spider venoms useage as toxins, transgenic development

1. Welcome

2. Spider Venom Peptides:
Structure, Pharmacology and Potential for control
of Insect pests
Ms. Shahanaz
10605
Chairperson
Dr. Vinay K.Kalia
Seminar Leader
Dr. Naresh Meshram

3. Contents
INTRODUCTION
SPIDER VENOM
CHEMISTRYAND PHARMACOLOGY OF SPIDER VENOM
STRUCTURE OF SPIDER VENOM PEPTIDES
SPIDER VENOM AS BIOINSECTICIDES
VENOM TOXIN TRANSGENES IN TRANGENICS
CONCLUSION

4. • Kingdom : Animalia
• Phylum : Arthropoda
• Class : Arachnida
• Order : Araneae
• Suborder : Araneomorphae
: Mygalomorphae
Two major suborders
 Araneomorphae (“modern”spiders)
 Mygalomorphae (“primitive” spiders)
 Araneomorphs represent >90% of all known spider species
 Mygalomorphs are more sustainable and convenient source of venom due
to their large venom glands . Longevity (Live for over 25 years )
Systematic position of spider
Mexican red-kneed tarantula
Brachypelma smithi

5.  Spiders are the most successful
venomous animals and they are the
most abundant terrestrial
predators
 Evolved 300 m. years ago
 Over 43,244 species identified
 Spiders are most specious venomous animal
 Spiders are active hunters and their bites to paralyze
to kill their prey before consuming it
American tarantula
Spiders: Master Insect Predators
(King and Hardy ,2013)

6.  Venomous animals produce a toxin that is
injurious or even lethal to another organism
 Inject venom into other organisms
 Venomous taxa = Cnidarians, Echinoderms,
Molluscs, Vertebrates and Arthropods
 A complex variety toxins produced by
organisms - delivered through fangs, sting,
teeth, fins and tentacles
 Use for predation, defence and competitor
deterrence
Venom…..?

7. • Venom = mixture of neurotoxic compounds
• Glands
– Basal segment of chelicerae ( Mygalomorph)
– Anterior prosoma (Araneomorph)
(Chaim et al. ,2011)
Spider Venom Glands

8. • Envenomated apparatus acts like a pressurized
hypodermic needle delivering venom
• Primary purpose of spider venom
• Rapidly immobile prey and deter the
predators
• Tackle large prey
• Crab spiders prey primarily on venomous
hymenopterans
Spider Venom

9. Most venomous spiders in the world
Australian funnel-web spider
Brazilian wandering spider
Considered the most toxic spider, an
indication of their deadly bite
Venom contains neurotoxin - PhTx3
which acts as broad-spectrum
 δ-ACTX toxins present in the venom
Presynaptic neurotoxins that (via
sodium channels)

10. Contd..
Brown Recluse
Mouse spider
They have a venomous bite which is
potentially deadly
These spiders are not aggressive, hence
the word recluse
Mouse spiders represent a potential
cause of serious envenomation
Venom contains tetrodotoxin causes a
slowing inactivation of TTX-sensitive
sodium currents

11. Contd..
Black widow
Goliath birdeater Tarantula (Annon , 2013)
It is one of the largest spiders in the
world that can cause panic and fear
These are found in South America
The venom is relatively toxic and its
effects are comparable to those of a wasp
sting
 Living throughout the world
Alpha-latrotoxin : a presynaptic
neurotoxin

12. Australian tarantula
Narcotized with ether and subsequently
submitted to direct current electric shock
(15 V)
Electrodes are placed on the prosoma of
adult spiders
The venom from fang tips is withdrawn
with a micropipette and immediately used.
 To avoid contamination with saliva, the
venom is collected only from the fang
tips
 The amount of venom per spider is
approximately 0.1 µL
How to Extract spider venom ???

13.  Venom components from only 174 (∼0.4%) species described
out of 43,244 extant species
Chemistry and pharmacology of spider venom
(King and Hardy, 2013)

14. Major classes of spider venom
Spider venoms
Salts and
Small
Organic
Compound
s
Linear
Cytolytic
Peptides
Disulfide-
Rich
Peptide
Neurotoxins
Enzymes
Large
Presynaptic
Neuro
toxins

15. o Ionic composition of spider venom indicates
o Low in Na+ (∼10 mM) and high in K+ (70–200 mM)
o Inverse to insect haemolymph - Na+/K+
o High K + causes depolarization of axonal fibres at injected site
o A wide range of small organic compounds <1 kDa
– Amino acids, (GABA, glutamate, taurine )and acylpolyamines,
biogenic amines - histamine and octopamine
– Neurotransmitter -acetylcholine
– Nucleosides – adenosine
– Nucleotide- ATP
o Limited phylum selectivity
o None of these compounds seriously pursued as insecticide leads
Salts and Small Organic Compounds
(Vassilevski et al.,2009)

16.  Dominant component of most spider venom
 Primary source of pharmacological diversity
 No cytolytic peptides reported in
Mygalomorphe
 Facilitates the action of SS- rich neuro toxins
 Broadly cytolytic and antimicrobial activity
 Non selectivity
 Weak insecticidal activity
Linear Cytolytic Peptides
(Saez et al., 2010)

17. • Dominant and major contributors to the venom’s
insecticidal activity
• Mass = 3.0 to 8.5 kDa
• 10 fold more potent than the cytolytic venom peptides
• SS rich peptides target presynaptic ion channel
or post synaptic receptors in PNS and CNS
• These peptides can deaden insect nervous system and
cause paralysis’ or overactivate NS and cause
convulsive paralysis.
• More than 1,000 unique peptides are present
Disulfide - Rich Peptides (SS rich)

18. (King and Hardy ,2013)
Molecular target of proteinaceous spider venoms

19. Distribution of disulfide bonds in venom toxins
(Windley et al.,2012)

20. Enzymes
• Collagenase
• Hyaluronidase
• Proteases
Degrade extra
cellular matrix
• Phospholipase A2
• Sphingomyelinase A
Destruction of
underline cell
membrane
Enzymes are large protein and used for insecticide
developments
(King and Hardy ,2013)

21. • Widow spiders (Latrodectus spp.) : latrotoxins
• Mass = 110 to 140 kDa
• Have remarkably different phylum selectivity
– Vertebrate-specific α-latrotoxin
– Crustacean-specific α-latrocrustatoxin
– Insect-specific α-, β-, γ-, δ- and ε-latroinsectotoxins
• Latroinsectotoxins are the most potent insecticidal toxins
• Low LD50 > 1 pmol g−1 = Lepidopterans and Dipterans
• Induce exhaustive release of neurotransmitter at NMJ
Large Presynaptic Neurotoxins
(Rohou et al.,2007)

22. Toxin Cabals
glutamate
activates
ionotropic
glutamate
receptors at prey
NMJs
immediately
acylpolyamines
access and
act as open-channel
blockers
“cabals”??
(King and Hardy 2013)

23. Insect synapse with molecular target of spider venom and
insecticides
(King and Hardy 2013)

24. Knots and Helices
Three-dimensional structures determined in 44 SV peptides
– Inhibitor cystine knot (ICK) motif- 39
– Helical cytolytic peptides -5
– Cysteine-rich secretory protein (CRISP) domains
– Prokineticin scaffolds
(Windley et al., 2012)
Structure of spider-venom peptides

25.  Antiparallel ß sheet stabilized by a cystine knot in spider
toxins, the ß sheet typically comprises only two ß strands
 ICK provides resistance to proteases chemicals, extremes of
pH ,organic solvents and high temperature stability
 Insect specific peptides
Inhibitor Cystine Knot (ICK)
(Windley et al.,2012)

26. • Predicted 10 million bio active spider venom peptides
• 800 characterized- 136 are insecticidal
Insecticidal Spider Venom Peptides (ISVP)
136
87
identified
33 Nav 33 Cav
11 Lipid
bilayer
7 Kca
2
Presynapti
c terminal
1 NMDA
receptor
41
unidentified
(Windley et al., 2012)

27. Spider toxins are highly selective receptor sites on insect
NaV channel
Pore blockers - neurotoxin tetrodotoxin (TTX)
Gating modifiers δ-Ctenitoxin
Spider toxins modulate neuronal excitability – paralysis
CaV channels ω-atracotoxin-Hv1a blocked calcium gated
channel
Insect KV Channels - κ-Hexatoxin-1
 Depolarization in axonal fiber
Contd ...

28. Distribution of spider venom peptides
(Windley et al., 2012)33Distribution of spider venom peptides

29. Delivery methods of ISVP in to insect haemolymph
(Herzig et al., 2014)

30. (King and Hardy, 2013)
Potential of Insecticidal Spider -Venom Peptides (ISVP)
 ISVP as
Bioinsecticides
 ISVP transgenes for
development of Insect
Resistant Traits
 ISVP transgenes in
enhancing efficacy of
entomopathogens

31.  Spider venoms are a complex toxins have specifically to kill
insects
 ISVP are active to the site of action in insect nervous system
 The X-ACTX-Hv1a toxin (Hvt), a component of the venom of
the (Hadronyche versuta) that is a calcium channel antagonist
I. Spider-Venom Peptides
as Bioinsecticides

32. Case Study I

33. Spider peptide toxins suitable as insecticide leads
(Windley et al.,2012)

34. Properties of insecticidal spider-venom peptides with
potential application as bioinsecticides
(King and Hardy, 2013)

35. Case Study II
The oral LD50 for OAIP-1 (Selenotypus plumipes) in the cotton
bollworm Helicoverpa armigera was 104.260.6 pmol/g, which
is the highest per os activity reported to date for an insecticidal
venom peptide.
OAIP-1 is equipotent with synthetic pyrethroids and it acts
synergistically with neonicotinoid insecticides.

36. Insecticidal activity of synthetic OAIP-1
(Hardy et al., 2013)
Comparisons of OAIP-1 with Pyrethroids
Insecticide Class of insecticide Strain Oral LD50
(nmol/g)
Bifenthrin Pyrethroid R1 20.6
S2 1.1
Deltamethrin Pyrethroid R 0.46
S 0.35
Etofenprox Pyrethroid R 55.9
S 0.31
Fenvelerate Pyrethroid R 49.1
S 7.8
OAIP- 1 Peptide - 0.10

37.  ISVP-expressing transgenic plants - resistance to insect pests
 ISVP transgenes appear to be good candidates for trait
stacking with Bt
Completely different mechanisms of action
Synergized by Bt, which causes lysis of midgut epithelial
cells and facilitate movement of ISVPs into the
haemolymph
II.
II. Spider-toxin Transgenes for Engineering
Insect-Resistant crops

38.  Transgenic expression of Hvt (Hadronyche versuta)in tobacco
effectively protected the plants from H. armigera and S. littoralis larvae
 Expressed as a fusion protein (Hvt- thiredoxin) in E. Coli
 The purified toxin fusion immobilized and killed Helicoverpa armigera
and Spodoptera littoralis with 100% mortality within 48 h.
Case Study III

39. • Transgenic expression of ω-ACTX-Hv1a toxin (Hvt) in tobacco
Spider venom incorporated insect resistant transgenic plants
(Khan et al., 2006)

40. Mortality of H. armigera on transformed tobacco
Plant line Larvae weight (mg)
Time after introduction (h)
0 72
Mortality %
Time after introduction (h)
24 48 72 Mean
NT 0.320 ± 0.006 3.506 ± 0.085 a 0 0 0 0.00 d
T1 0.309 ± 0.004 0.245 ± 0.008 b 0 33.3 86.7 40.0 b
T10 0.308± 0.005 0.234 ± 0.006 b 6.7 33.3 66.7 35.6 b
T15 0.308 ± 0.004 0.230 ± 0.005 b 0 13.3 53.3 22.2 c
T21 0.309 ± 0.005 0.221 ± 0.006 b 6.7 46.7 100 51.1 a
Mean 2.68 c 25.3 b 61.3 a
(Khan et al., 2006)
Mortality of S. lituralis on transformed tobacco
Plant line Larvae weight (mg)
Time after introduction (h)
0 72
Mortality %
Time after introduction (h)
24 48 72 Mean
NT 0.202±0.0025 1.667±0.0307 a 0 0 0 0.00 d
T1 0.207±0.0268 0.105±0.0055 c 0 60 100 53.33 b
T10 0.239±0.0041 0.148±0.0090 bc 7.7 38.5 92.3 46.11 b
T15 0.233±0.0044 0.185± 0.0186 b 0 30.8 76.9 35.56 c
T21 0.232±0.0034 0.107 ± 0.0082 bc 57.1 100 - 78.55 a
Mean 13.0 c 45.7 b 67.3 a

41. Case Study IV
 Peptide toxin from Macrothele gigas(Maggi 6) was cloned and
expressed in tobacco plants.
 5 groups (Neonatal to 6 instars) of Spodoptera frugiperda subjected to
detached leaf and whole plant bioassay .
 Mortality recorded on 3rd day for neonatal larvae and 7th day for
instar larvae.

42. Detached leaf toxicity Assays of tobacco expressing
Maggi 6 on Spodoptera frugiperda
(Campuzano et al., 2009)
Transgenic
Non Transgenic
 Transgenic lines were significantly more resistant than the
wild type plants.
 The oral ingestion and the translocation from the digestive
track to the haemolymph would depend on the insect
midgut specific conditions.

43. Case Study V
Fusion gene from Atrax robustus peptide - ω-ACTX
Ar1 sequence coding for an ω –atracotoxin and a
sequence coding for the Bt-toxin C-peptide .

44. Percent mortality at each larval instar for L. dispar fed on
the nontransgenic or on transgenic (ω-ACTX Ar1) poplar
(Cao et al., 2010)
Feeding choice test
 Effects of transgenic poplars on the mid gut proteinase
activity of the 5th instar larvae of L. dispar

45. III. Enhancing Entomopathogens using Spider-toxin
Transgenes
 Potency and speed of kill of M.
anisopliae improved by
engineering it to express the
spider-venom peptide AaIT.
 Transgenic fungus caused 50%
mortality of the tobacco
hornworm Manduca sexta and
the dengue vector Aedes aegypti

46.  Wild-type NPV are not competitive with chemical insecticides
 They take 5 to 10 days to kill their host
 Transgenes encoding insect-specific spider neurotoxins
 The development of a recombinant baculovirus
 Insertion of the gene encoding the toxin into the baculovirus genome
 Increasing the insecticidal potential of the viruses
 Baculovirus strain selected for gene insertion Autographa californica
nuclear polyhedrosis virus (AcNPV)
RECOMBINANT VIRUSES

47.  Incorporation of a transgene-encoding
μ-agatoxin-Aa1d
 ISVP American funnel-web spider Agelenopsis aperta
• FT50 and ST50 were compared in three lepidopteran
• Two genetically enhanced isolates of the Autographa
• californica nuclear polyhedrosis virus (AcMNPV) expressing
• insect-specific neurotoxin genes from the spiders Diguetia canities and
Tegenaria agrestis were evaluated for their commercial potential.
• insect pests, Trichoplusia ni, Spodoptera exigua
and Heliothis virescens
Case Study VI

48. FT50 and ST50 with T. ni and S. exigua larvae to enhanced
baculoviruses
Species Virus Average %
mortality
Average FT50
(hr)
Average ST50
(hr)
T. ni
Ac-E 2 35 68.9 82.2
Ac- Bb1 42 62.8 68.8
vAcDTx9.2 42 42.1 62.6
vAcTalTx-1 41 46.2 55.0
S. exigua
Ac-E2 28 62.4 78.3
Ac-Bb1 25 63.2 78.5
vAcDTx9.2 13 45.6 71.3
vAcTalTx-1 12 52.6 64.4
(Hughes et al., 1997)
FT50 and ST50 with T. ni and H. virescens larvae to
enhanced baculoviruses
Tests Virus Dilution % Mortality FT50
(± SE)
ST50
(± SE)
1
Ac-E 2 -1 100 58.3 (±1.6) 70.4 (± 0.9)
Ac- Bb1 -2 87 70.9 (± 1.4) 74.1 (± 1.4)
vAcDTx9.2 -2 95 37.1 (± 0.8) 65.7 (± 1.3)
vAcTalTx-1 -2 94 44.3 (± 0.8) 55.4 (± 0.9)
2
Ac-E2 -2 67 75.1 (± 2.2) 83.2 (± 1.1)
Ac-E2 -3 17 82.1(± 3.1) 87.4 (±1.7)
Ac-Bb1 -3 23 78.8 (± 1.9) 81.9 (± 2.4)
vAcDTx9.2 -3 25 45.4 (± 3.0) 74.1(± 2.1)
vAcTalTx-1 -3 18 49.8 (± 3.2) 66.1(± 3.4)

49.  High potency
 Novel target activity
 Structural stability
 Rapid speed to kill
 Lack of vertebrate toxicity
 Low production costs
 Activity against pests and disease vectors
Desirable qualities of Spider Venom Peptides
as Bioinsecticdes

50.  Have broad species specificity
 Have low toxicity to non target organisms
 Should remain in the environment to be effective
 Be cheap to produce
 Easy to formulate and deliver
 Accessible to small farmers and agribusinesses
Criteria to play competitive role in bioinsecticides market

51. .