rhabditida), several hun-
dred individuals were purchased from Biosuppliers, Ltd,
commercial insectary, Auckland. Whole organisms were
sonicated and then homogenized. (6) Common variable
Table 1 Summary of in vivo,in vitro,and
in silico evidence for the presence (+) or
absence ()) of cannabinoid receptors in
invertebrates (n.d. indicates no data).
Taxa Level IV Level III Level II Level I Level 0
Primates, rodents, etc. (+)
Sea squirt Ciona intestinalis (+)
n.d. n.d.
Sea urchin Strongylocentrus spp. n.d. (+)
n.d. n.d.
Earthworm Lumbricus terrestris n.d. (+)
n.d. n.d. (+)
Leech Hirudo medicinalis n.d. (+)
n.d. n.d.
Planaria Girardia tigina n.d. n.d. (+)
n.d. n.d.
Mussel Mytilus edulis n.d. (+)
Sea slug Aplysia californica n.d. (–)
n.d. n.d.
Fruit fly Drosophila melanogaster (–)
n.d. n.d. n.d.
Mosquito Anopheles gambiae (–)
n.d. n.d. n.d. n.d.
Honeybee Apis melifera n.d. (–)
n.d. n.d. (–)
Ant Formica pratensis n.d. (–)
n.d. n.d. (–)
Locust Shistocerca gregaria n.d. (+)
n.d. n.d. n.d.
Nematode Caenorhabditis elegans (–)
n.d n.d. n.d. n.d.
Mat nematode Panagrellus redivivus n.d. (+)
n.d. n.d. n.d.
Lobster Homarus americanus n.d. (+)
n.d. n.d. n.d.
Rock lobster Jasus edwardi n.d. (+)
n.d. n.d. n.d.
Velvet worm P. novaen.d.zelandiae n.d. (+)
n.d. n.d. n.d.
Hydra Hydra vulgaris n.d. (+)
n.d. (+)
Sea anomone Actinothoe albocincta n.d. (–)
n.d. n.d. n.d.
Sponge Tethya aurantium n.d. (–)
n.d. n.d. n.d.
Elphick & Egertova (2001);
Felder & Glass (1998);
Chaperon & Theibot (1999);
et al. (2003);
Matias et al. (2005);
current study;
Chang et al. (1993);
Schuel et al. (1987);
Chang et al. (1991);
Chang & Schuel (1991);
Schuel et al. (1991a);
Schuel et al. (1991b);
Schuel et al. (1994);
Berdyshev (1999);
Parkinson (1640);
Stefano et al. (1996);
Stefano et al. (1997b);
Elphick (1998);
Stefano et al. (1997a);
Salzet et al. (1997);
Stefano et al. (1998);
Matias et al. (2001);
Lenicque et al. (1972);
Buttarelli et al.
Howlett et al. (1990);
Acosta-Urquidi & Chase (1975);
McPartland et al. (2001);
McPartland (2004);
Howlett et al. (2000);
McPartland et al. (2000);
Waser (1999);
Egertova et al. (1998);
McPartland & Glass (2001);
Turkanis & Karler (1988);
Petrocellis et al. (1999).
sea anomone, Actinothoe albocincta (Cnidaria, Anthozoa),
one individual was collected from the intertidal zone of a
rocky pool at Piha Beach. Tissues from around the oral
disc were dissected, a combination of peri-oral sphincter
muscles, tentacles, and actinopharynx tissues. (7) Golf
ball sponge, Tethya aurantium (Porifera-Epipolasida-
Tethyidae), six individual colonies were collected from
the infralittoral zone at Piha Beach (Auckland, New
Zealand). Individuals were dissected in half and squeezed
to express cellular material from spiculae.
Binding assays
Organisms were cold-anaesthetized by chilling for
30 min at 4 C, then moved to a dissection tray atop a
bed of dry ice (frozen CO
) and instantly frozen or
asphyxiated with sublimated CO
gas. Dissected tissue
were immediately homogenized in ice-cold lysis buffer
(10 m
MOPS, 1 m
EDTA, 10 l
AEBSF pH 7.5), and
sequentially centrifuged to obtain a pellet of cell mem-
branes, as described previously (McPartland et al., 2001).
Membrane protein concentrations were determined via
standardized ‘Biorad D
’ assay following the manufac-
turers’ protocol. Ligand binding assays used established
methods (Kearn et al., 1999; McPartland et al., 2001),
utilizing [
H]CP55,940 (NEN Life Sciences), a synthetic
CB1 and CB2 receptor agonist. Nonspecific binding was
determined by displacement with SR141716A, a CB1
selective antagonist. Briefly, membrane samples (15–
40 lg) were incubated with 6.3 n
(158.00 Ci mmol
), in the presence or absence of
100 n
SR141716A in TME assay buffer (50 m
MgCl2, 1 m
EDTA, 5 mg mL
BSA for 90 min at
37 C. The incubation was terminated by addition of
200 lL ice cold TME buffer, and samples were filtered
though a printed filtermat A (GF-C) filter (Perkin Elmer)
presoaked in 0.1% polyetheleneimine, and washed twice
with 300 lL TME buffer on an Inotech cell harvester. The
filter was then dried prior to the addition of Multilex
(melt-on scintillant; Perkin Elmer) and was counted for
5 min in a Wallac Microbeta Trilux (Perkin Elmer).
Experiments were performed twice in triplicate. Positive
binding results were then extended by displacement
analysis for three representative organisms, L. terristus,
P. redivivus, and P. novae-zealandiae. Membranes were
incubated with 6.3 n
H]CP55,940 in the presence of
increasing concentrations of SR141716A (10
). Samples were filtered and counted as above.
Displacement curves were generated by nonlinear regres-
sion utilizing Graph Pad Prism, Version 4.0.
Systematic review
Publications regarding cannabinoid receptors and inver-
tebrates were located in peer-reviewed journals (n¼25),
chapters in edited books (n¼3), conference proceedings
abstracts (n¼2), and older, nonpeer reviewed sources
(n¼2). The total (n¼32) did not include conference
abstracts that were subsequently republished as peer-
reviewed articles, nor did the tally include dissertations.
In terms of levels of evidence, the literature distributed in
a skewed histogram: Level 0, n¼3; Level I, n¼6; Level
II, n¼14; Level III, n¼13; Level IV, n¼6. Note that
several publications reported several experiments, at
several levels of evidence. A summary of these publica-
tions is presented in Table 1, arrayed phylogenetically.
Binding assays
Specific, displaceable binding of [
H]CP55,940 was
observed in all deuterostomes, lophotrochozoans, and
ecdysozoans; no specific binding was observed in cnidar-
ian and poriferan species (Table 2). Nondisplaceable
binding levels were comparable between all tissues.
Percentage specific binding was highest in C. intestinalis
neural tissue and haemolymph, followed by tissues from
P. redivivus, L. terrestris, P. novae-zealandiae, and J. edwardi
(Table 2). Low receptor binding was observed in C. intes-
tinalis testis and intestine tissues.
In order to determine the binding characteristics of
these putative receptors, full competition binding assays
were performed for L. terristus,P. novae-zealandiae, and
P. redivivus (Fig. 1), Log IC50 values obtained from
displacement curves were )11.20 ± 0.12
,)10.93 ±
, and )11.21 ± 0.2
, respectively. For the pur-
poses of comparison, binding was performed under
identical conditions on rat cerebellum, which has estab-
lished high expression of CB1 receptors, and produced a
Log IC50 value of )10.17 ± 0.12
(Fig. 1).
Table 2 Concentrations of [
H]CP55,940 specific binding sites
observed in membrane preparations from seven invertebrates and
rat cerebellum.
Species, tissue fmol/mg Specific binding (%)
Tethya aurantium ND )2±8
Actinothoe albocincta ND )1±3
Peripatoides novae-zealandiae 90 ± 11 52 ± 3
Panagrellus redivivus 55±5 58±1
Jasus edwardi 104±3 41±2
Lumbricus terristus 78±5 56±2
Ciona intestinalis–neural tissue 134 ± 3 76 ± 10
Ciona intestinalis–intestine ND 11 ± 11
Ciona intestinalis–haemolymph 35 ± 2 60 ± 2
Ciona intestinalis–testis 30 ± 6 45 ± 14
Rat cerebellum 648 ± 16 48 ± 1
Results are presented as the mean ± SEM, for two experiments
performed in triplicate. The % specific binding represents the
proportion of the total binding observed that could be displaced by
100 n
SR141716A. Specific binding not significantly different to
zero was classed as no receptors being detected (ND).
Cannabinoid receptors in invertebrates 369
Tritiated ligand binding assays detected specific cannabi-
noid binding in all organisms tested except sea anemone
(A. albocincta) and sponge (T. aurantium). The receptors
were identified by binding of the nonspecific cannabi-
noid agonist [
H]CP55,940 displaced by the highly CB1
selective antagonist SR141716A, and therefore are con-
sistent with cannabinoid CB1, but not CB2 receptors. In
three of the organisms tested, earthworm (L. terristus),
velvet worm (P. novae-zealandiae), and mat nematode
(P. redivivus), sufficient tissue was available to carry out
competition binding assays, in comparison to a well
characterized CB1 ortholog in rat cerebellar tissue. In all
three organisms, a high-affinity binding interaction was
observed for SR141716A at various concentrations,
consistent with rat cerebellar tissue and characteristic of
CB1 receptors (Fig. 1).
The phylogenetic distribution of cannabinoid receptors
may address larger taxonomic questions. Invertebrate
taxonomy has recently been updated by the Ecdysozoa
hypothesis, which classifies protostome invertebrates by
their ability or inability to molt (Aguinaldo et al., 1997).
Under the Ecdysozoa arrangement, animals are grouped
into five clades: the Deuterostomes (vertebrates as well as
some invertebrates such as sea squirts and sea urchins),
Lophotrochozoans (protostomes that do not molt: anne-
lids, mollusks, platyhelminths), Ecdysozoans (protos-
tomes that molt: insects, crustaceans, nematodes,
onychophorans), Cnidarians (hydras, sea anemones,
and other nonbilaterian animals), and Poriferans (spon-
ges, primitive multicellular animals whose cells do not
form tissues or organs). Prior in vitro and in silico studies
indicated a lack of cannabinoid receptors in insects and
nematodes, which led McPartland et al. (2001) to propose
that cannabinoid receptors were secondarily lost in the
Ecdysozoa. However, our new binding studies suggest
that cannabinoid receptors are present in three species
representing three classes of animals within the Ecdyso-
zoa, P. novae-zealandiae (Ecdysozoa-Onychophora),
J. edwardi (Ecdysozoa-Crustacea), and P. redivivus
(Ecdysozoa-Nematoda). The presence of cannabinoid
receptors in onychophorans, crustaceans, and nematodes
does not contradict the Ecdysozoa hypothesis, but gives it
no support.
We also conducted ligand-binding studies upon a
lophotrochozoan, cnidarian, and sponge. These clades
had not been represented by good evidence in our
systematic review. Unique methods of reviewing the
literature (e.g. sourcing agricultural databases and hand-
scanning unindexed publications) proved successful. The
systematic review located 32 publications regarding
cannabinoid receptors in invertebrates, whereas 15 pub-
lications were cited by Elphick & Egertova (2001)
(including dissertations), and 14 publications were cited
by Salzet & Stefano (2002). The following discussion will
be presented under the Ecdysozoa arrangement, grouped
into five clades (see Table 1).
The high-affinity [
H]CP55,940 binding site we found in
C. intestinalis is presumably the cannabinoid receptor
ortholog that Elphick et al. (2003) characterized as the
descendant of a receptor that predated the CB1–CB2
duplication event. Our results with [
H]CP55,940 dis-
placed by the highly CB1 selective antagonist
SR141716A suggest the ancestral sequence may have
functioned more like present-day CB1 than CB2.
Another recent [
H]CP55,940 binding study of C. intes-
tinalis (Matias et al., 2005) reported a lower percentage
specific binding in cerebral ganglion than we report
herein. Evidence for cannabinoid receptors in sea urchins
included a slew of Level-II studies on Strongylocentrotus
purpuratus sperm cells (Berdyshev, 1999; Chang et al.,
1991; Chang & Schuel, 1991; Schuel et al.,
1991a,b,1994,1987), plus a Level-III study-specific bind-
ing of [
H]CP55,940, with dose–dependent displacement
by THC (Chang et al., 1993).
We found specific binding of [
H]CP55,940 in neural
membranes of L. terristus, extended by measuring the
potency of binding displacement with SR141716A (Log
IC50 ¼)11.20 ± 0.12
), comparable to measurements
performed under identical conditions on rat cerebellum
(Log IC50 ¼)10.17 ± 0.12). These results lends credi-
bility to Level-I evidence regarding earthworms
(Parkinson, 1640), and Level-II evidence regarding
planaria worms (Buttarelli et al., 2002; Lenicque et al.,
1972). Our evidence also reflects upon the controversy
involving a related annelid, the leech H. medicinalis.
Evidence suggests the leech expresses cannabinoid
receptors, based on Level-II studies (Stefano et al.,
1996,1997a,1998; Salzet et al., 1997; Matias et al.,
2001), and a tritiated ligand binding study that used
–14 –13 –12 –11 –10 –9 –8
Lumbricus terristus
Panagrellus redivivus
Rat c ereb ellum
940 bound)
(% of maximum)
Fig. 1 Displacement of 6.3 n
H]CP55,940 by increasing con-
centrations of SR141716A in 40 lgofLumbricus terristus,Peripatoides
novae-zealandiae, Panagrellus redivivus and rat cerebellum. Values are
expressed as mean ± SEM for values normalized to binding in the
absence of SR141716A in all cases.
an unusual ligand, [
H]anandamide (Stefano et al.,
1996). PCR cloning of a cannabinoid receptor gene
(Stefano et al., 1997b) has been contested (Elphick,
1998; Elphick & Egertova, 2001).
Studies on mollusks (mussel, Mytilus edulis and sea
slug, Aplysia californica) produced four positive studies
and one negative study: M. edulis cannabinoid suppres-
sion of dopamine release, antagonized by SR141716A
(Level II, Stefano et al., 1997a); M. edulis cannabinoid-
stimulated release of nitric oxide, blocked by SR141716A
(Level II, Stefano et al., 1998); M. edulis tritiated ligand
binding study (Level III, Stefano et al., 1996); A. califor-
nica cannabinoid suppression of nerve cell excitability
(Level II, Acosta-Urquidi & Chase, 1975); whereas
Howlett et al. (1990) reported no specific binding of
H]CP55,940 in A. californica (Level III). The binding
assay used by Howlett and colleagues may have lacked
power (displacement with weaker agonists, not
SR141716A), they also reported no binding in a verteb-
rate, which seems unlikely (a lamprey called Ichthyomy-
zon intercostus but probably I. unicuspis).
Level-IV studies indicate that at least two insects,
D. melanogaster and A. gambiae, lack cannabinoid
receptors (Elphick & Egertova, 2001; McPartland,
2004; McPartland et al., 2001). This agreed with one
tritiated ligand binding study that reported no specific
binding of [
H]CP55,940 or [
H]SR141716A in a panel
of species spanning the Insecta: D. melanogaster, Apis
mellifera, Gerris marginatus, Spodoptera frugiperda, and
Zophobas atratus (McPartland et al., 2001), but disagreed
with two positive tritiated ligand binding studies.
Egertova et al. (1998) reported 5% specific binding of
H]CP55,940 in the locust Schistocerca gregaria, but
questioned their findings. Howlett et al. (2000) detected
specific binding of [
H]CP55,940 in D. melanogaster,
although the binding was not displaced by CB1-specific
SR141716A or CB2-specific SR144528. In a Level-II
study, the moth Pieris brassicae demonstrated beha-
vioural changes when exposed to THC (Rothschild &
Fairbairn, 1980). But nearly identical behaviour was
elicited by cannabidiol (CBD), a ligand with little
affinity for CB receptors, suggesting behavioural
changes were not mediated by cannabinoid receptors.
Waser (1999) fed THC to the ant Formica pratensis and
no change in behaviour was noted, even though the
drug was absorbed and reached concentrations of
800 pg THC per ant brain. McPartland et al. (2000)
reported no change in behaviour in honeybee
(A. melifera) and ant (Formica sp.) that fed upon high-
THC plants vs. high-CBD plants.
Regarding crustaceans, THC suppressed neuromuscu-
lar junction activity in the lobster H. americanus (Level-II
evidence, Turkanis & Karler, 1988). This positive study
was supported by our tritiated ligand binding study
with the rock lobster J. edwardi. The only onychophoran
examined to date has been the velvet worm P. novae-
zealandiae, whose neural tissues exhibited high-affinity
binding of [
H]CP55,940 displaced by SR141716A, at
potencies consistent with rat cerebellar tissue. Our
tritiated binding assay also produced positive results with
the nematode P. redivivus (Fig. 1). This conflicts
with negative Level-IV studies of the related nematode
C. elegans (Elphick & Egertova, 2001; McPartland et al.,
2001). The discrepancy may be due to C. elegans’s
stripped-down genome, resulting in a high rate of
character loss (Copley et al., 2004); perhaps another
‘idiosyncrasy’ of C. elegans is the loss of cannabinoid
receptor genes.
De Petrocellis et al. (1999) presented strong evidence for
cannabinoid receptors in H. vulgaris (Cnidaria, Class
Hydroza), the most primitive animal with a nervous
system. Cannabinoids induced a Hydra feeding response,
blocked by SR141716A (Level II), and specific, displace-
able cannabinoid binding sites were detected with
H]SR141716A (Level III). This conflicts with our results
on a related species, A. albocincta (Cnidaria, Class Antho-
zoa). We cannot explain the discrepancy, although
A. albocincta was the most difficult specimen to identify
neural tissue for dissection, perhaps resulting in a Type-II
Systematic review of the literature uncovered no publi-
cations regarding evidence of cannabinoid receptors in
sponges. We found no specific binding of [
in T. aurantium (Porifera-Epipolasida-Tethyidae).
New information provided by our tritiated ligand binding
study, together with previous findings located in a
systematic review of the literature, indicates the presence
of cannabinoid receptors in all major subdivisions of
bilaterians (deuterostomes, lophotrochozoans, and
ecdysozoans). This information suggests cannabinoid
receptors evolved in the last common ancestor of
bilaterians. Conflicting data regarding Cnidarians (pre-
sent in Hydra, absent in A. albocincta) precludes us from
commenting upon the last common ancestor of eumeta-
zoans. No cannabinoid receptors are expressed in spon-
ges, which probably diverged before the origin of the
eumetazoan ancestor.
The most parsimonious explanation for the lack of
cannabinoid receptors in insects and some nematodes is
secondary loss, Secondary simplification in the Ecdyso-
zoa is also seen in Hox genes (de Rosa et al., 1999),
steroid receptors (Escriva et al., 2000), b-thymosin
Cannabinoid receptors in invertebrates 371
orthologs (Manuel et al., 2000), hedgehog genes (Kang
et al., 2003), and the Wnt gene family (Kusserow et al.,
2005). Loss of cannabinoid receptors is not a synapo-
morphic character shared by all ecdysozoans, however,
as shown by our results with P. novae-zealandiae,
J. edwardi, and P. redivivus. Selection pressures leading
to the loss of cannabinoid receptors in insects and some
nematodes is open to conjecture, as is the functional
relevance of this loss. Transgenic ‘knockout mice’ that
lack CB1 receptors have been generated, and they
survive and reproduce, but they suffer increased mor-
bidity and premature mortality (Zimmer et al., 1999).
CB1 knockout mice show greater aggression, anxiogenic-
like behaviour, depressive-like behaviour, anhedonia,
and they develop fear of newness (Martin et al., 2002).
Lack of cannabinoid receptors in knockout mice and in
insects demonstrates that the receptors may profoundly
alter consciousness, but the receptors did not evolve as a
prerequisite for consciousness. After all, cannabinoid
receptors are absent in honeybees, so the receptors are
not required for spatial memory, goal-directed desires,
elements of deception, and symbolic communication
through dance.
It is worth noting that insects continue to biosynthe-
size sn-2 arachidonyl glycerol (2-AG), an endogenous
ligand of CB1 and CB2, even in the absence of the
receptors (McPartland et al., 2001). This supports Hoyle
(1999), who hypothesized that there is greater evolu-
tionary pressure to conserve ligands than to conserve
receptors. However, insects do not biosynthesize anand-
amide (AEA), the other endogenous lignad of CB1 and
CB2. AEA is a metabolite of arachidonic acid that arises at
the sn-1 position of membrane phospholipids; 2-AG is
metabolized from arachidonic acid at the sn-2 position of
phospholipids (Mechoulam et al., 1998). Insects produce
very little arachidonic acid, especially at the sn-1 position
(Stanley-Samuelson & Pedibhotla, 1996). This may
explain the dearth of AEA in insects, but does it explain
the loss of receptors?
In summary, we hypothesize that the evolution of
cannabinoid receptors was linked to the evolution of
multi-cellular animals. This agrees with the current
employment of cannabinoid receptors in cell-to-cell
communication. The loss of cannabinoid receptors in
some organisms remains an enigma. Future studies are
clearly needed, particularly focusing upon the Nematoda
and Cnidaria, where enhanced phylogenetic resolution is
This work was funded by an unrestricted grant from GW
Pharmaceuticals, with additional financial support from a
UNITEC (NZ) AssocProf research grant. We thank Patricia
Pruitt, DVM, for Ciona dissections, Michelle Kelly, PhD,
for Tethya recommendations, and Katherine Blake-
Palmer for technical support.
Abbott, A. 1990. The switch that turns the brain on to cannabis.
New Scientist 127: 31.
Acosta-Urquidi, J. & Chase, R. 1975. The effects of delta9-
tetrahydrocannabinol on action potentials in the mollusc
Aplysia. Can. J. Physiol. Pharmacol. 53: 793–798.
Aguinaldo, A.M., Turbeville, J.M., Linford, L.S., Rivera, M.C.,
Garey, J.R., Raff, R.A. & Lake, J.A. 1997. Evidence for a clade
of nematodes, arthropods and other moulting animals. Nature
387: 489–493.
Berdyshev, E.V. 1999. Inhibition of sea urchin fertilization by
fatty acid ethanolamides and cannabinoids. Comp. Biochem.
Physiol. C. Pharmacol. Toxicol. Endocrinol. 122: 327–330.
Buttarelli, F.R., Pontieri, F.E., Margotta, V. & Palladini, G. 2002.
Cannabinoid-induced stimulation of motor activity in planaria
through an opioid receptor-mediated mechanism. Prog.
Neuropsychopharmacol. Biol. Psychiatry. 26: 65–68.
Chang, M.C., Berkery, D., Laychock, S.G. & Schuel, H. 1991.
Reduction of the fertilizing capacity of sea urchin sperm by
cannabinoids derived from marihuana. III. Activation of
phospholipase A2 in sperm homogenate by delta 9-tetrahy-
drocannabinol. Biochem. Pharmacol. 42: 899–904.
Chang, M.C., Berkery, D., Schuel, R., Laychock, S.G., Zimmer-
man, A.M., Zimmerman, S. & Schuel, H. 1993. Evidence for a
cannabinoid receptor in sea urchin sperm and its role in
blockade of the acrosome reaction. Mol. Reprod. Dev. 36: 507–
Chang, M.C. & Schuel, H. 1991. Reduction of the fertilizing
capacity of sea urchin sperm by cannabinoids derived from
marihuana. II. Ultrastructural changes associated with inhibi-
tion of the acrosome reaction. Mol. Reprod. Dev. 29: 60–71.
Chaperon, F. & Theibot, M. 1999. Behavioural effects of cannabi-
noid agents in animals. Critic. Rev. Neurobiol. 13: 243–281.
Copley, R.R., Aloy, P., Russell, R.B. & Telford, M.J. 2004.
Systematic searches for molecular synapomorphies in model
metazoan genomes give some support for Ecdysozoa after
accounting for the idiosyncrasies of Caenorhabditis elegans.
Evol. Dev. 6: 164–169.
De Petrocellis, L., Melck, D., Bisogno, T., Milone, A. & Di Marzo,
V. 1999. Finding of the endocannabinoid signalling system in
Hydra, a very primitive organism: possible role in the feeding
response. Neuroscience 92: 377–387.
de Rosa, R., Grenier, J.K., Andreeva, T., Cook, C.E., Adoutte, A.,
Akam, M., Carroll, S.B. & Balavoine, G. 1999. Hox genes in
brachiopods and priapulids and protostome evolution. Nature
399: 772–776.
Egertova, M., Cravatt, B.F. & Elphick, M.R. 1998. Phylogenetic
Analysis of Cannabionoid Signalling, p. 101.International Can-
nabinoid Research Society, Burlington, VT.
Elphick, M.R. 1998. An invertebrate G-protein coupled receptor
is a chimeric cannabinoid/melanocortin receptor. Brain Res.
780: 170–173.
Elphick, M.R. & Egertova, M. 2001. The neurobiology and
evolution of cannabinoid signalling. Philos. Trans. R. Soc. Lond.
B. Biol. Sci. 356: 381–408.
Elphick, M.R., Satou, Y. & Satoh, N. 2003. The invertebrate
ancestry of endocannabinoid signalling: an orthologue of
vertebrate cannabinoid receptors in the urochordate Ciona
intestinalis. Gene 302: 95–101.
Escriva, H., Delaunay, F. & Laudet, V. 2000. Ligand binding and
nuclear receptor evolution. Bioessays 22: 717–727.
Felder, C.C. & Glass, M. 1998. Cannabinoid receptors and their
endogenous agonists. Annu. Rev. Pharmacol. Toxicol. 38: 179–
Howlett, A.C., Bidaut-Russell, M., Devane, W.A., Melvin, L.S.,
Johnson, M.R. & Herkenham, M. 1990. The cannabinoid
receptor: biochemical, anatomical and behavioral character-
ization. Trends Neurosci. 13: 420–423.
Howlett, A.C., Mukhopadhyay, S., Wilken, G.H. & Neckamyer,
W.S. 2000. A cannabinoid receptor in Drosophila is pharma-
cologically unique. Soc. Neurosci. Abstracts 26: 2165.
Hoyle, C.H.V. 1999. Neuropeptide families and their receptors:
evolutionary perspectives. Brain Res. 848: 1–25.
Kang, D., Huang, F., Li, D., Shankland, M., Gaffield, W. &
Weisblat, D.A. 2003. A hedgehog homolog regulates gut
formation in leech (Helobdella). Development 130: 1645–1657.
Kearn, C.S., Greenberg, M.J., DiCamelli, R., Kurzawa, K. &
Hillard, C.J. 1999. Relationships between ligand affinities for
the cerebellar cannabinoid receptor CB1 and the induction of
GDP/GTP exchange. J. Neurochem. 72: 2379–2387.
Kusserow, A., Pang, K., Sturm, C., Hrouda, M., Lentfer, J.,
Schmidt, H.A., Technau, U., von Haeseler, A., Hobmayer, B.,
Martindale, M.Q. & Holstein, T.W. 2005. Unexpected com-
plexity of the Wnt gene family in a sea anemone. Nature 433:
Lenicque, P.M., Paris, M.R. & Poulot, M. 1972. Effects of some
components of Cannabis sativa on the regenerating planarian
worm Dugesia tigrina. Experientia 28: 1399–1400.
Manuel, M., Kruse, M., Muller, W.E. & Le Parco, Y. 2000. The
comparison of beta-thymosin homologues among metazoa
supports an arthropod-nematode clade. J. Mol. Evol. 51: 378–
Martin, M., Ledent, C., Parmentier, M., Maldonado, R. &
Valverde, O. 2002. Involvement of CB1 cannabinoid receptors
in emotional behaviour. Psychopharmacology 159: 379–387.
Matias, I., Bisogno, T., Melck, D., Vandenbulcke, F., Verger-
Bocquet, M., De Petrocellis, L., Sergheraert, C., Breton, C., Di
Marzo, V. & Salzet, M. 2001. Evidence for an endocannabi-
noid system in the central nervous system of the leech Hirudo
medicinalis. Brain Res. Mol. Brain. Res. 87: 145–159.
Matias, I., Di Marzo, V. & McPartland, J.M. 2005. Occurrence and
possible biological role of the endocannabinoid system in the
sea squirt Ciona intestinalis. J. Neurochemistry 93: 1141–1156.
McPartland, J.M. 2004. Phylogenomic and chemotaxonomic
analysis of the endocannabinoid system. Brain Res. Brain Res.
Rev. 45: 18–29.
McPartland, J.M., Di Marzo, V., De Petrocellis, L., Mercer, A. &
Glass, M. 2001. Cannabinoid receptors are absent in insects.
J. Comp. Neurol. 436: 423–429.
McPartland, J.M. & Glass, M. 2001. The nematocidal effects of
Cannabis may not be mediated by cannabinoid receptors. NZ
J. Crop Horticultural Sci. 29: 301–307.
McPartland, J.M., Clarke, R.C. & Watson, D.P. 2000. Hemp
Diseases and Pests. CABI Publishing, Wallingford, UK.
Mechoulam, R., Fride, E. & Di Marzo, V. 1998. Endocannabi-
noids. Eur. J. Pharmacol. 359: 1–18.
Parkinson, J. 1640. The Theater of Plants – an Universal and
Compleate Herbal, p. 42.Coates Co., London.
Rothschild, M. & Fairbairn, J.W. 1980. Ovipositing butterfly
(Pieris brassicae L.) distinguishes between aqueous extracts of
two strains of Cannabis sativa L. and THC and CBD. Nature 286:
Salzet, M., Salzet-Raveillon, B., Cocquerelle, C., Verger-
Bocquet, M., Pryor, S.C., Rialas, C.M., Laurent, V. &
Stefano, G.B. 1997. Leech immunocytes contain proopiome-
lanocortin: nitric oxide mediates hemolymph proopiomelano-
cortin processing. J. Immunol. 159: 5400–5411.
Salzet, M. & Stefano, G.B. 2002. The endocannabinoid system in
invertebrates. Prostaglandins Leukot Essent. Fatty Acids 66:
Schuel, H., Berkery, D., Schuel, R., Chang, M.C., Zimmerman,
A.M. & Zimmerman, S. 1991a. Reduction of the fertilizing
capacity of sea urchin sperm by cannabinoids derived from
marihuana. I. Inhibition of the acrosome reaction induced by
egg jelly. Mol. Reprod. Dev. 29: 51–59.
Schuel, H., Chang, M.C., Berkery, D., Schuel, R., Zimmerman,
A.M. & Zimmerman, S. 1991b. Cannabinoids inhibit fertiliza-
tion in sea urchins by reducing the fertilizing capacity of
sperm. Pharmacol. Biochem. Behav. 40: 609–615.
Schuel, H., Goldstein, E., Mechoulam, R., Zimmerman, A.M. &
Zimmerman, S. 1994. Anandamide (arachidonylethanola-
mide), a brain cannabinoid receptor agonist, reduces sperm
fertilizing capacity in sea urchins by inhibiting the acrosome
reaction. Proc. Natl. Acad. Sci. USA 91: 7678–7682.
Schuel, H., Schuel, R., Zimmerman, A.M. & Zimmerman, S.
1987. Cannabinoids reduce fertility of sea urchin sperm.
Biochem. Cell Biol. 65: 130–136.
Stanley-Samuelson, D.W. & Pedibhotla, V.K. 1996. What can we
learn from prostaglandins and related eicosanoids in insects?
Insect Biochem. Molec. Biol. 26: 223–234.
Stefano, G.B., Liu, Y. & Goligorsky, M.S. 1996. Cannabinoid
receptors are coupled to nitric oxide release in invertebrate
immunocytes, microglia, and human monocytes. J. Biol. Chem.
271: 19238–19242.
Stefano, G.B., Rialas, C.M., Deutsch, D.G. & Salzet, M. 1998.
Anandamide amidase inhibition enhances anandamide-
stimulated nitric oxide release in invertebrate neural tissues.
Brain Res. 793: 341–345.
Stefano, G.B., Salzet, B., Rialas, C.M., Pope, M., Kustka, A.,
Neenan, K., Pryor, S. & Salzet, M. 1997a. Morphine- and
anandamide-stimulated nitric oxide production inhibits pre-
synaptic dopamine release. Brain Res. 763: 63–68.
Stefano, G.B., Salzet, B. & Salzet, M. 1997b. Identification and
characterization of the leech CNS cannabinoid receptor:
coupling to nitric oxide release. Brain Res. 753: 219–224.
Turkanis, S.A. & Karler, R. 1988. Changes in neurotransmitter
release at a neuromuscular junction of the lobster caused by
cannabinoids. Neuropharmacology 27: 737–742.
Yamaguchi, F., Macrae, A.D. & Brenner, S. 1996. Molecular
cloning of two cannabinoid type 1-like receptor genes from
the puffer fish Fugu rubripes. Genomics 35: 603–605.
Waser, P. 1999. Effects of THC on brain and social organization
in ants. In: Marihuana and medicine (G. Nahas, N. Pace &
R, Cancro, eds). Humana Press, Totowa, N.J.
Zimmer, A., Zimmer, A.M., Hohmann A.G., Herkenham M. &
Bonner, T.I. 1999. Increased mortality, hypoactivity, and
hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc.
Natl. Acad. Sci. USA 96: 5780–5785.
Received 15 June 2005; revised 12 August 2005; accepted 23 August
Cannabinoid receptors in invertebrates 373
... Unlike most animals, insects lack cannabinoid receptors analogous to other animals (McPartland et al. 2001(McPartland et al. , 2006. Studies have reported, however, negative impacts of cannabinoids on the performance of insect herbivores (Rothschild et al. 1977;Rothschild and Fairbairn 1980). ...
... While negative impacts of cannabinoids on insect performance have been previously reported (Rothschild et al. 1977;Rothschild and Fairbairn 1980), insects lack a human analogue cannabinoid receptor (McPartland et al. 2001(McPartland et al. , 2006, so it is unlikely that the responses to variation in cannabinoid levels are driven by human-based pharmacological effect. Our findings suggest that cannabinoids possibly affect nutrient utilization and uptake in insect herbivores, yet the mechanism is unclear. ...
Full-text available
Agronomic management decisions can alter plant foliar traits, especially nutritional quality, with potential to influence plant–herbivore interactions. Herbivores balance consumption of plant tissue for nutritional gains related to growth and development while contending with plant traits that may deter herbivory or are toxic. This study evaluates the influence of management decisions on the foliar quality of industrial hemp (Cannabis sativa) and the impact on the performance of fall armyworm (Spodoptera frugiperda). We included three common management decisions in this study: fertilization rate, cultivar choice, and planting date. In a no-choice feeding trial, fall armyworm larvae were fed leaves of field-grown hemp from three different cultivars that received different rates of fertilizer and were planted on two different dates. We quantified levels of foliar nitrogen, the ratio of carbon to nitrogen, leaf mass per unit area, and concentrations of tetrahydrocannabinol and cannabidiol, and determined the influence of foliar quality on herbivore performance. Fertilization rate, cultivar, and the interactions of planting date with both fertilization rate and cultivar influenced multiple aspects of hemp foliar quality. Overall, fertilization had the largest influence on foliar quality and subsequent herbivore performance. Variation in foliar traits influenced herbivore performance. Foliar nitrogen had a positive impact on fall armyworm performance and the ratios of carbon and total cannabinoids to nitrogen had negative impacts on fall armyworm performance. Our findings show that management decisions in industrial hemp can affect plant–insect interactions through changes in foliar quality. These outcomes suggest that developing crop management recommendations for pest management will be important for a re-emerging crop like industrial hemp.
... In addition to pCBs and sCBs, there are naturally occurring cannabinoids (endocannabinoids or eCBs) in all major subdivisions of bilaterians, i.e., animals with bilateral symmetry [65]. For example, both AEA and 2-AG are major eCBs (see Table 1), and their structures are provided in Fig. 3 [66]. ...
Full-text available
Introduction: The legal and illicit use of cannabinoid-containing products is accelerating worldwide and is accompanied by increasing abuse problems. Due to legal issues, the USA will be entering a period of rapidly expanding recreational use of cannabinoids without the benefit of needed basic or clinical research. Most clinical cannabinoid research is focused on adults. However, the pediatric population is particularly vulnerable since the central nervous system is still undergoing developmental changes and is potentially susceptible to cannabinoid-induced alterations. Research design and methods: This review focuses on the systems medicine of cannabinoids with emphasis on the need for future studies to include pediatric populations and mother-infant dyads. Results and conclusion: Systems medicine integrates omics-derived data with traditional clinical medicine with the long-term goal of optimizing individualized patient care and providing proactive medical advice. Omics refers to large-scale data sets primarily derived from genomics, epigenomics, proteomics, and metabolomics.
... The endocannabinoid system has been found to play important roles in the functioning of various physiological systems in humans including the nervous system, the immune system and the cardiovascular system (Montecucco and Marzo, 2012;Cabral et al., 2015;Zou and Kumar, 2018). Interestingly, the endocannabinoid system is also present in non-mammalian animals, including invertebrates (Salzet and Stefano, 2002;McPartland et al., 2006;Silver, 2019;Clarke et al., 2021). This system also occurs in Hydra (De Petrocellis et al., 1999), the earliest animal described to date to have evolved a neural network. ...
Full-text available
Endocannabinoids play important roles in the functioning of various physiological systems in humans and non-mammalian animals, including invertebrates. However, information concerning their roles in physiological functions in members of the phylum Mollusca is scarce. Here the hypothesis that the endocannabinoids are involved in mediating settlement of marine invertebrates was tested. Two endocannabinoids [N-arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG)], and two endocannabinoid-like lipids [N-Oleoylethanolamide (OEA) and N-Palmitoylethanolamide (PEA)] were detected in the green mussel Perna viridis . In particular, 2-AG was present at significantly higher levels in unattached P. viridis compared with attached mussels. The in vivo level of 2-AG was inversely correlated with the attachment activity of P. viridis . Furthermore, exposure to synthetic 2-AG inhibited attachment of P. viridis in a reversible manner. Transcriptomic analysis suggested that up-regulation of 2-AG synthase (Phospholipase C-β, PLC-β) and down-regulation of its degrading enzyme (Monoacylglycerol lipase, MAGL) resulted in higher levels of 2-AG in unattached mussels. A putative mechanism for the negative regulation of mussel attachment by 2-AG is proposed that involves a Ca ²⁺ - Nitric oxide (NO)- cyclic guanosine monophosphate (cGMP) pathway. This study broadens our understanding of the evolution and roles of the endocannabinoid system in animals, and reveals an endogenous regulatory cue for mussel attachment.
... Ligand-receptor coevolution is a critical driving force of phylogenetic selection to increase fitness and survival by gain of function. 1 Evolutionarily, Hydra vulgaris (Cnidaria) first shows an ancestral ''endocannabinoid system'' (i.e., 2-arachidonoylglycerol and anandamide (AEA) content and the existence of SR141716-sensitive binding sites). 2 Similarly, cannabinoid (ant-)agonistbinding sites were broadly found in mollusks, annelids, as well as in some crustaceans, 1,3,4 with the first orthologs of the CB 1 cannabinoid receptor (CB 1 R) described in nematodes (such as the NPR-19 receptor in C. elegans 5 ). However, classical CB 1 Rs appear unique to chordates. ...
Introduction: In mammals, sn-1-diacylglycerol lipases (DAGL) generate 2-arachidonoylglycerol (2-AG) that, as the major endocannabinoid, modulates synaptic neurotransmission by acting on CB1 cannabinoid receptors (CB1R). Even though the insect genome codes for inaE, which is a DAGL ortholog (dDAGL), its products and their functions remain unknown particularly because insects lack chordate-type cannabinoid receptors. Materials and Methods: Gain-of-function and loss-of-function genetic manipulations were carried out in Drosophila melanogaster, including the generation of both dDAGL-deficient and mammalian CB1R-overexpressing flies. Neuroanatomy, dietary manipulations coupled with targeted mass spectrometry determination of arachidonic acid and 2-linoleoyl glycerol (2-LG) production, behavioral assays, and signal transduction profiling for Akt and Erk kinases were employed. Findings from Drosophilae were validated by a CB1R-binding assay for 2-LG in mammalian cortical homogenates with functionality confirmed in neurons using high-throughput real-time imaging in vitro. Results: In this study, we show that dDAGL is primarily expressed in the brain and nerve cord of Drosophila during larval development and in adult with 2-LG being its chief product as defined by dietary precursor availability. Overexpression of the human CB1R in the ventral nerve cord compromised the mobility of adult Drosophilae. The causality of 2-LG signaling to CB1R-induced behavioral impairments was shown by inaE inactivation normalizing defunct motor coordination. The 2-LG-induced activation of transgenic CB1Rs affected both Akt and Erk kinase cascades by paradoxical signaling. Data from Drosophila models were substantiated by showing 2-LG-mediated displacement of [3H]CP 55,940 in mouse cortical homogenates and reduced neurite extension and growth cone collapsing responses in cultured mouse neurons. Conclusions: Overall, these results suggest that 2-LG is an endocannabinoid-like signal lipid produced by dDAGL in Drosophila.
... Similar conclusions have been reached for endocannabinoid system in insects. Considering some evidence regarding the endocannabinoid system in non-insect invertebrates and research demonstrating the absence of the system in insects (Salzet et al., 2000;Elphick and Egertova, 2001;McPartland et al., 2005;Elphick, 2012), a secondary loss of endocannabinoid system in Ecdysozoa may be assumed. ...
The fruit fly Drosophila melanogaster brain is the most extensively investigated model of a reward system in insects. Drosophila can discriminate between rewarding and punishing environmental stimuli and consequently undergo associative learning. Functional models, especially those modelling mushroom bodies, are constantly being developed using newly discovered information, adding to the complexity of creating a simple model of the reward system. This review aims to clarify whether its reward system also includes a hedonic component. Neurochemical systems that mediate the ´wanting´ component of reward in the Drosophila brain are well documented, however, the systems that mediate the pleasure component of reward in mammals, including those involving the endogenous opioid and endocannabinoid systems, are unlikely to be present in insects. The mushroom body components exhibit differential developmental age and different functional processes. We propose a hypothetical hierarchy of the levels of reinforcement processing in response to particular stimuli, and the parallel processes that take place concurrently. The possible presence of activity-silencing and meta-satiety inducing levels in Drosophila should be further investigated.
Ensuring that food is safe and nutritious is a highly complex process, requiring national and international cooperation, multidisciplinary approaches, and active participation of the food industry. This chapter introduces chemical food safety, focusing on important food toxicological and regulatory approaches that minimize human risk from foodborne toxicity. The chapter is organized to discuss those compounds that are intentionally added to food, contaminants of food, and inherent toxicity of the food itself. Novel foods have received particular attention. Next we address the clinical and pathological adverse reactions to food, the mechanism of action of clinical disorders, and safety assessment. Finally, we address global and country safety assessment from a regulatory perspective and finish the chapter with a discussion of challenges and future developments in food safety.
Full-text available
This article provides a narrative review of the state of the science for both cyclic vomiting syndrome and cannabis hyperemesis syndrome along with a discussion of the relationship between these 2 conditions. The scope of this review includes the historical context of these conditions as well as the prevalence, diagnostic criteria, pathogenesis, and treatment strategies for both conditions. A synopsis of the endocannabinoid system provides a basis for the hypothesis that a lack of cannabidiol in modern high-potency Δ9-tetrahydrocannabinol cannabis may be contributory to cannabis hyperemesis syndrome and possibly other cannabis use disorders. In concluding assessment, though the publications addressing both adult cyclic vomiting syndrome and cannabis hyperemesis syndrome are steadily increasing overall, the state of the science supporting the treatments, prognosis, etiology, and confounding factors (including cannabis use) is of moderate quality. Much of the literature portrays these conditions separately and as such sometimes fails to account for the confounding of adult cyclic vomiting syndrome with cannabis hyperemesis syndrome. The diagnostic and therapeutic approaches are, at present, based generally on case series publications and expert opinion, with a very limited number of randomized controlled trials and a complete absence of Level 1 evidence within the cyclic vomiting literature overall as well as for cannabis hyperemesis syndrome specifically.
Full-text available
With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 °C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.
The “Endocannabinoid System” (ECS) was not discovered until early in the 1990s, as a result of research to understand the actions of Δ⁹-THC (THC) on the nervous system. Some of this work determined that THC works by binding to two endogenous membrane receptors that had not been identified prior. These endogenous membrane receptors were identified as G-protein coupled receptors (GPCR) and named Cannabinoid Receptor 1 (CB1) and Cannabinoid Receptor 2 (CB2). After the discovery of these receptors, it was a short path to find the endogenous ligands that pair with these receptors (Panagis et al. 2014).
Medical marijuana and the promise of medical advances with cannabinoids is a controversial topic. This book provides clinicians with credible, peer-reviewed science to advise patients on the use of cannabinoids in practice. From the history of cannabis to the recent discoveries, chapters include the science of cannabinoids, changes in the legal and regulatory landscape, and the emerging area of endocannabinoids. The book differentiates approved cannabinoids from cannabis and medical marijuana and stimulates clinicians to think about the risks and benefits of these two drugs. It provides the factual background for clinicians to lead the discussion on the continued use of marijuana, ongoing areas of research and future advances and development of new medications for treatment. An invaluable guide for all specialists in the pharmaceutical sciences, toxicologists, biochemists, neurologists, psychiatrists, addiction specialists, as well as primary care physicians, nurse practitioners, and regulators and policymakers.
Full-text available
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 δ‐tetrahydrocannabinol (δ‐THC). In humans, δ‐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 δ‐THC and Cannabis are not mediated through CB receptors.
Two subtypes of cannabinoid receptors have been identified to date, the CB, receptor, essentially located in the CNS, but also in peripheral tissues, and the CB2 receptor, found only at the periphery. The identification of Δ9-tetrahydrocannabinol (Δ9-THC) as the major active component of marijuana (Cannabis sativa), the recent emergence of potent synthetic ligands and the identification of anandamide and sn-2 arachidonylglycerol as putative endogenous ligands for cannabinoid receptors in the brain, have contributed to advancing cannabinoid pharmacology and approaching the neurobiological mechanisms involved in physiological and behavioral effects of cannabinoids. Most of the agonists exhibit nonselective affinity for CB1/CB2 receptors, and Δ9-THC and anandamide probably act as partial agonists. Some recently synthesized molecules are highly selective for CB2 receptors, whereas selective agonists for the CB1 receptors are not yet available. A small number of antagonists exist that display a high selectivity for either CB1 or CB2 receptors. Cannabinomimetics produce complex pharmacological and behavioral effects that probably involve numerous neuronal substrates. Interactions with dopamine, acetylcholine, opiate, and GABAergic systems have been demonstrated in several brain structures. In animals, cannabinoid agonists such as Δ9-THC, WIN 55,212-2, and CP 55,940 produce a characteristic combination of four symptoms, hypothermia, analgesia, hypoactivity, and catalepsy. They are reversed by the selective CB1 receptor antagonist, SR 141716, providing good evidence for the involvement of CB1-related mechanisms. Anandamide exhibits several differences, compared with other agonists. In particular, hypothermia, analgesia, and catalepsy induced by this endogenous ligand are not reversed by SR 141716. Cannabinoid-related processes seem also involved in cognition, memory, anxiety, control of appetite, emesis, inflammatory, and immune responses. Agonists may induce biphasic effects, for example, hyperactivity at low doses and severe motor deficits at larger doses. Intriguingly, although cannabis is widely used as recreational drug in humans, only a few studies revealed an appetitive potential of cannabimimetics in animals, and evidence for aversive effects of Δ9-THC, WIN 55,212-2, and CP 55,940 is more readily obtained in a variety of tests. The selective blockade of CB1 receptors by SR 141716 impaired the perception of the appetitive value of positive reinforcers (food, cocaine, morphine) and reduced the motivation for sucrose, beer and alcohol consumption, indicating that positive incentive and/or motivational processes could be under a permissive control of CB1-related mechanisms. There is little evidence that cannabinoid systems are activated under basal conditions. However, by using SR 141716 as a tool, a tonic involvement of a CB1-mediated cannabinoid link has been demonstrated, notably in animals suffering from chronic pain, faced with anxiogenic stimuli or highly motivational reinforcers. Some effects of SR 141716 also suggest that CB1-related mechanisms exert a tonic control on cognitive processes. Extensive basic research is still needed to elucidate the roles of cannabinoid systems, both in the brain and at the periphery, in normal physiology and in diseases. Additional compounds, such as selective CB1 receptor agonists, ligands that do not cross the blood brain barrier, drugs interfering with synthesis, degradation or uptake of endogenous ligand(s) of CB receptors, are especially needed to understand when and how cannabinoid systems are activated. In turn, new therapeutic strategies would likely to emerge.
After a single dose of 3H-LSD or THC (100 µg/ml), the maximum brain concentration of LSD reached 150 pg after 12 hours and that of THC reached 800 pg after 36 hours. Ants fed sugar water containing 100 µg/mL LSD or 1 mg/mL THC (the maximum that could be solubilized in water-Tween) presented impairment of social behavior. Behavioral reactions or the performance of individual ants were likewise altered.
What is the role of the cannabinoid system in invertebrates and can it tell us something about the human system? We discuss in this review the possible presence of the cannabinoid system in invertebrates. Endocannabinoid processes, i.e., enzymatic hydrolysis, as well as cannabinoid receptors and endocannabinoids, have been identified in various species of invertebrates. These signal molecules appear to have multiple roles in invertebrates; diminishing sensory input, control of reproduction, feeding behavior, neurotransmission and antiinflammatory actions. We propose that since this system worked so well, it was retained during evolution, and that invertebrates can serve as a model to study endogenous cannabinoid signaling.
The ovipositing female butterfly has proved a useful tool for separating closely related strains of various plant species. Using the large white (Pieris brassicae), we have been able to distinguish between the Turkish and Mexican strains of Cannabis sativa, and demonstrate that the butterfly is sufficiently sensitive to differentiate between purified THC (tetrahydrocannabinol) and CBD (cannabidiol) - two substances which taste and smell alike to the human observer.
In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography–mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5±0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4±42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5±3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4±1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. ∼42, ∼46 and ∼66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.
The background knowledge leading to the isolation and identification of anandamide and 2-arachidonoyl glycerol, the principal endocannabinoids is described. The structure–activity relationships of these lipid derivatives are summarized. Selected biochemical and pharmacological topics in this field are discussed, the main ones being levels of endocannabinoids in unstimulated tissue and cells, biosynthesis, release and inactivation of endocannabinoids, the effects of `entourage' compounds on the activities of anandamide and 2-arachidonoyl glycerol, their signaling mechanisms and effects in animals.
Anandamide, an endogenous cannabinoid signaling molecule, in a concentration dependent manner, initiates the release of nitric oxide (NO) from leech and mussel ganglia. SR 141716A, a cannabinoid antagonist, blocks the anandamide stimulated release of NO from these tissues. Methyl arachidonyl fluorophosphonate (MAFP), a specific anandamide amidase inhibitor, when administered to either ganglia with anandamide (10−6 M) did not increase the peak level of NO release but did significantly extend NO release from 12 to 18 min (P<0.05). Lower levels of anandamide (10−8 and 10−7 M) do not stimulate the release of significant amounts of NO from these tissues. However, in the presence of MAFP (2.5 nM), the lower anandamide concentrations were able to release significant peak amounts of NO. In mussel neural tissues, the peak NO release increased from 2.2±1.3 nM to 8.6±2.1 nM. Taken together, the results indirectly demonstrate the presence of anandamide amidase in these tissues, suggesting that the enzyme may serve as an endogenous regulator of anandamide action.
Eicosanoids are oxygenated metabolites of three C20 polyunsaturated fatty acids (20:3n-6, 20:4n-6, and 20:5n-3). While eicosanoids are very well known in mammalian systems, mostly due to their pharmaceutical interest, there is increasing recognition of the significance of these compounds in insects and other invertebrates. In this paper we consider four major concepts emerging from work on eicosanoids in invertebrates. First, the biological significance of eicosanoids extends far beyond their physiological and pathophysiological actions in human and veterinary medicine. Second, we can greatly improve our understanding of eicosanoids in insects by integrating our work on insects into ongoing studies of other invertebrates. Third, some eicosanoid actions may be fundamental to animals. Fourth, the biochemistry of eicosanoids in insects and other invertebrates can differ from expectations based on the mammalian background. Finally, we point to an uncharted frontier in insect studies-the biochemical mechanisms of eicosanoid action—by drawing attention to some of the work on eicosanoid receptors in mammalian systems.