Figure 1.
Isolation of an orally active insect toxin from spider venom.
(A) RP-HPLC chromatogram showing fractionation of crude venom from the Australian tarantula Selentypus plumipes. An asterisk highlights the fraction that displayed oral termiticidal activity. (B) Chromatogram from cation exchange fractionation of the active RP-HPLC fraction shown in (A). An asterisk highlights the fraction with oral termiticidal activity. (C). Insecticidal assay of native OAIP-1. The peptide was injected into larvae of the mealworm beetle (Tenebrio molitor) at a dose of 3 pmol/g or fed to termites (Coptotermes acinaciformis) at a dose of 350 nmol/g. Each column represents the mean ±SD of three replicates of 10 insects.
Figure 2.
(A) Sequence of transcript encoding the OAIP-1 prepropeptide precursor isolated from an S. plumipes venom-gland cDNA library. The 3′ and 5′ untranslated region (UTR), signal sequence, propeptide region, and mature toxin are labeled. The “GR” dipeptide sequence at the end of the mature toxin sequence is labeled AS (amidation signal) as it is a signal for C-terminal amidation. (B) Amino acid sequence of OAIP-1 prepropeptide precursor obtained from in silico translation of the cDNA sequence shown in panel (A). (C) Comparison of the amino acid sequence of the mature OAIP-1 toxin obtained from in silico translation of the venom-gland prepropeptide transcript with the N-terminal sequences obtained from Edman degradation of the native toxin at the APAF and APC protein sequencing facilities. (D) Alignment of OAIP-1 primary structure with the two closest hits obtained from a BLAST search against the ArachnoServer database. Identical residues are highlighted by white letters on a black background, while residues that are identical in two of the three sequences are shown on a gray background.
Figure 3.
Insecticidal activity of synthetic OAIP-1.
(A) Dose-response curves resulting from administration of sOAIP-1 to mealworms (larval T. molitor) via injection (▪) or feeding (□). (B) Dose-response curve resulting from feeding sOAIP-1 to cotton bollworms (larval H. armigera) (•). The calculated LD50 values are shown. (C) Mortality observed at 48 h after feeding 100 pmol imidacloprid, 100 pmol sOAIP-1, or a 50∶50 mixture of these compounds into H. armigera. Each data point is the mean ±SEM of three replicates of 10 individuals.
Figure 4.
Mortality of T. molitor larvae (mealworms) determined at 48 h after insects were simultaneously offered toxin-treated and untreated agar. The toxin concentration in the treated agar ranged from 1 mmol to 1 pmol, and the data represent the mean and SEM of three replicates of 10 individuals for each dose. The data correlate well with the oral toxicity of sOAIP-1 in a non-choice test (Fig. 3A); the mortality at the same dose in the choice test is approximately the same as that observed in the non-choice test. Mortality at all but the lowest two doses (10 and 1 pmol) was significantly greater than the untreated agar control (P<0.01). Columns represent the mean ±SD for three replicates of 10 insects for each dose.
Figure 5.
Phenotypic response of insects to OAIP-1.
T. molitor larvae (mealworms) were monitored 5, 30, and 60 min following injection of sOAIP-1 (horizontally striped, grey, and black bars, respectively). The response was scored relative to the control as excitatory (prolonged muscle spasms), excitation to the point of paralysis (spasms so severe the insect was unable to move independently), or death/moribund (dead or, if alive, the insect was unable to right itself when turned on its back). See Table S2 for details for the scoring matrix. No dose produced a depressed state at any of the time points. Columns represent the mean ±SEM of three replicates of 10 insects for each dose.
Figure 6.
(A) Thermal stability of sOAIP-1 over 7 days. Note that the data obtained at −20°C, 22°C, and 30°C overlap completely since OAIP-1 is 100% intact at these temperatures at all time points. OAIP-1 only degrades at temperatures of 37°C and higher. (B) Stability of sOAIP-1 over a range of different pH conditions. The toxin is least stable at alkaline pH. (C) A series of RP-HPLC chromatograms showing fractionation of undiluted hemolymph from H. armigera larvae (cotton bollworms) at various times following addition of 30 µg sOAIP-1 (highlighted in the solid box). Immediately before RP-HPLC fractionation, 30 µg of ω-HXTX-Hv1a (dashed box) was added to each sample for the purposes of quantification. In all experiments shown in panels A–C, intact OAIP-1 was identified using mass spectrometry.
Figure 7.
(A) Stereoview of the ensemble of 20 OAIP-1 structures. The three disulfide bonds and the N- and C-termini are labeled. (B) Schematic (Richardson) representation of OAIP-1. β-strands are colored blue and disulfide bonds are shown as red tubes. The four intercystine loops (loops 1–4) are labeled. (C) Overlay of OAIP-1 (blue) and the orthologous toxin U1-TRTX-Pc1a (orange). The intercystine loops and termini are labeled. (D) Overlay of OAIP-1 (blue) and the orthologous toxin U1-TRTX-Pc1a (orange). Residues Y11 and Y26 in U1-TRTX-Pc1a (red tubes) interact in such a way that loops 2 and 4 are brought into close proximity. The equivalent residues in OAIP-1, P10 and Y27 (light blue tubes), do not interact and consequently loops 2 and 4 are further apart.
Table 1.
Structural statistics for the ensemble of OAIP-1 structures1.
Figure 8.
Structural homologues of OAIP-1.
Alignment of the structure of OAIP-1 (orange) with the top six structural homologues (all shown in green) as ranked by the Dali server [43]. The activity of each structural homologue is indicated, as is the Z score and RMSD of the alignment. Disulfide bonds are shown as solid tubes and the N- and C-termini are labeled.
Table 2.
Comparison of OAIP-1 with pyrethroid insecticides.