toxins

Article Insect-Active Toxins with Promiscuous Pharmacology from the African Theraphosid Monocentropus balfouri

Jennifer J. Smith 1,†, Volker Herzig 1,†, Maria P. Ikonomopoulou 1,†,‡, Sławomir Dziemborowicz 2, Frank Bosmans 3, Graham M. Nicholson 2 and Glenn F. King 1,* 1 Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; [email protected] (J.J.S.); [email protected] (V.H.); [email protected] (M.P.I.) 2 School of Life Sciences, University of Technology Sydney, NSW, Sydney 2007, Australia; [email protected] (S.D.); [email protected] (G.M.N). 3 Department of Physiology & Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA; [email protected] * Correspondence: [email protected]; Tel.: +61-7-3346-2025 † These authors contributed equally to this work. ‡ Present address: QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia.

Academic Editor: Lourival D. Possani Received: 28 March 2017; Accepted: 28 April 2017; Published: 5 May 2017

Abstract: Many chemical insecticides are becoming less efficacious due to rising resistance in pest species, which has created much interest in the development of new, eco-friendly bioinsecticides. Since insects are the primary prey of most , their venoms are a rich source of insect-active peptides that can be used as leads for new bioinsecticides or as tools to study molecular receptors that are insecticidal targets. In the present study, we isolated two insecticidal peptides, µ/ω-TRTX-Mb1a and -Mb1b, from venom of the African Monocentropus balfouri. Recombinant µ/ω-TRTX-Mb1a and -Mb1b paralyzed both Lucilia cuprina (Australian sheep blowfly) and Musca domestica (housefly), but neither peptide affected larvae of Helicoverpa armigera (cotton bollworms). Both peptides inhibited currents mediated by voltage-gated sodium (NaV) and calcium channels in Periplaneta americana (American cockroach) dorsal unpaired median neurons, and they also inhibited the cloned Blattella germanica (German cockroach) NaV channel (BgNaV1). An additional effect seen only with Mb1a on BgNaV1 was a delay in fast inactivation. Comparison of the NaV channel sequences of the tested insect species revealed that variations in the S1–S2 loops in the voltage sensor domains might underlie the differences in activity between different phyla.

Keywords: insecticide; pharmacology; venom; sodium channel; calcium channel; spider

1. Introduction Spider venoms contain a plethora of bioactive peptides that target a diverse range of vertebrate and invertebrate voltage-gated ion channels [1,2]. Spider venoms evolved for two main purposes: defense against predators [3] and as a chemical weapon for prey capture [4,5]. Since insects are the predominant prey of most spiders, these have developed a range of toxins that are highly effective at incapacitating insects [6]. There are over 46,600 extant spider species [7], with the venoms of some spiders containing >1000 different peptides [8]. Thus, spider venoms are an ideal source of toxins that can be used to study insect ion channels, or as potential candidates for the development of insecticides [6,9]. The two major pharmacological targets of spider-venom peptides are voltage-gated calcium (CaV) channels and voltage-gated sodium (NaV) channels [6,10]. Both channels consist of

Toxins 2017, 9, 155; doi:10.3390/toxins9050155 www.mdpi.com/journal/toxins Toxins 2017, 9, 155 2 of 18 four homologous domains (DI–DIV), each of which contains six transmembrane helices (S1–S6). The four sets of S5–S6 helices come together to form the pore of the channel, while each S1–S4 region serves as an independent voltage sensor [11,12]. Many chemical insecticides target insect NaV channels and bind to the S5–S6 pore region, but these insecticides are becoming less useful due to increasing resistance in pest species [13–16]. However, spider venoms represent a source of novel toxins that act on insect NaV channels via a different mode of action to extant chemical insecticides. The majority of spider toxins are gating modifiers that interact with the voltage-sensing domains and not the pore region, and therefore pests resistant to current NaV channel insecticides would likely be sensitive to spider toxins [17]. In addition to NaV channels, insect CaV channels are an emergent insecticidal target, and they are another major target of spider toxins [9,18]. Indeed, a spider toxin that inhibits insect CaV channels has been developed into a bioinsecticide called SPEARTM that will become commercially available early 2017 [19]. This study describes the isolation, recombinant production, and characterization of the disulfide-rich peptides µ/ω-TRTX-Mb1a and µ/ω-TRTX-Mb1b (hereafter Mb1a and Mb1b) from venom of the African tarantula Monocentropus balfouri. Both peptides inhibited NaV and CaV channel currents in cockroach neurons as well as the cloned NaV channel from the German cockroach Blatella germanica. Mb1a and Mb1b are paralytic to the dipterans L. cuprina and M. domestica, but do not affect the lepidopteran H. armigera. We propose that variations between taxa in the S1–S2 loops of the NaV channel voltage-sensor domains underlie the differences in activity between phyla and mode of action of Mb1a and Mb1b. Ultimately, understanding how spider toxins bind to insect NaV and CaV channels will facilitate the rational design of bioinsecticides with better toxicity and selectivity profiles than the current arsenal of chemical insecticides.

2. Results

2.1. Isolation and Sequencing of µ/ω-TRTX-Mb1a and -Mb1b A screen of insecticidal activity by injection into sheep blowflies of fractions resulting from reversed-phase (RP) HPLC separation of M. balfouri venom revealed that the fraction eluting at ~35% solvent B concentration (Figure1) induced paralysis that was reversible within 24 h. Mass spectrometry revealed that this fraction consists of a single peptide with monoisotopic (M + H+) mass 4147.00 Da (Figure1, inset). N-terminal sequencing of this peptide revealed the first 36 residues as GVDKPGCRYMFGGCVQDDDCCPHLGCKRKGLYCAWD(A)(T), with residues 37 and 38 shown in parentheses due to sequencing ambiguities. The two C-terminal residues cannot be both A and T as this would yield a sequence whose mass does not correspond to that of the observed peptide. Therefore, two peptide sequences were derived that matched the observed 0 0 0 0 mass, one with GT-NH2 as the C-terminus and another with AS-NH2 . In an attempt to ascertain the two terminal residues, LC-MS/MS was performed on a nine-residue C-terminal fragment of reduced and alkylated native peptide liberated by tryptic digestion. Analysis of 0 0 the b- and y-ion series revealed three matches for GT-NH2 while only one match corresponded 0 0 0 0 to AS-NH2 therefore GT-NH2 is the most likely C-terminal sequence of the native peptide. Nevertheless, both peptide sequences were expressed for further characterization as the native sequence was not conclusively determined. Based on their activity (see below) and the species of origin, the two peptides were named in accordance with published nomenclature guidelines [20] as µ/ω-TRTX-Mb1a (sequence GVDKPGCRYMFGGCVQDDDCCPHLGCKRKGLYCAWDGT) and µ/ω-TRTX-Mb1b (sequence GVDKPGCRYMFGGCVQDDDCCPHLGCKRKGLYCAWDAS). Toxins 2017, 9, 155 3 of 18 Toxins 2017, 9, 155 3 of 18

FigureFigure 1.1. ((aa)) PhotoPhoto of of a a female femaleM. M. balfouri balfouri;(;b ()b Chromatogram) Chromatogram resulting resulting from from RP-HPLC RP-HPLC fractionation fractionation of M.of balfouriM. balfourivenom. venom. The peakThe peak highlighted highlighted in red in contains red contains the µ/ω the-TRTX-Mb1a/b µ/ω-TRTX-Mb1a/b peptide. peptide. The dotted The linedotted indicates line indicates the gradient the ofgradient solvent of B (90%solvent acetonitrile/0.1% B (90% acetonitrile/0.1% formic acid). formic Inset isacid). a MALDI-TOF Inset is a massMALDI-TOF spectrum mass of the spectrum isolated ofµ/ theω-TRTX-Mb1a/b isolated µ/ω-TRTX-Mb1a/b peptide. peptide.

2.2.2.2. RecombinantRecombinant ProductionProduction ofof Mb1aMb1a andand Mb1bMb1b RecombinantRecombinant Mb1aMb1a andand Mb1bMb1b werewere producedproduced byby overexpressionoverexpression inin thethe periplasmperiplasm ofof EscherichiaEscherichia colicoli usingusing thethe systemsystem wewe previouslypreviously optimizedoptimized forfor expressionexpression ofof disulfide-richdisulfide-rich venom peptides [[21].21]. TheThe fusionfusion protein protein was was the the major major soluble soluble protein protein expressed expressed after after induction, induction, and cleavage and cleavage of the fusionof the proteinfusion protein with tobacco with tobacco etch virus etch (TEV) virus protease (TEV) protease liberated liberated free recombinant free recombinant Mb1a (Figure Mb1a2, top (Figure inset) 2, and top Mb1binset) (notandshown). Mb1b (not RP-HPLC shown). purification RP-HPLC of purification the cleaved peptidesof the cleaved resulted peptides in elution resulted profiles consistingin elution ofprofiles two peaks consisting (Figure 2of) with two the peaks correct (Figure mass ( m2)/ zwithcalculated: the correct 4147.8 mass Da, observed: (m/z calculated: 4147.9 Da) 4147.8 (Figure Da,2, bottomobserved: inset). 4147.9 Re-injection Da) (Figure of each 2, bottom peak separately inset). Re-inj resultedection in of the each reappearance peak separately of two peaksresulted (data in notthe shown),reappearance suggesting of two possible peakscis (data-trans notisomerization shown), suggesting of the proline possible residue cis at-trans position isomerization 5 or 22. The of final the yieldproline for residue both peptides at position was ~1005 or 22.µg The per final litre ofyield bacterial for both culture. peptides The was recombinant ~100 µg per peptides litre of were bacterial used forculture. all in The vitro recombinant and in vivo assays.peptides were used for all in vitro and in vivo assays.

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FigureFigure 2. Recombinant 2. Recombinant production production of Mb1a. of Semi-preparativeMb1a. Semi-preparative RP-HPLC RP-HPLC chromatogram chromatogram of recombinant of Mb1aFigurerecombinant released 2. Recombinant by Mb1a TEV released protease production by cleavage TEV protease of of theMb1a. cleavage MBP-Mb1a Semi of the-preparative fusion MBP-Mb1a protein RP-HPLC fusion (see protein Materials chromatogram (see andMaterials Methods of and Methods for more details). The dotted line indicates the gradient of solvent B (90% forrecombinant more details). Mb1a The released dotted by line TEV indicates protease the cleavage gradient of the of solvent MBP-Mb1a B (90% fusion acetonitrile/0.043% protein (see Materials TFA). acetonitrile/0.043% TFA). Top inset: SDS-PAGE gel showing pre-cleaved MBP-Mb1a fusion protein Topand inset: Methods SDS-PAGE for more gel showing details). pre-cleaved The dotted MBP-Mb1a line indicates fusion proteinthe gradient (lane 1)of and solvent remaining B (90% MBP (lane 1) and remaining MBP after cleavage (lane 2). Lane M contains molecular markers (masses in afteracetonitrile/0.043% cleavage (lane 2). TFA). Lane Top M contains inset: SDS-PAGE molecular gel markers showing (masses pre-cleaved in kDa). MBP-Mb1a Bottom inset: fusion MALDI-TOF protein kDa). Bottom inset: MALDI-TOF mass spectrum of pure recombinant Mb1a. mass(lane spectrum 1) and remaining of pure recombinant MBP after cleavage Mb1a. (lane 2). Lane M contains molecular markers (masses in kDa). Bottom inset: MALDI-TOF mass spectrum of pure recombinant Mb1a. 2.3. Insecticidal Activity of µ/ω-TRTX-Mb1a and -Mb1b 2.3. Insecticidal Activity of µ/ω-TRTX-Mb1a and -Mb1b 2.3. InsecticidalBoth Mb1a Activity and Mb1b of µ/ω caused-TRTX-Mb1a fast, but and fully -Mb1b reversible, paralysis of sheep blowflies (L. cuprina) withBoth median Mb1a paralytic and Mb1b dose caused (PD50) fast, values but in fully the range reversible, of 5600–5800 paralysis pmoL/g of sheep at 30 blowfliesmin post-injection (L. cuprina ) Both Mb1a and Mb1b caused fast, but fully reversible, paralysis of sheep blowflies (L. cuprina) with(Figure median 3). paralytic No lethal dose effects (PD were50) values observed, in the and range paralysis of 5600–5800 was fully pmoL/greversed atafter 30 24 min h. post-injectionComplete with median paralytic dose (PD50) values in the range of 5600–5800 pmoL/g at 30 min post-injection (Figureparalysis3). No was lethal also effects seen for were both observed, Mb1a and and Mb1b paralysis at 60 wasmin fullypost-injection reversed into after house 24 h. flies Complete (M. (Figure 3). No lethal effects were observed, and paralysis was fully reversed after 24 h. Complete paralysisdomestica was), alsowithseen partial for recovery both Mb1a observed and Mb1b after at24 60h (Mb1a: min post-injection 3/5 recovered; into Mb1a: house 4/5 flies recovered). (M. domestica In ), paralysis was also seen for both Mb1a and Mb1b at 60 min post-injection into house flies (M. withcontrast, partial recoveryinjection of observed up to 73.4 after nmoL/g 24 h of (Mb1a: Mb1b into 3/5 cotton recovered; bollworms Mb1a: (H. 4/5 armigera recovered). larvae) Indid contrast, not domesticainduce ),any with paralytic partial effects.recovery No observed lethality afteror significant 24 h (Mb1a: changes 3/5 recovered; in the weight Mb1a: gain 4/5 of recovered). the larvae In injection of up to 73.4 nmoL/g of Mb1b into cotton bollworms (H. armigera larvae) did not induce any contrast,occurred injection within theof up 72-h to observation 73.4 nmoL/g period. of Mb1b into cotton bollworms (H. armigera larvae) did not paralyticinduce any effects. paralytic No lethality effects.or No significant lethality changesor significant in the changes weight gainin the of weight the larvae gain occurred of the larvae within theoccurred 72-h observation within the period.72-h observation period.

Figure 3. Insecticidal effects of recombinant (a) Mb1a and (b) Mb1b. Toxins were injected intra-thoracically into L. cuprina blowflies and paralytic effects measured at 0.5, 1, 2 and 24 h post injection. PD50 values for each time-intervals (±S.E.M.) are indicated. Hill slopes for Mb1a were 2.01 FigureFigure(0.5 3.h),3. 2.24 InsecticidalInsecticidal (1.0 h), 2.53 effectseffects (2.0 h), of and recombinantrecombinant for Mb1b: 2.23 ( a )(0.5 Mb1a h), 2.74 and (1.0 ( bh),) Mb1b.Mb1b.2.98 (2.0 Toxins h). Toxins No lethalitywere were injected injectedwas intra-thoracicallyintra-thoracicallyobserved, and the intointo paralytic L.L. cuprinacuprina effects blowfliesblowflies caused by and bo paralytic th toxins wereeffects effects fully meas measured reversibleured at atwithin 0.5, 0.5, 1, 1,24 2 2h.and and 24 24 h hpost post injection. PD50 values for each time-intervals (±S.E.M.) are indicated. Hill slopes for Mb1a were 2.01 injection. PD50 values for each time-intervals (±S.E.M.) are indicated. Hill slopes for Mb1a were 2.01 2.4.(0.5(0.5 Activity h), h), 2.24 2.24 of (1.0 (1.0Mb1a h), h), 2.53and 2.53 Mb1b (2.0 (2.0 h), h),on and andInsect forfor Na Mb1b:Mb1b:V Channel 2.232.23 Currents (0.5(0.5 h), 2.74 (1.0 h), h), 2.98 2.98 (2.0 (2.0 h). h). No No lethality lethality was was observed, and the paralytic effects caused by both toxins were fully reversible within 24 h. observed,At a concentration and the paralytic of 1 effectsµM, both caused Mb1a by both(Figure toxins 4a) wereand fullyMb1b reversible (Figure 4b) within reversibly 24 h. inhibited endogenous sodium channel currents (INa) in dorsal unpaired median (DUM) neurons isolated from 2.4. Activity of Mb1a and Mb1b on Insect NaV Channel Currents 2.4.the Activity American of Mb1a cockroach and Mb1b Periplaneta on Insect americana NaV Channel. Mb1aCurrents rapidly inhibited 91.5 ± 3.0% of the current (n = At a concentration of 1 µM, both Mb1a (Figure 4a) and Mb1b (Figure 4b) reversibly inhibited At a concentration of 1 µM, both Mb1a (Figure4a) and Mb1b (Figure4b) reversibly inhibited endogenous sodium channel currents (INa) in dorsal unpaired median (DUM) neurons isolated from endogenous sodium channel currents (I ) in dorsal unpaired median (DUM) neurons isolated from the American cockroach Periplaneta americanaNa . Mb1a rapidly inhibited 91.5 ± 3.0% of the current (n =

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the AmericanToxins 2017, cockroach9, 155 Periplaneta americana. Mb1a rapidly inhibited 91.5 ± 3.0% of the current5 of 18 (n = 5; τon = 16.1 s; Figure4b,d), while Mb1b was slightly less potent, inhibiting current by 84.3 ± 3.7% (n = 3; τon =5; 18.7 τon = s; 16.1 Figure s; Figure4c,e). 4b,d), Tail currentswhile Mb1b were was unaffected slightly less by potent, Mb1a inhibiting and Mb1b current (Figure by 484.3b,c). ± 3.7% (n = To3; τ investigateon = 18.7 s; Figure if current 4c,e). inhibitionTail currents was were caused unaffected by a toxin-induced by Mb1a and Mb1b shift in(Figure the voltage- 4b,c). dependence To investigate if current inhibition was caused by a toxin-induced shift in the voltage- of NaV channel activation, INa/V curves were generated before (Figure5b) and after (Figure5c) dependence of NaV channel activation, INa/V curves were generated before (Figure 5b) and after addition of 100 nM Mb1a. No shift in INa/V curves was observed following perfusion with toxin (Figure 5c) addition of 100 nM Mb1a. No shift in INa/V curves was observed following perfusion with (Figuretoxin5d). (Figure To determine 5d). To determine if toxin-induced if toxin-induced block block was voltage-dependent,was voltage-dependent, the the peak peak currentcurrent in the presencethe presence of 100 nM of 100 Mb1a nM was Mb1a plotted was plotted as a percentage as a percentage of the of control the control current current (Figure (Figure5e). 5e). There There was no significantwas no changesignificant in thechange extent in the of extent inhibition of inhibition for INa fordepolarizations INa depolarizations ranging ranging from from− −4040 mVmV to to 0 0 mV, indicatingmV, indicating that toxin-induced that toxin-induced block isblock voltage-independent is voltage-independent over over this this range. range.

Figure 4. Effects of 1 µM Mb1a and Mb1b on P. americana DUM neuron INa. (a) The depolarizing Figure 4. Effects of 1 µM Mb1a and Mb1b on P. americana DUM neuron INa.(a) The depolarizing voltage test protocol (Vtest) used to elicit INa at 0.1 Hz. (b,c) Representative superimposed current voltage test protocol (Vtest) used to elicit INa at 0.1 Hz. (b,c) Representative superimposed current traces elicited by Vtest prior to (black lines), and 3 min following, exposure to 1 µM Mb1a ((b), red traces elicited by Vtest prior to (black lines), and 3 min following, exposure to 1 µM Mb1a ((b), red line) line) and Mb1b (c, red line). Solid gray lines represent INa recorded 3 min after perfusion with and Mb1btoxin-free (c, redsolution, line). while Solid dashed gray lines gray representlines representINa recordedzero current. 3 min (d,e after) Timecourse perfusion of the with block toxin-free of solution,INa by while 1 µM dashed Mb1a (d gray) and lines Mb1b represent (e). Average zero normalized current. ( dpeak,e) Timecourse INa before (open of the circles), block during of INa (redby1 µM Mb1acircles), (d) and and Mb1b after ((bluee). Average circles) perfusion normalized with peak 1 µMI toxin.Na before Values (open represent circles), the duringmean ± S.E.M (red circles),of 5 (d) and afteror (blue 3 (e) circles)experiments. perfusion The rate with constant 1 µM for toxin. association Values of representthe toxin to the themean channel± (S.E.Mτon) was of calculated 5 (d) or 3 (e) experiments.using Equation The rate (1) constantdefined in for the association Materials and of theMethods. toxin toThere the channelwas no significant (τon) was difference calculated in using Equationcurrent (1) inhibition defined by in Mb1a the Materials and Mb1b and (p = Methods.0.169). There was no significant difference in current inhibition by Mb1a and Mb1b (p = 0.169). Mb1a and Mb1b were also tested on the cloned BgNaV1 channel from the German cockroach (Blattella germanica), heterologously expressed in Xenopus oocytes. (Note that the orthologous P. Mb1aamericana and Na Mb1bV channel were has also never tested been onfunctionally the cloned expresse BgNadV in1 channeloocytes.) Similar from the to their German activity cockroach on (BlattellaAmerican germanica cockroach), heterologously NaV channels, application expressed of in Mb1aXenopus or Mb1boocytes. to BgNa (NoteV1 caused that a thereduction orthologous in P. americanapeak BgNaNaVV1channel currents has with never no shift been in functionallythe GNa-V curve expressed (Figure in6a). oocytes.) However, Similar application to their of activityMb1a on Americancaused cockroach inhibition Na ofV fastchannels, inactivation, application which ofwas Mb1a not orseen Mb1b with to Mb1b BgNa (FigureV1 caused 6b). aTherefore, reduction the in peak C-terminal residues ′GT′ are responsible for the inhibition of fast inactivation seen with Mb1a, likely BgNaV1 currents with no shift in the GNa-V curve (Figure6a). However, application of Mb1a caused inhibitionmediated of fast by an inactivation, additional interaction which was with not the seen domain with IV Mb1b voltage-sensor (Figure6b). [12]. Therefore, the C-terminal residues 0GT0 are responsible for the inhibition of fast inactivation seen with Mb1a, likely mediated by an additional interaction with the domain IV voltage-sensor [12]. Toxins 2017, 9, 155 6 of 18 Toxins 2017, 9, 155 6 of 18

Figure 5. Effect of 100 nM Mb1a on the voltage-dependence of activation of P. americana DUM Figure 5. Effect of 100 nM Mb1a on the voltage-dependence of activation of P. americana DUM neuron neuron NaV channels. (a) Families of NaV channel currents were elicited by depolarizing test pulses NaV channels. (a) Families of NaV channel currents were elicited by depolarizing test pulses to +40 mV to +40 mV from a holding potential of −90 mV in 10-mV steps. Representative superimposed families from a holding potential of −90 mV in 10-mV steps. Representative superimposed families of INa are of INa are shown prior to (b), and 5 min after (c), application of 100 nM Mb1a. Currents were shown prior to (b), and 5 min after (c), application of 100 nM Mb1a. Currents were generated using the generated using the test pulse protocol shown in panel a. (d) Normalised INa-V relationships before test pulse protocol shown in panel a. (d) Normalised INa-V relationships before (open circles), and after (open(red circles circles), and and shaded), after (red application circles and of 100 shaded), nM Mb1a. application Data were of fitted100 nM using Mb1a. Equation Data were (1) (see fitted Materials using Equationand Methods). (1) (see Currents Materials recorded and inMethods). the presence Current of toxins recorded were normalised in the presence against maximumof toxin were peak normalised against maximum peak INa in controls (red solid curve) and maximum peak INa in the INa in controls (red solid curve) and maximum peak INa in the presence of toxin (red dashed curve). presence(e) Linear of regression toxin (red analysis dashed ofcurve). the data (e) Linear using Equation regression (2) analysis (see Materials of the data and Methods)using Equation revealed (2) (see that Materials and Methods) revealed that inhibition of INa was voltage-independent over the range −40 to inhibition of INa was voltage-independent over the range −40 to 0 mV in the presence of 100 nM Mb1a. 0Data mV pointsin the presence are the mean of 100± nMS.E.M Mb1a. of 3 experiments.Data points are the mean ± S.E.M of 3 experiments.

2.5. Effect of Mb1a and Mb1b on Insect CaV Channel Currents 2.5. Effect of Mb1a and Mb1b on Insect CaV Channel Currents At a concentration of 1 µM, Mb1a and Mb1b rapidly and irreversibly inhibited both At a concentration of 1 µM, Mb1a and Mb1b rapidly and irreversibly inhibited both mid-/low-voltage-activated (M-LVA) and high-voltage-activated (HVA) CaV channel currents mid-/low-voltage-activated (M-LVA) and high-voltage-activated (HVA) CaV channel currents endogenously present in P. americana DUM neurons (Figures 7 and 8). Mb1a was slightly more endogenously present in P. americana DUM neurons (Figures7 and8). Mb1a was slightly more potent than Mb1b on both types of CaV channel currents, with 45% of M-LVA CaV channel currents potent than Mb1b on both types of CaV channel currents, with 45% of M-LVA CaV channel currents and 48% of HVA CaV channel currents inhibited by Mb1a (Figures 7b and 8b, respectively) and 48% of HVA CaV channel currents inhibited by Mb1a (Figures7b and8b, respectively) compared compared with 27% inhibition of M-LVA currents and 34% inhibition of HVA CaV channel currents with 27% inhibition of M-LVA currents and 34% inhibition of HVA CaV channel currents by Mb1b by Mb1b (Figures 7c and 8c, respectively). Mb1a did not shift the IBa/V curve (Figure 9a–d), and its (Figures7c and8c, respectively). Mb1a did not shift the IBa/V curve (Figure9a–d), and its inhibition inhibition of CaV channel currents was voltage-independent (Figure 9e). of CaV channel currents was voltage-independent (Figure9e).

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Figure 6. (a) Effect of 200 nM Mb1a (left) or Mb1b (right) on BgNaV1. Normalized conductance- Figure 6. (a) Effect of 200 nM Mb1a (left) or Mb1b (right) on BgNaV1. Normalized conductance- voltage (G-V) relationships (G/Gmax) are shown by closed circles and steady-state inactivation (SSI) voltage (G-V) relationships (G/Gmax) are shown by closed circles and steady-state inactivation (SSI) relationships (I/Imax) by open circles, before (black) and after (red) toxin addition. Normalization was relationships (I/Imax) by open circles, before (black) and after (red) toxin addition. Normalization performed relative to the peak current before toxin addition. Solid and dashed lines depict, was performed relative to the peak current before toxin addition. Solid and dashed lines depict, respectively, the G-V and SSI curves fit using the standard Boltzmann equation. Oocytes were respectively, the G-V and SSI curves fit using the standard Boltzmann equation. Oocytes were depolarized in 5-mV steps from a holding potential of –90 mV up to 5 mV for 50 ms, followed by a depolarized in 5-mV steps from a holding potential of –90 mV up to 5 mV for 50 ms, followed depolarizing pulse to −15 mV for 50 ms. Peak current from the initial step series was converted to by a depolarizing pulse to −15 mV for 50 ms. Peak current from the initial step series was converted toconductance conductance and and normalized normalized to tocreate create the the G-V G-V relationship relationship while while peak peak current current from from the the following following – −1515 mV mV voltage voltage depolarization depolarization step step was was normalized normalized to to yield yield the the SSI SSI relationship. relationship. Mb1a Mb1a caused caused a adecrease decrease in in peak currentcurrent and and increase increase in persistentin persistent current, current, while while Mb1b Mb1b reduced reduced peak current peak withoutcurrent affectingwithout affecting persistent persistent current (n current= 4; error (n = bars 4; error represent bars represent S.E.M) (b) S.E.M) Representative (b) Representative examples ofexamples the effects of the effects of Mb1a (left) and Mb1b (right) on BgNaV1 current when depolarized to –15 mV, with of Mb1a (left) and Mb1b (right) on BgNaV1 current when depolarized to –15 mV, with black and red tracesblack correspondingand red traces tocorresponding the current before to the and current after toxin before application, and after respectively.toxin application, Each set respectively. of traces is takenEach set from of antraces individual is taken oocyte from usedan individual to generate oocyte the data used shown to generate in panel the (a ).data Note shown that the in persistentpanel (a). currentNote that seen the in persistent the traces current in panel seen (b) doesin the not traces result in frompanel inhibition (b) does not of fast result inactivation from inhibition by Mb1a of andfast Mb1binactivation sinceBgNav1 by Mb1a inherently and Mb1b possesses since BgNav1 these characteristics inherently possesses at mildly these depolarizing characteristics voltages at [ 22mildly–24]. Toxinsdepolarizing 2017, 9, 155 voltages [22–24]. 8 of 18

Figure 7. Cont.

Figure 7. The effects of 1 µM Mb1a and Mb1b on P. americana M-LVA CaV channel currents. (a)

M-LVA IBa were elicited by 100-ms depolarizing test pulses to –20 mV from a holding potential of –90 mV. (b,c) Representative superimposed M-LVA IBa showing tonic block of M-LVA CaV channel currents before (black lines) and following (red lines) a 3 min perfusion, with (b) 1 µM Mb1a and (c) 1 µM Mb1b. Dashed gray lines represent zero current. (d,e) Timecourse of inhibition of normalized

peak M-LVA CaV channel currents by (d) 1 µM Mb1a and (e) 1 µM Mb1b. Data are mean ± S.E.M, n =

4. Average normalized peak IBa before (open circles), during (red circles), and after (blue circles) perfusion with 1 µM toxin. Data are mean ± S.E.M of 5 (panel d) or 3 (panel e) experiments. Inhibition

of IBa by Mb1a was significantly greater than for Mb1b (p = 0.008).

Toxins 2017, 9, 155 8 of 18

Toxins 2017, 9, 155 8 of 18

Figure 7. The effects of 1 µM Mb1a and Mb1b on P. americana M-LVA CaV channel currents. (a) Figure 7. The effects of 1 µM Mb1a and Mb1b on P. americana M-LVA CaV channel currents. (a) M-LVA M-LVA IBa were elicited by 100-ms depolarizing test pulses to –20 mV from a holding potential of –90 IBa were elicited by 100-ms depolarizing test pulses to –20 mV from a holding potential of –90 mV. mV. (b,c) Representative superimposed M-LVA IBa showing tonic block of M-LVA CaV channel (b,c) Representative superimposed M-LVA IBa showing tonic block of M-LVA CaV channel currents currents before (black lines) and following (red lines) a 3 min perfusion, with (b) 1 µM Mb1a and (c) 1 before (black lines) and following (red lines) a 3 min perfusion, with (b) 1 µM Mb1a and (c) 1 µM Mb1b. µM Mb1b. Dashed gray lines represent zero current. (d,e) Timecourse of inhibition of normalized Dashed gray lines represent zero current. (d,e) Timecourse of inhibition of normalized peak M-LVA peak M-LVA CaV channel currents by (d) 1 µM Mb1a and (e) 1 µM Mb1b. Data are mean ± S.E.M, n = CaV channel currents by (d) 1 µM Mb1a and (e) 1 µM Mb1b. Data are mean ± S.E.M, n = 4. Average 4. Average normalized peak IBa before (open circles), during (red circles), and after (blue circles) normalized peak I before (open circles), during (red circles), and after (blue circles) perfusion with 1 perfusion with 1Ba µM toxin. Data are mean ± S.E.M of 5 (panel d) or 3 (panel e) experiments. Inhibition µM toxin. Data are mean ± S.E.M of 5 (panel d) or 3 (panel e) experiments. Inhibition of IBa by Mb1a of IBa by Mb1a was significantly greater than for Mb1b (p = 0.008). wasToxins significantly 2017, 9, 155 greater than for Mb1b (p = 0.008). 9 of 18

Figure 8. Effect of 1 µM Mb1a and Mb1b on P. americana HVA CaV channel currents. (a) M-LVA IBa Figure 8. Effect of 1 µM Mb1a and Mb1b on P. americana HVA CaV channel currents. (a) M-LVA were elicited by 100-ms depolarizing test pulses to +20 mV from a holding potential of −90 mV. (b,c) IBa were elicited by 100-ms depolarizing test pulses to +20 mV from a holding potential of −90 mV. Representative superimposed HVA IBa showing tonic block of HVA CaV channel currents before (b,c) Representative superimposed HVA I showing tonic block of HVA Ca channel currents before (black lines) and following a 3 min perfusionBa (red lines) with (b) 1 µM Mb1aV and (c) 1 µM Mb1b. µ µ (blackDashed lines) gray and lines following represent a 3 zero min current. perfusion (d,e) (red Timecourse lines) with of inhibition (b) 1 M of Mb1anormalized and (peakc) 1 HVAM Mb1b.

DashedCaV graychannel lines currents represent by (d) zero 1 µM current. Mb1a and (d ,(e) 1 Timecourse µM Mb1b. Data of inhibition are mean ± of S.E.M, normalized n = 4. Average peak HVA

CaV channelnormalized currents peak IBa by before (d)1 (openµM Mb1acircles), and during (e) 1(redµM circles), Mb1b. and Data after are (blue mean circles)± S.E.M, perfusionn = 4.with Average 1 normalizedµM toxin. peak DataIBa arebefore mean (open± S.E.M circles), of 5 experiments during (red (panel circles), d) or 3 and experiments after (blue (panel circles) e). Inhibition perfusion of with 1 µMIBa toxin. by Mb1a Data was are significantly mean ± S.E.M greater of5 than experiments for Mb1b ( (panelp = 0.007).d) or 3 experiments (panel e). Inhibition of

IBa by Mb1a was significantly greater than for Mb1b (p = 0.007).

Toxins 2017, 9, 155 9 of 18 Toxins 2017, 9, 155 10 of 18

Figure 9. Effect of 1 µµMM Mb1aMb1a onon thethe voltage-dependencevoltage-dependence of activation of P. americanaamericana DUM neuron CaV channels. (a) Families of CaV channel currents were elicited by depolarizing test pulses to +40 mV CaV channels. (a) Families of CaV channel currents were elicited by depolarizing test pulses to +40 mV from a holding potential of –90 mV in 10-mV steps. Representative superimposed families of INa are from a holding potential of –90 mV in 10-mV steps. Representative superimposed families of INa are shown prior to to ( (bb),), and and 5 5 min min after after (c (),c ),application application of of1 µM 1 µM Mb1a. Mb1a. Cu Currentsrrents were were generated generated using using the test pulse protocol shown in panel a. (d) Normalised IBa-V relationships before (open circles) and the test pulse protocol shown in panel a.(d) Normalised IBa-V relationships before (open circles) and after (red circlescircles andand shaded)shaded) applicationapplication ofof 1 1µ µMM Mb1a Mb1a generated generated using using the the pulse pulse protocol protocol in in panel panela. Dataa. Data were were fitted fitted using using Equation Equation (2) (see (2) Materials (see Mate andrials Methods). and Methods). Currents recordedCurrents inrecorded the presence in the of presence of toxin were normalised against maximum peak IBa in controls (red solid curve) and toxin were normalised against maximum peak IBa in controls (red solid curve) and maximum peak IBa inmaximum toxin (red peak dashed IBa curve).in toxin ( e(red) Linear dashed regression curve). analysis (e) Linear of the regression data using analysis Equation of (2) the (see data Materials using Equation (2) (see Materials and Methods) revealed that inhibition of IBa by 100 nM Mb1a was and Methods) revealed that inhibition of IBa by 100 nM Mb1a was voltage-independent over the range –40voltage-independent to +10 mV. Data points over the are range mean –40± S.E.M, to +10n mV.= 4. Data points are mean ± S.E.M, n = 4.

3. Discussion

3.1. Promiscuous Pharmacology of Mb1a/1b The closest closest homologues homologues of ofMb1a Mb1a and-Mb1b and-Mb1b are the are spider the spidertoxins ω toxins-TRTX-Hg1aω-TRTX-Hg1a (71% identity) (71% identity)[25,26], β[-TRTX-Cm2a25,26], β-TRTX-Cm2a (68% identity) (68% identity) [27] and [ 27ω-TRTX-Pm1a] and ω-TRTX-Pm1a (68% identity) (68% identity) [28] (Figure [28] (Figure 10), all 10 of), allwhich of which are active are activeon vertebrate on vertebrate voltage-gated voltage-gated ion ch ionannels. channels. None of None these of to thesexins toxinshave been have tested been testedagainst against insects. insects. It is interesting It is interesting to note to note that that the the Ca CaV Vchannelchannel inhibitors inhibitors ωω-TRTX-Hg1a-TRTX-Hg1a and ω-TRTX-Pm1a-TRTX-Pm1a also also modulate Na V channelchannel currents currents by by reducing peak current and delaying fast inactivation [[28,29],28,29], akin to thethe actionaction ofof Mb1aMb1a onon BgNaBgNaVV1 (Figure 66).). The C-terminusC-terminus of ofω -TRTX-Pm1aω-TRTX-Pm1a was was found found to be to critical be critical for its activity:for its activity: a C-terminally a C-terminally truncated analoguetruncatedwas analogue almost was inactive almost on inactive both Ca Vonand both Na CaV channelsV and NaV [28 channels]. Similarly, [28]. we Similarly, found that we thefound activity that ofthe Mb1a/1b activity of was Mb1a/1b influenced was byinfluenced the C-terminal by the region,C-terminal with region, the putative with nativethe putative toxin, native Mb1a, beingtoxin, moreMb1a, potent being atmore inhibiting potent Ca atV inhibitingchannels than CaV Mb1b.channels Furthermore, than Mb1b. the Furthermore, two C-terminal the residues two C-terminal of Mb1a areresidues responsible of Mb1a for are its inhibitionresponsible of for fast its inactivation inhibition inof BgNafast inactivationV1, as this was in notBgNa observedV1, as this with was Mb1b. not Itobserved is interesting with Mb1b. that the It delayis interesting in inactivation that the appearsdelay in toinactivation be simply dueappears to the to lossbe simply and/or due addition to the ofloss a and/or methyl addition group of of the a methyl side chains group of of G the and side T, respectively,chains of G and compared T, respectively, to those compared of AS. The to ability those of AS. The ability of Mb1a to inhibit peak BgNaV1 currents and delay fast inactivation contrasts with that of the toxin PnTx4(5-5) from Phoneutria nigriventer, which enhances peak current in addition to causing delayed inactivation of BgNaV1 [30].

Toxins 2017, 9, 155 10 of 18

of Mb1a to inhibit peak BgNaV1 currents and delay fast inactivation contrasts with that of the toxin ToxinsPnTx4(5-5)Toxins 2017 2017, ,9 9, ,155 from155 Phoneutria nigriventer, which enhances peak current in addition to causing delayed1111 of of 18 18 inactivation of BgNaV1 [30]. FastFast inactivationinactivation ofof NaNaVV channelschannels isis mediatedmediated byby domaindomain IV,IV, andand spiderspider toxinstoxins interactinginteracting withwith Fast inactivation of NaV channels is mediated by domain IV, and spider toxins interacting with the the S3–S4 extracellular loop in channel domain IV (DIV) have been found to inhibit fast inactivation S3–S4the S3–S4 extracellular extracellular loop loop in channel in channel domain domain IV (DIV) IV (D haveIV) beenhave found been found to inhibit to inhibit fast inactivation fast inactivation [31,32]. [31,32]. The loss of inhibition of fast inactivation by Mb1b suggests that the C-terminal ′GT′ residues The[31,32]. loss The of inhibition loss of inhibition of fast inactivation of fast inactivation by Mb1b by suggests Mb1b suggests that the C-terminalthat the C-terminal0GT0 residues ′GT′ residues in Mb1a inin Mb1aMb1a facilitatefacilitate itsits interactioninteraction withwith thethe domaindomain IVIV voltagevoltage sensorsensor ofof BgNaBgNaVV1.1. Notably,Notably, Mb1aMb1a diddid facilitate its interaction with the domain IV voltage sensor of BgNaV1. Notably, Mb1a did not delay notnot delaydelay fastfast inactivationinactivation ofof P.P. americanaamericana NaNaVV channelchannel currents,currents, eveneven thoughthough thethe DIVDIV S3–S4S3–S4 DIVDIV looploop fast inactivation of P. americana NaV channel currents, even though the DIV S3–S4 DIV loop is identical isis identicalidentical inin thethe PP.. americanaamericana andand B.B. germanicagermanica NaNaVV channels.channels. However,However, numerousnumerous recentrecent studiesstudies in the P. americana and B. germanica NaV channels. However, numerous recent studies have revealed have revealed that the extracellular S1–S2 loops can be important sites for toxin recognition [24,33– thathave the revealed extracellular that the S1–S2 extracellular loops can S1–S2 be important loops can sites be for important toxin recognition sites for [toxin24,33 –recognition38]. A comparison [24,33– 38]. A comparison of the DIV S1–S2 region of the P. americana and B. germanica channels reveals only of38]. the A DIVcomparison S1–S2 region of the of DIV the S1–S2P. americana region andof theB. P. germanica americanachannels and B. germanica reveals only channels a single reveals difference only a single difference (K1634Q; Figure 11), which may be at least partly responsible for the ability of (K1634Q;a single difference Figure 11), (K1634Q; which may Figure be at 11), least which partly may responsible be at least for thepartly ability responsible of Mb1a tofor inhibit the ability the fast of Mb1aMb1a toto inhibitinhibit thethe fastfast inactivationinactivation ofof B.B. germanicagermanica NaNaVV channels.channels. inactivation of B. germanica NaV channels.

FigureFigure 10. 10. SequenceSequence alignment alignment of of Mb1a Mb1aMb1a and and Mb1b Mb1b withwiwithth theirtheir closestclosestclosest homologues.homologues.homologues. Amino Amino acids acids identicalidenticalidentical to toto Mb1a Mb1aMb1a are areare in inin bold boldbold and andand differences differencesdifferences areare highlightedhighlighted inin gray.gray. CysteinesCysteines areareare coloured colouredcoloured blue. blue.blue.

FigureFigure 11.11. AlignmentAlignment ofof thethe S1–S2S1–S2 regionsregions fromfrom eacheach ofof thethe fourfour domainsdomains (DI–DIV)(DI–DIV) inin thethe NaNaVV Figure 11. Alignment of the S1–S2 regions from each of the four domains (DI–DIV) in the NaV channelschannels fromfrom P.P. americanaamericana (UniProt(UniProt D0E0C1),D0E0C1), B.B. germanicagermanica (UniProt(UniProt O01307),O01307), andand H.H. armigeraarmigera channels from P. americana (UniProt D0E0C1), B. germanica (UniProt O01307), and H. armigera (deduced (deduced(deduced fromfrom thethe publishedpublished genogenome).me). TheThe S1–S2S1–S2 extracellularextracellular loopsloops fromfrom thethe fourfour domainsdomains areare from the published genome). The S1–S2 extracellular loops from the four domains are coloured colouredcoloured purplepurple (DI),(DI), redred (DII),(DII), blueblue (DIII)(DIII) andand greengreen (DIV).(DIV). DifferencesDifferences inin aminoamino acidacid sequencesequence purple (DI), red (DII), blue (DIII) and green (DIV). Differences in amino acid sequence between species between species are highlighted in grey. The boundaries of S1 and S2 are based on the recently arebetween highlighted species in are grey. highlighted The boundaries in grey. of S1The and bounda S2 areries based of onS1 the and recently S2 are determined based on the structure recently of determineddetermined structurestructure ofof thethe P.P. americanaamericana NaNaVVPaSPaS channelchannel [39].[39]. the P. americana NaVPaS channel [39].

InhibitionInhibition ofof peakpeak IINaNa withoutwithout aa shiftshift inin thethe IINaNa//VV curve,curve, asas seenseen withwith Mb1aMb1a andand Mb1b,Mb1b, maymay Inhibition of peak INa without a shift in the INa/V curve, as seen with Mb1a and Mb1b, may indicateindicate aa simplesimple porepore blockingblocking memechanismchanism ofof action.action. However,However, mostmost NaNaVV channelchannel spiderspider toxinstoxins areare indicate a simple pore blocking mechanism of action. However, most Na channel spider toxins are gatinggating modifiersmodifiers [11,40][11,40] thatthat interactinteract withwith thethe voltagevoltage sensorsensor domainsdomains [11],[11],V andand numerousnumerous spiderspider gating modifiers [11,40] that interact with the voltage sensor domains [11], and numerous spider toxins toxinstoxins suchsuch asas µ-TRTX-Hs2aµ-TRTX-Hs2a (huwentoxin(huwentoxin IV;IV; [41])[41]) andand µ-TRTX-Hd1aµ-TRTX-Hd1a [42][42] havehave beenbeen foundfound toto such as µ-TRTX-Hs2a (huwentoxin IV; [41]) and µ-TRTX-Hd1a [42] have been found to inhibit NaV inhibitinhibit NaNaVV channelschannels byby interactinginteracting withwith thethe DIIDII voltvoltageage sensorsensor withoutwithout causingcausing aa shiftshift inin thethe channels by interacting with the DII voltage sensor without causing a shift in the voltage-dependence voltage-dependencevoltage-dependence ofof channelchannel activation.activation. Therefore,Therefore, itit isis likelylikely thatthat Mb1aMb1a andand Mb1bMb1b inhibitinhibit insectinsect NaNaVV channelschannels viavia gatinggating modimodification.fication. ComparedCompared toto NaNaVV channels,channels, muchmuch lessless isis knownknown aboutabout howhow spiderspider toxinstoxins interactinteract withwith CaCaVV channelschannels [43].[43]. SpiderSpider toxinstoxins thatthat affectaffect CaCaVV channelschannels areare generallygenerally thoughtthought toto actact asas gatinggating modifiers,modifiers, althoughalthough somesome toxinstoxins suchsuch asas ωω-AGTX-Aa3a-AGTX-Aa3a andand ωω-SGTX-Sf1a-SGTX-Sf1a areare believedbelieved toto bebe porepore blockersblockers [44[44–46].–46]. Mb1aMb1a andand Mb1bMb1b bothboth inhibitinhibit peakpeak CaCaVV channelchannel currentscurrents

Toxins 2017, 9, 155 11 of 18

of channel activation. Therefore, it is likely that Mb1a and Mb1b inhibit insect NaV channels via gating modification. Compared to NaV channels, much less is known about how spider toxins interact with CaV channels [43]. Spider toxins that affect CaV channels are generally thought to act as gating modifiers, although some toxins such as ω-AGTX-Aa3a and ω-SGTX-Sf1a are believed to be pore blockers [44–46]. Mb1a and Mb1b both inhibit peak CaV channel currents without shifting the IBa/V curve, consistent with them being pore blockers. However, further experiments will be required to determine the binding sites of these two peptides on insect CaV and NaV channels.

3.2. Phyletic Selectivity of Mb1a/1b Several spider toxins have been found to paralyze or kill the dipterans L. cuprina and M. domestica, with activities ranging from 198 pmoL/g (LD50;U1-AGTX-Ta1a) to 2229 pmoL/g (PD50; µ-SGTX-Sf1a) on L. cuprina, and 77 pmoL/g (LD50; ω-HXTX-Hv1a) to 1380 pmoL/g (LD50; µ-AGTX-Aa1b) on M. domestica [47–50]. With PD50 values of 5600–5800 pmoL/g, Mb1a and Mb1b are only moderately potent against L. cuprina compared to other spider toxins that affect dipterans. Notably, Mb1a and Mb1b were inactive against H. armigera, suggesting they do not modulate the activity of H. armigera NaV or CaV channels. Since a BAC library of the H. armigera genome has been published [51,52], we used this to deduce the sequence of the H. armigera NaV channel and compared it to that of B. germanica and P. americana channels on which Mb1a and Mb1b are active (Figure 11). The S3–S4 loops are identical in all four voltage-sensor domains of the of P. americana, B. germanica and H. armigera NaV channels, indicating that these regions are not responsible for the differences in activity. However, the S1–S2 loops of all four domains of the H. armigera NaV channel contain variations in amino acid sequence compared to both cockroach species (Figure 11). Thus, Mb1a and Mb1a possibly provide additional examples of insecticidal toxins where differences in phyletic selectivity are due to taxonomic variations in the sequences of the S1–S2 loops [24,37].

4. Conclusions Mb1a and Mb1b add to the expanding repertoire of spider toxins that are active against insects. Due to a high level of homology with toxins active on vertebrates, Mb1a and Mb1b may have an activity on vertebrate NaV or CaV channels that would render them unsuitable candidates for bioinsecticide development. However, the closest vertebrate-active homolog is only 71% identical with 11 differences in amino acid sequence (Figure 10), and it is well known that even small differences in venom-peptide sequences can alter their channel and species selectivity. Even if Mb1a and Mb1b turn out to be vertebrate active, differences in their mechanism of action make them useful tools for studying insect NaV channels. Moreover, Mb1a and Mb1b contribute to the growing body of evidence derived from toxins which suggests that phyletic selectivity can be achieved by targeting the voltage-sensor domains of insect NaV channels.

5. Materials and Methods

5.1. Venom Collection Venom from a single female Monocentropus balfouri was extracted using mild electrical stimulation [53], then the venom was lyophilized and kept frozen until reconstituted and used for the experiments.

5.2. Fractionation of Crude Venom

1 mg of dried M. balfouri venom was fractionated using a Jupiter C18 analytical RP-HPLC column (25 cm × 4 mm, 5 µm, Phenomenex Australia Pty. Ltd., Sydney, Australia) connected to a Shimadzu Prominence HPLC system. The flow rate was 1 mL/min. Varying gradients of solvent A (0.1% formic acid) and solvent B (90% acetonitrile/ 0.1% formic acid) were used. Isocratic elution with 5% solvent B Toxins 2017, 9, 155 12 of 18 was used for the first 5 min, followed by 5–20% solvent B over 5 min, 20–40% solvent B over 40 min, 40–80% solvent B over 5 min, then 80% solvent B for 2 min.

5.3. Sequencing of Active Peptide N-terminal Edman sequencing, including the prior reduction and alkylation of Mb1a/b with TCEP/iodoethanol, was performed by the Australian Proteome Analysis Facility (APAF). The sample (30 µL) was loaded onto a precycled, biobrene-treated discs and subjected to 40 cycles of Edman N-terminal sequencing. The analysis was run using the pulsed liquid method with a modified begin cycle to include extra washes. Automated Edman degradation was carried out using an Applied Biosystems 494 Procise Protein Sequencing System. Performance of the sequencer was assessed routinely with 10 pmoL β-lactoglobulin standard.

5.4. Recombinant Production of Mb1a and Mb1b Recombinant Mb1a and Mb1b were produced by expression in the periplasm of Escherichia coli using a previously described protocol [21]. Briefly, a synthetic gene encoding Mb1a and Mb1b was cloned into a variant of the pLic-MBP expression vector by GeneArt (Invitrogen, Regensburg, Germany). Codons were optimized for expression in E. coli. The modified pLic-MBP vector encodes a MalE signal sequence for periplasmic export of the fusion protein, a His6 tag for affinity purification, a maltose-binding protein (MBP) fusion tag to aid solubility, and a tobacco etch virus (TEV) protease recognition site directly preceding the toxin sequence. The plasmids encoding Mb1a and Mb1b were transformed via heat shock into E. coli strain BL21(λDE3) for production of recombinant toxin. Protein expression and purification were performed as previously described with minor modifications. In summary, a 50 mL overnight starter culture grown in Luria-Bertani broth at 37 ◦C with shaking (~220 rpm) was used to inoculate a 2 liter culture the following day. After the culture reached an OD600 of ~1.0, toxin gene expression was induced with 500 µM IPTG. Cells were grown at 30 ◦C overnight before centrifugation for 15 min at 10,500 g to obtain the cell pellet. The pellet was then reconstituted in a minimal amount of TN buffer (20 mM Tris, 250 mM NaCl, pH 8), then cells were lysed at 27 kpsi using a constant-pressure cell disruptor (TS Series Cell Disrupter, ConstantSystems Ltd., Daventry, UK). The cell lysate was passed over a column containing Ni-NTA Superflow resin (Qiagen Pty Ltd., Chadstone, VIC, Australia) and weakly bound proteins were eluted with 15 mM imidazole in TN buffer. The MBP-toxin fusion protein was then eluted with 300 mM imidazole in TN buffer, after which the eluate was spun in a 30 kDa cut-off centrifugal filter to remove the imidazole and concentrate the protein to 5 mL. The resulting solution was diluted to 10 mL with TN buffer, then the fusion protein was cleaved overnight at room temperature using ~100 µg TEV protease in 0.6 mM and 0.4 mM reduced and oxidised glutathione, respectively [21]. 0.1% TFA was then added to precipitate the cleaved His6-MBP protein; the sample was then centrifuged at 17,000 g to pellet the precipitant. The supernatant was filtered with a 0.45 µm syringe filter (EMD Millipore, Billerica, MA, USA) before purification of recombinant Mb1a or Mb1b using semi-preparative RP-HPLC (Phenomenex Jupiter C4 column; 250 × 10 mm, 10 µm; flow rate 5 mL/min). Recombinant toxin was eluted using a gradient of 10–45% solvent B (0.043% TFA in 90% acetonitrile) in solvent A (0.05% TFA in water) over 30 min. Further purifications were performed using an Agilent C18 column (ZORBAX 300SB; 250 × 9.4 mm, 5 µm; flow rate 3 mL/min) with appropriate solvent gradients.

5.5. Insecticidal Assays

5.5.1. Sheep Blowflies and House Flies Mb1a and Mb1b were dissolved in insect saline (see [54] for composition) and injected into the ventro-lateral thoracic region of adult sheep blowflies (Lucilia cuprina) or house flies (Musca domestica) according to methods described previously [23]. Briefly, a maximum of 2 µL was injected per fly using a 1.0 mL Terumo Insulin syringe with a fixed 29-gauge needle fitted to an Arnold hand micro-applicator Toxins 2017, 9, 155 13 of 18

(Burkard Manufacturing Co. Ltd., Rickmansworth, UK). Flies were individually housed in 2 mL tubes and paralytic effects determined 0.5, 1, 2 and 24 h after injection. For L. cuprina, three replicates were performed and for each replicate six doses of Mb1a or Mb1b (n = 10 flies per dose) was used, along with appropriate controls (insect saline; n = 20 flies each). Dose-response data were fitted using the sigmoidal dose-response (variable slope) function in Prism 6. For M. domestica, a single replicate (n = 5) of one dose of Mb1a or Mb1b was injected (Mb1a: 15 nmoL/g, Mb1b: 11.7 nmoL/g). PD50 and LD50 values were calculated as previously described [55]. We defined “complete paralyzed” flies as those that could move their appendages (legs and proboscis), but were unable to fly or drag their body forward when placed onto a flat bench. At earlier times post-injection, or at lower doses, we observed “incomplete paralyzed” flies that had uncoordinated movement (e.g., due to some extremities twitching or being paralyzed) but which were nevertheless able to move their body along a flat bench even though they could not fly.

5.5.2. Cotton Bollworms Cotton bollworms (i.e., Helicoverpa armigera larvae) were obtained from AgBiTech Pty Ltd. (Glenvale, QLD, Australia). Toxins were injected into the lateral thoracic region and larvae observed for paralytic or lethal effects at 0.5, 1, 3, 24, 48, and 72 h after injection. Larvae were kept in standard 6-well plates and fed on artificial diet (AgBiTech, Clifford Gardens, Australia). Larval weight was measured 24, 48 and 72 h after injection.

5.6. Patch Clamp Electrophysiology Using P. americana Neurons DUM neurons were isolated from unsexed adult P. americana as described previously [56]. Briefly, terminal abdominal ganglia were removed and placed in normal insect saline (NIS) containing (in mM): NaCl 180, KCl 3.1, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) 10, D-glucose 20. Ganglia were then incubated in 1 mg/mL collagenase (type IA) for 40 min at 29 ◦C, washed twice in NIS, resuspended in NIS supplemented with 4 mM MgCl2, 5 mM CaCl2, 5% foetal bovine serum and 1% penicillin/streptomycin (NIS+; Life Technologies, Mulgrave, VIC, Australia), then triturated through a fire-polished Pasteur pipette. The resultant cell suspension was then distributed onto 12-mm diameter glass coverslips pre-coated with 2 mg/mL concanavalin A (type IV). DUM neurons were maintained in NIS+ at 29 ◦C and 100% humidity. Ionic currents were recorded from DUM neurons in voltage-clamp mode using the whole-cell patch-clamp technique employing version 10.2 of the pCLAMP data acquisition system (Molecular Devices, Sunnyvale, CA, USA). Data were filtered at 10 kHz with a low-pass Bessel filter with leakage and capacitive currents subtracted using P–P/4 procedures. Digital sampling rates were set between 15 kHz and 25 kHz depending on the length of the protocol. Single-use 0.8–1.5 MΩ electrodes were pulled from borosilicate glass and fire-polished prior to current recordings. Liquid junction potentials were calculated using JPCALC, and all data were compensated for these values. Cells were bathed in external solution through a continuous pressurized perfusion system at 1 mL/min, while toxin solutions were introduced via a wide-bore gravity-fed perfusion needle at ~80 µL/min (Automate Scientific, San Francisco, CA, USA). All experiments were performed at ambient temperature (20–23 ◦C). To record sodium currents (INa), the external bath solution contained (in mM): NaCl 80, CsCl 5, CaCl2 1.8, tetraethylammonium chloride 50, 4-aminopyridine 5, HEPES 10, NiCl2 0.1, CdCl2 1, adjusted to pH 7.4 with 1 M NaOH. The pipette solution contained (in mM): NaCl 34, CsF 135, MgCl2 1, HEPES 10, 0 0 ethylene glycol-bis(2-aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA) 5, and ATP-Na2 3, adjusted to pH 7.4 with 1 M CsOH. Note that in these and other electrophysiology experiments, peptides were tested at concentrations that yielded 50–75% inhibition of currents. Two subtypes of CaV channel currents have been observed in P. americana DUM neurons: high-voltage-activated (HVA) and mid/low-voltage-activated (M-LVA) CaV channel currents [23,57]. Notwithstanding differences in the kinetic and pharmacological properties of M-LVA and HVA CaV channels, there is no mechanism for recording one current in isolation from the other, as no peptide or Toxins 2017, 9, 155 14 of 18 small molecule inhibitors have been developed that block one type of current and not the other [58]. Therefore, depolarizing voltage command pulses to different levels were used to investigate the actions of Mb1a and Mb1b on M-LVA and HVA CaV channels [23,57]. CaV channel currents were evoked by 100-ms depolarising pulses from a membrane holding potential (Vh) of –90 mV to potentials at 7-s intervals to −20 mV for generation of predominantly M-LVA CaV channel currents and to +20 mV to evoke mainly HVA CaV channel currents [57]. Previous studies revealed significant rundown of CaV currents when calcium was used as a charge carrier, but much less when barium was used instead [58]; thus, we replaced CaCl2 with BaCl2 in all CaV channel experiments. The external bath solution for barium current (IBa) recordings contained (in mM): sodium acetate 140, TEA-bromide 30, BaCl2 3, HEPES 10, adjusted to pH 7.4 with 1 M TEA-OH. The external solution also contained 300 nM tetrodotoxin to block NaV channels. Pipette solutions contained (in mM): sodium acetate 10, CsCl 110, TEA-bromide 50, ATP-Na2 2, CaCl2 0.5, EGTA 10, HEPES 10, adjusted to pH 7.4 with 1 M CsOH. To eliminate any influence of differences in osmotic pressure, all internal and external solutions were adjusted to 400 ± 5 mOsmol/L with sucrose. Experiments were rejected if leak currents exceeded 1 nA or if currents showed signs of poor space clamping. Peak current amplitude was analyzed offline using AxoGraph X v1.5.3 (Molecular Devices, Sunnyvale, CA, USA). All curve-fitting was performed using Prism 6 (GraphPad Software Inc., San Diefo, CA, USA). All data are mean ± SEM of n independent experiments. On-rates (τon) were calculated using the following Equation (1):

Y = Y0 + (A − Y0) × (1 − exp(−K × t)) (1) where Y0 is the maximal peak INa, A is the minimum peak INa, K is the rate constant and t is time. The on-rate (τon) was subsequently determined from the inverse of the rate constant (K). The data for voltage-dependence of channel activation, for all channel types, were fitted using the following current-voltage (I/V) curve formula Equation (2):

  1  I = gmax 1 − (V − Vrev) (2) 1 + exp[(V − V1/2)/s] where I is the amplitude of the current at a given test potential V, gmax is the maximal conductance, V1/2 is the voltage at half-maximal activation, s is the slope factor and Vrev is the apparent reversal potential. Differences in current inhibition between toxins was analysed using one-way ANOVA, with a probability of p < 0.05 being considered statistically significant.

5.7. Two–Electrode Voltage-Clamp Electrophysiology

BgNaV1 [59] cRNA was synthesized using T7 polymerase (mMessage mMachine kit, Life Technologies, Carlsbad, CA, USA) after linearizing the fully-sequenced DNA with NotI. BgNaV1 was expressed in Xenopus oocytes together with the TipE subunit [60] (1:5 molar ratio), and studied following a 1-day incubation after cRNA injection. Cells were incubated at 17 ◦C in ND96 consisting of (in mM) NaCl 96, KCl 2, HEPES 5, MgCl2 1, CaCl2 1.8, pH 7.6 with NaOH supplemented 50 µg/mL gentamycin, then studied using two-electrode voltage-clamp recording techniques (OC-725C, Warner Instruments, Hamden, CT, USA) with a 150-µL recording chamber. Data were filtered at 4 kHz and digitized at 20 kHz using pClamp software (Molecular Devices, Sunnyvale, CA, USA). Microelectrode resistances were 0.5–1 MΩ when filled with 3 M KCl. The external recording solution consisted of ND96. All experiments were performed at room temperature (~22 ◦C). Leak and background conductances, identified by blocking the channel with tetrodotoxin, were subtracted for all currents shown. Voltage-activation relationships were obtained by measuring steady-state currents and calculating conductance. Protocols for other measurements are described in the figure legends. After addition of toxin to the recording chamber, the equilibration between the toxin and the channel was monitored using weak depolarizations elicited at 5-s intervals. For all channels, voltage-activation Toxins 2017, 9, 155 15 of 18 relationships were recorded in the absence and presence of toxin. Off-line data analysis was performed using Clampfit 10 (Molecular Devices, Sunnyvale, CA, USA) and Origin 8.0 (Originlab, Northampton, MA, USA).

Acknowledgments: The authors thank the Australian Grains Research & Development Corporation (GRDC; Project UQ00048, Kingston, Australia) for financial support, Arnd Schlosser (Bremen, Germany) for venom extraction, Michael Scheller (Mainz, Germany) for providing the spider, Geoff Brown (Department of Agriculture, Fisheries and Forestry, Brisbane, Australia) for supply of blowflies, and Ke Dong (Michigan State University, East Lansing, MI, USA) for sharing the BgNaV1/TipE clones. This research was facilitated by access to the Australian Proteome Analysis Facility (Sydney, Australia), which is supported under the Australian Government’s National Collaborative Research Infrastructure Strategy. G.F.K. is supported by a Principal Research Fellowship (APP1044414) from the Australian National Health & Medical Research Council (Canberra, Australia). Author Contributions: Jennifer J. Smith, Volker Herzig, Maria P. Ikonomopoulou, Sławomir Dziemborowicz, Frank Bosmans and Glenn F. King conceived and designed the experiments; Jennifer J. Smith, Volker Herzig, Maria P. Ikonomopoulou, Sławomir Dziemborowicz and Frank Bosmans performed the experiments; Jennifer J. Smith, Volker Herzig, Sławomir Dziemborowicz, Frank Bosmans and Graham M. Nicholson analyzed the data; Frank Bosmans, Graham M. Nicholson and Glenn F. King contributed reagents/materials/analysis tools; Jennifer J. Smith, Volker Herzig, Maria P. Ikonomopoulou, Sławomir Dziemborowicz, Frank Bosmans, Graham M. Nicholson and Glenn F. King wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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