Toxicon 38 (2000) 133±138 www.elsevier.com/locate/toxicon
Short communication
Paralizing activity of the Parawixia bistriata crude venom in termites: a new bioassay A.C.K. Fontana a, M.A.R. CairraÄo b, c, A.J. Colusso c, W.F. Santos c,*, J. Coutinho-Netto a a
Department of Biochemistry, Neurochemistry Laboratory, RibeiraÄo Preto School of Medicine, University of SaÄo Paulo, Av. Bandeirantes 3900, 14049, RibeiraÄo Preto, SaÄo Paulo, Brazil b Psychobiology Post-graduation Program, Neurobiology and Venoms Laboratory, Faculty of Philosophy, Sciences and Letters, University of SaÄo Paulo, Av. Bandeirantes 3900, 14049, RibeiraÄo Preto, SaÄo Paulo, Brazil c Department of Biology, Neurobiology and Venoms Laboratory, Faculty of Philosophy, Sciences and Letters, University of SaÄo Paulo, Av. Bandeirantes 3900, 14049, RibeiraÄo Preto, SaÄo Paulo, Brazil Received 2 September 1998; accepted 1 April 1999
Abstract Spider venoms have high speci®city to neuronal elements. Therefore, the use of venom has been important in the characterisation of mammal and insect nervous systems. The evaluation of insect paralysis has been an important tool for distinguishing the biological eects of venom. In this study we describe the paralysing eect of a spider crude venom (Parawixia bistriata ) in termites, utilising a new bioassay. The crude venom of P. bistriata caused an irreversible and dose-dependent paralysis in the animals in the following doses: 2.10ÿ5 U; 2.10ÿ4 U; 2.10ÿ3 U; 2.10ÿ2 U and 0.12 U (1 U=1 gland). This bioassay will allow for easy and direct evaluation of biological eects from dierent venoms and puri®ed fractions. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Spider venom; Paralysis; Termites; Glutamatergic receptors
* Corresponding author. Tel.: +55 16 6023657; fax: +55 16 6023666. E-mail address:
[email protected] (W.F. Santos) 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 3 3 - 6
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1. Introduction Spiders, scorpions and solitary wasps are important predators of arthropod pests (Riechert and Lockley, 1984; Nyfeller and Benz, 1987; Nyfeller et al., 1987a,b; Provencher et al., 1988). The natural development of insect resistance to chemical control encourages the search for new toxins and natural insecticides (Blagbrough et al., 1992; Quistad et al., 1992). Animal venoms have high anity and speci®city to neuronal receptors, transporters and ion channels. Thus, they are important tools in the characterisation of mammal and insect nervous systems (Usherwood, 1994). There are toxins that have been useful for studying functional properties of the dierent subtypes of calcium channels in neurons, e.g., toxins of the spider Agelenopsis sp. and the snail Conus sp. (Uchitel, 1997). The spider toxin DTX9.2 from the venom of the weaving spider Diguetia canities acts upon the voltage-dependent sodium channels of insect nerve membranes (Bloomquist et al., 1996). The study of a large number of potassium channels cloned recently and their physiologic roles has been aided greatly by the discovery of speci®c blockers, such as the heteropodatoxin peptides isolated from venom of the spider Heteropoda venatoria (Sanguinetti et al., 1997), and kappa-conotoxin PVIIA, from the venom of the snail Conus purpurascens (Savarin et al., 1998). Bioassays, with the analysis of paralysis and death, are important tools for distinguishing the biological eects of venom in insects are bioassays with the analysis of paralysis and death (Zlotkin et al., 1971; Zlotkin, 1983; Friedel and Nentwing, 1989; Quistad et al., 1992; BoeveÂ, 1994; Escoubas et. al., 1995). In this study we describe the paralysing eects of Parawixia bistriata spider venom in termites, utilising a new bioassay. P. bistriata commonly preys upon coee tree parasites in the RibeiraÄo Preto region. The spiders were collected in cerrados of Cajuru/SP, Brazil, and sacri®ced by freezing. Venom glands were extracted, macerated and centrifuged at 3000 g, 48C, 3 min. The supernatant was collected and dissolved in a range of dilutions with 0.9% saline (0.12; 2.10ÿ2; 2.10ÿ3, 2.10ÿ4 and 2.10ÿ5 Units of venom). In this study, 1 Unit of venom (1 U) corresponds to the venom contained in 1 venom gland of P. bistriata and 4.4 mg of protein, measured according to Lowry et al. (1951) and modi®ed by Hartree (1972). The injection volume was 2 ml for each animal. The termites (Sintermes sp) were collected at the RibeiraÄo Preto University Campus. A capillary glass tube was tightly stretched after heating, resulting in two ``needles''. The volume was calibrated in 1 ml divisions at the external wall. The termites were held gently, and injected in the anus. The ¯uid meniscus indicated that the volume of liquid had been injected. There was no observable expelling of liquid after the injection. The termites were kept in a Petri dish and observed at 5, 10, 30, 60, 120, 720 and 1440 min after the injections. Behaviour classi®cations, adapted from Boeve (1994), consisted in the following: normal (N; normally walking and body moving, mandible responsive to stimulation, continuous movement of the antennas), paralysis (P; incapacity to shift whole body, sometimes with uncoordinated contractions of the legs, mandible and antennas responsive) or dead (D; total absence of movement. If the mandible and antennas were not responsive, the animal was placed on its back, its
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thorax and abdomen were touched, and the antenna were pinched to con®rm whether the insect was dead or not). Death could not be distinguished from full paralysis. This does not alter our results, however, since our goal to observe the evolution of the venom eect is reached either way. The target insect species (Termitidae ) was selected because it is readily available and easily collected. Termite average body weight is 0.148 2 0.015 g, and the described behavioural parameters are readily observed. The injections of P. bistriata venom in termites produced the following results: the termites progressively and irreversibly became immobilised, or they even died after the injection, in a dose-dependent manner. Fisher exact test was used to verify statistical signi®cance between (P+D) and N states against the control group. Table 1 shows the results of the bioassays, where 85.8% of control animals remained normal up to 1440 min. Paralysis was observed at injections of 2.10ÿ5 U and 2.10ÿ4 U (not statistically signi®cant, data not shown). The concentration of 2.10ÿ3 U produced 16.7% of animal paralysis 10 min after the injection. The percentage of animals paralysed increased with time, and 41.7% was dead after 120 min. Concentrations of 2.10ÿ2 U and 0.12 U produced 30.8 and 46.2% of animal paralysis, respectively, after only 5 min (Table 1). Paralysis was always irreversible. We observed in our bioassays a dose-dependent paralysis of termites injected with P. bistriata venom. Spider and wasp venoms possess various acylpolyamine toxins in their venom glands, which paralyse insects by blocking the nerve-muscle signal transduction of glutamatergic synapses (Itagaki et al., 1997). It is known that polyamine amide toxins of spiders (e.g., arg-636) and parasitic wasps (PhTx-343) target the insect postjunctional and extrajunctional glutamatergic receptors (Jackson and Usherwood, 1988; Blagbrough et al., 1992; Usherwood, 1994). The crude venom of Parawixia bistriata (1.25.10ÿ3 U) evoked a strong inhibition on the Na+-speci®c binding of 3H-L-glutamate in rat brain
Table 1 Percentages of Normal state (N), Paralysis (P) and Death (D) of termites injected with 2 ml of saline (control) or VPb=2.10ÿ3 U and 2.10ÿ2 U and 0.12 U (N=13 for all)
Time (min) 5 10 30 60 120 720 1440 a
Control (0.9% saline) Nb P D 100 100 100 100 100 92.8 85.8
0 0 0 0 0 7.1 0
0 0 0 0 0 0 14.2
2.10ÿ3 U (VPb)a
2.10ÿ2 U (VPb)
0.12 U (VPb)
N
P
D
N
P
D
N
P
D
91.7 83.3 33.3 33.3 0 0 0
8.3 16.7 66.7 66.7 58.3 16.7 16.7
0 0 0 0 41.7 83.3 83.3
69.2 38.6 46.1 0 0 0 0
30.8 61.5 46.1 69.2 0 0 0
0 0 7.7 30.8 100 100 100
53.8 15.4 0 0 0 0 0
46.2 84.6 46.2 38.6 0 0 0
0 0 53.8 61.4 100 100 100
VPb=crude venom of P. bistriata, concentrations are indicated. ( ) p < 0.05; () p < 0.001; one-sided Fisher exact test. N=normal, P=paralysis, D=dead (termite behaviours). b
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synaptosomal membranes. This concentration also inhibited 60% of GABA uptake and stimulated four-fold L-glutamate uptake in rat brain synaptosomes (Fontana, 1997). These data suggest a glutamatergic antagonism as a possible explanation for the insect paralysis, despite the dierences between mammals and insect glutamatergic receptors (Usherwood, 1994). Otherwise, there is evidence of some vertebrate/invertebrate structural homology. Donly et al. (1997) reported on the strong amino acid sequence identity of insect glutamate transporters with known members of the vertebrate Na+- and K+-dependent amino acid transporter family. Insect paralysis may also be due to blockage of ion channels. The spider toxin DTX9.2 induced a rapid paralysis when injected into insects, acting upon the voltage-dependent sodium channel of insect nerve membranes (Bloomquist et al., 1996). In our bioassays the parameters ED50 (the eective dose to paralyse 50% of the treated termites) and LD50 (lethal dose for 50% of the animals) were calculated for 60 min according to Karber (1937): ED50=0.33 U (980 ng of protein/100 mg of termite body weight) and LD50=0.6 U (1780 ng/ 100 mg). DeDianous et al. (1987) observed the locomotion activity of several insects after Androctonus australis venom injection, and they calculated the following parameters for 60 min: LD50=2 ng/100 mg of body weight and ED50=3 ng/100 mg for Musca domestica; LD50=24 ng/100 mg and ED50=46 ng/ 100 mg for Periplaneta americana; LD50=289 ng/100 mg and ED50=290 ng/ 100 mg for Grillus domesticus. It is dicult to compare our ED50 and LD50 values with others, due to dierences in the methodologies used. It is important to note that the injection needle reaches up to the rectal ampoule, a region where water and salt absorption occurs. In this region, enzyme activity is absent, or poor at best. Activity has been described for some cellulases and cellulase-like enzymes at the end of the intestine, next to the rectal ampoule (GrasseÂ, 1982). Thus, this suggests the absence of venom degradation in the injected region. However, it is not known how much venom is absorbed. In conclusion, the crude venom of P. bistriata, when injected into termites, caused irreversible and dose-dependent paralysis. This methodology is especially useful, considering the ease of termite collection; inoculation without dermal damage to the animal, or the need for special equipment, like automatic microinjectors (Escoubas et al., 1995). The behavioural parameters used to describe the eects are readily observed, producing reliable and reproducible results. The methods described above represent a new tool in the study of the neurotoxins potentially acting in insect nervous systems, as well the investigation of arthropod venoms. This termite bioassay certainly can be used as an aide in the fractionating of venom in biochemical puri®cation as part of the search for neuroactive puri®ed compounds.
Acknowledgements We thank CNPq and CAPES for grant support, Prof. LuõÂ s de Souza (FMRP-
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USP) for statistical assistance and Vera L.A. EpifaÄnio and Silvia E. EpifaÄnio (FMRP-USP) for technical support.
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