Natural Born Insect Killers: Spider-Venom Peptides And Their Potential For Managing Arthropod Pests

Article (PDF Available)inOutlooks on Pest Management 24(1):16-19 · February 2013with 852 Reads
DOI: 10.1564/v24_feb_05
Abstract
A number of arthropod predators such as centipedes, scorpions, spiders, and wasps employ venom for incapacitating prey. Spiders, in particular, are the most successful venomous animal and with the possible exception of predatory beetles they are the most abundant terrestrial predators. Since spiders are the most efficient insect killers on the planet, it is not surprising that their venoms have proved to be a rich source of natural insecticidal compounds. Spiders use venom to paralyze and ultimately kill their prey, and consequently it is rich in insecticidal neurotoxins. The venoms are a complex chemical cocktail of inorganic salts, small organic molecules, peptides, and proteins that act together on virtually every component of the synaptic machinery in the central or peripheral nervous system of envenomated prey. This approach is tantamount to "shock-and-awe" at the molecular level. The dominant components of most spider venoms are disulfide-rich peptides that modulate the activity of neuronal ion channels. These peptides are small, typically comprising 30–40 amino acid residues, and more than 1000 unique peptides can be present in the venom from a single spider. These peptides rapidly incapacitate envenomated prey either by "deadening" the nervous system and causing flaccid paralysis or "over-activating" the nervous system and inducing convulsive paralysis. Many are highly selective for invertebrates. The range of activities exhibited by these peptides is extraordinary and includes modulation of glutamate receptors, transient receptor potential channels, calcium-activated potassium channels, and voltage-gated calcium, sodium, and potassium channels. While in many cases these peptides act on the same molecular target as extant chemical insecticides, in other cases these peptides have novel pharmacologies that have allowed identification of new insecticide targets. A key factor that enhances the potential of these peptides for arthropod pest control is their unusual three-dimensional structure. Most spider-venom peptides contain an architectural motif known as an "inhibitor cystine knot" in which a ring formed by two disulfide bridges and the intervening sections of peptide backbone is pierced by a third disulfide bond to create a pseudo-knot. This "trick" of tying themselves in a knot makes these peptides resistant to harsh solvents, extremes of temperature and pH, and, most importantly, proteases that might otherwise degrade the peptides in the body of arthropod prey. Thus, insecticidal spider-venom peptides should be stable during long periods of storage as well as in the field, and they should degrade to innocuous breakdown products (amino acids).
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NATURAL BORN INSECT KILLERS
1 6 O u t l o o k s o n Pe s t M a n a ge m e n t Fe b r u a r y 2 0 1 3 DOI: 10.1564/v24_feb_05
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NATURAL BORN INSECT KILLERS: SPIDER-VENOM PEPTIDES AND
THEIR POTENTIAL FOR MANAGING ARTHROPOD PESTS
Maria Ikonomopoulou and Glenn King
#
from the Institute for Molecular Bioscience, University of Queensland
examine the potential of insecticidal peptides from spider venom for control of arthropod pests.
#
Address for
correspondence: Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St
Lucia, QLD 4072, Australia; Phone: +61 7 3346-2025; FAX: +61 3346-2101; Email: glenn.king@imb.uq.edu.au
were planted in 29 countries, representing 10% of all crop-
land (Gatehouse et al., 2011). The introduction of insect-
resistant GM crops carrying an insecticidal protein (known as
d-endotoxin, Cry toxin, or simply Bt) from the bacterium
Bacillus thuringiensis dramatically reduced insecticide use and
in many cases improved crop yields (King & Hardy, 2013).
Although there have been few reports of field resistance to
Bt, there is concern that its constitutive expression in trans-
genic plants will ultimately expedite resistance development.
Consequently, there is much interest in engineering Bt plants
that express additional insecticidal-toxin genes that act via
different and possibly synergistic mechanisms, an approach
known as pyramiding or trait stacking.
Spiders: a goldmine of natural insecticidal
toxins
A number of arthropod predators such as centipedes, scor-centipedes, scor-
pions, spiders, and wasps employ venom for incapacitating
prey. Spiders, in particular, are the most successful venomous
animal and with the possible exception of predatory beetles
they are the most abundant terrestrial predators (Windley et
al., 2012). Since spiders are the most efficient insect killers on
the planet, it is not surprising that their venoms have proved
to be a rich source of natural insecticidal compounds.
Spiders use venom to paralyze and ultimately kill their
prey, and consequently it is rich in insecticidal neurotoxins.
The venoms are a complex chemical cocktail of inorganic
salts, small organic molecules, peptides, and proteins (Kuhn-
Nentwig et al., 2011; King & Hardy, 2013) that act together
on virtually every component of the synaptic machinery in
the central or peripheral nervous system of envenomated prey
(Figure 1). This approach is tantamount to “shock-and-awe”
at the molecular level.
The dominant components of most spider venoms are
disulfide-rich peptides that modulate the activity of neuronal
ion channels. These peptides are small, typically comprising
30–40 amino acid residues, and more than 1000 unique
peptides can be present in the venom from a single spider
(King & Hardy, 2013). These peptides rapidly incapacitate
envenomated prey either by “deadening” the nervous system
and causing flaccid paralysis or “over-activating” the nervous
system and inducing convulsive paralysis. Many are highly
selective for invertebrates (King & Hardy, 2013). The range
of activities exhibited by these peptides is extraordinary and
includes modulation of glutamate receptors, transient recep-
tor potential channels, calcium-activated potassium channels,
Keywords: arthropod pest, insecticide, insecticide resistance, GM crops, spider,
insecticidal peptide
Arthropod pests
The human population is projected to increase to 9.3 billion
in 2050 (Bloom, 2011), creating unprecedented challenges
in the supply and distribution of food. Most of the required
increase in food production will have to come from improved
crop yields since there is limited scope for increasing the
amount of cultivated land. The control of arthropod pests is
thereby of paramount importance as they are one of the major
causes of crop loss; despite intensive control measures, they
reduce world crop yields by about 14% annually, resulting in
economic losses of more than $100 billion (Pimental, 2009).
Arthropod pests also vector a diverse array of livestock and
plant pathogens, as well as pernicious human diseases such as
malaria, dengue, yellow fever, filariasis, and Chagas disease.
Current methods of controlling arthropod
pests
Chemical insecticides are the dominant method for control-
ling arthropod pests in both the agricultural and public
health arenas. However, two key developments have led to
an inexorable decline in the arsenal of commercially avail-
able insecticides. First, the continual use over many decades
of a very small panel of insecticide classes has inevitably led
to the evolution of resistance in key arthropod pests; more
than 600 species of insects and mites are now resistant to one
or more classes of chemical insecticide (Bass & Field, 2011).
Second, legislative decisions such as the 1996 U.S. Food Qual-
ity Protection Act, which require more stringent review of
insecticides by regulatory authorities, has led to de-registra-
tion or use cancellation for a number of previously successful
classes of chemical insecticides (King & Hardy, 2013). For
example, between January 2005 and December 2009 the U.S.
EPA de-registered or limited the use of 169 insecticides, while
only 9 new insecticides were registered during the same time
period (King & Hardy, 2013).
The decreasing effectiveness of many chemical insecticide
led to renewed interest in the 1990s in alternative methods of
arthropod pest control. By far the most successful alternative
approach has been the introduction of genetically modified
(GM) crops. In 2010, 148 million hectares of GM crops
NATURAL BORN INSECT KILLERS
O u t l o o k s o n Pe s t M a n a g e m e n t Fe b r u a r y 2 0 1 3 1 7
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and voltage-gated calcium, sodium, and potassium channels.
While in many cases these peptides act on the same molecular
target as extant chemical insecticides (Figure 1), in other cases
these peptides have novel pharmacologies that have allowed
identification of new insecticide targets (King, 2007).
A key factor that enhances the potential of these peptides
for arthropod pest control is their unusual three-dimensional
structure. Most spider-venom peptides contain an architectural
motif known as an “inhibitor cystine knot” in which a ring
formed by two disulfide bridges and the intervening sections
of peptide backbone is pierced by a third disulfide bond to
create a pseudo-knot (Figure 2). This “trick” of tying them-
selves in a knot makes these peptides resistant to harsh
solvents, extremes of temperature and pH, and, most impor-
tantly, proteases that might otherwise degrade the peptides in
the body of arthropod prey (Saez et al., 2010; King & Hardy,
2013). Thus, insecticidal spider-venom peptides should be
stable during long periods of storage as well as in the field,
and they should degrade to innocuous breakdown products
(amino acids).
Deployment of spider-venom peptides for
control of arthropod pests
In contrast with chemical insecticides, spider-venom peptides
are unlikely to be topically active since they would have to
penetrate the insect exoskeleton in order to access molecular
targets in the insect nervous system. Moreover, since spiders
employ a hypodermic-needle-like fang to inject venom into
prey, there is no evolutionary selection pressure that might
lead to accrual of oral activity. While some insecticidal spider-
venom peptides have been demonstrated to be orally active,
most are 100-fold less potent when fed to insects compared
to when they are injected (King & Hardy, 2013). Thus, the
commercial potential of spider-venom peptides would be
significantly enhanced by approaches that improved their oral
activity or by the use of vector-based delivery methods (such
incorporation of toxin transgenes into entomopathogens) that
entirely removed the requirement for oral activity.
One promising option is to fuse spider-venom peptides
with a carrier protein that facilitates their illicit transport
across the insect gut. The best studied example is Galanthus
nivalis agglutinin (GNA), a mannose-specific lectin from the
snowdrop plant. Following ingestion, GNA binds to glyco-
proteins in the insect digestive tract and is then transported
across the gut epithelium into the hemolymph; over a period
of several hours, the protein accumulates in the insect gut,
Malpighian tubules, hemolymph, and central nervous system
(Fitches et al., 2001; Fitches et al., 2012). Thus, GNA can be
fused to insecticidal spider-venom peptides to enhance their
transport across the gut to their sites of action in the nervous
system, thereby enhancing their oral activity. This approach
has been used to massively enhance the oral insecticidal activ-
ity of several spider-venom peptides (Fitches et al., 2012).
Figure 1. Schematic of insect cholinergic and glutamergic synapses showing the primary molecular targets of spider-venom components (green
boxes) and major classes of chemical insecticides (red boxes). Spider venoms contain enzymes that facilitate the access of peptide neurotoxins
to their molecular targets by degrading the sheath around myelinated axons as well as the extracellular matrix protecting the synaptic cleft. The
?-latrotoxins found in the venom of widow spiders cause massive neurotransmitter release by promoting synaptic vesicle exocytosis. Abbreviations:
AChE, acetylcholinesterase; Ca
V
, voltage-gated calcium channel; CNS, central nervous system; Glu, glutamate; GluR, ionotrophic glutamate receptor;
K
V
, voltage-gated potassium channel; nAChR, nicotinic acetylcholine receptor; Na
V
, voltage-gated sodium channel; NMJ, neuromuscular junction.
Adapted from (King & Hardy, 2013).
1 8 O u t l o o k s o n Pe s t M a n a g e m e n t Fe b r u a r y 2 0 1 3
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NATURAL BORN INSECT KILLERS
Alternatively, since spider-venom peptides are essentially
gene-encoded mini-proteins, genes encoding these peptides
could be engineered into a variety of entomopathogens (Figure
2), which would mitigate two of the potential disadvantages of
spider-venom peptides. First, in this scenario, the spider-venom
peptides would be produced systemically in the insect host after
pathogen infection, and hence lack of oral activity would no
longer be an impediment to toxin deployment. Second, the
phylum selectivity of the spider-venom peptide would become
unimportant as the range of affected insects would be deter-
mined primarily by the host range of the entomopathogen.
Off-target effects, particularly on predators and parasitoids,
could be limited by choosing entomopathogens with restricted
host selectivity; for example, the fungus Metarhizium acridum
exclusively infects grasshoppers in the suborder Caelifera,
making it valuable as a locust-specific bioinsecticide.
Transgenic baculovirus have been engineered that express
insecticidal venom peptides from sea anemones, scorpions
or spiders; in all cases, the toxin transgene reduced the time
between virus application and cessation of feeding or death.
Notably, however, the most dramatic improvement in insec-
ticidal activity resulted from incorporation of a transgene
encoding a spider-venom peptide (Maggio et al., 2010). In
addition to baculoviruses, the potency and speed of kill of
entomopathogenic fungi could also be enhanced by engineer-
ing them to express insecticidal spider-venom peptides. For
example, the time as well as the dose required for Metarhizium
anisopliae to kill the tobacco hornworm Manduca sexta and
the dengue vector Aedes aegypti were reduced when the
fungus was engineered to express the scorpion-venom peptide
AaIT (Wang & St Leger, 2007).
Transgenic plants
An alternative genetic approach for deployment of insec-
ticidal spider-venom peptides is to incorporate transgenes
encoding these toxins into crop plants. For example, trans-
genes encoding the venom peptide w-hexatoxin-Hv1a from
the Australian funnel-web spider Hadronyche versuta have
been engineered into cotton, tobacco, and poplar plants
(King & Hardy, 2013). This peptide is a specific blocker of
insect neuronal voltage-gated calcium channels (see Figure
1), but it has no activity on vertebrates. Tobacco plants have
also been engineered to express Magi-6, a peptide from the
venom of the related hexathelid spider Macrothele gigas
(Hernández-Campuzano et al., 2009). All of these transgenic
plants have significantly enhanced resistance to lepidop-
teran crop pests. For example, the mortality of 2nd instar
Helicoverpa armigera (cotton bollworms) fed on transgenic
tobacco expressing w-hexatoxin-Hv1a was 75–100% after
three days compared to 0% for larvae fed on untransformed
plants (Khan et al., 2006). Remarkably, it has even been
claimed that transgenic cotton expressing w-hexatoxin-
Hv1a is as effective as Monsantos pyramided Bollgard II
®
cotton for controlling major pests of cotton (Omar & Ali
Chatha, 2012).
As mentioned previously, there is currently much interest
in engineering Bt plants that express additional insecticidal-
toxin genes that act via different and possibly synergistic
mechanisms. Transgenes encoding insecticidal spider-venom
peptides might be good candidates for trait stacking with Bt
for several reasons. First, they have entirely different mecha-
nisms of action. Second, they are likely to be synergized by
Bt due to its ability to induce lysis of midgut epithelial cells
(Soberon et al., 2007), which might be expected to promote
movement of spider-venom peptides across the gut into the
insect hemoceol. Finally, whereas Bt toxins are specific for
the insect orders Lepidoptera, Coleoptera, Hymenoptera, and
Diptera, spider-venom peptides with complementary selectiv-
ity, particularly against sap-sucking hemipterans, could be
selected for trait stacking.
Figure 2. Schematic of the various insect control options available for spider-venom peptides. The peptides could be used as standalone insecticides
incorporated into sprays or baits, or transgenes encoding the peptides could be engineered into crops or entomopathogens such as viruses and
fungi. The central panel is a schematic of the spider-venom peptide w-hexatoxin-Hv1a, with the three disulde bonds that stabilise the peptide by
forming an inhibitor cystine knot motif highlighted in green. Spider photos provided by Bastian Rast. Adapted from (King & Hardy, 2013).
NATURAL BORN INSECT KILLERS
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Summary
In summary, disulfide-rich spider-venom peptides are promis-
ing insecticide leads due to their invertebrate selectivity, stabil-
ity, ease of production, and in many cases novel pharmacology.
Moreover, it should be possible to use transgenes encoding these
peptides as insect-resistant plant traits or as a means of improv-
ing the efficacy of bacterial and fungal entomopathogens.
Acknowledgements
The authors acknowledge financial support from the Austral-
ian Research Council.
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A) lectins within tomato moth (Lacanobia oleracea) larvae;
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to snowdrop lectin magnifies the oral activity of insecticidal
w-hexatoxin-Hv1a peptide by enabling its delivery to the central
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1–86, Elsevier.
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(2010) Spider toxins and their potential for insect control, in
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(Gilbert LI and Gill SS eds) pp 101–123, Academic Press, London.
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Glenn King is a Professorial Research Fellow in the Institute for Molecular
Bioscience (IMB) at the University of Queensland. He completed his BSc and
PhD at the University of Sydney before undertaking postdoctoral studies as
a NHMRC CJ Martin Fellow at the University of Oxford. After stints as a
research and teaching academic the University of Sydney and the University
of Connecticut, he moved to a full-time research position at the IMB in 2007.
Glenn founded the agricultural biotechnology company Vestaron Corpora-
tion (ww.vestaron.com) that is developing insecticides based on spider-venom
peptides. Maria Ikonomopoulou is a Postdoctoral Fellow at the IMB work-
ing on the discovery and characterization of novel insecticidal spider-venom
peptides. Maria obtained her Bachelor of Animal Science at the University of
West Macedonia in Greece and then an MSc in Zoology at the University of
Tasmania before completing her PhD in the eld of sea turtle endocrinology
and toxicology at the University of Queensland.
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  • About 3 billion tons of pesticides are applied each year in the world. However, despite this large amount of pesticide applied worldwide, pests, insects, weeds and plant pathogens destroy about 40° of all crops.
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    Over 10,000 arthropod species are currently considered to be pest organisms. They are estimated to contribute to the destruction of ~14% of the world’s annual crop production and transmit many pathogens. Presently, arthropod pests of agricultural and health significance are controlled predominantly through the use of chemical insecticides. Unfortunately, the widespread use of these agrochemicals has resulted in genetic selection pressure that has led to the development of insecticide-resistant arthropods, as well as concerns over human health and the environment. Bioinsecticides represent a new generation of insecticides that utilise organisms or their derivatives (e.g., transgenic plants, recombinant baculoviruses, toxin-fusion proteins and peptidomimetics) and show promise as environmentally-friendly alternatives to conventional agrochemicals. Spider-venom peptides are now being investigated as potential sources of bioinsecticides. With an estimated 100,000 species, spiders are one of the most successful arthropod predators. Their venom has proven to be a rich source of hyperstable insecticidal mini-proteins that cause insect paralysis or lethality through the modulation of ion channels, receptors and enzymes. Many newly characterized insecticidal spider toxins target novel sites in insects. Here we review the structure and pharmacology of these toxins and discuss the potential of this vast peptide library for the discovery of novel bioinsecticides.
  • Article
    Full-text available
    Over 10,000 arthropod species are currently considered to be pest organisms. They are estimated to contribute to the destruction of ~14% of the world's annual crop production and transmit many pathogens. Presently, arthropod pests of agricultural and health significance are controlled predominantly through the use of chemical insecticides. Unfortunately, the widespread use of these agrochemicals has resulted in genetic selection pressure that has led to the development of insecticide-resistant arthropods, as well as concerns over human health and the environment. Bioinsecticides represent a new generation of insecticides that utilise organisms or their derivatives (e.g., transgenic plants, recombinant baculoviruses, toxin-fusion proteins and peptidomimetics) and show promise as environmentally-friendly alternatives to conventional agrochemicals. Spider-venom peptides are now being investigated as potential sources of bioinsecticides. With an estimated 100,000 species, spiders are one of the most successful arthropod predators. Their venom has proven to be a rich source of hyperstable insecticidal mini-proteins that cause insect paralysis or lethality through the modulation of ion channels, receptors and enzymes. Many newly characterized insecticidal spider toxins target novel sites in insects. Here we review the structure and pharmacology of these toxins and discuss the potential of this vast peptide library for the discovery of novel bioinsecticides.
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    When fed in semi-artificial diet the lectins from snowdrop (Galanthus nivalis: GNA: mannose-specific) and jackbean (Canavalia ensiformis: Con A: specific for glucose and mannose) were shown to accumulate in vivo in the guts, malpighian tubules and haemolymph of Lacanobia oleracea (tomato moth) larvae. Con A, but not GNA, also accumulated in the fat bodies of lectin-fed larvae. The presence of glycoproteins which bind to both lectins in vitro was confirmed using labelled lectins to probe blots of polypeptides extracted from larval tissues. Immunolocalisation studies revealed a similar pattern of GNA and Con A binding along the digestive tract with binding concentrated in midgut sections. Binding of lectins to microvilli appeared to lead to transport of the proteins into cells of the gut and malpighian tubules. These results suggested that both lectins are able to exert systemic effects via transport from the gut contents to the haemolymph across the gut epithelium. The delivery of GNA and Con A to the haemolymph was shown to be dependent on their functional integrity by feeding larvae diets containing denatured lectins. Con A, but not GNA, was shown to persist in gut and fat body tissue of lectin-fed larvae chased with control diet for three days. Con A also shows more extensive binding to larval tissues in vitro than GNA, and these two factors are suggested to contribute to the higher levels of toxicity shown by Con A, relative to GNA, in previous long term bioassays.
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    Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.
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    The world is currently in the midst of the greatest demographic upheaval in human history. Dramatic reductions in mortality, followed (but with a lag) by equally marked reductions in fertility, resulted in a doubling of world population between 1960 and 2000. A further increase of 2 to 4.5 billion is projected for the current half-century, with the increase concentrated in the world’s least developed countries. Despite alarmist predictions, historical increases in population have not been economically catastrophic. Moreover, changes in population age structure have opened the door to increased prosperity. Demographic changes have had and will continue to have profound repercussions for human well-being and progress, with some possibilities for mediating those repercussions through policy intervention.