Spider venom: enhancement of venom efficacy mediated by different synergistic strategies in Cupiennius salei
Zoological Institute, University of Bern, Baltzerstrasse 6, CH-3012 Bern, Switzerland
* Author for correspondence (e-mail: lucia.kuhn{at}zos.unibe.ch)
Accepted 14 March 2005
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Summary |
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Key words: neurotoxin, Drosophila melanogaster, bioassay, synergism, multicomponent venom
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Introduction |
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The Central American spider Cupiennius salei (Keyserling 1877)
uses its venom as economically as possible. The amount of venom injected
varies depending on size, activity, defense behaviour and venom sensitivity of
a prey item (Malli et al.,
1999; Wigger et al.,
2002
; Wullschleger and
Nentwig, 2002
). This optimal venom dosage is continued on the
biochemical level through positive interactions among various venom components
(Kuhn-Nentwig et al., 1998
;
Wullschleger et al., 2004
). In
C. salei venom, proteins with molecular masses above 10 kDa have been
identified. Additionally, disulfide-rich neurotoxins, highly cationic peptides
with molecular masses between 3-10 kDa, and low molecular mass compounds, such
as ions, biogenic amines, polyamines and neurotransmitters, are present
(Kuhn-Nentwig et al.,
2004
).
To date, toxicological information and sequence data for the neurotoxins
CSTX-1 and CSTX-9, and the neurotoxic two-chain enhancer peptide CSTX-13 have
been reported (Kuhn-Nentwig et al.,
2004; Wullschleger et al.,
2004
). Furthermore, antimicrobially and cytolytically acting
cupiennins have been identified. These highly cationic,
-helical,
cysteine-free peptides may play a dual role in the venom: protection of the
venomous apparatus against microbial invaders and, after venom injection into
prey, an enhancement of the paralytic effect of the neurotoxins
(Kuhn-Nentwig, 2003
).
Insecticidal activities of similar cytolytically acting peptides have also
been reported for the spider Lycosa carolinensis
(Yan and Adams, 1998
) and the
ant Pachycondyla goeldii (Orivel
et al., 2001
). Beyond these insecticidal activities, additional
synergistic interactions with neurotoxins have been demonstrated for the
spiders Oxyopes kitabensis (Corzo
et al., 2002
) and C. salei
(Kuhn-Nentwig et al.,
2004
).
Only limited information is available about possible synergistic effects of
low molecular mass substances with neurotoxins immediately after venom
injection into a prey item (Chan et al.,
1975; Inceoglu et al.,
2003
; Wullschleger et al.,
2004
). It was previously shown that histamine and taurine
facilitate the neurotoxic activity of CSTX-1 from C. salei
(Kuhn-Nentwig et al., 1998
).
Accordingly, it was also hypothesised that µ-agatoxins, which are
disulfide-bridged short peptides modifying Na+ channels, enhance
the short-term action of
-agatoxins (acylpolyamines). Both agatoxins
have been identified in the venom of the spider Agelenopsis aperta.
Furthermore, additive interactions among different
-agatoxins, which
are disulfide-bridge-rich voltage activated Ca2+ channel
inhibitors, have been reported for A. aperta
(McDonough et al., 2002
;
Adams, 2004
). However, these
findings were mainly obtained through neurophysiological investigations and
bioassays of different venom components without considering their
physiological ratios as they occur in the venom.
In the study reported here, we analysed interactions among the main low
molecular mass components and the most important identified peptides
(Kuhn-Nentwig et al., 2004) as
well as interactions between these peptides. Finally, we compared the
paralytic activity of crude venom to the synergistic activity caused by
defined venom components. The findings show multiple interactions of venom
components in different molar ratios and help us to understand the complex
nature of spider venom.
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Materials and methods |
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Bioassays
The LD50 bioassays were performed according to Escoubas et al.
(1995) using 1-3 day old
Drosophila melanogaster female flies. The injection was always
applied into the mesothorax laterally and the injected volume was 0.05 µl
of 0.1 mol l-1 ammonium acetate, pH 6.1. As control, for each assay
20 flies were injected with this solution. All further injections with
different components were also carried out in 0.05 µl of 0.1 mol
l-1 ammonium acetate, pH 6.1 (four concentrations, 20 flies each).
Mortality rates were recorded 24 h after injection. LD50 bioassays
were performed with (1) crude venom, (2) CSTX-1, and (3) CSTX-1 in combination
with CSTX-9, CSTX-13, cupiennin 1a, histamine and KCl in their physiological
venom concentrations. The physiological venom concentrations and the
LD50 values of the main low molecular components as well as of the
most important identified peptides in the venom of C. salei are given
in Tables 1 and
2.
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Interactions between low molecular mass components and peptides
Interactions between venom peptides and low molecular mass components were
investigated in bioassays with 0.315 pmol CSTX-1 mg-1 fly, 7.95
pmol CSTX-9 mg-1 fly and 5.0 pmol cupiennin 1a mg-1 fly.
We tested the peptides alone or in combination with histamine (5.7 mmol
l-1), taurine (0.07 mmol l-1), and KCl (215 mmol
l-1) in their physiological venom concentrations. For statistical
analyses the flies were divided in two independent series of 15 groups each
with five flies (N=30 groups). For every series 20 flies were
injected as control.
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In a third series of experiments we have analysed the synergistic activity
of CSTX-13 on the neurotoxins CSTX-1 and CSTX-9 as well as on the cytolytic
cupiennin 1a. (i) Bioassays were performed with 0.315 pmol CSTX-1
mg-1 fly alone and in combination with 0.035 pmol CSTX-13
mg-1 fly (non-toxic concentration) corresponding to their molar
ratio in the crude venom (9:1) (repetition of
Wullschleger et al., 2004).
(ii) Next, the mortality of 7.95 pmol CSTX-9 mg-1 fly was compared
with the mortality of 7.95 pmol CSTX-9 mg-1 fly combined with
either 0.035 pmol CSTX-13 mg-1 fly (molar ratio of 227:1) or (iii)
4.97 pmol CSTX-13 mg-1 fly (physiological molar ratio 1.6:1),
respectively. (iv) Finally, the mortality of 5.0 pmol cupiennin 1a
mg-1 fly was compared with the mortality of 5.0 pmol cupiennin 1a
mg-1 fly combined with either 1.25 pmol CSTX-13 mg-1 fly
(physiological molar ratio 4:1) or 2.78 pmol CSTX-9 mg-1 fly
(physiological molar ratio 1.8:1), respectively
(Table 2). For statistical
analyses the flies were divided in two independent series of 12 groups each
with five flies (N=24 groups). Twenty flies were used as control for
each series of bioassay.
Calculations and statistics
Mortality rates corresponds to the number of dead flies out of a total of
N=150 flies [2 x (15 x 5)] for interactions between low
molecular mass components and peptides and N=120 flies [2 x (12
x 5)] for interactions between peptides.
LD50 calculations were done using Proban software (Version 1.1, Jedrychowski, 1991, shareware). The relative mortality of D. melanogaster was arcsinus square root-transformed and treated as the dependent variable, whereas the venom components or the co-injected peptides were treated as nominal independent variables. The experiments were analysed using generalised linear models. The means of the nominal independent variables venom components or co-injected peptides, respectively, were compared pairwise by the Fisher LSD method. Fulfilment of model assumptions was checked by visual inspection of the residuals distribution for every statistical test conducted. Statistics were done with S-PLUS 6.0 Professional software.
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Results |
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In a second series of experiments, a possible cooperativity between CSTX-1 and CSTX-9 was analysed first according to their molar ratio in the venom of 5.7:1 (0.315 pmol CSTX-1 mg-1 fly: 0.06 pmol CSTX-9 mg-1 fly). Injection of 0.06 pmol CSTX-9 mg-1 fly had no effect on the flies. No enhanced mortality was observed between the injection of 0.315 pmol CSTX-1 mg-1 fly alone (16% mortality; 19 dead out of 120) and in combination with CSTX-9 (17% mortality; 20 dead out of 120). Secondly, cupiennin 1a and CSTX-9 appear in the venom in a molar ratio of 1.8:1 (5.0 pmol cupiennin 1a mg-1 fly: 2.78 pmol CSTX-9 mg-1 fly). Injection of CSTX-9 in this concentration is non-toxic. Furthermore, a mortality rate of 56% (67 dead out of 120) by injection of cupiennin 1a alone is not increased by co-injection with CSTX-9 (mortality of 61%; 73 dead out of 120) (Fig. 3A).
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In a third series of experiments possible enhancer effects of CSTX-13 on the insecticidal activity of CSTX-9 and cupiennin 1a, corresponding to their venom concentrations, were investigated. The molar ratio of CSTX-9 and CSTX-13 in the venom is 1.6:1 (7.95 pmol CSTX-9 mg-1 fly: 4.97 pmol CSTX-13 mg-1 fly). CSTX-13 alone was used in the above-mentioned concentration and was non-toxic. Co-injection of both enhanced the mortality caused by CSTX-9 alone from 37% to 98% (44 to 118 dead out of 120; P<0.001). Surprisingly, co-injection of CSTX-9 and CSTX-13 even in a molar ratio of 227:1 (7.95 pmol CSTX-9 mg-1 fly: 0.035 pmol CSTX-13 mg-1 fly) increased the mortality nearly as much (91%; 109 dead out of 120; P<0.001, not shown). By contrast, co-injection of cupiennin 1a (5.0 pmol mg-1 fly) and CSTX-13 (1.25 pmol mg-1 fly) in their physiological venom ratio of 4:1 failed to increase the mortality rate significantly (50%; 60 dead out of 120), above that of cupiennin 1a alone (56%; 67 dead out of 120) (Fig. 4).
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Discussion |
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The strategy of combining a high K+ concentration with specific
neurotoxins in the prevenom, thus enhancing paralytic activity, was first
reported for the scorpion Parabuthus transvaalicus
(Inceoglu et al., 2003). It was
hypothesised that the potassium equilibrium potential is locally shifted and
the resulting paralytic effects are further amplified through the presence of
peptide toxins, inhibiting ion channels that are responsible for the
regeneration of the K+ equilibrium potential. C. salei
exhibits a comparable strategy but uses a 2.7-fold higher concentration of
potassium ions in its venom than P. transvaalicus to enhance its
venom efficacy. This high K+ ion concentration can also provoke a
nerve depolarisation thus affecting Ca2+ channels, which are in
turn inhibited by CSTX-1, a known L-type calcium channel inhibitor
(Mafra et al., 2001
).
As for cupiennin 1a, no synergistic effect of KCl on its insecticidal
activity was detected. In contrast to the neurotoxins CSTX-1, CSTX-9 and
CSTX-13, the cytolytically active cupiennin 1a adopts an -helical
conformation in the presence of negatively charged membranes, accumulates at
the membrane surface and inserts itself into the lipid bilayer resulting in a
destruction of cell membranes
(Kuhn-Nentwig et al., 2002
).
Histamine, a neurotransmitter in insect nerve systems
(Nässel, 1999
) and
present in the spider venom, caused a significant mortality increase of 20%
when co-injected with CSTX-1, but was less effective in combination with
CSTX-9 or CSTX-13 in Drosophila flies
(Wullschleger et al., 2004
).
Taurine, a neuromodulator in insects
(Bicker, 1991
) and also present
in the spider venom had no paralytic effect when co-injected with CSTX-1,
CSTX-9 or CSTX-13. By contrast, we were previously able to show in a blow fly
bioassay that the neurotoxicity of CSTX-1 was enhanced by both taurine and
histamine when injected in its physiological venom concentrations
(Kuhn-Nentwig et al., 1998
).
This could indicate that synergistic interactions are highly species and
neurotoxin specific, despite the close relationship between both fly
families.
Interactions between peptides
As shown previously, cupiennin 1a dramatically enhances the efficacy of the
neurotoxins CSTX-1 and CSTX-13 although it is applied in a completely
non-toxic concentration, or even 20-fold below its LD50
(Kuhn-Nentwig et al., 2004;
Wullschleger et al., 2004
).
This synergistic effect was additionally proven for the neurotoxin CSTX-9
(Fig. 2). Positive insecticidal
cooperativity between the cytolytically active oxyopinins and the neurotoxin
Oxytoxin 1 is also reported for the spider Oxyopes kitabensis
(Corzo et al., 2002
). There is
evidence that these highly cationic peptides, found in the venom of O.
kitabensis and C. salei, afford diverse neurotoxins better
access to their targets through their cytolytic activities. By contrast, the
insecticidal activity of cupiennin 1a was definitely not enhanced when
administered with the neurotoxins CSTX-9 or CSTX-13. The dramatic enhancer
effect of the two-chain peptide CSTX-13 on the insecticidal activity of CSTX-1
in a concentration 440-fold below its LD50 value as reported
recently (Wullschleger et al.,
2004
) could also be enlarged on the neurotoxin CSTX-9.
Furthermore, the synergistic activity of CSTX-13 and the neurotoxins CSTX-1
and CSTX-9 is highly specific.
The strong synergistic activities generated by cupiennin 1a and CSTX-13 are
based on their physiological venom concentrations, which implies that both
components were applied in non-toxic concentrations together with the
neurotoxins CSTX-1 or CSTX-9. In contrast to these results, CSTX-9 did not
enhance the neurotoxic activity of CSTX-1 when co-injected corresponding to
its molar ratio in the venom. However, co-injection of CSTX-1 and CSTX-9, both
in toxic concentrations, increased the toxicity by more than 50% when compared
with the theoretical toxicity sum of CSTX-1 and CSTX-9 injected alone, thus
exhibiting a positive cooperativity. These findings are in agreement with
other reports in which positive cooperativities between different neurotoxins
were demonstrated by applying the components in a 1:1 molar ratio or in
concentrations in which both components alone cause intoxications, or by using
toxins from different sources (Bindokas et
al., 1991; Herrmann et al.,
1995
; Shu and Liang,
1999
; Regev et al.,
2003
; Adams,
2004
).
LD50 bioassays
Corresponding to their venom concentrations, injection of a combination of
CSTX-1, CSTX-9, CSTX-13, cupiennin 1a, histamine and KCl into
Drosophila flies resulted in an LD50 value which is
4.5-fold lower than a single injection of CSTX-1. Taking this and the
LD50 value obtained by injection of native venom into account, the
venom components mentioned above are responsible for up to 57% of the crude
venom efficacy. Obviously, still other unknown components are important in the
envenomation process and cause at least 43% of the toxicity of C.
salei venom.
The interactions of different venom components presented here are extremely
complex: histamine and taurine seem to enhance the activity of CSTX-1 highly
specifically, and their effects are, in part, species specific. The high
K+ ion concentration in the venom facilitates the neurotoxin
activity, but not that of cupiennin 1a. However, this group of cytolytic
peptides dramatically enhances the activity of the neurotoxins. In addition,
the neurotoxins are further amplified by the two-chain enhancer CSTX-13.
Differences in LD50 values obtained by injection of crude venom
into various arthropods (Kuhn-Nentwig et
al., 1998) lead us to assume that the interactions presented here
cannot be generalised and are only validated for Drosophila
melanogaster.
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Acknowledgments |
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References |
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Adams, M. E. (2004). Agatoxins: ion channel specific toxins from the American funnel web spider, Agelenopsis aperta. Toxicon 43,509 -525.[CrossRef][Medline]
Bicker, G. (1991). Taurine-like immunoreactivity in photoreceptor cells and mushroom bodies: a comparison of the chemical architecture of insect nervous systems. Brain Res. 560,201 -206.[CrossRef][Medline]
Bindokas, V. P., Venema, V. J. and Adams, M. E.
(1991). Differential antagonism of transmitter release by
subtypes of -agatoxins. J. Neurophysiol.
66,590
-601.
Chan, T. K., Geren, C. R., Howell, D. E. and Odell, G. V. (1975). Adenosine triphosphate in tarantula spider venoms and its synergistic effect with the venom toxin. Toxicon 13, 61-66.[CrossRef][Medline]
Corzo, G., Villegas, E., Gómez-Lagunas, F., Possani, L.
D., Belokoneva, O. S. and Nakajima, T. (2002). Oxyopinins,
large amphipathic peptides isolated from the venom of the wolf spider
Oxyopes kitabensis with cytolytic properties and positive
insecticidal cooperativity with spider neurotoxins. J. Biol.
Chem. 277,23627
-23637.
Escoubas, P., Palma, M. F. and Nakajima, T. (1995). A microinjection technique using Drosophila melanogaster for bioassay - guided isolation of neurotoxins in arthropod venoms. Toxicon 33,1549 -1555.[CrossRef][Medline]
Herrmann, R., Moskowitz, H., Zlotkin, E. and Hammock, B. D. (1995). Positive cooperativity among insecticidal scorpion neurotoxins. Toxicon 33,1099 -1102.[CrossRef][Medline]
Inceoglu, B., Lango, J., Jing, J., Chen, L., Doymaz, F., Pessah,
I. N. and Hammock, B. D. (2003). One scorpion, two venoms:
prevenom of Parabuthus transvaalicus acts as an alternative type of
venom with distinct mechanism of action. Proc. Natl. Acad. Sci.
USA 100,922
-927.
Kuhn-Nentwig, L. (2003). Antimicrobial and cytolytic peptides of venomous arthropods. Cell. Mol. Life Sci. 60,2651 -2668.[CrossRef][Medline]
Kuhn-Nentwig, L., Schaller, J. and Nentwig, W. (1994). Purification of toxic peptides and the amino acid sequence of CSTX-1 from the multicomponent venom of Cupiennius salei (Araneae: Ctenidae). Toxicon 32,287 -302.[CrossRef][Medline]
Kuhn-Nentwig, L., Bücheler, A., Studer, A. and Nentwig, W. (1998). Taurine and histamine: low molecular compounds in prey hemolymph increase the killing power of spider venom. Naturwissenschaften 85,136 -138.[CrossRef][Medline]
Kuhn-Nentwig, L., Müller, J., Schaller, J., Walz, A.,
Dathe, M. and Nentwig, W. (2002). Cupiennin 1, a new family
of highly basic antimicrobial peptides in the venom of the spider
Cupiennius salei (Ctenidae). J. Biol. Chem.
277,11208
-11216.
Kuhn-Nentwig, L., Schaller, J. and Nentwig, W. (2004). Biochemistry, toxicology and ecology of the venom of the spider Cupiennius salei (Ctenidae). Toxicon 43,543 -553.[CrossRef][Medline]
Mafra, R. A., Kuhn-Nentwig, L., Araújo, D. A., Beirão, P. S. and Cruz, J. S. (2001). Effect of CSTX-1, a toxin from Cupiennius salei spider, on L-type calcium currents. XVI FESBE Annual Meeting, Abstract #20.030, p.237 .
Malli, H., Kuhn-Nentwig, L., Imboden, H. and Nentwig, W.
(1999). Effects of size, motility and paralysation time of prey
on the quantity of venom injected by the hunting spider Cupiennius
salei. J. Exp. Biol.
202,2083
-2089.
McDonough, S. I., Boland, L. M., Mintz, I. M. and Bean, B.
P. (2002). Interactions among toxins that inhibit N-type and
P-type calcium channels. J. Gen. Physiol.
119,313
-328.
Nässel, D. R. (1999). Histamine in the brain of insects: a review. Microsc. Res. Tech. 44,121 -136.[CrossRef][Medline]
Orivel, J., Redeker, V., Le Caer, J.-P., Krier, F.,
Revol-Junelles, A.-M., Longeon, A., Chaffotte, A., Dejean, A. and Rossier,
J. (2001). Ponericins, new antibacterial and insecticidal
peptides from the venom of the ant Pachycondyla goeldii.
J. Biol. Chem. 276,17823
-17829.
Regev, A., Rivkin, H., Inceoglu, B., Gershburg, E., Hammock, B. D., Gurevitz, M. and Chejanovsky, N. (2003). Further enhancement of baculovirus insecticidal efficacy with scorpion toxins that interact cooperatively. FEBS Lett. 537,106 -110.[CrossRef][Medline]
Shu, Q. and Liang, S. P. (1999). Purification and characterization of huwentoxin-II, a neurotoxic peptide from the venom of the Chinese bird spider Selenocosmia huwena. J. Peptide Res. 53,486 -491.[CrossRef][Medline]
Wigger, E., Kuhn-Nentwig, L. and Nentwig, W. (2002). The venom optimisation hypothesis: a spider injects large venom quantities only into difficult prey types. Toxicon 40,749 -752.[CrossRef][Medline]
Wullschleger, B. and Nentwig, W. (2002). Influence of venom availability on a spider's prey-choice behaviour. Funct. Ecol. 16,802 -807.[CrossRef]
Wullschleger, B., Kuhn-Nentwig, L., Tromp, J., Kämpfer, U.,
Schaller, J., Schürch, S. and Nentwig, W. (2004).
CSTX-13, a highly synergistically acting two-chain neurotoxic enhancer in the
venom of the spider Cupiennius salei (Ctenidae). Proc.
Natl. Acad. Sci. USA 101,11251
-11256.
Yan, L. and Adams, M. E. (1998). Lycotoxins,
antimicrobial peptides from venom of the wolf spider Lycosa
carolinensis. J. Biol. Chem.
273,2059
-2066.