Review
Pharmacology and biochemistry of spider venoms
Lachlan D. Rash, Wayne C. Hodgson*
Monash Venom Group, Department of Pharmacology, PO Box 13E, Monash University, Victoria 3800, Australia
Abstract
Spider venoms represent an incredible source of biologically active substances which selectively target a variety of vital
physiological functions in both insects and mammals. Many toxins isolated from spider venoms have been invaluable in helping
to determine the role and diversity of neuronal ion channels and the process of exocytosis. In addition, there is enormous
potential for the use of insect speci®c toxins from animal sources in agriculture. For these reasons, the past 15±20 years has seen
a dramatic increase in studies on the venoms of many animals, particularly scorpions and spiders. This review covers the
pharmacological and biochemical activities of spider venoms and the nature of the active components. In particular, it focuses
on the wide variety of ion channel toxins, novel non-neurotoxic peptide toxins, enzymes and low molecular weight compounds
that have been isolated. It also discusses the intraspeci®c sex differences in given species of spiders. q 2001 Elsevier Science
Ltd. All rights reserved.
Keywords: Spider venom; Neurotoxins; Ion channels; Enzymes; Necrotic arachnidism; Sex differences
Contents
1. Spiders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
2. Spider venoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
3. Venom components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
3.1. Neurotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
3.2. Glutamate receptor toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
3.3. Calcium channel toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
3.4. Sodium channel toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
3.5. Potassium channel toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
3.6. Chloride channel toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
3.7. Toxins that stimulate the release of neurotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
3.8. Toxins affecting cholinergic transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
3.9. Other neurotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4. Non neurotoxic peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
5. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
6. Low molecular weight components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
6.1. Biogenic amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
6.2. Free amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
6.3. Other low MW components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
7. Necrotic arachnidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
7.1. Necrotic arachnidism in Australia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
8. Sex-linked variation in venom composition and activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Toxicon 40 (2002) 225±254
0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0041-0101(01)00199-4
www.elsevier.com/locate/toxicon
* Corresponding author. Tel.: 161-3-99054861; fax: 161-3-99055851.
E-mail address: wayne.hodgson@med.monash.edu.au (W.C. Hodgson).
1. Spiders
Spiders and humans have been sharing habitats for as
long as humans have existed. In that time spiders have
evoked numerous and varied responses from people includ-
ing fascination, wariness and, as any bona ®de arachno-
phobe will attest to, debilitating terror. They have secured
a place in the mythology of many cultures throughout the
world as well as in modern popular culture, from nursery
rhymes to horror movies.
Spiders are an ancient group with the oldest known fossil
records dating 300 million years, from the Carboniferous
period. Excluding insects, spiders are the most successful
invertebrates on land, with nearly 40,000 species having
been described world-wide (Platnick, 1993). Spiders can
be found in nearly every terrestrial habitat from seashores
to alpine regions above the tree line, from deserts to tropical
rain forests, and are found in all levels of these environ-
ments. Many have readily adapted to living in close associa-
tion with man. Most spiders are not naturally aggressive and
are generally harmless to humans. However, several species
of spider are responsible for harmful and even fatal bites.
When spider bites do occur they are usually accidental
and/or defensive, such as when a spider hiding in clothing
or bed linen is trapped against the skin.
There are two main groups of spiders: the Orthognatha or
mygalomorph (primitive or trapdoor spiders), whose cheli-
cera project forward from the cephalothorax, while the fangs
move downwards; and the Labidognatha or araneomorphs,
whose chelicera are positioned vertically, and together with
the fangs move laterally like pincers. Most spiders feed
predominantly on insects and other arthropods with some
larger species readily catching and eating frogs, lizards,
snakes, small birds and rodents. Prey is either ambushed
or tackled and grasped with the fangs (hunting spiders) or
caught in silk snares (web builders). Whatever the method of
capture the ultimate demise of the prey is often brought
about by the injection of venom. Once the prey is secured
all spiders begin digestion externally, regurgitating diges-
tive ¯uids onto or into the prey before swallowing the liquid
meal using their strong sucking stomach.
2. Spider venoms
Given the age of spiders as an evolutionary group and the
minimal changes in their morphology over time, it is possi-
ble that the use of venom was developed very early (Bettini
and Brignoli, 1978). The primary purpose of spider venom
is to paralyse or kill prey, and it may also play a role in
pre-digestion of the intended meal. It may be that a spiders
venom also serves as a self defence mechanism against
predators. Indeed, it has been suggested that some spiders
do not inject venom when subduing prey but only defen-
sively (Minton, 1974). Whatever its purpose the use of
venom has been integral in the successful evolution of
spiders, possibly more so than their use of silk (Bettini
and Brignoli, 1978).
In general, spider venoms, especially those from tarantu-
las, are clear, colourless liquids that are easily soluble in
water (Bucherl, 1971; Norment and Foil, 1979; Savel-
Neimann, 1989). Minton (1974) states that most spider
venoms are neutral or alkaline, although some are acidic.
The latter is the case with the mygalomorphs Atrax robustus,
Dugesiella hentzi and Eurypelma californicum, all of
which have venoms with pHs of 4±5.5 (Wiener, 1961;
Schanbacher et al., 1973a; Savel-Neimann, 1989). Upon
lyophilisation, spider venoms have been reported as ranging
in colour from whitish to whitish-pink, -yellow or -grey
(Wiener, 1961; Bucherl, 1971). The dried venom of true
spiders (araneomorphs) is reportedly stable for years in
vacuum while mygalomorph venoms are more hygroscopic
and will liquefy if left in contact with air, but will keep for a
few months in vacuo (Bucherl, 1971).
3. Venom components
Like the venoms of other animals, such as snakes and
scorpions, venoms from spiders are heterogeneous, not
only between species but also within species. They are
made up of complex mixtures of biologically active and
inactive substances. The major constituents of spider
venoms are protein, polypeptide and polyamine neurotox-
ins, enzymes, nucleic acids, free amino acids, monoamines
and inorganic salts (Jackson and Parks, 1989).
3.1. Neurotoxins
As the primary purpose of their venom is to paralyse prey,
spiders produce a variety of toxins which affect the nervous
system. Most, if not all, spider neurotoxins characterised to
date have been found to be either proteins, peptides or acyl-
polyamines (McCormick and Meinwald, 1993), and have a
variety of actions throughout the nervous system. Two
important characteristics of nervous tissue are the excitabil-
ity of the cell membrane and the ability to transmit the
electrical signal across a synapse. The majority of spider
toxins are therefore targeted to either neuronal receptors,
neuronal ion channels or presynaptic membrane proteins
involved in neurotransmitter release. Thus neurotoxins
isolated from spider venoms can be classi®ed according to
their mode of action: toxins affecting glutamatergic trans-
mission; calcium (Ca
21
), sodium (Na
1
), potassium (K
1
)
and chloride (Cl
2
) channel toxins; toxins that stimulate
transmitter release and toxins blocking postsynaptic choli-
nergic receptors. Following is a description of the normal
physiology of these target sites and the known spider toxins
affecting them.
The structure of neurotoxins isolated so far and their
molecular evolution are beyond the scope of this review
and have recently been reviewed by Escoubas et al. (2000).
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
226
3.2. Glutamate receptor toxins
L-Glutamate (glutamic acid) is the principal excitatory
transmitter in the mammalian central nervous system
(CNS) (Greenamyre and Porter, 1994), the main sources
being glucose, via the Krebs cycle, and glutamine
synthesised in glial cells. It is stored in synaptic vesicles
and released by Ca
21
-dependent exocytosis. The action of
glutamate is terminated by its removal from the synapse via
high af®nity transporters located on both neurones and glial
cells. There are several types of glutamate receptor broadly
classi®ed as the ionotropic and metabotropic glutamate
receptors, iGluR and mGluR respectively. The ionotropic
receptors are further divided into NMDA (N-methyl-D-
aspartate) and non-NMDA (consisting of AMPA
(a-amino-3-hydroxy-5-methyl-isoxazole)
and
kainate)
subtypes, being named after their sensitivity to selective
agonists. The ionotropic receptors are ligand-gated ion
channels that are cation (K
1
, Na
1
and Ca
21
) selective and
are oligomeric assemblies of either four or ®ve subunits
each with three transmembrane domains (the exact number
of subunits making up functional receptors is a matter of
intense debate and is reviewed by Dingledine et al., 1999).
The NMDA receptor consists of NR1 and NR2 subunits, has
slow kinetics, is highly permeable to Ca
21
and has a modu-
latory site sensitive to the endogenous polyamines spermine
and spermidine, the presence of which potentiates the
response to glutamate. AMPA and kainate receptors are
made up of the GluR1-7 and KA1-2 types of subunit, are
both sensitive to the amino acid quisqualic acid, have fast
kinetics and low permeability to Ca
21
. NMDA and AMPA
receptors are generally co-localised and widely distributed
whereas kainate receptors have a more restricted
distribution. The metabotropic receptors are G-protein-
coupled receptors linked to either inositol (1,4,5) tris-
phosphate (IP
3
) formation and intracellular Ca
21
release or
inhibition of adenylate cyclase and are found both pre- and
post-synaptically.
Glutamate is also an important transmitter in the CNS
and the main transmitter in the neuromuscular junction of
invertebrates (Osborne, 1996) with both an excitatory and
inhibitory function. There are several types of glutamate
receptors recognised in invertebrates. The excitatory, or
depolarising, D-response is mediated by a cation (Na
1
)
selective channel with fast kinetics that is sensitive to quis-
qualate, being similar to the mammalian AMPA receptor.
The inhibitory, or hyperpolarising, H-response is mediated
by anion (Cl
2
) selective channels with a slow prolonged
action and no known mammalian counterpart. Both
receptors are found in the CNS and neuromuscular junc-
tion. The ®rst description of an NMDA receptor in an
invertebrate, found in cray®sh visual interneurons, was
reported by Pfeiffer-Linn and Glantz (1991), suggesting a
third type of invertebrate glutamate receptor. Given the
importance of glutamate as a transmitter in invertebrates,
particularly its involvement in locomotion, it is not surpris-
ing that spiders possess toxins that target glutamatergic
transmission.
In the early 1980s, a toxin (JSTX, Joro spider toxin) was
isolated from the venom of the orb weaving spider Nephila
clavata. In the lobster neuromuscular junction, JSTX
irreversibly suppressed excitatory post synaptic potentials
(EPSP) and depolarisation elicited by exogenous glutamate
without affecting inhibitory postsynaptic potentials or
depolarisation due to aspartate (Kawai et al., 1982a).
JSTX was also found to block glutamatergic synapses in
mammalian brain (Kawai et al., 1982b). Similar activity
was found in venom gland extracts from the spider Araneus
ventricosus (Kawai et al., 1983). Shortly after, JSTX and
another glutamate receptor blocker NSTX (Nephila spider
toxin from the spider Nephila maculata) were chemically
characterised (Aramaki et al., 1986). At about the same time
Usmanov et al. (1983) found that venom from Argiope
lobata irreversibly blocked responses of locust muscle to
exogenous glutamate. A 636 Da toxin (named argiopine)
(Fig. 1) was subsequently isolated and found to be closely
related to JSTX and NSTX (Grishin et al., 1986).
Since then, several glutamate receptor antagonists (called
argiotoxins) have been isolated from the venoms of the
North American orb weaving spiders Argiope trifasciata,
Argiope ¯orida and Araneus gemma, one of which (Arg
636; Fig. 1) was subsequently found to be identical to
argiopine (Usherwood et al., 1984). Usmanov et al. (1985)
found that postsynaptic inhibition of glutamatergic trans-
mission at the locust neuromuscular junction was a common
property of eight other spiders belonging to the family
Araneidae (orb weavers).
The chemical structures of many of these toxins have now
been elucidated and they form a family of closely related
compounds, the acylpolyamines, or polyamine amides
(Fig. 1). These toxins contain an aromatic acyl end group
and a polyamine chain (McCormick and Meinwald, 1993).
To date, in all polyamine amide toxins isolated from
Araneidae spiders, the acyl head group is connected to the
polyamine tail by one or two of the amino acids asparagine,
ornithine and v-methyllysine (McCormick and Meinwald,
1993; Schulz, 1997). In general the amino acid containing
polyamine amides appear to have a similar action at inver-
tebrate neuromuscular junctions, causing an open channel
(use-dependent), irreversible or slowly reversible, non-
competitive block of quisqualate sensitive glutamate recep-
tors (Jackson and Parks, 1989). However, there have been
con¯icting reports on their selectivity and speci®city for
vertebrate glutamate receptors. JSTX-3 (Fig. 1) has been
reported to be selective for non-NMDA ionotropic receptors
(Kawai et al., 1991) whereas argiotoxin-636 has been found
to preferentially inhibit both NMDA (Priestly et al., 1989)
and non-NMDA (Ashe et al., 1989) receptors. Nemeth et al.
(1992) found that the argiotoxins and a-agatoxins (see
below) are speci®c inhibitors of NMDA receptors in rat
brain, whereas JSTX was an equipotent inhibitor of
NMDA- and kainate-evoked [Ca
21
]
i
increase in rat
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
227
cerebellar granule cells. In contrast, argiotoxin-636 equally
inhibited responses to both NMDA and kainate in Xenopus
oocytes injected with total rat brain RNA (Usherwood et al.,
1992). More recent work suggests that the sensitivity of
AMPA receptors to argiotoxins is subunit dependent
(Herlitze et al., 1993; reviewed by Usherwood and
Blagbrough, 1994).
A second class of acylpolyamine toxins has been
identi®ed in the venom of a variety of spiders outside the
family of orb weavers. Non-amino acid containing
acylpolyamines have been isolated from the funnel-web
spiders Agelenopsis aperta and Hololena curta, a trapdoor
spider (Hebestatis theveniti), and two tarantulas,
Harpactirella sp. and Aphonopelma chalcodes. The
a-agatoxins, isolated from A. aperta, are selective and
reversible, non-competitive antagonists of NMDA recep-
tors in mammalian brain (Parks et al., 1991). In the chick
CNS, venom from Hololena curta irreversibly blocked
non-NMDA mediated transmission, the responsible toxin
reported as having a molecular weight of 5±10 kDa (Jack-
son et al., 1987). Ten paralytic acylpolyamines and one
insecticidal peptide (collectively known as curtatoxins)
were subsequently isolated from the venom of H. curta
(Quistad et al., 1991), two of the acylpolyamines (HO
489
and HO
505
; Fig. 1) and the peptide (m-AgaII) having
previously been described in the venom of A. aperta. The
polyamine toxins isolated from Hebestatis theveniti
(Hettoxins) were paralytic to lepidopteran larvae.
However, the mechanism of action was not investigated
(Skinner et al., 1990). The structures and pharmacology
of the acylpolyamine spider toxins have been thoroughly
reviewed by Usherwood and Blagbrough (1991);
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
228
Fig. 1. Generalised structure of polyamine amide spider toxins (modi®ed from Usherwood and Blagbrough, 1991) and structures of several key
polyamine toxins from spider venoms.
McCormick and Meinwald (1993). In addition, Hisada et
al. (1998) recently outlined the generalised structures of
toxins (types A±D) isolated from the Nephila spiders in a
paper reporting the identi®cation of a ®fth structure type
(type-E).
In addition to glutamate receptor antagonists, a new class
of toxin affecting glutamatergic transmission was recently
reported (Mafra et al., 1999). PhTx-4, a family of seven
individual toxins, was isolated from the venom of the
Brazilian armed spider (Phoneutria nigriventer) and found
to inhibit glutamate uptake in rat cerebrocortical synapto-
somes in a dose-dependent manner. One of these toxins
Tx4(5±5) with potent insecticidal activity has subsequently
been found to reversibly antagonise the NMDA receptor
while having little effect on AMPA, kainate or GABA
induced currents in rat hippocampal neurons (Figueiredo
et al., 2001).
3.3. Calcium channel toxins (Table 1)
Calcium channels are present in many tissues throughout
the body, and are of particular importance in the nervous
system where the entry of Ca
21
into the nerve terminal plays
a key role in the release of neurotransmitters. In addition to
voltage-activated calcium channels (the focus of this
section), the permeability of membranes to calcium is
increased via ligand-gated ion channels such as the
NMDA receptor (discussed in Section 3.2) and by a family
of intracellular calcium release channels referred to as
ryanodine receptors (Sutko et al., 1997). This family of
receptors are involved in the release of intracellular Ca
21
from the sarcoplasmic (muscle) or rough endoplasmic
reticulum (non-muscle).
There are currently six recognised subtypes of voltage-
activated Ca
21
channel (T-, L-, N-, P-, Q- and R-types)
based on their electrophysiological properties and sensitiv-
ity to various activators/inhibitors and ions (Uchitel, 1997).
However, in some cells it is dif®cult to separate the P and Q
components of a current therefore the term `P/Q-type'
current is often used when referring to either one (Alexander
and Peters, 1999). T-type channels are activated by small
depolarisations (low-voltage activated) and undergo fast
inactivation but slow deactivation and are involved in the
generation of pacemaker activity in neurons and cardiac
muscle. L- and N-type channels are characterised by high-
voltage activation and fast deactivation. L-type channels
inactivate slowly, are blocked by dihydropyridines and are
found in cardiac and vascular smooth muscle, neuronal cell
bodies and proximal dendrites of many central neurons. In
contrast, N-type (neuronal) channels have a moderate rate of
inactivation, are characterised by their sensitivity to
v-conotoxin GVIA (from the venom of the cone snail
Conus geographus) and mediate neurotransmitter release
at synaptic endings. P- and Q-type channels are moderate
voltage-activated Ca
21
channels with fast deactivation, are
found in central and peripheral neurons and are also
involved in transmitter release. Q-type channels have a
moderate rate of inactivation while P-type channels are
non-inactivating. The most recent addition to the voltage-
activated Ca
21
channels is the R-type, which is also acti-
vated at moderate voltages but undergoes fast inactivation
and deactivation and is resistant to known pharmacological
agents. Mammalian voltage-gated calcium channels are
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
229
Table 1
Spider toxins affecting calcium channels
Toxin
Spider
Channel selectivity
Reference/s
PLTXs I±III
P. tristes
Non-selective Drosophila Ca
21
currents
Branton et al., (1987); Leung et al., (1989)
Hololena toxin
H. curta
Non-inactivating Drosophila Ca
21
currents Bowers et al., (1987); Leung et al., (1989)
v-Aga IA-IC
A. aperta
L
Bindokas and Adams, (1989); Venema et al., (1992)
v-Aga IIA and IIB
A. aperta
N
Adams et al., (1990); Venema et al., (1992)
v-Aga IIIA
A. aperta
L and N (P)
Mintz et al., (1991); (1992a); Ertel et al., (1994)
v-Aga IIIB and IIID A. aperta
N (L and P)
Ertel et al., (1994)
v-Aga IVA and IVB A. aperta
P/Q
Mintz et al., (1992b); Adams et al., (1993)
FTX
A. aperta
P
Llinas et al., (1989); (1992)
v-GsTx-SIA
G. spatulata
P, N and Q
Lampe et al., (1993); Piser et al., (1995)
PhTx3); 3-3 and 3-4 P. nigriventer
P/Q
Guatimosin et al., (1997); Miranda et al., (1998)
PhTx3); 3-2 and 3-5 P. nigriventer
L
LeaÄo et al., (1997)
CNS2103
D. okefenokiensis L and R
Kobayashi et al., (1992)
CSTX1
C. salei
L
Cruz et al. in Kuhn-Nentwig and Nentwig, (1997)
SNX-325
S. ¯orentina
N-type selective at nM []s
Newcomb et al., (1995)
Agelenin
A. opulenta
P
Hagiwara et al., (1991); (1999)
DW13.3
F. hibernalis
Non selective
Sutton et al., (1998a)
SNX-482
H. gigas
E
Newcomb et al., (1998)
v-atracotoxin
H. versuta
Insect VACCs
Wang et al., (1999)
HWTX-1
S. huwena
N
Peng et al., (2001)
Hanatoxin 1 and 2
G. spatulata
rabbit brain a
1A
subunit (non-L-type)
Li-Smerin and Swartz, (1998)
made up of several subunits; a1, a2, b, d. Skeletal muscle
channels also contain a g subunit. The a1 subunit of calcium
channels forms the calcium conducting pore. Several a1
subunits have now been cloned from a variety of inverte-
brates and show high structural homology with their verte-
brate counterparts (i.e. the same four repeat structure, each
containing six transmembrane domains) (Hall et al., 1994).
The insect a1 subunits characterised so far share some
sequence identity and pharmacology with the mammalian
L-type channels, for example their sensitivity to phenylalk-
ylamines (Zheng et al., 1995; Lee et al., 1997; MacPherson
et al., 2001). However, as with insect sodium channels
(discussed in Section 3.4), there are suf®cient structural
differences for insecticidal compounds to discriminate
between phyla (Hall et al., 1994).
The presence of Ca
21
channel blocking toxins in spider
venoms was suggested with the discovery that the venoms
of three spiders, including Agelenopsis aperta, caused an
irreversible presynaptic block at the Drosophila neuromus-
cular junction (Branton et al., 1987). Several peptide toxins
were isolated from the spider Plecteurys tristes (PLTXs
I±III, MW 6±7 kDa) (Branton et al., 1987) and one from
Hololena curta (Hololena toxin, MW 16 kDa) (Bowers et
al., 1987) the activities of which were consistent with long
lasting speci®c block of presynaptic Ca
21
channels. This
mode of action was con®rmed when Leung et al. (1989)
found that Hololena toxin selectively blocked non-inactivat-
ing Ca
21
current while PLTX±II blocked both inactivating
and non-inactivating Ca
21
currents in cultured Drosophila
neurones. The complete primary structure of PLTX±II has
been determined revealing a unique post-translational
modi®cation, palmitoylation of the C-terminal threonine
(Branton et al., 1992).
Further work on venom from A. aperta resulted in the
discovery of the v-agatoxins, a family of unrelated peptide
toxins with differing selectivity for neuronal Ca
21
channels
(reviewed by Olivera et al., 1994; Uchitel, 1997). Type I
agatoxins (v-AgaIA, IB and IC) are heterodimeric peptides
of similar size (MW 7.5 kDa) with ®ve disulphide bonds.
v-AgaIA potently blocks voltage sensitive Ca
21
channels in
insects, blocks neurotransmission at the frog neuromuscular
junction (Bindokas and Adams, 1989) and suppresses high
threshold Ca
21
currents in rat dorsal root ganglion cells
(Scott et al., 1990) thereby affecting Ca
21
channels from
both vertebrates and invertebrates. However, v-AgaIA and
IB failed to inhibit binding of v-conotoxin GVIA to chick
synaptosomal membranes (Adams et al., 1990). In addition
to v-AgaIA having no effect on potassium-stimulated Ca
21
entry into chick synaptosomes (Venema et al., 1992), this
suggests that the type-I agatoxins have no activity at N-type
high voltage-activated Ca
21
channels making them
relatively selective for L-type channels.
Type II v-agatoxins (IIA and IIB, ~10 kDa), which have
limited homology with type I toxins (only 43% at NH
2
terminal end), also cause presynaptic block of insect neuro-
muscular transmission. Unlike type I v-agatoxins, type II
v-agatoxins inhibit the binding of v-conotoxin GVIA to
chick synaptosomes (Adams et al., 1990) and potently
block the potassium-stimulated Ca
21
entry into chick
synaptosomes (Venema et al., 1992) hence their classi®ca-
tion as N-type channel blockers. v-Agatoxin IIIA, an
8.5 kDa peptide, was found to be an equipotent blocker of
L- and N-type Ca
21
channels (Mintz et al., 1991), also
causing a partial (~40%) voltage-dependent block of
P-type channels (Mintz et al., 1992a). During the puri®ca-
tion of v-AgaIIIA, several homologous peptides
(v-AgaIIIB, IIIC and IIID) were discovered (Ertel et al.,
1994). v-AgaIIIB and IIID have similar actions to IIIA
with no activity at the insect neuromuscular junction, no
effect on atrial type-T channels and are equipotent inhibitors
of v-conotoxin GVIA binding to rat synaptic membranes.
On the contrary, v-AgaIIIA is a more potent blocker of
locust Ca
21
channels and 100-fold more potent at blocking
L-type channels, making v-AgaIIIB and IIID 100-fold more
selective for N-type Ca
21
channels. Finally, v-AgaIVA, a
48 amino acid peptide unrelated to v-agatoxins I±III, is a
potent blocker of P/Q-type Ca
21
channels (K
d
of ~2 nM for
P-type and ~90 nM for Q-type) in mammalian systems,
however has no effect on T-, L- or N-type channels
(Mintz et al., 1992b). Although having similar potency at
P-type channels (K
d
3 nM) v-AgaIVB, which shares 71%
homology with IVA, is eight times slower in blocking the
current (Adams et al., 1993).
In addition to the peptide v-agatoxins, a low molecular
weight (200±400 Da) fraction of A. aperta venom blocks
Ca
21
channels (now known as P-type after the tissue in
which they were ®rst described) in guinea-pig cerebellar
Purkinje cells, in squid giant synapse and in Xenopus
oocytes injected with rat brain mRNA (Llinas et al., 1989;
Lin et al., 1990). The active toxin, named FTX, was identi-
®ed as a polyamine (Llinas et al., 1992) and a structure
proposed but not con®rmed. A toxin (FTX-3.3; Fig. 1)
claimed to have the identical structure as FTX, along with
an amide-containing analogue (sFTX-3.3), have been
synthesised (Blagbrough and Moya, 1994; Moya and
Blaghbrough, 1994) and shown to block P-type Ca
21
chan-
nels in rat cerebellar Purkinje cells (Dupere et al., 1996).
v-Grammotoxin SIA (v-GsTx-SIA) is a 36 amino acid
peptide (MW 4.1 kDa) with three disulphide bridges
isolated from the venom of the South American Rose taran-
tula, Grammostola spatulata. v-GsTx-SIA produced
concentration-dependent inhibition of K
1
-evoked
45
Ca
21
in¯ux into rat and chick brain synaptosomes suggesting
that it is a blocker of presynaptic N- and P-type voltage-
activated channels in vertebrates (Lampe et al., 1993). Piser
et al. (1995) con®rmed the action of v-GsTx SIA at N- and
P-type channels, in rat cultured hippocampal neurons, and
found that it also blocked Q-type but not L-type Ca
21
chan-
nels. Using rat cerebellar Purkinje neurons, as well as rat and
frog sympathetic neurons, McDonough et al. (1997)
concluded that v-grammotoxin potently inhibits P- and
N-type channels by impeding channel gating and that it
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
230
binds to different or additional sites than v-AgaIVA on
P-type channels. In addition to a relatively non-speci®c
action at voltage-activated Ca
21
channels, v-GsTx SIA
has subsequently been shown to interact with and modify
the gating of voltage-gated K
1
channels, raising the possi-
bility of a voltage-sensing domain common to voltage-gated
ion channels (Li-Smerin and Swartz, 1998).
Venom from the Brazilian `armed' spider contains
another family of peptides with Ca
21
channel blocking
activity. Three fractions toxic to mice, PhTx1, 2 and 3,
were isolated from P. nigriventer venom (Rezende et al.,
1991). Fraction PhTx3 was further separated into six neuro-
toxic peptides (Tx3-1±6) which induced varying types and
degrees of paralysis after intracerebroventricular (i.c.v.)
injection in mice (Cordeiro et al., 1993). Tx3-3 and Tx3-4
potently inhibited Ca
21
in¯ux into rat cerebrocortical synap-
tosomes (IC
50
s of 0.32 and 7.9 nM, respectively) via chan-
nels sensitive to v-AgaIVA but not v-conotoxin GVIA,
suggesting an action at P/Q-type Ca
21
channels (Guatimosin
et al., 1997; Miranda et al., 1998). Toxins Tx3-2 and Tx3-5
had no effect on T-type Ca
21
current, however irreversibly
inhibited L-type current by 80 and 100% respectively, in the
pituitary cell line GH3 (LeaÄo et al., 1997). Using cDNA
sequence analysis and patch clamp studies, Kalapothakis
et al. (1998a) con®rmed the previously published (Cordeiro
et al., 1993) amino acid sequence of Tx3-2 and its action on
L-type Ca
21
channels.
Many other Ca
21
channel toxins, with varying selectivity,
from a wide spectrum of spider species have been isolated in
the last 8 years. A polyamine Ca
21
channel antagonist
(CNS2103) isolated from the venom of the hunting spider
Dolomedes okefenokiensis reversibly blocks both L-type
and dihydropyridine resistant (R-type) channels in
N1E-115 neuroblastoma cells with no effect on T-type
currents or voltage-activated Na
1
or K
1
channels
(Kobayashi et al., 1992). Venom from another hunting
spider, Cupiennius salei, contains a 74 amino acid peptide
toxin (CSTX-1) which inhibits high-threshold Ca
21
chan-
nels (L-type) at glutamatergic synapses (unpublished obser-
vations of Cruz, J., Lacerda BeiraÄo, P.S., and LeaÄo, R.M.,
cited in Kuhn-Nentwig and Nentwig, 1997). Venom from
Segestria ¯orentina yielded the peptide toxin SNX-325,
which has no effect on Na
1
or K
1
channels but inhibits
most Ca
21
channels at micromolar concentrations.
However, at nanomolar concentrations SNX-325 is a selec-
tive blocker of N-type Ca
21
channels (Newcomb et al.,
1995).
Agelenin, isolated from the venom of Agelena opulenta,
is a 35 amino acid peptide containing six cysteine residues
which appears to block P-type Ca
21
channels (Hagawira et
al., 1990, 1991). DW13.3, a novel peptide from Filistata
hibernalis, causes potent blockade of most native Ca
21
channels with the exception of the T-type channel (Sutton
et al., 1998a). The 41 amino acid peptide SNX-482, from the
African tarantula Hysterocrates gigas, blocks human class E
Ca
21
channels (made up of a1E ion-conducting subunits
believed to be responsible for the characteristics of R-type
channels) expressed in a mammalian cell line. SNX-482
also inhibits a native R-type Ca
21
current in rat neurohypo-
physeal nerve terminals but has no effect on R-type currents
in several other types of rat central neurons (Newcomb et
al., 1998). A family of peptide neurotoxins (36±37 amino
acids in length), the v-atracotoxins, isolated from the Blue
Mountains funnel-web spider, Hadronyche versuta, display
selectivity for blocking insect voltage-gated Ca
21
channels
making them promising lead compounds for the develop-
ment of new pesticides (Wang et al., 1999).
HWTX-I is a 33 amino acid peptide with three disulphide
bonds isolated from the Chinese bird spider Selenocosmia
huwena (Liang et al., 1993; Zhang and Liang, 1993).
HWTX±I was found to be toxic to mice (i.p. LD
50
0.7 mg/
kg) and inhibited indirectly-evoked twitches in the mouse
phrenic nerve diaphragm preparation without affecting
directly-evoked twitches. In addition to the amino acid
sequence, the three-dimensional structure of HWTX±I
was determined using
1
H NMR (Quet al., 1995, 1997).
The action of HWTX±I on mouse phrenic nerve diaphragm
was found to be slowly reversible upon prolonged repeated
washing with the recovery time being decreased when
HWTX±I and tubocurarine were added together. It was
suggested that HWTX±I was a blocker of postsynaptic nico-
tinic receptors (Zhouet al., 1997). However, these ®ndings
were not supported by binding studies (Liang et al., 2000)
and subsequent experiments on three electrically stimulated
in vitro preparations suggested that the toxin acted presy-
naptically to inhibit the release of neurotransmitters (Liang
et al., 2000). Using patch-clamp studies, Peng et al. (2001)
recently found that HWTX±I potently and selectively inhi-
bits N-type high-voltage-activated Ca
21
channels in prosta-
glandin E1 differentiated NG108-15 cells. A second toxin,
huwentoxin-II, with neuromuscular blocking activity in the
mouse phrenic nerve diaphragm and reversible paralytic
activity in cockroaches, was recently isolated from
S. huwena, however the mechanism of action has not yet
been determined (Shuand Liang, 1999).
3.4. Sodium channel toxins (Table 2)
The movement of sodium ions across excitable cell
membranes, either via ligand-gated channels (eg. nicotinic
cholinoceptors and non-NMDA glutamate receptors) or
voltage-gated channels, is responsible for the rapid trans-
mission of impulses along and between excitable cells.
Voltage-gated sodium channels allow the co-ordination of
processes such as locomotion and cognition, and are often
the most abundant ion channels present in nerve and muscle
cells (Marban et al., 1998). This makes Na
1
channels prime
targets for paralytic neurotoxins. This section focuses on
spider toxins that target voltage-gated Na
1
channels while
toxins interacting with ligand-gated Na
1
channels are
discussed in Sections 3.2 and 3.8. Like most other
voltage-gated ion channels, Na
1
channels are made up of
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
231
several subunits. The main a subunit, consisting of four
repeats each with six transmembrane domains, determines
the selectivity and properties of the channel and is the only
subunit required for function (Marban et al., 1998). At rest-
ing membrane potentials Na
1
channels are in a closed state,
activating or opening upon depolarisation of the membrane.
Once open, the channels undergo inactivation, or assume a
non-conducting state despite continued depolarisation of the
membrane, before deactivating and returning to a resting
state (Conley and Brammar, 1999). There are currently
nine recognised subtypes of mammalian Na
1
channel,
however there is no of®cial scheme for their classi®cation
(Alexander and Peters, 1999), with an unof®cial scheme
only recently proposed (Conley and Brammar, 1999;
Goldin, 1999). The majority of known Na
1
channels are
selectively and reversibly blocked by nanomolar concentra-
tions of tetrodotoxin (TTX) and saxitoxin (STX). There are,
however, several isoforms of voltage-gated Na
1
channels
that are relatively resistant to TTX, the cardiac Na
1
channel
NaH1 (Conley and Brammar, 1999) and two channels, SNS
and NaN, expressed in sensory neurons of rat, mouse and
human dorsal root ganglion (Did-Hajj et al., 1999). Studies
on insect Na
1
channels have revealed that they are structu-
rally, physiologically and pharmacologically very similar to
their vertebrate counterparts. However, there are also phar-
macological differences as evidenced by the isolation of
insect speci®c Na
1
channels toxins from the venoms of
several species of scorpion (reviewed by Zlotkin, 1999).
For the purpose of this review Na
1
channels will be broadly
classi®ed as either TTX-sensitive (TTX-S) or TTX-resistant
(TTX-R).
Three families of spider toxins are known to act at Na
1
channels (Adams et al., 1989; ArauÂjo et al., 1993a;
Nicholson et al., 1994, 1998), with the venom or toxins of
several other spiders displaying activity consistent with Na
1
channel interactions.
In conjunction with the discovery of the use-dependent
glutamate receptor antagonists the a-agatoxins, a second
class of peptide toxins was isolated from the venom of the
spider Agelenopsis aperta. The m-agatoxins (m-Aga-I±VI)
cause a slowly induced, irreversible, excitatory paralysis in
house¯ies. They were found to act presynaptically, inducing
repetitive ®ring of the nerve, an action blocked by TTX
(Adams et al., 1989). The solution structures of m-Aga-I
and -IV were elucidated and found to have the same overall
fold as many phylogenetically diverse peptide toxins selec-
tive for ion channels (Omecinsky et al., 1996). Fraction
PhTx2, isolated from the venom of P. nigriventer, is toxic
to mice causing excitatory symptoms including salivation,
lachrimation, priapism, convulsions, spastic paralysis of the
limbs and death upon i.c.v. injection (Rezende et al., 1991).
PhTx2 was subsequently shown to inhibit Na
1
channel inac-
tivation and shift the activation voltage to more negative
potentials in frog skeletal muscle (ArauÂjo et al., 1993a).
Nine peaks were obtained upon further separation of the frac-
tion PhTx2 (Tx2-1±9) of which toxins 2-1, 2-5, 2-6 and 2-9
were sequenced (Cordeiro et al., 1992). Toxins Tx2-5 and
Tx2-6 were found to have the same effects on Na
1
channel
inactivation and activation, voltage dependence and inactiva-
tion time constant as the whole fraction (ArauÂjo et al., 1993b).
The cDNAs of Tx2-1 and Tx2-5 were recently cloned
con®rming the previously published amino acid sequences
and identifying two new putative toxins Pn2-1A and Pn2-
5A (Kalapothakis et al., 1998b). Fraction PhTx2 has also
recently been found to cause progressive myonecrosis of
the mouse phrenic nerve diaphragm (Mattiello-Sverzuta and
Cruz-Hoȯing, 2000). The morphological changes induced by
PhTx2 include swelling of the sarcoplasmic reticulum, mito-
chondrial damage, disorganisation of sarcomeres, zones of
hypercontraction and rupture of the plasma membrane. The
authors suggest that the ability of PhTx2 to increase the
permeability of Na
1
channels can account for the myonecro-
sis as the morphological changes observed were similar to
those caused by osmotic disturbances.
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
232
Table 2
Spider toxins affecting sodium channels
Toxin
Spider
Na
1
channel action
Reference/s
m-Aga-I±VI
A. aperta
Induce repetitive ®ring of nerves;
TTX sensitive (TTX-S)
Adams et al., (1989);
Omecinsky et al., (1996)
PhTx2; (Tx2-5 and 2-6)
P. nigriventer
inhibit inactivation
Araujo et al., (1993a)
Pn2-1A and Pn2-5A
P. nigriventer
putative Na
1
channel toxins
Kalapothakis et al., (1998b)
d-atracotoxin Hv1a
H. versuta
inhibit inactivation
Nicholson et al., (1994); (1996)
d-atracotoxin Ar1a
A. robustus
inhibit inactivation
Nicholson et al., (1996); (1998)
d-atracotoxin like
M. bradleyi
inhibit inactivation
Rash et al., (2000)
Curtatoxin II
H. curta
identical structure to m-Aga-II
Quistad et al., (1991)
Lyc IV
L. erythrognatha
probable Na
1
channel activity
Cruz et al., (1994)
DTX9.2
D. canities
TTX-S insect Na
1
channels
Bloomquist et al., (1996)
Arg-636/argiopine
Argiope sp.
inhibits Na
1
currents
Scott et al., (1998)
whole venom
Ancylometes sp.
" freq of MEPPs and depolarise
muscle ®bre membrane; TTX±S
GreÂgio et al., (1999)
PcTx1
P. cambridgei
blocks H
1
-gated Na
1
channels
Escoubas et al., (2000)
Two mammalian selective toxins, robustoxin (RTX) and
versutoxin (VTX), from the Australian funnel-web spiders
Atrax robustus and Hadronyche versuta, respectively, are 42
amino acid peptides with four disulphide bonds which also
interact with Na
1
channels. In rat dorsal root ganglion cells
under voltage clamp conditions, VTX had no effect on TTX-
R Na
1
currents or K
1
currents. However, VTX did cause
dose-dependent slowing or removal of TTX-S Na
1
current
inactivation, reduced peak TTX-S Na
1
current and
increased the rate of recovery from inactivation (Nicholson
et al., 1994). VTX also shifted the voltage dependence of
Na
1
channel activation in the hyperpolarising direction with
both VTX and RTX causing partial activation of
22
Na
1
¯ux
as well as inhibiting batrachotoxin-activated
22
Na
1
¯ux in
rat DRG neurons (Nicholson et al., 1996). RTX was
subsequently found to have similar effects on Na
1
channel
activation and inactivation as VTX and the toxins renamed
d-atracotoxin-Ar1 and -Hv1a, respectively, re¯ecting the
subfamily (i.e. Atracinae) to which the spiders belong
(Nicholson et al., 1998).
Venom from the male Eastern mouse spider M. bradleyi
(Fig. 2), of Australia, appears to contain a neurotoxin that
facilitates neurotransmitter release in both smooth and
skeletal muscle preparations. The activity of the venom in
the chick biventer cervicis muscle preparation was inhibited
by A. robustus antivenom. In addition, experiments in rat
dorsal root ganglion cells under voltage clamp conditions
suggested that a component in the venom modi®es
TTX-sensitive sodium channel gating in a manner similar
to the d-atracotoxins (Rash et al., 2000a).
The curtatoxins (I±III) are peptide toxins from another
Agelenid spider (Hololena curta) which have been isolated,
sequenced and found to cause rapid, irreversible ¯accid
paralysis in crickets (Stapleton et al., 1990). Curtatoxin II
has an identical sequence to the peptide toxin m-Aga III
from the venom of A. aperta, however the mechanism of
action of the toxins from H. curta has not been con®rmed
(Quistad et al., 1991). The venom of the Brazilian wolf
spider Lycosa erythrognatha increased the duration of
compound action potentials and produced long-lasting
post-potentials in the bullfrog sciatic nerve, actions which
were essentially irreversible and attributed to a peptide (Lyc
IV) of approximately 8 kDa with a probable action at Na
1
channels (Cruz et al., 1994). An insecticidal peptide,
DTX9.2, from the venom of a weaving spider Diguetia
canities induced rapid paralysis in insects, caused hyperex-
citation of sensory nerve and neuromuscular preparations
from house¯y larvae, and depolarised the membrane of
cockroach giant axons. All the in vitro actions of DTX
were prevented or reversed by TTX suggesting an action
at voltage-sensitive Na
1
channels of insect nerve
membranes (Bloomquist et al., 1996). In addition to antag-
onising glutamate receptors, argiotoxin-636 was shown to
reduce the neuronal excitability of rat cultured DRG cells
partly by inhibiting voltage-activated Na
1
currents in a
manner dependent on repeated activation (Scott et al.,
1998). In the rat phrenic nerve diaphragm, venom from a
South American hunting spider (Ancylometes sp.), increased
the amplitude of indirectly evoked twitches, depolarised
muscle ®bre membranes and increased the frequency of
miniature endplate potentials. The depolarisation of muscle
®bre membranes was prevented by low Na
1
Tyrode solution
and TTX, which also inhibited the increase in miniature
endplate potentials suggesting the presence in the venom
of another toxin that activates voltage-sensitive Na
1
channels (GreÂgio et al., 1999).
A novel class of sodium channels appears to be blocked
by a recently isolated toxin (PcTX1) from the venom of the
tarantula Psalmopoeus cambridgei (Escoubas et al., 2000).
The 40-amino acid toxin potently and selectively blocks the
ASIC1a subclass proton-gated sodium channels (a member
of the Acid Sensing Ion Channel family) that are expressed
in sensory neurons from dorsal root ganglion and in the
central nervous system.
3.5. Potassium channel toxins (Table 3)
The largest and most diverse family of ion channels are
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
233
Fig. 2. The male (a) and female (b) Eastern Mouse spider (Missulena bradleyi). scale bar 2 cm.
those selective for K
1
(Garcia et al., 1997), which is not
surprising given that K
1
channels regulate a wide variety of
cellular processes throughout the body. Potassium channels
are divided into two broad families, voltage-gated and
inwardly rectifying channels (Jan and Jan, 1994). Potassium
channels, like voltage-gated Na
1
and Ca
21
channels, are
made up of four repeats (in this case individual subunits),
each with six transmembrane domains, forming the ion pore
and several accessory subunits (Robertson, 1997). Voltage-
gated K
1
currents of native cells have been classi®ed as
either rapidly inactivating, transient outward `A-type' or
delayed rectifying, non-inactivating currents. Cloned
voltage-gated (K
V
) K
1
channel subunits are divided into
several subfamilies, those best characterised represent
mammalian counterparts of the Drosophila channels Shaker
(K
V
1), Shab (K
V
2), Shaw (K
V
3) and Shal (K
V
4) (Garcia et
al., 1997;Conley and Brammar, 1999). Although the genes
for many subunits have been cloned, there is still a substantial
gap in the correlation between cloned K
V
channels and native
currents (Robertson, 1997). However, the recent discovery of
toxins selective for K
1
channels from a variety of animal
venoms, including spiders, is helping to close this gap (Harvey
and Anderson, 1991; Garcia et al., 1994; Schweitz et al., 1995;
Sanguinetti et al., 1997; Diochot et al., 1999).
Two peptides, hanatoxins 1 and 2, isolated from the venom
of the Rose tarantula Grammostola spatulata (now Phrixotri-
cus spatulata), were shown to inhibit rat brain Shab- (Kv2.1)
and Shal-related K
1
channels expressed in Xenopus oocytes
whilst Shaker-, Shaw- and eag channels were relatively insen-
sitive (Swartz and Mackinnon, 1995). As mentioned
previously, the Ca
21
-channel blocker v-grammotoxin SIA,
from the same spider, was additionally found to interact
with voltage-gated K
1
channels. Likewise, the hanatoxins
were found to interact with voltage-sensitive Ca
21
channels
(Li-Smerin and Swartz, 1998). The K
V
4 family of channels are
also targeted by peptide toxins from two other spiders. In rat
ventricular myocytes, the heteropodatoxins (HpTx 1±3) from
Heteropoda venatoria prolonged the action potential duration
by blocking the transient outward K
1
current (I
to
) (Sanguinetti
et al., 1997). They were also found to block cloned K
V
4.2 but
not K
V
1.4 channels expressed in Xenopus oocytes and have
similar effects on channel gating as many of the Na
1
channel
toxins previously described, slowing current activation and
inactivation, as well as shifting the voltage-dependence of
inactivation to more positive potentials. The I
to
current in rat
ventricular myocytes and underlying channels, K
V
4.2 and
K
V
4.3, were also potently and selectively blocked by two
toxins (phrixotoxins, PaTx1 and PaTx2) from the venom of
the Chilean ®re tarantula Phrixotrichus auratus (Diochot et
al., 1999). Marvin et al. (1999) suggested that SGTx1, puri®ed
from the venom of the African tarantula Scodra griseipes, had
a similar mechanism of action to the hanatoxins after ®nding
the peptide partially (~40%) and reversibly inhibited both fast
transient and delayed recti®er K
1
currents in rat cerebellar
granule cells.
Using whole-cell patch clamping on GH3 cells,
Kushmerick et al. (1999) showed that toxin Tx3-1 from P.
nigriventer reversibly inhibits A-type K
1
currents without
affecting delayed rectifying, inward-rectifying and Ca
21
-
sensitive K
1
currents or T- and L-type Ca
21
channels.
Studies on the intra- and extracellular application of argio-
toxin-636 found that it reduced the excitability of rat DRG
neurons partly by inhibiting Na
1
channels, as mentioned
previously, in addition to dose-dependently inhibiting
voltage-activated K
1
channels without affecting the voltage
dependence of activation or steady-state inactivation (Scott
et al., 1998). More recently, argiotoxin was found to block
outward currents through the strong inward rectifying chan-
nel Kir2.1, probably via the insertion of the polyamine tail
lengthwise into the channel pore (Lee et al., 1999).
3.6. Chloride channel toxins (Table 3)
Chloride channels are involved in a multitude of
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
234
Table 3
Spider toxins affecting potassium and chloride channels
Spider
Channel/current inhibited
Reference/s
Potassiun channel Toxins
Hanatoxin 1 and 2
G. spatulata (now Phrixotrichus
spatulata)
Shab related (Kv2.1) and Shal related K
1
channels
Swartz and MacKinnon, (1995)
v-grammotoxin SIA
G. spatulata
voltage gated K
1
channels
Li-Smerin and Swartz, (1998)
HpTx (1±3)
H. venatoria
Kv4.2 channels
Sanguinetti et al., (1997)
PaTx 1 and 2
H. versuta
Kv4.2 and 4.3 channels
Diochot et al., (1999)
SGTx1
S. griseipe
fast transient and delayed recti®er currents
Marvin et al., (1999)
Tx3-1
P. nigriventer
A-type K
1
current
Kushmerick et al., (1999)
Arg 636
Argiope sp.
Kir2.1 channels
Scott et al., (1998); Lee et al.,
(1999)
Chloride channel toxins
Arg 636
Argiope sp.
inhibit Ca
21
-activated Cl
2
currents
Sutton et al., (1998b)
sFTX3.3
synthetic analogue of FTX
inhibit Ca
21
-activated Cl
2
currents
Sutton et al., (1998b)
FTX3.3
synthetic A. aperta toxin
inhibit Ca
21
-activated Cl
2
currents
Sutton et al., (1998b)
physiological processes including transepithelial transport,
cell volume control and acidi®cation of intracellular orga-
nelles (Jentsch et al., 1999). They are also important for the
regulation of cellular excitability as components of
receptors for the inhibitory neurotransmitters GABA, in
the vertebrate CNS (Bormann, 1988), and glutamate, in
the invertebrate nervous system (Osborne, 1996). Although
important, Cl
2
channels are not crucial for the initiation and
propagation of action potentials or transmission at the
neuromuscular junction and few toxins have been found
which affect them. However, Sutton et al. (1998b) have
recently shown that the polyamine, sFTX3.3 inhibits both
voltage-activated Ca
21
currents and Ca
21
-activated Cl
2
currents (I
Cl(Ca)
) in rat cultured dorsal root ganglion neurons,
whilst synthesised FTX and argiotoxin-636 inhibit I
Cl(Ca)
but
not the Ca
21
currents.
3.7. Toxins that stimulate the release of neurotransmitters
Spiders of the genus Latrodectus (family Theridiidae) are
found throughout the world (Bucherl, 1971). Likewise,
latrodectism, or envenomation by a spider of this genus, is
a world-wide phenomenon (Maretic, 1978). Human enve-
nomation by Latrodectus sp. spiders consists of a character-
istic set of symptoms including; severe pain radiating from
the bite site to the muscles of the back, abdomen and lower
limbs, restlessness, respiratory dif®culty, abdominal rigid-
ity, transient hypertension, profuse sweating, salivation,
nausea and vomiting (Duchen and Gomez, 1984).
Latrodectus venom has been found to cause massive
neurotransmitter release from a variety of nerves in both
vertebrates and invertebrates (Longnecker et al., 1970;
Frontali et al., 1972; Kawai et al., 1972; Cull-Candy et al.,
1973; Pinto et al., 1974) resulting in blockade of nerve
transmission leading to muscle paralysis (Henkel and
Sankaranarayanan, 1999). The effects on nerves from
vertebrates, insects and crustaceans have been attributed to
several high molecular weight proteins selective for each
type of animal (Frontali et al., 1976; Ornberg et al., 1976;
Fritz et al., 1980). The vertebrate selective toxin, a 130 kDa
protein, was named a-latrotoxin (a-LTX) (Tzeng and
Siekevitz, 1978) and the names latroinsectotoxins (LITs)
and a-latrocrustatoxin (a-LCT) have been proposed for
the insect and crustacean selective toxins, respectively
(Grishin, 1998).
Whole venom from black widow spiders and a-latrotoxin
cause transmitter release and depletion of synaptic vesicles
(Harvey, 1990). This may be explained by several possible
actions of a-latrotoxin at the presynaptic membrane.
Finkelstein et al. (1976) found that the component active
at the neuromuscular junction interacts irreversibly with
arti®cial phospholipid membranes to form non-selective
cation channels, a property which allows the in¯ux of extra-
cellular Ca
21
into the nerve terminal leading to vesicular
exocytosis. It has been noted that a-latrotoxin-stimulated
exocytosis at neuromuscular synapses is independent of
extracellular Ca
21
(Ceccarelli et al., 1979; Henkel and
Betz, 1995) whereas secretion of catecholamines from rat
adrenal chromaf®n cells is dependent on extracellular
calcium (Barnett et al., 1996), suggesting different modes
of action of a-latrotoxin.
A receptor for a-latrotoxin was isolated from bovine
solubilised brain membranes (Petrenko et al., 1990) and
found to bind to synaptotagmin, a synaptic vesicle-speci®c
membrane protein involved in exocytosis (Petrenko et al.,
1991). The isolated receptor was subsequently identi®ed as
a member of the neurexins, a family of single transmem-
brane domain, synaptic cell surface proteins (Ushkaryov et
al., 1992) the interaction with which is dependent on
calcium (Davletov et al., 1995). The role of neurexins,
speci®cally neurexin 1a as the calcium-dependent receptor
for a-latrotoxin has recently been con®rmed (Geppert et al.,
1998; Sugita et al., 1999). A second, calcium-independent
receptor for a-latrotoxin (CIRL or latrophilin) was also
isolated from bovine brain membranes by af®nity
chromatography (Krasnoperov et al., 1996) and found to
be a member of the secretin family of G-protein coupled
receptors (Lelianova et al., 1997). Overall the mechanism of
action of a-latrotoxin appears to consist of several
components, at low concentrations it stimulates exocytosis
by binding to the receptors neurexin and/or latrophilin,
while at higher concentrations it forms cation channels
directly increasing cytosolic calcium (Henkel and
Sankaranarayanan, 1999).
Venom gland extract from another theridiid spider,
Steatoda paykulliana, at mg/ml concentrations, also
increased the conductance of arti®cial lipid membranes
and at higher concentrations stimulated the release of neuro-
transmitter from PC12 cells, but did not contain a-latrotoxin
(Cavalieri et al., 1987).
3.8. Toxins affecting cholinergic transmission
In vertebrates, acetylcholine (ACh) is well established as
a transmitter in autonomic and motor neurons as well as
being an important central neurotransmitter (Florey,
1967). ACh is also a major neurotransmitter in the central
nervous system of invertebrates (Osborne, 1996). Receptors
for ACh are broadly classi®ed into nicotinic and muscarinic
subtypes, depending on their sensitivity to nicotine and
muscarine, respectively. A third type of ACh receptor, the
mixed nicotinic/muscarinic receptor is present in inverte-
brates (Osborne, 1996). Nicotinic receptors are ligand-
gated ion channels made up of ®ve subunits, each with
four membrane spanning domains, whilst muscarinic recep-
tors belong to the G-protein-coupled receptor superfamily.
Venom from the orb weaving spider, Argiope lobata, not
only caused postsynaptic block of glutamatergic synapses
but also reversibly blocked cholinergic transmission in frog
skeletal muscle (Usmanov et al., 1983). In a subsequent
study, Usmanov et al. (1985) found that the ability to post-
synaptically block cholinergic synapses was common
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
235
to seven other spiders from the family Araneida. They also
separated the venom of A. lobata and found seven toxic
fractions, of which one was selective for vertebrate neuro-
muscular transmission while the other six affected glutama-
tergic transmission of the locust. Nicotine-induced, but not
high K
1
-induced, secretion of catecholamines and ATP
from bovine chromaf®n cells was inhibited by v-agatoxin
IVA, which also reversibly blocked nicotine-induced inward
current suggesting that, in addition to blocking P-type Ca
21
channels, it also inhibits the neuronal nicotinic channel
(Granja et al., 1995). The polyamine amide JSTX-3, but
not argiotoxin-636, was also found to affect cholinergic
transmission causing voltage-dependent inhibition of ACh-
activated currents in rat phaeochromocytoma PC12 cells
(Liuet al., 1997).
3.9. Other neurotoxins
The potential use of spider toxins as tools for studying
physiological processes, identifying and characterising
neuronal ion channels and as new selective insecticides
has led to the isolation and characterisation of many novel
peptides. In response to the latter potential use of spider
venoms, there has been a shift in the emphasis of toxicity
assays toward using insects rather than more traditionally
used vertebrate laboratory animals (Bettini and Brignoli,
1978; Quistad et al., 1992; Atkinson et al., 1996; Manzoli-
Palma et al., 2000). However, mouse toxicity assays are still
widely used as a means of detecting novel neurotoxins
active on the vertebrate CNS (Escoubas et al., 1998). In
addition to the neurotoxins previously described, for
which distinct mechanisms of action have been reported,
many neurotoxic peptides have been isolated but as yet,
no mode of action determined.
The isolation from the East African tarantula, Pterino-
chilus sp., of one of at least 12 peptides toxic to mice was
reported by Bachmann (1982). Fraction A4/4 is a 77 amino
acid peptide that, like most of the toxic peptides isolated
from spiders thus far, appears to be tightly folded with
four disulphide bonds and causes death in mice by respira-
tory paralysis. The mode of action was not investigated but a
presynaptic action was suggested as unlikely, due to the
rapid reversibility of sublethal doses.
While characterising the venom of the tarantula
Eurypelma californicum, Savel-Neimann (1989) isolated
and sequenced a 38 amino acid peptide, ESTX, lethal to
cockroaches. A 39 amino acid peptide, identical to an
isoform of ESTX, and another unrelated protein toxin
showing similarities to toxin Tx2-9 from P. nigriventer,
have been isolated from the Mexican red knee tarantula
Brachypelma smithii (Kaiser et al., 1994). Escoubas et al.
(1997) isolated two novel peptide neurotoxins (lasiotoxins 1
and 2) from the venom of the tarantula Lasiodora
parahybana, which were lethal to mice and share 74%
homology with toxins isolated from the tarantulas
E. californicum and B. smithii. Upon initial fractionation
of the venom, this group noticed an interesting partition of
the vertebrate and invertebrate toxicity into different frac-
tions with little overlap. The insecticidal fractions contained
low molecular weight components while the vertebrate
toxicity was concentrated in peptide components of
3.7±7.3 kDa. Nine insecticidal peptides were isolated from
the venom of the trapdoor spider Aptostichus schlingeri, of
which six were sequenced (Skinner et al., 1992). Seven of
the isolated toxins caused ¯accid paralysis in tobacco horn-
worm (Manduca sexta) and beet armyworm (Spodotera
exigua) larvae and the sequenced peptides contained three
or four disulphide bonds. Two insecticidal peptides, Tx4(7)
and Tx4(6-1), which had no effects in mice, were isolated
from toxin fraction four of P. nigriventer venom (Figueiredo
et al., 1997). The peptide Tx4(6-1) and pooled fraction
(Tx4) stimulated the release of glutamate from cockroach
muscle slices. PhTx1, also from P. nigriventer, is lethal to
mice causing neurotoxic symptoms (tail elevation, excita-
tion and spastic paralysis) upon i.c.v. injection (Rezende et
al., 1991). In the mouse phrenic nerve diaphragm prepara-
tion, PhTx1 had no effect on indirectly evoked twitch height,
resting membrane potential or miniature end-plate poten-
tials. However, it did induce morphological changes in
nerves (vacuolization) and 20±30% of muscle ®bres
(vacuolisation with damaged mitochondria and sarcoplas-
mic reticulum) suggesting both neurotoxic and myotoxic
actions, although the mechanism remains unclear
(Mattiello-Sverzut et al., 1998).
4. Non neurotoxic peptides (Table 4)
The majority of the toxic proteins or peptides isolated to
date from spider venoms are either neurotoxins or enzymes.
However, several peptides displaying novel activities have
been discovered.
A necrotoxic peptide of 6.7 kDa appears to be the
predominant toxic component of venom from the female
Arkansas tarantula Dugesiella hentzi (Lee et al., 1974).
The toxin was found to bring about histological changes at
the site of injection and in the heart, where it produced
lesions typi®ed by acute focal areas of myocardial necrosis.
Chan et al. (1975) noticed that the co-administration of
adenosine 5
0
-triphosphate, also present in the venom in
substantial quantities, had a synergistic effect with the
necrotoxin, signi®cantly decreasing the LD
50
values when
injected into mice. The venom of the Singapore tarantula
Corecnemius validus contains another myotoxic peptide,
Covalitoxin-I, which was recently isolated, sequenced and
chemically synthesised, and causes necrosis of mouse
skeletal muscle (Balaji et al., 1999a). Two other peptides
(Covalitoxins II and III) from C. validus venom have been
isolated, sequenced and synthesised, however no toxicity or
functional studies have been reported as yet (Balaji et al.,
1999b).
In addition to the neurotoxic actions of P. nigriventer
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
236
venom, it has been found to contract rabbit vascular smooth
muscle (Antunes et al., 1993) and cause an increase in the
vascular permeability of rabbit and rat skin (Antunes et al.,
1992) by activating the local tissue kallikrein±kininogen±
kinin system (Marangoni et al., 1993). A series of peptides
with activity accounting for these actions on smooth muscle
have been isolated. PNV1 is a 13.9 kDa protein of approxi-
mately 125 amino acids with two disulphide bonds which
causes contractions in rabbit pulmonary arteries and mesen-
teric veins and has an N-terminal sequence different from
other peptides so far isolated from P. nigriventer venom
(Marangoni et al., 1993). A second large peptide, PNV2
(102 amino acids and 12.1 kDa), which contracts rabbit
vascular smooth muscle was isolated and found to have
four disulphide bonds and an N-terminal sequence different
to that of PNV1 (Bento et al., 1993). The peptide PNV3,
isolated by Bento et al. (1995), appears to be responsible for
the increase in microvascular permeability in rabbit skin and
has an N-terminal sequence which shares 60±70% homol-
ogy with toxins Tx2.1, Tx2.5 and Tx2.6 from the same
spider. Finally, PNV4, a 16.6 kDa peptide of 147 residues,
is one of two fractions, generated by reverse-phase HPLC of
P. nigriventer venom, responsible for causing relaxation of
the rabbit corpus cavernosum (Rego et al., 1996).
Short peptides that potentiate the action of kinins, both in
vitro and in vivo, have been found in the venoms of snakes
(Ferreira, 1965; Ferreira et al., 1992, 1995) and scorpions
(Ferreira et al., 1993) and led to the development of angio-
tensin converting enzyme (ACE) inhibitors used clinically
in the treatment of some forms of hypertension (Opie and
Kowolik, 1995). In 1990, Sosnina et al. (1990) isolated two
peptides from the venom of the European black widow
spider Latrodectus tredecimguttatus that inhibited the
activity of ACE. An undecapeptide (BPP-S) that signi®-
cantly potentiates the effects of bradykinin on smooth
muscle and inhibits ACE in vitro was also isolated from
the venom of Scaptocosa raptoria, a Brazilian wolf spider
(Ferreira et al., 1996). Two kinin-like peptides (peptide-S
and peptide-R), which were almost equipotent with brady-
kinin in contracting guinea-pig isolated ileum, were subse-
quently isolated from the venom of the same spider (Ferreira
et al., 1998). Kinin-like peptides have also been found in the
venoms of many other animals including snakes, wasps and
frogs (Schachter and Thain, 1954; Yasahura et al., 1973;
Ferreira and Henriques, 1992).
The lycotoxins (I and II, 25 and 27 amino acids, respec-
tively) are two novel peptides isolated from the venom of
the North American wolf spider Lycosa carolinensis with a
broad range of actions (Yan and Adams, 1998). In insect
body wall muscles, lycotoxin I caused complete loss of cell
membrane potential and blockade of neuromuscular trans-
mission. At high concentrations lycotoxin I lyses rabbit
erythrocytes, causing 55% haemolysis. Both lycotoxins I
and II caused ef¯ux of calcium ions from, and prevented
sequestration of
45
Ca
21
by, rat brain synaptosomes. In
addition, both peptides possess potent antimicrobial activity
against both prokaryotic and eukaryotic cells in plate growth
inhibition assays. The lycotoxins have predicted secondary
structures of amphipathic a-helices, a con®guration
characteristic of the antimicrobial pore-forming peptides
adenoregulin, dermaseptins and magainins. Indeed, the abil-
ity to form pores in biological membranes can account for
all the observed actions of lycotoxins I and II (Yan and
Adams, 1998). An antibacterial peptide was also found in
the venom of the wolf spider Lycosa singoriensis (Xuet al.,
1989). In addition, ®ve antibacterial peptides of 3±4 kDa
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
237
Table 4
Non-neurotoxic peptides from spider venoms
Toxin/Spider
Action/effect
References
D. hentzi
necrotoxin, myocardial necrosis
Lee et al., (1974); Chan et al., (1975)
Covalitoxin1: C. validus
myotoxin, skeletal muscle necrosis
Balaji et al., (1999a)
PNV1: P. nigriventer
contracts vascular smooth muscle
Marangoni et al., (1993)
PNV2: P. nigriventer
contracts vascular smooth muscle
Bento et al., (1993)
PNV3: P. nigriventer
increases microvascular permeability
Bento et al., (1995)
PNV4: P. nigriventer
relaxes rabbit corpus cavernosum
Rego et al., (1996)
L. tredecimguttatus
inhibits angiotensin converting enzyme (ACE)
Sosnina et al., (1990)
BPP-S: S. raptoria
potentiates bradykinin/inhibits ACE
Ferreira et al., (1996)
peptide-S: S. raptoria
kinin-like peptide
Ferreira et al., (1998)
peptide-R: S. raptoria
kinin-like peptide
Ferreira et al., (1998)
Lycotoxins I and II: L. carolinensis
antimicrobial activity
Yan and Adams, (1998)
L. singoriensis
antibacterial activity
Xuet al., (1989)
C. salei
®ve antibacterial peptides
Haeberli et al., (2000)
SHLP-1: S. huwena
lectin-like peptide
Liang and Pan, (1995)
A. schlingeri, Aphonopelma sp., Filistata sp.,
L. deserta, and P. tristis
cytotoxic to mouse neuroblastoma cells
Cohen and Quistad, (1998)
Salticidae (jumping spider venom)
cytotoxic to mouse and insect cells
Cohen and Quistad, (1998)
L. reclusa
cytotoxic to human neutrophils
Majeski et al., (1977)
have recently been isolated from the venom of C. salei
(Haeberli et al., 2000). The activity of antibacterial peptides
is thought to be due to a lytic action on bacterial cell walls
but the mode of lysis remains unknown.
Lectins are carbohydrate-binding proteins, found primar-
ily in plant seeds, which can cause agglutination of erythro-
cytes. Several lectins have been isolated from snake venoms
(Ogilvie et al., 1986; Bruno, 1990). Liang and Pan (1995)
isolated and sequenced the ®rst lectin-like peptide from
spider venom. The venom of the Chinese bird spider S.
huwena was found to contain substantial amounts of a 32
amino acid peptide (S. huwena lectin-like peptide-I,
SHLP-I) that agglutinated both mouse and human erythro-
cytes. SHLP-I showed homology with a fragment of great
nettle lectin as well as the neurotoxin HWTX-I, but
displayed no toxicity when administered to mice.
Cohen and Quistad (1998) conducted a study examining
the cytotoxic activity of venom from 26 species of spider
and four other arthropods on mammalian and insect cultured
cells. The insect cell line Sf9 was more sensitive to the
venom of spiders from the families Araneidae, Lycosidae
and Oxyopidae whilst venom from Aptostictnus schlingeri,
Aphonopelma sp., Filistata sp., Loxosceles deserta and
Plectreurys tristis were preferentially toxic to the mouse
neuroblastoma cell line N1E-115. Venom from the jumping
spiders (family Salticidae) were found to be the most
cytotoxic, being equipotent in insect Sf9 and mouse N1E-
115 cells with IC
50
values less than 1 mg venom protein per
ml in both lines. Although no toxic components were
isolated and characterised, this study introduced another
novel activity of spider venoms, which may be in part due
to enzymes or toxins already described. Previously, Majeski
et al. (1977) had reported that venom from the brown recluse
spider, Loxosceles reclusa, was directly cytotoxic to human
neutrophils at high concentrations as well as inhibiting
neutrophil chemotaxis toward complement derived
chemotaxins.
5. Enzymes (Table 5)
Enzymes are an important and common component of the
venom of many animals including bees, wasps, snakes and
lizards, with several possible functions. In this respect the
venom of spiders is no different, containing a wide variety of
enzymatic activities.
Hyaluronidase or hyaluronidase activity has been found
in the venom of many spiders, both mygalomorphs and
labidognaths. Nearly 50 years ago the venoms of the
Brazilian spiders Lycosa raptoria and Ctenus nigriventer
(now Phoneutria nigriventer) were reported to contain a
considerable amount of a hyaluronidase-like substance simi-
lar to the testicular enzyme (Kaiser, 1956). Shortly after,
Cantore and Bettini (1958) found hyaluronidase activity in
the venom of the European widow spider L. tredecimgutta-
tus (cited in Geren and Odell, 1984). Schanbacher et al.
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
238
Table 5
Enzyme activities found in spider venoms
Enzyme activity
Spider
Reference/s
Hyaluronidase
L. raptoria (now S. raptoria)
Kaiser, (1956)
P. nigriventer
Kaiser, (1956)
L. tredecimguttatus
Cantore and Bettini, (1958) (cited in Geren and Odell, (1984))
D. hentzi
Schanbacher et al., (1973a)
L. reclusa
Wright et al., (1973); Geren et al., (1976)
L. laeta
Schenone and Suarez, (1978)
L. rufescens
Young and Pincus, (2001)
C. salei
Kuhn-Nentwig et al., (1994)
A. robustus
Sutherland, (1978)
E. californicum
Savel-Niemann, (1989)
L. cylindrata/murina and L. godeffroyi
Rash and Hodgson, unpublished observations
Phosphodiesterase
A. robustus, Aphonopelma cratus and L. mactans Russell, (1966)
Alkaline phosphatase
L. reclusa
Heitz and Norment, (1974); Norment et al., (1979)
Esterase
L. reclusa
Wright et al., (1973); Norment et al., (1979)
ATPase
L. reclusa
Geren et al., (1976)
L. laeta
Schenone and Suarez, (1978)
Sphingomyelinase D
L. reclusa
Forrester et al., (1978)
Kininase (endopeptidase)
L. tredecimguttatus
Akhunov et al., (1981)
Collagenase
N. edulis, E. transmarina and I. Immanis
Atkinson and Wright, (1992)
Peptide isomerase
A. aperta
Shikata et al., (1995)
Phospholipase A
female H. versutus
Sheumack et al., (1984)
Eresus niger
Usmanov and Nuritova, (1994)
Proteases
see text for discussion on the presence of
protease activity in spider venoms
(1973a) identi®ed hyaluronidase as a major constituent of
venom from the tarantula D. hentzi. They subsequently
isolated the enzyme and found it to have a molecular weight
of approximately 39 kDa (Schanbacher et al., 1973b). Since
then hyaluronidase activity has been found in the venom of
Loxosceles reclusa, L. laeta and L. rufescens (Wright et al.,
1973; Geren et al., 1976; Schenone and Suarez, 1978;
Young and Pincus, 2001), the wandering spider Cupiennius
salei (Kuhn-Nentwig et al., 1994), Lycosa godeffroyi and
Lampona cylindrata/murina (Rash and Hodgson, unpub-
lished observations). In addition, hyaluronidases have
been isolated from the venom of the Sydney funnel-web
spider A. robustus (Sutherland, 1978) and the tarantula
E. californicum (Savel-Neimann, 1989). The substrate for
hyaluronidase is the mucopolysaccharide, hyaluronic acid,
which is a major constituent of the extracellular matrix
(Kreil, 1995). This has lead several researchers to postulate
that the hyaluronidase in animal venoms facilitates the
spread of other venom components by hydrolysing connec-
tive tissue (Schanbacher et al., 1973b; Sutherland, 1978).
Venoms from L. raptoria and C. nigriventer were
reported to possess proteolytic activity similar to that of
trypsin hydrolysing casein and ®brin (Kaiser, 1956). In addi-
tion, Kaire (1963) found a heat stable, neutral protease in the
venom of A. robustus. Three fractions, possibly different
forms of the one enzyme, displaying proteolytic but not
esterase activity were puri®ed from the venom of Pampho-
beteus roseus (Mebs, 1972). There have been several
con¯icting reports as to the presence of protease activity
in brown recluse spider venom. L. reclusa venom, obtained
by extraction of the dissected venom apparatus, was
reported to be devoid of proteolytic activity using casein
and haemoglobin as substrates (Geren et al., 1973). In
contrast, Jong et al. (1979), using venom obtained by elec-
trostimulation, isolated a proteolytic enzyme of 29.6 kDa
which hydrolysed the amide linkage of amino acids contain-
ing aliphatic, aromatic or basic side chains. Using the same
assay system as Jong et al. (1979); Rekow et al. (1983)
found protease activity in the cephalothorax and abdomen
extracts but not in venom apparatus extracts or the puri®ed
toxin from L. reclusa. Perret (1977) showed that the proteo-
lytic activity of tarantula venoms was due to the contamina-
tion of milked venom with digestive secretions expelled at
the time of milking. In support of this, Kuhn-Nentwig et al.
(1994) found proteolytic activity in the venom of C. salei
upon `normal' milking, which was completely lacking when
the chelicera had been carefully cleaned or when venom
from dissected glands was tested. The authors suggest that
the lack of venom protease activity is common to all spiders
and that when it is detected it is due to contamination during
the milking process.
In contrast to this view, two metalloproteinases from the
venom of Loxosceles intermedia have recently been identi-
®ed and characterised (Feitosa et al., 1998). Zymogram
analysis of venom, obtained by electrical stimulation, indi-
cated a 28 kDa band with ®bronectinolytic and ®brinogen-
olytic activity and a 35 kDa molecule with gelatinolytic
activity. Furthermore, two gelatinolytic molecules were
detected in the venom of L. intermedia following treatment
with trypsin (Veiga et al., 2000). The enzymes were
identi®ed as large serine-proteases with molecular weights
of 85- and 95-kDA. The authors suggested that upon enve-
nomation these zymogens may be activated by tissue
proteases and may act synergistically with other venom
components to induce necrosis. The venom used in both
of these studies was from the same source and obtained by
electrostimulation of the cephalothorax again raising the
question of contamination with digestive secretions.
However, Veiga et al. (2000) suggested that the narrow
proteolytic effect of the enzymes they have characterised
supports the fact that they are venom enzymes, as digestive
enzymes originating in the stomach of the spider would be
expected to have broad substrate speci®cities. In support of
proteolytic enzymes being constituents of spider venoms,
using zymogram analysis and venom gland extracts, we
have identi®ed several bands of activity (from ,18.5 to
around 32.5 kDa) in the venoms of the Australian spiders
Lycosa godeffroyi and Lampona cylindrata/murina against
both casein and gelatine, but not ®brinogen (Authors unpub-
lished observations). The use of venom gland extracts mini-
mises the likelihood of contamination with digestive
enzymes, although the possibility of contamination from
glandular tissue or adhering muscle remains. At this point
the studies providing evidence that spider venom itself
contains proteolytic enzymes still harbour the possibility
of some kind of contamination, be it from surrounding
tissues or digestive secretions. Perhaps in the not too distant
future the ever expanding cDNA libraries will answer this
question once and for all.
In a study examining the enzyme activity of several
species of snakes and arthropods, Russell (1966) found
phosphodiesterases in the venom of A. robustus,
Aphonopelma cratus, and the North American black
widow, Latrodectus mactans. The venoms of the brown
spiders L. laeta and L. reclusa were found to lack both
phosphodiesterase and collagenase activity (Suarez et al.,
1971; Wright et al., 1973; Schenone and Suarez, 1978).
However, they did contain enzymes with the ability to
hydrolyse the nucleotide ATP (Geren et al., 1976; Schenone
and Suarez, 1978). Wright et al. (1973) reported the
presence of esterase activity in the venom of L. reclusa
which was also shown by ¯uorometric assay to contain
alkaline phosphatase activity (Heitz and Norment, 1974).
The presence of these enzyme activities in L. reclusa
venom was later con®rmed when Norment et al. (1979),
using electrophoretic assays, detected two protein bands
with alkaline phosphatase activity and three bands exhibit-
ing esterase activity. In a study on L. reclusa venom-induced
lysis of red blood cells, Forrester et al. (1978) partially
puri®ed a haemolytic fraction that caused degradation of
the sphingomyelin component of red blood cell membranes.
This observation was found to be due to sphingomyelinase
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
239
D-type activity rather than phospholipase C or phospholi-
pase A
2
activity. This toxic enzyme will be discussed further
in the section on necrotic arachnidism.
A bradykinin-inactivating enzyme (kininase) has been
isolated from the venom of L. tredecimguttatus (Akhunov
et al., 1981). Further characterisation of the enzyme found
it to be a thiol endopeptidase which cleaved the -Pro(7)-
Phe(8)-bonds of both bradykinin and angiotensin I. In the
same study it was noted that whole venom had no proteoly-
tic activity against casein or haemoglobin (Akhunov et al.,
1996). These results are interesting as speci®c non-toxic
kininases are useful tools for studying the mammalian
kallikrein±kinin system and have potential as therapeutic
agents.
Studies on the enzymic activities of venom from several
species of spider have failed to detect the presence of
collagenase activity (Kaiser and Raab, 1967; Suarez et al.,
1971; Wright et al., 1973; Schenone and Suarez, 1978).
Conversely, Atkinson and Wright (1992) reported the
presence of measurable amounts of collagenase activity in
the milked venom of three Australian spiders, Nephila
edulis, Eriophora transmarina (orb weavers) and Isopeda
immanis (huntsman spider), and in midgut extracts of all 13
species tested.
In biological systems the translation and synthesis of
proteins only involves the use of L-form amino acids
(Branden and Tooze, 1991), however D-amino acid contain-
ing peptides have been isolated from a variety of higher
organisms (Kreil, 1994). Following the discovery of a
D-form amino acid (Ser
46
) in the P-type calcium channel
toxin v-agatoxin-TK (also designated as v-AgaIVB)
(Kuwada et al., 1994), a novel peptide isomerase, which
speci®cally inverts the chirality of Ser
46
, was isolated and
characterised (Shikata et al., 1995).
In several studies on venom from Loxosceles species
spiders and D. hentzi, neither phospholipase A nor phospho-
lipase C activities were found (Suarez et al., 1971;
Schanbacher et al., 1973a; Schenone and Suarez, 1978).
Conversely, in a study comparing the venoms of several
species of Australian funnel-web spiders, Sheumack et al.
(1984) detected phospholipase A activity only in the venom
of female Atrax versutus (now Hadronyche versutus).
Another phospholipase A, named component EnPA, has
subsequently been identi®ed in Eresus niger spider venom
and found to have anticoagulant activity (Usmanov and
Nuritova, 1994).
Therefore, spider venoms appear to contain a wide variety
of enzyme activities. Several of these enzymes, such as the
hyaluronidases of many venoms and the peptide isomerase
of A. aperta venom, may play a speci®c role in the enveno-
mation process or the post-translational modi®cation of
peptide toxins. Minton (1974) suggests that, as spiders
feed on the juices and lique®ed tissues of their prey, spider
venoms may have originally been digestive secretions and
may retain this function to varying degrees, thereby
explaining the abundance of enzymes present.
6. Low molecular weight components
In addition to the wide spectrum of biologically active
proteins, peptides and polyamine amides, spider venoms
contain a variety of low molecular weight components
displaying pharmacological activities, including biogenic
amines, free amino acids, polyamines, nucleotides and
inorganic salts.
6.1. Biogenic amines
Welsh and Batty (1963) found the amine 5-hydroxytryp-
tamine (5-HT) in the venoms or venom apparatus of a
variety of arthropods including seven species of Brazilian
spiders from the families Theraphosidae, Lycosidae and
Ctenidae. Venom gland extracts of the black widow spider
L. tredecimguttatus were also reported to contain 5-HT
(Pansa et al., 1972), as was the venom from male A.
robustus, the female of which lacked 5-HT but contained
trace amounts of 5-methoxytryptamine (Duf®eld et al.,
1979). Using gas chromatography and mass spectrometry,
Duf®eld et al. (1979) also detected and quantitated the
amines tyramine and octopamine in the venoms of both
male and female A. robustus.
Histamine is another biogenic amine commonly found in
arthropod venoms (Bettini and Brignoli, 1978; von Sicard et
al., 1989; Matuszek et al., 1992) and substantial amounts
have been detected in spider venoms. For example, L.
erythrognatha, P. nigriventer (Fischer and Bohn, 1957),
C. salei (Kuhn-Nentwig et al., 1994), Lampona cylindrata/
murina
1
(Rash et al., 2000b) and Lycosa godeffroyi (Rash et
al., 1998).
The vasoconstrictor action of Hololena curta venom on
rat thoracic aorta was found to be due to the presence of a
substance identi®ed by HPLC and electrochemical detection
as the catecholamine noradrenaline (Frew et al., 1994).
Noradrenaline has also been detected in the venom of
male, but not female, white-tailed spiders (L. cylindrata/
murina) (Rash et al., 2000b). Venom from female A.
robustus, in addition to the Australian huntsman spiders
Delana cancerides and Isopeda immanis, have also been
reported to display direct activity at adrenoceptors.
However, the agents responsible for this activity were not
identi®ed (Morgans and Carroll, 1976; Korszniak and Story,
1993).
Most of these biogenic amines have demonstrated roles as
neurotransmitters in the insect nervous system (Osborne,
1996), their presence in the venoms of spiders serving
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
240
1
The original publication refers to the white-tailed spider as
Lampona cylindrata. However, Platnick (2000) revised the genus
raising Lampona to family status, noted that the distribution of the
two most common species of Lampona (cylindrata and murina)
overlaps and that the spiders are almost indistinguishable. For this
reason spiders previously identi®ed as L. cylindrata will be referred
to as L. cylindrata/murina.
several possible functions. Amines such as 5-HT and hista-
mine are well established agents of pain production suggest-
ing a possible role in self defence, or in the facilitation of
uptake of other venom components by increasing local
blood ¯ow and cell permeability (Welsh and Batty, 1963;
Kuhn-Nentwig et al., 1998). As many of the polyamine
toxins so far isolated from spiders are use-dependent block-
ers of neurotransmission, the presence of a variety of known
insect neurotransmitters, in particular glutamate and
g-aminobutyric acid, in their venoms may accelerate the
paralytic actions of these toxins.
6.2. Free amino acids
A wide variety of free amino acids have been detected in
the venoms of many spiders. Particularly common are the
amino acid neurotransmitters g-aminobutyric, glutamic and
aspartic acids, which have been found in the venom of
several mygalomorphs, D. hentzi, E. californicum and
A. robustus (GABA only) (Gilbo and Coles, 1964;
Schanbacher et al., 1973a; Savel-Neimann, 1989) as well
as araneomorphs, P. nigriventer and L. tredecimguttatus
(Fischer and Bohn, 1957; Bettini and Maroli, 1978).
The amino acid taurine occurs in the venom of L.
tredecimguttatus (Bettini and Maroli, 1978) and is present
in high concentrations in C. salei venom (Kuhn-Nentwig et
al., 1994). Kuhn-Nentwig et al. (1998) subsequently discov-
ered that higher levels of taurine and histamine in insect
haemolymph increased the sensitivity of the insect to the
peptide toxin CSTX-1 from the venom of C. salei. They
concluded that the role of these compounds was to act
synergistically with other toxins increasing their lethality.
Other amino acids so far detected in spider venoms include
glycine, serine, threonine, lysine, glutamine, alanine,
arginine, asparagine, leucine and histidine (Fischer and
Bohn, 1957; Gilbo and Coles, 1964; Bettini and Maroli,
1978; Duf®eld et al., 1979).
6.3. Other low MW components
The venoms of three tarantulas, D. hentzi, an
Aphonopelma sp. from Arizona, and E. californicum, have
been described as containing the adenine nucleotides ATP,
ADP and AMP (Chan et al., 1975; Savel-Neimann, 1989).
As mentioned previously, ATP was found to have a syner-
gistic effect with the necrotoxin isolated from D. hentzi
venom (Chan et al., 1975). The purine derivatives adeno-
sine, guanosine, inosine and 2,4,6-trihydroxypurine were
recently reported in the venom of Latrodectus menavodi
(Horni et al., 2001). Venom from female L. cylindrata/
murina has been shown to have activity at adenosine recep-
tors which was inhibited upon exposure to adenosine deami-
nase but this venom component has not yet been isolated
(Rash et al., 2000b).
Substantial amounts of citric acid are present in the
venoms of A. robustus (Duf®eld et al., 1979), the tarantula
Grammostola cala and brown recluse spider L. reclusa,
among other arthropods (Fenton et al., 1995). In addition,
Fenton et al. (1995) found that citrate inhibits bee venom
phospholipase A
2
. However, no role for citrate in the venom
of spiders was suggested.
In addition to the polyamine amide compounds discussed,
the free polyamines spermine, spermidine, cadaverine and
putrescine have been detected in the venoms of A. robustus
(Duf®eld et al., 1979), D. hentzi, Aphonopelma sp. and
Aphonopelma emilia (Cabbiness et al., 1980). Savel-
Neimann (1989) detected trace amounts of free cadaverine
and spermidine in female E. californicum venom as well as
compounds containing spermine and aromatic molecules.
However, the author also points out that in the above studies,
which reported substantial concentrations of free polya-
mines, the venom used was hydrolysed with 6 M HCl and
that it cannot be excluded that those polyamines are released
from polyamine amides upon hydrolysis.
Other low molecular weight components found in spider
venoms thus far include glucose, lactic and phosphoric
acids, glycerol, urea and the inorganic ions sodium, potas-
sium, calcium, magnesium, chloride and phosphorous
(Wiener, 1961; Savel-Neimann, 1989; Kuhn-Nentwig et
al., 1994) some of which may be present to stabilise other
venom components, particularly neurotoxic peptides or
enzymes.
7. Necrotic arachnidism
Necrotic arachnidism is the development of necrotic cuta-
neous lesions following the bites of several species of
spider, particularly of the genus Loxosceles. The link
between skin ulceration and spider bites was suggested by
Schmaus (1929), but was not con®rmed in South America
until 1947 when Macchiavello (1947) associated the bite of
Loxosceles laeta to the `gangrenous spot of Chile', and 1957
in North America when Atkins et al. (1957) attributed necro-
tic arachnidism in the midwest to Loxosceles reclusa.
The envenomation syndrome of Loxosceles sp. spiders
consists of local necrotic lesions and, less commonly,
systemic reactions. The initial bite is reportedly painless,
often going unnoticed, with mild to severe pain (probably
due to ischaemia), itching, swelling and tenderness preced-
ing the development of a blister surrounded by an area of
ischaemia. Over a few days the lesion becomes a dull, blue-
violet colour and the centre sinks, hardens and usually drops
off leaving an ulcer which can take 6±8 weeks to heal. Bites
in areas of thicker fatty tissue such as the thighs, abdomen
and buttocks result in the most severe cases of local necrosis
and scarring. The rare cases of lethal Loxosceles envenoma-
tion, mainly in children, are due to systemic reactions, the
symptoms of which include fever, malaise, weakness,
nausea, vomiting and haematological disturbances such as
haemolytic anaemia, thrombocytopenia and consumption
coagulopathy (Futrell, 1992).
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
241
Early studies on the necrotic actions of venom from
L. reclusa found that rats were relatively resistant, while
mice, guinea-pigs and rabbits were susceptible to the
venom, with only rabbits and guinea-pigs developing the
dermonecrotic lesions seen in cases of human envenomation
(Morgan, 1969). As a result, rabbits have been used exten-
sively in studying the pathology and underlying mechanism
of Loxosceles induced dermonecrosis. The histopathologic
changes resemble cutaneous Arthus reaction (a type III
immune response involving the development of an in¯am-
matory lesion, characterised by induration, erythema,
oedema, haemorrhage and necrosis) and include oedema,
thickening of blood vessel endothelium, polymorphonuclear
leukocyte in®ltration, intravascular coagulation, vasodilata-
tion, destruction of blood vessel walls and haemorrhage
(Futrell, 1992). The accumulation of polymorphonuclear
leucocytes is required for the development of necrotic
lesions (Smith and Micks, 1970) and appears to be related
to the presence of circulating complement (Ward and
Cochrane, 1965). Exactly how the complement pathway is
involved is a topic of much debate (discussed by Futrell,
1992).
As mentioned previously, Forrester et al. (1978) partially
puri®ed a haemolytic fraction from L. reclusa venom that
also displayed sphingomyelinase D activity, both activities
being coincident within the electrophoretic pattern upon
further characterisation. The sphingomyelinase D
component (MW 31±32 kDa) has subsequently been
puri®ed and can account for most of the toxic actions of
whole L. reclusa venom, being able to induce necrotic
lesions, haemolyse red blood cells, cause platelet aggrega-
tion and cause death in laboratory animals (Babcock et al.,
1981; Kurpiewski et al., 1981). L. reclusa venom-induced
platelet aggregation appears to involve serotonin release
(Kurpiewski et al., 1981) and serum amyloid P component
(Rees, 1989; Gates and Rees, 1990), not C-reactive protein
as originally suggested (Rees et al., 1988). However,
C-reactive protein was reported to be required for venom-
induced lysis of human erythrocytes (Hufford and Morgan,
1981). The mechanism of action of L. reclusa venom and the
roles of C-reactive protein, serum amyloid P and serotonin
release are still not fully understood. However, the
dermonecrosis has been suggested to result from a direct
action of sphingomyelinase D on cell membranes at the
site of envenomation (Futrell, 1992).
There are seven species of Loxosceles found in Brazil
(Mota and Barbaro, 1995) with L. intermedia, L. gaucho
and L. laeta being the most common and clinically
important (Braz et al., 1999). The venoms of these three
species are antigenically cross-reactive and upon
SDS±PAGE analysis the dermonecrotic activity lies in the
major protein band of each with molecular weights of
35 kDa for L. gaucho and 32 kDa for both L. intermedia
and L. laeta (Barbaro et al., 1997). Tambourgi et al.
(1997) found the necrotoxic protein from L. intermedia
venom to be 35 kDa and further separated it into three
components P1, P2 and P3. The N-terminal sequence of
P2 shares 78% identity and 91% similarity with the sphin-
gomyelinase D isolated from the venom of L. reclusa,
suggesting that venoms of Loxosceles sp. spiders contain a
relatively homogenous family of toxic proteins.
Experimental envenomation of rabbits, by inducing the
Agelenid spider Tegenaria agrestis to bite a shaved patch of
skin, caused necrotic lesions closely resembling those
produced by Loxosceles venom (Vest, 1987a). T. agrestis,
also known as the hobo spider and the aggressive house
spider, has been implicated in several cases of human enve-
nomation resulting in dermonecrosis from the north west
United States (Vest, 1987b). Lesions resulting from
T. agrestis envenomation are characterised by induration,
blistering and necrotic ulcers requiring from 45 days to 3
years to heal. Systemic symptoms can include headache,
nausea, weakness, fatigue, dizziness, temporary memory
loss and vision impairment, with more severe cases some-
times resulting in aplastic anaemia, intractable vomiting and
profuse diarrhoea (Vest, 1987b).
The venom of two South African crab spiders of the
species Sicarius (testaceus and albospiosus) have also
been reported to cause tissue necrosis and haemorrhagic
lesions in rabbits suggesting that these spiders are also
potentially harmful to humans (Newlands, 1982; Van Aswe-
gen et al., 1997).
7.1. Necrotic arachnidism in Australia
The possibility that necrotising arachnidism occurs in
Australia was ®rst raised in the late 1970s and early 1980s
with the report of several cases of full thickness skin loss
following presumed envenomation (Sutherland, 1983a). In
no case was a creature seen to bite the victim. However,
circumstantial evidence suggested spiders as the likely
cause with Lycosa and Lampona species being singled out
as likely suspects (Sutherland, 1983b). Reports of three
further cases of substantial skin loss (one involving massive
dermonecrosis covering much of the victims right leg)
following suspected spider bites (Spring, 1987; Ibrahim et
al., 1989) highlighted the case for necrotic arachnidism
being a potential health issue in Australia (Sutherland,
1987). In these reports, as above, no offending creature
was felt or seen to bite the victims, sparking a large debate
as to the true cause of such a syndrome (Kemp, 1990;
Sutherland, 1990; Beardmore, 1991; Harvey and Raven,
1991; Hayman and Smith, 1991; Raven and Harvey, 1991;
Sutherland, 1991). After catching a small, brown, cigar
shaped spider (possibly L. cylindrata/murina) in the act of
biting her inner thigh, a 33 year-old woman developed an
acutely painful ulcerous lesion which required 3 weeks to
begin healing (Gray, 1989). Gray (1989) also reported treat-
ing three other cases of local ulceration following the bite of
a small brown spider. In contrast, White et al. (1989)
presented 36 cases of con®rmed spider bites, including
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
242
eight by L. cylindrata/murina, none of which resulted in
local ulcers or necrosis.
To date, cases of positively identi®ed spider bites result-
ing in local tissue damage in Australia are rare. However,
several species of spider are still implicated, the prime
suspects being the white-tailed spider L. cylindrata/murina
(Skinner and Butler, 1995; Pincus et al., 1999; Woo and
Smart, 1999; Fig. 3), and the black house or window spider
Badumna insignis (Macmillan, 1989; Pincus et al., 1999).
In an editorial comment, White (1999) points out that until
Pincus et al. (1999) published 14 cases of de®nite or
suspected spider bites resulting in necrosis, only four
cases of tissue injury associated with spider bite in Austra-
lia had been published, and in none of these cases was the
spider formally identi®ed. He also notes that in only three
of the cases reported by Pincus et al. (1999) was the spider
positively identi®ed. The two cases involving the white-
tailed spider, L. cylindrata/murina, resulted in shallow or
small ulcers that healed within a month and were described
by White as `at the mild end of the necrotising arachnidism
spectrum'. Some authors agree that bites by L. cylindrata/
murina may produce small localised lesions or ulcers
(Raven and Harvey, 1991; Sutherland, 1991) but doubt
that the unexplained extensive skin loss sometimes seen
in Australia can be attributed to the action of spider
venom (Raven and Harvey, 1991). White et al. (1995)
acknowledges that there is `a poorly de®ned clinical entity
consisting of unexplained skin blistering and ulceration' in
Australia and has suggested that some individuals may be
particularly sensitive to spider venoms. In a follow-up
study to Pincus et al. (1999); Young and Pincus (2001)
detected hyaluronidase and protease activity in venom
extracts from Loxosceles rufescens, B. insignis and L.
cylindrata/murina. Interestingly, only venom extract
from Loxosceles rufescens, and the abdominal extracts of
B. insignis and L. cylindrata/murina, displayed sphingo-
myelinase activity.
In Australia, most of the cases where spider bite has been
associated with necrotic lesions, or indeed, a spider seen to
bite before the development of local necrosis, the spider was
identi®ed by the victim, not by an arachnologist. The
problem with this is illustrated in a letter to the editor of
Toxicon by Russell and Gertsch (1983) where they describe
ticks, assassin bugs, and even crickets and grasshoppers as
being brought to the hospital as `spiders' by patients bearing
a necrotic lesion. Clinicians and arachnologists are now
stressing the importance of witnessing the bite and having
the offending animal positively identi®ed by an expert if we
are to determine, without doubt, the creature or creatures
responsible for `necrotic arachnidism' in Australia (White,
1987; Raven and Harvey, 1991; Pincus et al., 1999; White,
1999; Isbister and Gray, 2000a). Interestingly, a recent
prospective study involving 52 bites by L. cylindrata/
murina, where the identity of the spider was con®rmed by
an arachnologist, reported the following clinical effects:
local pain in all victims, the presence of a persistent lump
(44%), red area (56%) or both (27%) but no cases of ulcera-
tion or necrosis (Isbister and Gray, 2000b).
8. Sex-linked variation in venom composition and
activity
The resulting symptoms from envenomation by many
creatures can vary dramatically from case to case. There
are several factors that can contribute to the severity of a
venomous bite or sting, such as the species of the offending
creature, the site of envenomation and the susceptibility of
the victim. The quantity and/or quality of venom injected
are factors that should also be considered. Within a given
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
243
Fig. 3. Female (a) and male (b) white-tailed spider (Lampona cylindrata/murina). scale bar 1 cm.
species, the two latter aspects have been shown to vary
according to the geographic origin, size, age and sometimes
sex of the creature in question. For example venom from
female Buthus eupeus scorpions was found to be twice as
toxic to mice and guinea-pigs as venom from males
(Kashnikova, 1979). Despite the relative paucity of direct
comparison studies, the venoms of spiders appear to be no
exception, with differences between the venom of male and
female spiders of the same species having been noted for
many years. This is mainly because one sex proves to be
toxic to humans or mammals while the other does not. This
is the case with the theridiid spider Latrodectus mactans, the
notorious black widow, in which the venom of the female
spider was lethal to rats, while venom from males was
essentially non toxic (D'Amour et al., 1936). Maretic et
al. (1964) reported similar ®ndings for another theridiid
spider, Steatoda paykulliana, where guinea-pigs bitten by
female but not male spiders developed symptoms analogous
to those of latrodectism.
In contrast, the reverse is true for the Sydney funnel-web
spider A. robustus. Where the male spider has been respon-
sible for 14 human fatalities (Gray and Sutherland, 1978),
whereas venom from female A. robustus seems to lack
signi®cant amounts of the lethal neurotoxin, robustoxin
(Sheumack et al., 1984). In toxicity assays in new-born
mice, female A. robustus venom was found to be 19 times
less potent than venom from the male. In addition, the
venoms of male and female A. robustus displayed substan-
tially different HPLC pro®les and polyacrylamide gel
isoelectric focusing patterns. This re¯ects the degree of
sex-related variation of M. bradleyi venom, where the
male contained a potent neurotoxin with activity in avian
and mammalian nerve-muscle preparations whereas this
component appeared to be absent from female M. bradleyi
venom (Rash et al., 2000a). Interestingly, venoms from male
and female Blue Mountains funnel-web spiders Hadronyche
versutus (formerly Atrax versutus) were found to have very
similar LD
50
s in new born mice (0.7 and 0.5 mg/kg s.c.,
respectively) and HPLC pro®les (Sheumack et al., 1984).
However, isoelectric focusing patterns of venom from both
sexes revealed several differences in their respective protein
components.
In characterising the venom of an African tarantula
Scodra griseipes bred in captivity, CeÂleÂrier et al. (1993)
noted several gender differences. Female spiders produced
more venom which was of higher toxicity to mice and
contained three more electrophoretic bands than venom
from male spiders. Conversely, male S. griseipes venom
had a higher protein content than female venom. A higher
protein content in venom from male spiders as compared to
female spiders was also reported for both venom gland
extracts and venom obtained by electro-stimulation of the
sac spider Cheiracanthium furculatum (Croucamp and
Veale, 1999). Aside from the differences in biogenic
amine content and adenosine receptor activity of male and
female L. cylindrata/murina venom (Rash et al., 2000b) and
the apparent neurotoxic activity peculiar to venom from the
male, the protein content and SDS±PAGE patterns were
found to be very similar (authors unpublished observations).
Like L. cylindrata/murina, venom from males and females
of an Australian Lycosa sp. had almost identical
SDS±PAGE patterns. However, the female Lycosa sp.
venom contained signi®cantly more protein and myotoxic
activity than venom from the male (authors unpublished
observations). An analogous situation was noted for the
venom of the South American brown spider L. intermedia
(de Oliveira et al., 1999). Both male and female
L. intermedia had a similar pattern of bands upon
SDS±PAGE analysis. However, venom from female spiders
had a two-fold higher protein content, was richer in the
dermonecrotic toxin F35 and induced both complement-
dependent haemolysis and dermonecrosis more potently
than did venom from male L. intermedia. These ®ndings
supported the results of Kent et al. (1984) who reported
that although adult male and female L. reclusa spiders had
the same venom composition, the relative abundance of the
components differed. Finally, in a study examining the
variability of venom from several tarantulas, using HPLC
and MALDI-TOF-MS, Escoubas et al. (1999) found both
qualitative and quantitative differences in six of eight
species examined. These observations indicate that sex is
a substantial factor in the intraspeci®c variation of spider
venoms.
In general, there appears to be a trend toward the female
of the species having more toxic venom than that of the
male, which, given the maternal responsibilities, longer
life span of female spiders and the relatively short life of
the mature male, would make sense for the survival of the
species. However, the toxicity assays used in many of the
above studies were conducted on mammals rather than
insects. The latter constitute the main diet and therefore
presumably the speci®c target of spider venoms. As a result,
one must exercise caution in hypothesising on the possible
reasons for the sometimes marked gender differences
observed in spider venoms. Therefore, until more compar-
isons are made between venoms from male and female
spiders in light of their relevance to prey capture and survi-
val the purpose of the differences observed thus far remains
unclear.
9. Conclusions
Over millions of years spiders have evolved toxins, with
a variety of targets, affecting primarily the nervous systems
of their prey. As illustrated in this review, the majority of
the components of spider venom are either neurotransmit-
ters (excitatory and inhibitory amino acids), neurotoxins
themselves or components that aid in the stability, delivery
or effectiveness of these paralysing toxins to their site of
action such as enzymes, inorganic ions and nucleotides.
Given the dependence of neurotransmission on the
L.D. Rash, W.C. Hodgson / Toxicon 40 (2002) 225±254
244
movement of ions across membranes it is not surprising
that most of the neurotoxins speci®cally target neuronal
ion channels. It is likely that, with further work, neurotox-
ins with as yet unidenti®ed actions will prove to act at ion
channels of some description.
Spider venom neurotoxins are of interest for several
reasons. Due to the similarity of ion channels from both
the natural invertebrate prey and vertebrates, and the amaz-
ing selectivity of spider neurotoxins, they have already been
instrumental in our understanding of channel structure and
function. As the trend to screen spider toxins for more selec-
tive ligands progresses, they will surely continue to provide
new tools and lead compounds for the design of selective ion
channel activators and blockers with therapeutic potential.
In addition, toxins which discriminate between insect and
vertebrate channels may not only give us access to envir-
onmentally friendly insecticides but knowledge of the struc-
tural differences allows the possibility to design new, highly
selective, non-peptide insecticides. As discussed in the ®nal
section of this review, the sex of a spider or indeed many
venomous creatures plays a substantial role in the composi-
tion of venom and should not be overlooked in the search for
the next captopril or novel ion channel.
Acknowledgements
We gratefully acknowledge support from the Monash
University Faculty of Medicine Research Initiatives Scheme
and the Australian Research Council Small Grants Scheme.
We are grateful to David Humfrey for spider photography.
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