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b/d-PrIT1, a highly insecticidal toxin from the venom of the Brazilianspider Phoneutria reidyi (F.O. Pickard-Cambridge, 1897)

Leida Caleg�ario de Oliveira a, c, Fabiana V. Campos b, Suely Gomes Figueiredo d,Marta N. Cordeiro e, Beatriz R. Adaime c, Michael Richardson e, Adriano M.C. Pimenta b,Marie-France Martin-Eauclaire f, Paulo S.L. Beir~ao b, Maria Elena De Lima b, *

a Departamento de Farm�acia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Rua da Gl�oria, 187 e Centro, 39100-000 Diamantina, MG, Brazilb Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazilc Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazild Departamento de Ciencias Fisiol�ogicas, Centro de Ciencias da Saúde, Universidade Federal do Espírito Santo, Avenida Marechal Campos, 1468, 29043-900,Maruípe, Vit�oria, ES, Brazile Centro de Pesquisa e Desenvolvimento “Prof. Carlos Ribeiro Diniz”, Fundaç~ao Ezequiel Dias, Rua Conde Pereira Carneiro, 80, 30550-010 Belo Horizonte,MG, Brazilf Aix-Marseille University, CNRS UMR 7286, CRN2M, School of Medicine, Bd Pierre Dramard, 13916 Marseille Cedex 20, France

a r t i c l e i n f o

Article history:Received 3 February 2015Received in revised form8 June 2015Accepted 16 July 2015Available online 26 July 2015

Keywords:Spider venomsVoltage-gated sodium channelsAnti-insect toxinsPhoneutria reidyi

a b s t r a c t

A potent insecticidal toxin, b/d-PrIT1, molecular mass of 5598.86 [MþH]þ, was characterized fromPhoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidalspider toxins from the genus Phoneutria. b/d-PrIT1 was very toxic (LD50 ¼ 4 nmol/g) to flies (Muscadomestica), but not to mice (Mus musculus). Kinetic studies showed that 125I-b/d-PrIT1 binds to twodistinct sites in insect sodium channels, with close affinity (Kd1 ¼ 34.7 pM and Kd2 ¼ 35.1 pM). Its as-sociation is rather fast (t1/2(1) ¼ 1.4 min, t1/2(2) ¼ 8.5 min) and its dissociation is a slower process (t1/2(1) ¼ 5.4 min, t1/2(2) ¼ 32.8 min). On rat brain synaptosomes b/d-PrIT1 partially competed (~30%) withthe beta-toxin 125I-CssIV, but did not compete with the alpha-toxin of reference 125I-AaII, nor with thebeta-toxin 125I-TsVII. On cockroach nerve cord synaptosomes, b/d-PrIT1 did not compete with the anti-insect toxin 125I-LqqIT1, but it competed (IC50 ¼ 80 pM) with the “alpha-like” toxin 125I-BomIV. Incockroach neurons, b/d-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodiumchannel activation to hyperpolarizing potentials. These results indicate two different binding sites for b/d-PrIT1, leading to two different pharmacological responses. b/d-PrIT1 is one of the most toxic spidertoxins to insects without apparent toxicity to mammals, and provide new model for the development ofinsecticides.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Wandering “armed” spiders belong to the genus Phoneutria andconstitute the second major cause of human envenomation inBrazil, after scorpion stings. Intense local pain is the main clinicalmanifestation after Phoneutria spp bite and may be followed bylocal oedema and hyperemia (Bucaretchi et al., 2000). The symp-toms may also include: tremors, convulsions, lachrymation, spasticparalysis, priapism, salivation, cardiac arrhythmia, visual

disturbance and cold sudoresis (for review see Borges et al., 2009).Venom proteomes of three Brazilian Phoneutria species (Pho-

neutria nigriventer, Phoneutria reidyi, and Phoneutria keyserlingi)were compared (Richardson et al., 2006) using two-dimensionalgel electrophoresis and chromatographic analyse. Nearly 400peptides and proteins in pure state (1.2e27 kDa) have been isolatedfrom these three species, and over a hundred of them had theircomplete or partial amino acid sequences determined (Richardsonet al., 2006). P. reidyi and P. keyserlingi showed a comparable rangeof homologous neurotoxins, similar to those previously isolatedfrom P. nigriventer, which have been shown to act on neuronal ionicCa2þ, Naþ and Kþ- channels, and NMDA-type glutamate receptors(Richardson et al., 2006; Borges et al., 2009). In addition, two new

* Corresponding author.E-mail addresses: melenalima@icb.ufmg.br, lima.mariaelena@gmail.com

(M.E. De Lima).

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families of small toxins (3e4 kDa), some larger protein components(>10 kDa), and two serine proteinases from P. nigriventer venomhave been described (Richardson et al., 2006).

The main P. nigriventer toxins currently studied that are activeon mammalian ionic channels include: 1) PnTx1, a reversible in-hibitor of voltage-gated sodium channels that binds to a m-con-otoxin related site (Martin-Moutot et al., 2006); 2) PnTx2-6, thatmarkedly delays the fast inactivation kinetics of neuronal-typesodium channels (Matavel et al., 2009) and is interestingly able topotentiate erectile function (Nunes et al., 2008); 3) PnTx3 andPnTx3-4, that block with different activities/affinities voltage-activated calcium channels (Le~ao et al., 2000; Dos Santos et al.,2002); and 4) PnTx3-1, a blocker of the inactivating current of po-tassium channels (IA) (Kushmerick et al., 1999). Several publicationshave described different pharmacological activities for many ofthese toxins, and reviewed information may be found elsewhere(Gomez et al., 2002; Borges et al., 2009; De Lima et al., 2015; Nuneset al., 2013). Phoneutria venoms are also known for their hightoxicity to insects and some of their neurotoxins have beenextensively investigated (For review see De Lima et al., 2015). Fewstudies have focused on the contents of Phoneutria reidyi or Pho-neutria keyserlingi venoms. P. reidyi, found in some Brazilian statessuch as Amazonas, Amap�a, Par�a and Roraima, produces a complexmixture of neurotoxic polypeptides (Richardson et al., 2006).

Some P. nigriventer insect-toxins are active on neuronal ionchannels and NMDA-type glutamate receptors (De Lima et al.,2002; Figueiredo et al., 2001). Two of them, PnTx4(6-1) andPnTx4(5-5), are highly insecticidal, as verified on Diptera and Dic-tyoptera, being inactive when intracerebroventricularly (ic) injec-ted in mice, even at higher doses (Figueiredo et al., 1995, 2001;Oliveira et al., 2003). They were demonstrated to act on voltage-gated sodium (Nav) channels by increasing the sodium currententry, and by slowing down the inactivation of these channels, onisolated nerve cord axon from cockroaches (De Lima et al., 2002).This pharmacological answer characterizes an alpha-type effect(Billen et al., 2010). However, they are able to displace, at differentdegrees, Nav channel activation to hyperpolarizing potentials, acharacteristic beta-type effect (De Lima et al., 2002; Stankiewiczet al., 2004; Billen et al., 2010).

Nav channels consist of a 260 kDa pore-forming a subunit,associated or not to auxiliary subunits. The a subunit contains fourhomologous domains (I-IV), each made of six transmembrane (TM)segments (S1eS6) (Catterall, 1986; Cest�ele and Catterall, 2000;Payandeh et al., 2011). Nav channels increase sodium perme-ability in excitable tissues, during the initial rapidly raising phase ofaction potential. These channels are themolecular targets of severalgroups of neurotoxins, which strongly alter channel function bybinding to specific receptor sites. Due to their high affinity andspecificity, neurotoxins that affect both permeation and gatingproperties, constitute powerful tools to study the structure andfunction of Nav channels and to design selective drugs and in-secticides (Catterall et al., 2007). Among them, some arachnidneurotoxins, in particular from scorpion venoms, are able to spe-cifically identify insect Nav channels (Zlotkin et al., 1985, 1991; DeLima et al., 1986, 2002; Cest�ele et al., 1997; Gordon et al., 1992) ordistinct mammalian neuronal subtypes (Gilles et al., 1999, 2000),indicating structural differences in toxin binding sites, which arefrequently described on Nav voltage-sensor modules, made of theTM (trans-membrane domains) from 1 to 4 (Rogers et al., 1996;Bosmans et al., 2008; Song et al., 2011; Bende et al., 2014). On theother hand, many commercially available insecticides, such as py-rethroids, interact with the channel pore module made of TM 5 and6 (Bende et al., 2014). Pyrethroids constitute a class of sodiumchannels gating modifiers, highly active on insects. However, it iswell established that knockdown resistance represents a threat to

the continued usefulness of these insecticides in the control ofmany pest species (Zhao et al., 2000). For this reason, the design ofnew molecules that specifically target insect pests, without affectother animals, requires a better knowledge on the interactionsbetween the toxins and their receptor sites. In addition, informa-tion about resistance to possible insecticides derived fromarthropod toxins is still unavailable.

In the present study, we obtained and investigated the phar-macological properties of b/d-PrIT1, a toxin isolated from thevenom of the Brazilian spider Phoneutria reidyi, on the insect cen-tral nervous system, in order to define the sensitivity of Nav chan-nels to this toxin.

2. Material and methods

2.1. Toxins and reagents

Vydac C4 preparative, Resource S and Pep RPC columns werepurchased from Amersham Pharmacia Biotech and Pharmacia. The“classical” alpha-toxins AaH II from Androctonus australis, the “beta-like” toxin TsVII (Tityus serrulatus), the contractile anti-insect toxinLqqIT1 (Leiurus quinquestriatus quinquestriatus), the “alpha-like”toxin BomIV (Buthus occitanus mardochei) and the “classical” betatoxin CssIV (Centruroides suffusus suffusus) were provided by Dr.Marie France Martin-Eauclaire, Aix-Marseille-University, Marseille,France. All reagents used were from analytical grade.

2.2. Venom

Venoms from male and female adult P. reidyi spiders were agenerous gift from Butantan Institute (S~ao Paulo, Brazil). They werecollected from specimens captured in the hydroelectric reservoirsvicinities of Tucurui (State of Par�a), Samuel (State of Roraima) andBalbina (State of Amazonas), in Brazil. They were obtained byelectrical stimulation of the spider's fangs, as described by Barrioand Vital Brazil (1949). The venoms (5e12 mL/spider, 160 mg/mL)were immediately transferred to siliconized glass tubes on ice,diluted with the same volume of distilled water and centrifuged(4000 g, 10 min at 4 �C), to remove insoluble materials and cellulardebris. The supernatant was lyophilized and stored at �18 �C.Protein content was determined by a modified method of Lowryet al. (1951) using bovine serum albumin (BSA) as standard.

2.3. Isolation of the insecticidal toxin

The purification method of P. reidyi insecticidal toxin followedthat described by Richardson et al. (2006). Aliquots of lyophilizedvenom (25e30 mg) were dissolved in 2 mL of aqueous 0.1% tri-fluoroacetic acid (TFA) and centrifuged again at 4000 g for 10min atcold temperature to remove insoluble material. The brownish yel-low supernatant was applied onto a preparative Vydac C4 column(2.2 � 25 cm) equilibrated with 0.1% TFA in water (solvent A).Solvent B was 100% acetonitrile containing 0.1% TFA. The columnwas eluted at a flow rate of 5 mL/min with the following gradientsystem: 0e20 min, 100% A; 20e30 min, 0e20% B; 30e110 min;20e40% B; 110e130 min, 40e50% B; 130e150 min, 50e70% B. Thepresence of peptides or proteins in the eluate was detected bymeasuring the UV absorption at 214 nm. Fractions containingpeptides or proteins were manually collected and lyophilized. Thelyophilized fractions obtained from preparative Reverse-PhaseHPLC (RP-HPLC) were then dissolved in 2 mL of 10 mM sodiumphosphate buffer pH 6.1 and subjected to ion-exchange FPLC on aResource S column (6.4 mm � 30 mm, Amersham PharmaciaBiotech) equilibrated in the same buffer. After sample application,the columnwas initially washed with the starting buffer for 10 min

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and then eluted with a gradient of 0e0.5 M NaCl in the same bufferat a flow rate of 1 mL/min over a period of 45 min. Polypeptideswere detected by absorbance at 214 nm. Venom components ob-tained from these steps were then desalted by RP-FPLC on analyt-ical Pep RPC columns (C18, HR 5/5, Pharmacia LKB) using a gradientof acetonitrile in 0.1% TFA. Fractions homogeneity was assessedusing mass spectrometry as described below.

2.4. Mass spectrometry analysis

MALDI-TOF (Matrix-Assisted Laser Desorption Ionization e TimeOf Flight) analyses were performed on a Voyager-DETMRP Bio-Spectrometer Workstation (Perseptive Biosystems, local) as previ-ously described (Pimenta et al., 2001). Briefly, samples diluted in 50%acetonitrile/0.1% TFA were analysed in positive linear mode byMALDI-TOFMS. Samples (0.5 mL, variable concentrations) werespotted on the target, followed by 0.5 mL of CHCA (m-cyano-4-hydroxycinnamic acid) matrix solution (60% acetonitrile/0.3% TFA),and allowed to dry at room temperature. A 337 nm nitrogen laserwas used to desorb the samples. Mass spectra were obtained by anaverage of 256 laser pulses. Calibrations were made using standardkits (Sequazyme™ Peptide Mass, Perseptive Biosystems), withAngiotensin I (1297.51); ACTH, 1e17 clip (2094.46); ACTH, 1839 clip(2466.72); ACTH, 7e38 clip (3660.19); Insulin, bovine (5734.59);Thioredoxin, E. coli (11674.48) and Apomyoglobin, horse (16 952.56).

2.5. Determination of the amino acid sequence

For sequence determination, the toxin (5 nmol) was reducedand alkylated as previously described (Friedman et al., 1970), anddesalted on a narrow-bore system from Applied Biosystems (140ASolvent Delivery System and 759A Absorbance Detector) equippedwith a C8 column (Brownlee RP300, 7 mm, 2.1 � 220 mm). Aminoacid sequences of both native and S-pyridylethylated peptides weredetermined using an Applied Biosystems 476A sequencer.

2.6. Toxicity tests

The toxicity of toxins was assessed using 4-day-old house flies(Musca domestica, Diptera; 20 ± 2 mg), and albino Swiss mice (Musmusculus, 20 ± 2.0 g). Insects were chilled for 3e5 min at 4 �C andthen injected via the thoracic cavity as previously described(Figueiredo et al., 1995), delivering volumes of 0.5 or 1.0 mL con-taining known amounts of each fraction. Control animals receivedphysiological saline alone. The following signs of intoxication wereassessed: excitability, salivation, trembling of the legs and body,jerking of the limbs, faint, loss of ability to walk or fly and death.

Effects in mice of both genders (18e22 g) were assayed using ic(intracerebroventricular) injection of the samples (5 mL), asdescribed by Rezende et al. (1991). The presence of possibleneurotoxic signs (excitation, salivation, lachrymation, priapism,spastic or flaccid paralysis, scratching, tail elevation) or death wasobserved after injection. Control animals received physiologicalsaline alone. For the crude (whole) venom and toxin the medianlethal dose (LD50) was calculated using probit analysis (COMBISTATProgram, version 5.0, http://www.edqm.eu/en/combistats-635.html) from the results obtained following the injections at 7different dose levels, using 12 mice and 15 house flies at each doselevel. The experiments were performed twice. Animals were ob-tained from the Animal facilities of Instituto de Ciencias Biol�ogicasfrom Universidade Federal de Minas Gerais.

All animal procedures (mice and rats) were conducted inaccordance with the Biomedical Research Guidelines for the Careand Use of Laboratory Animals (1996), as stated by the BrazilianCollege of Animal Experimentation (COBEA).

2.7. Preparation of synaptosomes from rat brain

Adult Wistar rats (200e250 g) were decapitated, the brain wasrapidly removed and homogenized 1:10 (w/v) in a solution con-taining (inmM): 320.0 sucrose,1.0 EDTA, 0.25 Dithiothreitol, pH 7.4.Homogenates were first centrifuged (1000 g for 10 min) and thesynaptosomes were purified from the supernatant by discontin-uous Percoll-density gradient centrifugation (Dunkley et al., 1988).The isolated nerve terminals were suspended in ice-cold Tyrode'ssolution pH 7.4 containing (in mM): 136.0 NaCl; 5.0 KCl; 2.5KH2PO4; 2.0 CaCl2; 1.0MgSO4; 5.0 Glucose; 25.0 Tris-HCl and storedon ice until needed. The protein content of this synaptosomalpreparation was determined by Lowry method (Lowry et al., 1951).

2.8. Insect synaptosomal preparation

Synaptosomal preparation was made from nerve cords of adultcockroaches (Periplaneta americana) according to established pro-cedures (De Lima et al., 1989). Nerve cords of adult cockroacheswere extracted and homogenized in a Potter-Elvehjem apparatus1:10 (w/v) in a solution containing (in mM): 140.0 NaCl, 5.4 KCl, 2.8CaCl2, 1.3 MgSO4, 20.0 HEPES, 5.0 Glucose, pH 7.4. Homogenateswere first centrifuged (1000 g for 10 min) and the supernatant wascollected and centrifuged (30 000 g for 30 min). The pellet was re-suspended and the protein content of this synaptosomal prepara-tion was determined by Lowry method.

2.9. Radioiodination of toxins

The toxins b/d-PrIT1, AaH II (“classical” alpha-toxin), CssIV(“classical” beta-toxin), BomIV (“alpha-like” toxin), TsVII (“beta-like” toxin) and LqqIT1 (alpha anti-insect toxin) from spider (Pho-neutria reidyi) and scorpion (Androctonus australis, Centruroidessuffusus, Buthus occitanus mardochei, Tityus serrulatus and Leiurusquinquestriatus quinquestriatus) venoms, respectively, werelabelled using the lactoperoxidase method (Rochat et al., 1977). Inbrief, 5 nmol of toxin, 0.5 mCi carrier-free 125IeNa (Amersham, UK),1.25 mg lactoperoxidase (Sigma Chem. Co., USA) and 15 mL H202(diluted 1/50 000) reacted during 2 min in 50 mL of 50 mM phos-phate buffer (pH 7.4). Free iodide was separated from labelledtoxins using a Dowex 1-X8 resin (Serva-Heidelberg, Germany)washed with phosphate buffer (pH 7.4) containing 20 mg/mL BSA.

2.10. Competition and binding experiments

Binding assays were carried out by centrifugation according toHulme (1992). Assays were performed at 37 �C, for 30 min, with25e250 mg/mL of synaptosomes from rat brain or cockroach nervecord incubated with radiolabelled toxins in a medium (200 mL finalvolume, in mM: 140 choline chloride, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4,10 glucose, 25 HEPES, pH 7.4, supplemented with 0.2% BSA). Freeligands were separated by centrifugation (13 000 g, 5 min) from thebound ligands and washed twice by addition of ice-cold washingbuffer (as assay buffer, except that HEPES was reduced to 5 mM andBSA was added at 7%). Non-specific binding was calculated byincubating the samples in the presence of an excess (1000�) ofunlabelled toxin. After washing twice with 1.2 mL ice-coldedwashing buffer, the bound radioactivity was counted in a Gammaradiation counter (1275, LKB).

To characterise the b/d-PrIT1 binding sites on Nav channels fromcockroach and/or rat brain synaptosomes, competition assays withdifferent radiolabelled scorpion toxins were performed. Rat brainsynaptosomes (25e250 mg/mL) were incubated with radiolabelled125I-AaII, 125I-CssIV, 125I-TsVII, 125I-LqqIT1 or 125I-BomIV (10�10 Mfinal concentration) andwith an excess of native b/d-PrIT1 (1000�).

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For association assays, cockroach nerve cord synaptosomes(120 mg/mL) were incubated for 10 min, 37 �C, and were diluted in abottle containing the radiolabelled toxin 125I-b/d-PrIT1 (10�10 M).Samples of 200 mL were analyzed at different times (0, 0.5, 1, 3, 6, 9,12, 15, 20, 30, 40, 60 and 90 min) and the reaction was stopped byaddition of 1.2 mL ice-colded washing buffer. After washing twicewith 1.2 mL (ice-colded washing buffer), the radioactivity bound tothe tubes was measured in a Gamma radiation counter (1275, LKB).Non-specific binding was calculated by incubating the sampleswith an excess of unlabelled b/d-PrIT1 toxin (10�7 M).

For the dissociation assay, 120 mg/mL cockroach nerve cordsynaptosomes were incubated for 10 min, 37 �C. This preparationwas incubated with 125I-b/d-PrIT1 (10�10 M final) at 37 �C, 30 min.Samples of 200 mL were then taken at different times (1, 3, 5, 7, 9,12,15, 18, 22, 26, 30, 35, 40, 45, 50, 60, 70, 90 and 120min), centrifugedand counted in a Y-counter apparatus.

2.11. Electrophysiological study

2.11.1. Dissociation of neurons from adult cockroach (P. americana)Metathoracic ganglia were excised from P. americana adults and

the neurons were dissociated as described by Pinnock and Sattelle(1987). The ganglia were briefly surface-sterilized in 30% ethanol,and rinsed in a cockroach saline of the following composition (inmM): NaCl 214.0, KCl 3.1, MgCl2 4.0, CaCl2 5.0, sucrose 50.0 and TES10.0, pH 7.2. This solution contained penicillin (1%) and strepto-mycin (1%). The isolated ganglia were desheathed and transferredto saline containing collagenase (1 mg/mL, Sigma type I) and hy-aluronidase (1 mg/mL, Sigma). The tissue surrounding the neuronswas weakened by exposing ganglia to these enzymes for 40 min.Ganglia were then removed, washed 3 times in cockroach salineand left for a further 15 min in cockroach saline supplemented withfetal calf serum FCS (Sigma) to a concentration of 10% (final) byvolume. After a further rinse in serum-containing saline, neuronswere dispersed by gently crushing the ganglia in an Eppendorf tubewith a pipette. This mix was submitted to centrifugation and thepellet was then transferred to concanavalin A (1 mg/mL) coatedcoverslips in Petri dishes containing media cockroach saline, FCS(10% by volume), gentamicin (5 mg/mL), penicillin and streptomycin(1%). The cells weremaintained at room temperature overnight andsubsequently used in patch-clamp experiments.

2.11.2. Electrophysiological recordingSodium currents from dissociated ganglia metathoracic neurons

were recorded using the whole cell patch clamp technique. Pipetteswere filled with a solution containing (in mM): 90.0 CsCl, 80.0 CsF,15.0 NaCl, 1.0 MgCl2, 5.0 EGTA, 10.0 HEPES, pH 7,4 adjusted withCsOH, and had resistances from 1.5 to 2.5 MOhm. The bath solutioncontained (in mM): 100.0 NaCl, 100.0 TEA-Cl, 3.1 CsCl, 2.0 CaCl2, 7.0MgCl2, 1.0 CdCl2, 10.0 HEPES, pH 7.4. The neuron under study waskept under continuous focal perfusion with the bath solution (flowrate of 0.2 mL/min), with or without b/d-PrIT1, as appropriate. Theneurons were kept at a holding potential of �90 mV and depolar-ized with a voltage step to �10 mV for 30 ms every 5 s. After sta-bilization of the current, 1 mM b/d-PrIT1 was added to the perfusingsolution. To investigate the voltage-dependence of the b/d-PrIT1effect, the cell under investigation was held at �80 mV and, pre-ceding the test pulse, the membrane was hyperpolarized for100 mse�100 mV. Test pulses ranging from �90 to þ100 mV for40mswere applied in the presence or absence of 200 nM b/d-PrIT1.The peak currents were fitted to the Boltzmann-Ohm equation:

I ¼ gðmaxÞ*f1=1þ exp½ðVm� VaÞ=k�g*ðVm� VrÞ (1)

where g(max) is the maximal conductance, Vm is the membrane

potential, Va is the potential where half maximal conductance isattained, Vr is the reversal potential and k is the slope factor.

Currents were recorded with an Axopatch 200B amplifieroperated by PClamp6 software (Axon Instruments). Currents werelow-pass filtered (cutoff frequency of 5 kHZ) and sampled at10 kHZ. Leak and capacitive currents were subtracted using a P/6protocol.

2.12. Statistics

Data were evaluated using One Way Anova Test (GraphPadPrism, version 5). Differences were considered statistically signifi-cant at *p < 0.05.

3. Results and discussion

3.1. Chemical characterization

In an early study, the venoms of the Brazilian spiders of thegenus Phoneutria were compared, and the whole venom of Pho-neutria reidyiwas reported to be approximately 20 timesmore toxicfor certain insects than that of P. nigriventer (Richardson et al.,2006). Differences between the RP-HPLC profiles of these twovenoms were also observed. P. reidyi contains a much higher con-centration of components in the region where the eluting aceto-nitrile gradient has a concentration of 32e37%. These componentswere described as more potent for insects (house flies, crickets andcockroaches) than for mice (Figueiredo et al., 1995, 2001). In thiswork, we isolated a highly insecticidal toxin and studied its mode ofaction.

Fig. 1A shows the profile of the preparative RP-HPLC obtainedfor the crude venom of P. reidyi (30 mg) on a Vydac C4 column. Atleast 56 fractions were identified in this chromatogram. Taking intoconsideration the above mentioned previous work and the resultsof our biological assays (toxicity against M. domestica vs lack ofactivity in M. musculus), we selected the fraction in focus (IF-1) tocontinue the purification process. This lyophilized fraction wasthen subjected to cationic ionexchange FPLC (Fig. 1B). The fractionexhibiting the highest toxicity to insects (IF-II; arrow in Fig. 1B) waseluted as a single symmetric peak on C18 column Pep RPC (Fig. 1C),and the material obtained was named b/d-PrIT1 (Phoneutria reidyiInsecticidal Toxin 1) partially in according to the nomenclatureproposed by King et al. (2008), considering the greek letters, thatmeans the beta and alpha-activities on sodium channels. Massspectrometry analysis revealed that b/d-PrIT1 was highly pure withan observedmolecular mass of 5598.86 [MþH]þ (data not shown).

Table 1 displays the N-terminal amino acid sequence of b/d-PrIT1, after Edman degradation, compared to those of other Pho-neutria toxins previously purified. Few residues remain to be ob-tained and could not be assessed bymolecular mass measurementsnor similarity comparisons. The lack of venom for further purifi-cation, due to the scantiness of Phoneutria reidyi specimens aroundthe reservoirs, did not allow their complete identification. Theanalysis of the 40 initial amino acid residues showed a Cys residueas the first amino acid. This point is in accordance with an insec-ticide activity, since anti-insect toxins isolated from Phoneutrianigriventer venom show Cys residues at N- and C-terminals(Figueiredo et al. 1995, 2001; Oliveira et al., 2003). The “Multialign”programwas used to look for sequence similarities (Corpet,1988). Apercentage of similarity was calculated using the partial b/d-PrIT1amino acid sequence obtained. This sequence presents 90% ofsimilarity with the toxin PKT�20C2, isolated from Phoneutria key-serlingi, and 85% of similarity with Pn4B, 77% of similarity withPnT�4(5-5), 65% of similarity with PnT�4(6-1) and PnT�4-3, fromPhoneutria nigriventer. As this partial sequence is highly conserved

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among known anti-insect spider toxins, wemight suppose that b/d-PrIT1 is likely to contain 10½ cysteines cross-linked by five disul-phide bridges, which preserve a similar scaffold.

3.2. Biological characterization

3.2.1. In vivo testsAs previously described for P. nigriventer anti-insect toxins

(Figueiredo et al., 1995, 2001; Oliveira et al., 2003), b/d-PrIT1 didnot cause any apparent toxic effects in mammal (mice, M. muscu-lus), even at a dose of 30 mg per mouse (intracerebroventricularinjection). b/d-PrIT1 caused excitatory effects, as those produced ininsects by the “contractile” anti-insect toxins LqqIT1 from thescorpion Leiurus quinquestriatus quinquestriatus, AaHIT from thescorpion Androctonus australis and the m-agatoxins from the spiderAgelenopsis aperta (Zlotkin et al., 1985; Skinner et al., 1992; Adamset al., 1989). b/d-PrIT1, tested in two experiments, showed hightoxicity to flies (M. domestica) and low toxicity to cockroaches(P. americana), when injected by intrathoracic via. Its calculatedLD50 was 4 pmol/g of fly (M. domestica) and 230 pmol/g of cock-roach (P. americana). b/d-PrIT1 was approximately 12 times moretoxic than the crude venom (22.5 ng/g versus 278 ng/g of fly). TheLD50 values already known for P. nigriventer anti-insect-toxins are:192 pmol/g of fly for PnTx4-3 (Oliveira et al., 2003), 40 pmol/g forPnTx4(6-1) (Figueiredo et al., 1995) and 90 pmol/g for PnTx4(5-5)(Figueiredo et al., 2001). Comparing the toxicity values of b/d-PrIT1, with insect toxins from other species of spider on Arachno-Server Database (http://arachnoserver.org/advancedsearch.html),we observed that b/d-PrIT1 can be placed among the three mostpotent peptide insect toxins, i.e. U1-Cyrtautoxin-As1c from Apo-mastus sehlingeris, LD50 ¼ 2.4 pmol/g of the mot, Manduca sexta,U1-theraphotoxin-Sp1a from Selenotypus plumipes,LD50 ¼ 1.84 pmol/g of mealworm beetle, Tenebrio molitor and a-latroinsectoxinin Lt1a from Latrodectus tredecimguttatuts,LD50 ¼ 0.11 and 0.45 pmol/g of fly, M. domestica, and of moth,Galleria mellonela, respectively. In comparison, the contractile andhighly toxic anti-insect toxin AaHIT1 from the venom of the scor-pion Androctonus australisHector has an LD50 of 2.6 pmol/g of fly,M.domestica (De Dianous et al., 1987). However, any comparison be-tween these toxicity values must consider the variations betweenassay conditions and the different species tested. For crude venoms,it is also imperative to consider the venom batch, which can varyaccording to the site where animals were collected, or according tothe gender, feeding behaviour and age of the spiders. In addition,the response among different species may vary. For these reasons,comparisons taking into account only the mol/g-of-insect relationhave to be cautious.

Thus, b/d-PrIT1 constitutes one of the most potent anti-insecttoxin so far purified and, up to now, the most active describedamong the anti-insect toxins from Phoneutria.

3.2.2. In vitro testsScorpion neurotoxins affecting Nav channels were classified into

two major classes: the “classical” alpha-toxins, which block thesodium current fast inactivation; and the beta-toxins, whichmodify the voltage dependence of activation (Couraud et al., 1982;Cest�ele and Catterall, 2000). The “alpha-like” toxin group revealsrelatively high toxicity against mice and inhibits sodium channelinactivation on both mammalian and insect neuronal preparations.Nevertheless, it shows no competition with scorpion “classical”alpha-toxins or scorpion beta-toxin binding to rat brain sodiumchannels (Vargas et al., 1987; Gordon et al., 1996). Unlike the verylow toxicity to insects revealed by the “classical” alpha-toxin, the“alpha-like” toxins show high toxicity to insects (Gordon et al.,1996; Sauti�ere et al., 1998).

Fig. 1. A e Preparative RP-HPLC profile obtained for the crude venom of Phoneutriareidyi. The amount of protein injected onto the column (Vydac C4) was 30 mg. Peptidesor proteins in the eluate were detected by measuring the UV absorption at 214 nm (0 a2.0 Optical Density Unit Full Scale). The peak IF-I containing the toxin of interest ishighlighted. Other experimental details are given in the Material and Methods section.B e Cationic exchange profile of the lyophilized fraction obtained after RP-HPLC(Fig. 1A). This step was performed using FPLC, Resource S (GE HealthCare) column,10 mM sodium phosphate buffer pH 6.1. Polypeptides were detected by absorbance at214 nm (0 a 1.0 Optical Density Unit Full Scale). The arrow indicates the peak IF-IIcontaining the toxin of interest. Other experimental details are given in the Materialand Methods section. C e RP-FPLC profile of the lyophilized and desalted fractionobtained from the cationic exchange (Fig. 1B). This step was performed using a C18 PepRPC HR 5/5 (Pharmacia) column in RP-FPLC. The presence of peptides or proteins wasdetected by measuring the UV absorption at 214 nm (0 a 1.0 Optical Density Unit FullScale). The arrow indicates the toxin of interest, b/d-PrIT1. Other experimental detailsare given in the Material and methods section.

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Due to structural homologies of b/d-PrIT1 with the insecticidaltoxin PnTx4(6-1) from P. nigriventer (Table 1), which was alreadycharacterized as an “alpha-like” toxin (De Lima et al., 2002), thepossibility that b/d-PrIT1 could also belong to this class wasenvisaged. Competition experiments on synaptosomes from ratbrain and insect nerve cords were performed, using different typesof radioiodinated toxins previously well characterized at thepharmacological level. Among them were: the “classical” alphatoxin AaH II and the “classical” beta toxin CssIV, which bindrespectively to site 3 and 4 on rat brain synaptosomes; the “alpha-like” toxin BomIV and the “beta-like” toxin TsVII, which respec-tively bind to sites 3 and 4 on insect synaptosomes; and the“contractile” anti-insect toxin LqqIT1, which is highly specific to thesite 4 on insect synaptosomes.

b/d-PrIT1 was not able to displace 125I- AaH II, 125I-TsVII or 125I-LqqIT1 from their binding sites at 10�7 M concentrations (notshown). PrIT1 was only able to slightly compete with 125I-CssIV(about 30% of the radioactivity was displaced) on rat brain synap-tosomes (Fig. 2A). This result was confirmed by performing anisolated competition experiment between 125I-CssIV versus b/d-PrIT1 (10�7 M), n ¼ 3 (not shown). However, b/d-PrIT1 fully dis-placed the “alpha-like” toxin BomIV from its binding site on cock-roach nerve cord synaptosomes with an IC50 of 8 � 10�11 M(Fig. 2B). According to these results, we can claim that b/d-PrIT1

behaves like an “alpha-like” toxin and binds preferentially to site 3on the insect Nav channel, located on the Nav channel domain IV(Rogers et al., 1996; Bosmans et al., 2008). It has been previouslyshown that the highly specific BomIV binds to the Nav channeldomain IV from cockroach neurons, leading to a decrease of NaVchannel fast inactivation (Gordon et al., 1996).

The reverse experiments (125I-b/d-PrIT1 bound at 10�10 M oncockroach synaptosomes) were carried out with the toxins BomIV,LqqIT and TsVII as competitors. Among these toxins, only BomIV(10�7 M final) was able to partially displace 125I-b/d-PrIT1 from itsbinding site (not shown), in accordance with the results obtainedabove. The displacement was of 47%, suggesting that only the 125I-b/d-PrIT1 binding site shared by BomIV on the domain IV wasdisplaceable. The better understanding of the interaction domainsof b/d-PrIT1 in sodium channels could explain this incompletedisplacement by BomIV.

According to literature, these “alpha-anti-insect toxins” act byinhibiting the fast inactivation of Nav channels (Gordon and Zlotkin,1993; Gordon et al., 1996; Bende et al., 2014). The binding of the“classical” alpha toxins is highly specific to the mammalian Navchannels, and it is dependent on the membrane potential, whilethat of the alpha toxins active on insect Nav channels are not(Gordon et al., 1996; Cest�ele and Gordon, 1998). The binding of b/d-PrIT1 to cockroach synaptosomes was found to be independent on

Table 1Phoneutria insecticidal toxins. The cysteine residues are depicted in red (Penaforte et al., 2000).

Fig. 2. Competition assay of b/d-PrIT1 with 125I-CssIV (A) or 125I-BomIV (B). In A, 125I-CssIV (10�10 M final concentration) was bound to its receptor site on rat brain synaptosomes(100 mg) and incubated at 37 �C with increasing concentrations of native CssIV (C) or b/d-PrIT1 (B). In B, 125I-BomIV (10�10 M final concentration) was bound to its receptor site oncockroach nerve cord synaptosomal preparation (50 mg) and incubated with increasing concentrations of native BomIV (C) or b/d-PrIT1 (B). After 30 min, the reactionwas stopped,as indicated in Material and Methods. The data illustrate one experiment. Three isolated experiments of competition taking only the highest dose of b/d-PrIT1 (i.e.10�7 M) against125I-CssIV confirmed this result (test one way anova, P < 0.05, not shown).

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the membrane potential, since a high potassium concentration(50 mM) does not change the binding data (not shown).

Despite the conclusion that b/d-PrIT1 is an “alpha-insect toxin”,it partially displaced (Fig. 2A) 125I-CssIV (a “classical” beta toxin) onthemammalian neuronal Nav channels, displaying some features ofthe beta toxins. Other toxins able to displace both site 3 and 4 toxinshave already been described in the literature. The first one was theanti-insect toxin AaH IT4 from Androctonus australis Hector (Loretet al., 1991), and its structurally related Lqhb1, isolated from thescorpion Leiurus quinquestriatus hebraeus venom (Gordon et al.,2003). Corzo et al. (2005) purified a spider toxin from Para-coelotes luctuosus that binds to site 4 on insect Nav channels,although it modulates the channel inactivation (a site-3 effect).Also, it was shown that beta/delta Agatoxins affect both the acti-vation and inactivation processes of the insect Nav channel (Billenet al., 2010). In addition, several other spider neurotoxins, such as

delta atracotoxins, are known to target both insect and vertebrateNav channels (Nicholson et al., 2004). The voltage dependence ofactivation was shifted toward more hyperpolarized potentials (site4-like effect) and a non-inactivating persistent Naþ current wasinduced (site 3-like effect). It was proven using “paddle motif”chimera that some spider and scorpion toxins are able to bindextracellular loops of different Nav channel domains, thus leadingto various pharmacological effects (Bosmans et al., 2008, 2011).

Association kinetics shows that the 125I-b/d-PrIT1 binding tocockroach synaptosomes is highly specific (Fig. 3). A high rate of95% of the bound radioactivity could be displaced by the unlabelledtoxin. However, association and dissociation experiments sug-gested the possible existence of more than a single site. Afterlinearization, association data (Fig. 4A inset) showed half-time (t1/2)rates of 1.4 and 8.5 min and a Kobs of 0.240 and 0.039 for the 1st and2nd site, respectively. The binding was reversible (Fig. 4B) and semilogarithmic plots (Fig. 4B inset) yielded a Koff of 0.128 and a Kon of3.67 � 109 M�1 min�1 (for the first site) and a Koff of 0.021 and Konof 6.01 � 108 M�1 min�1 (for the second site). The t1/2 for thedissociation was 5.4 and 32.8 min for the first and second sites,respectively. The equilibrium dissociation constant (KD) calculatedfrom the kinetic data (KD ¼ k�1/kþ1) were 34.7 pM (1st site) and35.1 pM (2nd site). The Kd values of the two sites are very close,suggesting similar affinities of b/d-PrIT1 for the two possiblebinding sites. These results, compiled in Table 2, demonstrate thehigh-affinity interaction between b/d-PrIT1 and the synaptosomalpreparation from cockroach nerve cords. Analysis of the t1/2 ratesshows a fast association and a relatively slow dissociation of b/d-PrIT1. However, the heterogeneity of the ligand or the binding sitescannot be excluded and can affect the b/d-PrIT1 kinetics studies (DeLima et al., 1989).

3.3. Electrophysiological experiments

To investigate the physiological consequences of the binding ofb/d-PrIT1 to insect sodium channels, electrophysiological experi-ments were carried out using isolated neurons from the cockroachmetathoracic ganglion. Following application of b/d-PrIT1 (1 mM),the decaying phase of the sodium current progressively slowed

Fig. 3. Binding of 125I-b/d-PrIT1 to nerve cord of cockroach synaptosomal preparation.125I-b/d-PrIT1 (10�10 M) was added to nerve cord synaptosomal preparation (40 mg/mL), and the membrane-bound radioactivity was determined after the indicated timesat 37 �C. Total binding (C). Nonspecific binding (о) estimated in the presence of10�7 M unlabeled b/dPrIT1 was subtracted to make clear the specific binding (;).

Fig. 4. Kinetics of 125I-b/dPrIT1 binding to nerve cord of cockroach synaptosomal preparation. A: Association kinetics. 125I-b/dPrIT1 (10�10 M) was added to nerve cord synaptosomalpreparation (40 mg/mL), and the membrane-bound radioactivity was determined after the indicated times at 37 �C. Nonspecific binding estimated in the presence of 10�7 Munlabeled b/dPrIT1 was subtracted. Insert: linear semi logarithmic plots of association kinetic data. B: Dissociation kinetics. At equilibrium, native b/dPrIT1 (10�7 M) was added, andthe remaining specifically bound radioactivity was measured at the indicated times. Insert: linear semi logarithmic plots of dissociation kinetic data. In A and B, points were fittedwith exponential equation with one or two parameters. The data illustrated are the results of one of three independent experiments.

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down, indicating an inhibition of sodium current inactivation(Fig. 5A). In control conditions, the inactivated Naþ current fol-lowed a bi-exponential function, and its faster rate constant, whichaccounted for 70% of the decay, became 26 times the rate constantin the presence of the toxin. No alteration was noticed on the timecourse of activation. Similar electrophysiological effects areobserved with scorpion alpha toxins. Thus, the electrophysiologicalexperiments are in accordance with competition binding data andconsistent with the hypothesis that b/d-PrIT1 binds to site 3 of Navchannels. This site was first identified on the extracellular loopsS3eS4 of domain IV (Rogers et al., 1996; Cest�ele and Catterall, 2000;Bosmans et al., 2008; Song et al., 2011; Bende et al., 2014). Both“classical” alpha-toxins and the “alpha-like” toxins inhibit Navchannels inactivation (Cest�ele et al., 1999).

Another effect of the toxin was a marked increase (210%) in thecurrent amplitude in test pulses to �10 mV (Fig. 5A). The currentamplitude measured at different membrane potentials (I�V),showed that b/d-PrIT1 increases the sodium current densitybetween þ10 mV and �50 mV (Fig. 5B). In the control conditions,

the sodium current density reached its maximum valuearound þ5 mV, while, in the presence of b/d-PrIT1, the maximumvalue was around �5 mV. Furthermore, by fitting the cur-rentevoltage relationship, it was possible to determine that Va, thepotential that gave half of maximal channel activation, shifted from16.6 mV towards more negative potentials, with but a small (17%)increase of maximal conductance. This indicates that, on isolatedmetathoracic ganglion neurons, the toxin b/d-PrIT1 shifts the Navchannels activation to more negative potentials (hyperpolarizingeffect). This can be considered as a beta-type effect (Fig. 5B).

Previous ligand binding and electrophysiological studies usingthe “classical” beta toxin CssIV from the Mexican scorpion Cen-truroides suffusus first described the beta-toxins as toxins thatinteract with the Nav channel voltage-sensor of domain II, and mapthe binding site 4 on extracellular loops S1eS2 and S3eS4 ofdomain II (Cest�ele and Catterall, 2000; Cest�ele et al., 2006). Betatoxins facilitate and stabilize the outward conformation of thisvoltage-sensor, thus favouring the NaV opening at more negativepotentials. New structural studies achieved with the procaryoticNav channels show that the S5eS6 loop in each subunit is in closeproximity to the S1eS2 and S3eS4 loops of the adjacent subunit(Payandeh et al., 2011), and thus, that the S5eS6 extracellular loopof the neighbouring subunit (in clockwise direction) could beinvolved in the toxin binding site.

Also recently, the involvement of the domain III voltage-sensorin the binding of beta toxins was demonstrated, suggesting acoupling between the voltage sensors in neighbouring domainsduring channel activation, due to allosteric mechanisms (Bosmanset al., 2008; Campos et al., 2008; Song et al., 2011). A structuralmotif called the “paddle motif” was identified in each of the fourNav channel voltage sensor modules. The transfer of these regionsfrom Nav to Kv channels has resulted in functional Kv channelssensitive to animal toxins described as Nav channel modulators(Bosmans et al., 2008, 2011). Toxins interacting with the paddlemotifs from domain I to domain III alter channel opening whereasthose targeting the domain IV commonly inhibit fast inactivation.However, it has also been shown that some toxins could bind to

Fig. 5. Effects of b/dPrIT1 on Naþ currents on dissociated adult cockroach neurons. A: Naþ currents elicited in response to depolarization steps e control and experimental (in thepresence of 1 mM b/d-PrIT1) conditions (n ¼ 5). To compare the kinetics of activation and inactivation, the control inward current was multiplied by a factor (f ¼ 3.45) to match itspeak value with the current in the presence of the toxin (dotted line); B: Naþ current density� voltage in control (open circles, n ¼ 3) and in presence of 200 nM b/d-PrIT1 (closedcircles, n ¼ 3). The solid traces were obtained by fitting the data points to Equation (1) (see Material and methods). The parameters that gave the best fits were: g(max) ¼ 383 ± 21and 450 ± 26 pS/pF, Vr ¼ 28.6 ± 1.4 and 29.1 ± 1.4 mV, k ¼ 10.8 ± 2.0 and 12.3 ± 1.9 mV, Va ¼ 2.6 ± 3.6 and �14.0 ± 3.3 mV, for control and experimental currents, respectively.

Table 2Kinetic parameters obtained from binding assays of b/d-PrIT1 in cockroach nervecord synaptosomal preparations.

Association Site 1 t1/2 ¼ 1.4 minR2 ¼ 0.82kobs ¼ � 0.240

Site 2 t1/2 ¼ 8.5 minR2 ¼ 0.83kobs ¼ � 0.039

Kon Site 1 3.67� 109M�1s�1

Site 2 6.01� 108M�1s�1

Dissociation Site 1 t1/2 ¼ 5.4 minR2 ¼ 0.82koff ¼ 0.128

Site 2 t1/2 ¼ 32.8 minR2 ¼ 0.91koff ¼ 0.021

Kd (Kd ¼ koff/kon) Site 1 3.47 � 10�11 MSite 2 3.51 � 10�11 M

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several paddle motifs of the Nav channel. It was the case for TsVII, a“beta-like” scorpion toxin from the Brazilian scorpion Tityus ser-rulatus, which promotes opening of the neuronal rNav1.2a channelby primarily interacting with the paddle motif in the domain IIvoltage sensor (Bosmans et al., 2008). However, electrophysiolog-ical experiments with the rNav1.2a/Kv2.1 chimeras revealed thatTsVII could also bind to the paddle motifs from domain III and IV(Bosmans et al., 2008). It was also the case for the TarantulaThrixopelma pruriens toxins ProTx-I and ProTx-II, which were ableto recognize both the rNav1.2a domain II and IV paddle motifs(Bosmans et al., 2008; Bende et al., 2014).

It is interesting to consider potential mechanisms that mayunderlie the specificity of spider toxins. So far, previous studies oninsecticidal toxins from various Phoneutria sub-species venoms (DeLima et al., 2002; Stankiewicz et al., 2004 and the present work) arein favour of a commonmode of action. All these toxins bind to site 3of Nav channels on cockroach synaptosomes, with a predominant“alpha-like” effect (delaying the inactivation), showing higher orlower “beta type” effects (displacement of the opening to morenegative potentials). Thus, it is not surprising to find here that b/d-PrIT1 exhibits the same complex mode of action. In the light ofrecent papers in the literature, we suggest that b/d-PrIT1 binds toseveral binding sites, which could be located in the same channel,promoting complex pharmacological effects. b/d-PrIT1 perfectlyrecognizes BomIV binding site on the insect Nav channel voltage-sensing domain IV, since it completely displaces 125I-BomIV fromits binding site with a pM affinity and blocks the channel fastinactivation process. However, the 125I-b/d-PrIT1 binding to insectsynaptosomes is only partially displaced by BomIV (not shown) andit suggests the presence of two independent binding sites. Theobservation that the beta-toxin 125I-CssIV is partially displaced byb/d-PrIT1 on rat brain synaptosomes suggests that b/d-PrIT1 alsobinds to the channel voltage-sensing domains II and/or III (or theboth), promoting the voltage sensor activation.

Several mutations in the insect Nav channels have been attrib-uted to the emergence of resistance to the conventional insecticides(for review see Zlotkin et al., 2000). Several of these mutations (asKDR and super KDR) seem, in fact, to increase the insect suscepti-bility to some arthropod toxins. Although we have no informationwhether the insect toxins could cause resistance in insects, theiruse or of some derivative, in associationwith other compounds thataffect insect Nav channels could be considered as a potentialstrategy to obtain new tools for fighting against the resistant pests,as already suggested (Gordon, 1997). On the other hand, the toxinspresenting high specificity to insect sodium channels representgood candidates to design new specific insecticides.

Acknowledgements

We greatly thank Dr. Marcelo M. Santoro (in memoriam) for themathematic analyses, Dr. Marcelo Porto Bemquerer for supportingsequence experiments, Dr. C�elia de F�atima Barbosa for giving sup-port to the LD50 calculations, Dr. Thiago Verano Braga and Dr.Alessandra Matavel for suggestions and Júlio C. dos Reis andZulema J. Oliveira for their technical assistance. This study wassupported by grants from CNPq (Conselho Nacional de Desenvol-vimento Científico e Tecnol�ogico), CAPES (Coordenaç~ao de Pessoalde Nível Superior), INCTTOX (Instituto Nacional de Ciencia e Tec-nologia em Toxinas), PRPq-UFMG (Pr�o-Reitoria de Pesquisa daUFMG) and FAPEMIG (Fundaç~ao de Amparo �a Pesquisa do Estado deMinas Gerais).

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