Insect Toxin Research Articles

Baculovirus-mediated expression of a Chinese scorpion neurotoxin improves insecticidal efficacy

An Buthus martensii Karsch Insect Toxin (BmK IT) gene was inserted into the genome of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to construct a recombinant baculovirus, AcMNPV-BmK IT. The expression of BmK IT was confirmed using RT-PCR, dot blot and SDS-PAGE analysis. Dose-lethal time responses to Spodoptera exigua larvae were compared between wild-type baculovirus AcMNPV and recombinant virus AcMNPV-BmK IT. At the concentration of 1 × 107 PlBs/mL, the median lethal time of recombinant baculovirus (LT50 = 73.6 h) on third instar S. exigua larvae showed an improvement of 13.2% over the efficacy of wild type virus (LT50 = 84.8 h) during a 192 h infection.

Ecological mechanisms and prospects for utilization of toxins from parasitic hymenopterans

Insects in the order Hymenoptera defend themselves, attack prey and regulate hosts using toxins that are effective in small quantities. In this study, advances in the researches on parasitic hymenopteran toxins are summarized in terms of the production, categories, components, properties, ecological functions and mechanisms. The glands that produce venoms derive from the ectoderm tissue and evolve from the accessory glands of the reproductive system. Venoms are excreted by the poison gland or acid gland of mature female wasps and stored in reservoirs. The components of insect toxins are very complicated, and hymenopteran venoms contain alkanes, alcohols, aldehydes, ketones, organic acids, esters, lactones, proteins, polypeptides, enzymes, amines and other compounds. Toxins of parasitic hymenoptera play an important adaptive role. They can increase the probability of successful oviposition by paralyzing hosts, enhancing offspring survival by inhibiting host development and immunoreaction, and improving the nutrition available for their progeny by disturbing the hosts’ physiological response. Venoms of the ectoparasitoids often lead to arrested development, permanent paralysis and even death of hosts. These toxins are usually broad-spectrum and act on the central nervous system or at the neuro-muscular junction. While most endoparasitoids are koinobionts, these parasitoids can regulate the physiology and development of the hosts, but no longer paralyze the hosts permanently. Also, they kill the hosts in a concealed but safe position after the hosts cocoon or build their pupal cells. Venoms of koinobiont parasitoids can contain polydnaviruses (PDV) that regulate the growth and development of the hosts by inhibiting the immune system and influencing the metamorphosis of hosts. Thus, PDVs are commensal and mutualistic, but non-pathogenic, with parasitoids at the molecular level. Promising prospects for the utilization of insect toxins, especially as medicines or specific bioinsecticides, are discussed. Because insect toxins are mixtures of complex ingredients and are usually produced in small quantities, isolation and purification of all the ingredients with bioactivity are needed for biochemical and toxicological research and for practical application.

Effect of location of the His-tag on the production of soluble and functional Buthus martensii Karsch insect toxin

The low yield and poor folding efficiency in vivo of soluble and active recombinant cysteine-rich proteins expressed in Escherichia coli are a major challenge for large-scale protein production and purification. Expression vectors containing Buthus martensii Karsch insect toxin (BmK IT) fused to the C terminus of the intein Ssp DnaB were constructed in an attempt to overcome this problem. Following purification and intein self-cleavage, the fusion protein His6-intein-IT produced insoluble BmK IT, while intein-IT-His6 generated soluble and properly folded BmK IT. This result indicated that the positioning of the His6 tag has a key role in the production of soluble and functional BmK IT.

Colour biases are more than a question of taste

Unpalatable insects often advertise their defences to predators using conspicuous colours, such as red and yellow. Perhaps not surprisingly, birds show unlearned biases against warningly coloured food. These biases are particularly evident when other components of insect warning displays, such as sounds and odours, are also present. Quinine, a bitter-tasting toxic chemical, can also enhance unlearned biases against red and yellow prey in naive birds. However, whether this behaviour is performed specifically in response to quinine (which is chemically similar to many insect toxins) or can be elicited by other bitter-tasting chemicals is not known. The aim of our experiments was twofold. First, we investigated whether Bitrex, a bitter-tasting, nontoxic, man-made chemical, elicits colour biases similar to those elicited by quinine. Second, since avoidance learning can be affected by the number of bitter chemicals present in a prey population, we investigated whether the presence of both quinine and Bitrex enhanced unlearned biases against red crumbs compared to either chemical alone. We found that only quinine elicited attack biases against red prey, and there was no evidence that quinine and Bitrex in combination produced a stronger bias against red crumbs than quinine alone. These results indicate that colour biases incited by defence chemicals are chemical specific and may occur only in response to natural or toxic chemicals.

Transgenic cotton expressing synthesized scorpion insect toxin AaHIT gene confers enhanced resistance to cotton bollworm (Heliothis armigera) larvae

A synthetic scorpion Hector Insect Toxin (AaHIT) gene, under the control of a CaMV35S promoter, was cloned into cotton via Agrobacterium tumefaciens-mediated transformation. Southern blot analyses indicated that integration of the transgene varied from one to more than three estimated copies per genome; seven homozygous transgenic lines with one copy of the T-DNA insert were then selected by PCR and Southern blot analysis. AaHIT expression was from 0.02 to 0.43% of total soluble protein determined by western blot. These homozygous transgenic lines killed larvae of cotton bollworm (Heliothis armigera) by 44-98%. The AaHIT gene could used therefore an alternative to Bt toxin and proteinase inhibitor genes for producing transgenic cotton crops with effective control of bollworm.

Bacillus thuringiensis Vip3 mutant proteins: Insecticidal activity and trypsin sensitivity

Vip3 is a novel insect toxin isolated from Bacillus thuringiensis (Bt), and could be used as an alternative toxin for Bt δ-endotoxins for transgenic insect control. Vip3 mutants with deletion, addition or mutations at the very end of the C-terminus were generated. The deletion and addition of a few amino acid residues at the C-terminus totally abolished the insecticidal activity. The mutation of the last two residues from IK to LG also resulted in the total loss of its insecticidal activity; however, the mutation from IK to LR increased its activity substantially against beet armyworm. Interestingly, all the inactive mutants were found to be highly sensitive to trypsin digestion, while the active mutant generated a trypsin-resistant polypeptide of 62-kDa upon trypsin digestion. However, this 62-kDa polypeptide expressed in E. coli from the 5' end truncated vip3 gene was biologically inactive and sensitive to trypsin digestion. Thus, the N-terminal part of the protein is required to form the 62-kDa trypsin resistant core. This study suggested that the 62-kDa peptide core could be a hallmark of active insecticidal Vip3 proteins.

Identification of insect-selective and mammal-selective toxins from Parabuthus leiosoma venom

Venoms were collected from two scorpion species: Parabuthus leiosoma and Parabuthus pallidus from Kenya. Subcutaneous injection and oral toxicity tests of crude and pure fractions of scorpion venoms were done in Mus musculus (mice), Chilo partellus and Busseola fusca. The highest activity against C. partellus was found in P. leiosoma venom (LC 50 0.689 mg/50 mg body weight). Bioassay-guided purification by a combination of cation-exchange (CE) and reverse-phase high-performance liquid chromatography (RP-HPLC) led to the isolation of three toxic peptides. A lepidopteran-selective toxin ( P. leiosoma insect toxin, Plit) was isolated, and the partial N-terminal amino acid sequence (-KDGYPVDNANCKYE-) plus the molecular weight (6688.5 Da) determined. A peptide with significant insect toxicity coupled with mild effects on mice ( P. leiosoma toxin, Plt) was isolated, and the partial N-terminal amino acid sequence (-LCEKFKVQRLVELNCVD-) plus the molecular weight (6742.5 Da) was determined. Another toxin with anti-mammalian activity ( P. leisoma mammal-selective toxin, Plmt), and N-terminal partial amino acid sequence of ADVPGNYPLDKNGNRYY- plus a molecular weight of 7145.5 Da was also isolated. Comparison of the partial N-terminal amino acid sequences with other toxins revealed that Plit shows high homology to other known insect toxins. Similarly, Plmt shows high homology with several birtoxin-like anti-mammalian toxins. Plt does not exhibit homology with any known scorpion toxin with combined anti-insect and anti-mammalian activity.

Phenylamides in plants

Phenylamides, secondary metabolites, conjugates of aliphatic polyamines or arylmonoamines and hydroxycinnamic acids, combining the properties of both parent compounds, are a subject of increasing interest, as follows from their ubiquitous distribution and multiplicity of functions. In this paper the occurrence, distribution, localization, metabolism, as well as chemical structure and properties of phenylamides underlying their physiological functions are briefly reviewed, with an emphasis on their involvement in the developmental and stress-defense phenomena of plants. Basic knowledge on phenylamides is supplemented with recent data pointing to the structural similarity of plant phenylamides with those found in insect toxins. This finding challenges the view of phenylamides as plant-specific molecules and suggests their role as a common defense tool in various organisms.

Distribution of the endophytic fungus, Neotyphodium occultans in naturalized Italian ryegrass in western Japan and its production of bioactive alkaloids known to repel insect pests

AbstractEndophytic fungi belonging to the genus Neotyphodium that form mutualistic associations with grasses of the subfamily Pooideae are ecologically and agriculturally important, producing mycotoxins that affect grazing animals and some insect pests. After our first report of naturalized Italian ryegrasses (Lolium multiflorum, annual ryegrass) infected with a Neotyphodium endophyte in Japan, the identity of this endophyte, the nature of its association with host grasses and its distribution, as well as the possible presence of toxic alkaloids have been of interest. To answer these questions we conducted surveys covering the western part of Japan to determine the distribution of infected Italian ryegrasses. Further, to identify the endophyte we used microscopy to determine its distribution in infected grasses and we applied DNA analysis using the ribosomal RNA gene (rDNA) to compare strains of this endophyte obtained from different geographic locations. Finally, we examined seed obtained from endophyte‐infected Italian ryegrasses using high‐performance liquid chromatograph (HPLC) and gas chromatograph. These studies indicated that naturalized Italian ryegrass in all three islands surveyed; Honshu, Kyushu and Shikoku, was frequently infected with N. occultans. N‐formyl loline, an insect toxin, and peramine, a feeding deterrent were detected in the infected seed samples, but neither ergovaline nor lolitrem B (causal agents of grazing animal toxicity) were detected. These chemical analyses indicated that N. occultans infection of Italian ryegrasses may provide some influence on the insect fauna of the area through the chemicals active against insects, but was unlikely to be toxic to grazing animals.

Efficient In Vitro Refolding and Characterization of a New Peptide from the Scorpion Buthotus saulcyi Venom Produced in Escherichia coli

The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying the insect sodium channel gating, as well as being relevant to several other applications. In order to carry out structure/activity studies, the functional expression of such polypeptides is required. In the work reported here, the cDNA of a new peptide from the venom of the scorpion Buthotus saulcyi was cloned and sequenced. It codes for a 64 residues peptide (BsaulI) with 8 highly-conserved cysteines. This peptide shares high sequence similarity with depressant insect toxins of other scorpion species. Large amounts of insoluble BsaulI protein were expressed in Escherichia coli. Purification of this peptide was carried out under denaturing conditions. Renaturation was performed by pulsed dilution of the denatured BsaulI in the refolding buffer. Production of refolded Bsaul1, however, is approximately an order of magnitude higher than that obtained with similar scorpion depressant toxins. Intrinsic fluorescence, far-UV circular dichroism spectra and biological activity assays indicate that the peptide adopts a folded structure.

CDNA Cloning, Sequence Analysis and Molecular Modeling of a New Peptide from the Scorpion Buthotus saulcyi Venom

In this study, the cDNA of a new peptide from the venom of the scorpion, Buthotus saulcyi, was cloned and sequenced. It codes for a 64 residues peptide (Bsaul1) which shares high sequence similarity with depressant insect toxins of scorpions. The differences between them mainly appear in the loop1 which connects the beta-strand1 to the alpha-helix and seems to be functionally important in long chain scorpion neurotoxins. This loop is three amino acids longer in Bsaul1 compared to other depressant toxins. A comparative amino acid sequence analysis done on Bsaul1 and some of alpha-, beta-, excitatory and depressant toxins of scorpions showed that Bsaul1 contains all the residues which are highly conserved among long chain scorpion neurotoxins. Structural model of Bsaul1 was generated using Ts1 (a beta-toxin that competes with the depressant insect toxins for binding to Na(+) channels) as template. According to the molecular model of Bsaul1, the folding of the polypeptide chain is being composed of an anti-parallel three-stranded beta-sheet and a stretch of alpha- helix, tightly bound by a set of four disulfide bridges. A striking similarity in the spatial arrangement of some critical residues was shown by superposition of the backbone conformation of Bsaul1 and Ts1.

A new toxin from the sea anemone Condylactis gigantea with effect on sodium channel inactivation

A new peptide toxin exhibiting a molecular weight of 5043 Da (av.) and comprising 47 amino acid residues was isolated from the sea anemone Condylactis gigantea. Purification of the peptide was achieved by a multistep chromatographic procedure monitoring its strong paralytic activity on crustacea (LD 50 approx. 1 μg/kg). Complete sequence analysis of the toxic peptide revealed the isolation of a new member of type I sea anemone sodium channel toxins containing the typical pattern of the six cysteine residues. From 11 kg of wet starting material, approximately 1 g of the peptide toxin was isolated. The physiological action of the new toxin from C. gigantea CgNa was investigated on sodium currents of rat dorsal root ganglion neurons in culture using whole-cell patch clamp technique ( n = 60 ). Under current clamp condition (CgNa) increased action potential duration. This effect is due to slowing down of the TTX-S sodium current inactivation, without modifying the activation process. CgNa prolonged the cardiac action potential duration and enhanced contractile force albeit at 100-fold higher concentrations than the Anemonia sulcata toxin ATXII. The action on sodium channel inactivation and on cardiac excitation-contraction coupling resemble previous results with compounds obtained from this and other sea anemones [Shapiro, B.I., 1968. Purification of a toxin from tentacles of the anemone C. gigantea. Toxicon 5, 253–259; Pelhate, M., Zlotkin, E., 1982. Actions of insect toxin and other toxins derived from the venom of scorpion Androtonus australis on isolated giant axons of the cockroach Periplaneta americana. J. Exp. Biol. 97, 67–77; Salgado, V., Kem, W., 1992. Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels. Toxicon 30, 1365–1381; Bruhn, T., Schaller, C., Schulze, C., Sanchez-Rodriquez, J., Dannmeier, C., Ravens, U., Heubach, J.F., Eckhardt, K., Schmidtmayer, J., Schmidt, H., Aneiros, A., Wachter, E., Béress, L., 2001. Isolation and characterization of 5 neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima. Toxicon, 39, 693–702]. Comprehensive analysis of the purified active fractions suggests that CgNa may represent the main peptide toxin of this sea anemone species.

Beyond the spore – past and future developments of Bacillus thuringiensis as a biopesticide

Formulated and sporulated cultures of Bacillus thuringiensis (Bt) are widely used as foliar sprays as part of integrated pest management strategies against insect pests of agricultural crops. Although in several cases the presence of the spore has been shown to improve the activity of the product, other Bt-based insecticides have been developed in which the spore is absent. The most notable of these are transgenic plants expressing just the insect toxin gene from the bacterium. This paper will discuss these developments, and the advantages and disadvantages of having the spore present.

Expression, Renaturation and Functional Analysis of an Excitatory Insect-Specific Toxin from Scorpion Buthus martensii Karsch

The cDNA of BmK IT-AP, an excitatory insect toxin from the scorpion Buthus martensi Karsch that has an analgesic effect on mammalian cells, was expressed in E. coli in the form of an inclusion body. Following denaturation and reduction, the recombinant protein was renatured and purified by liquid chromatography. The authenticity of the recombinant product was confirmed by bioassay and its electrophysiological effect on insect sodium channel.

Novel insect resistance in Brassica napus developed by transformation of chitinase and scorpion toxin genes

Transgenic plants with introduced pest-resistant gene offer an efficient alternative insect control. The novel insect-resistant gene combination, chitinase(chi) and BmkIT(Bmk), containing an insect-specific chitinase gene and a scorpion insect toxin gene was introduced into Brassica napus cultivar via Agrobacterium-mediated transformation. Fifty-seven regenerated plantlets with kanamycin-resistance were obtained. Transgenic plants were verified by Southern blot analysis. Enzyme-linked immunosorbent assay (ELISA) and bioassay of artificial inoculation with diamondback moth (Plutella maculipenis) (DBM) larvae indicated that some of the transgenic plants were high-level expression for both chitinase and scorpion toxin proteins and performed high resistance against the tested pest infestation. The genetic analysis of T(1) progeny confirmed that the inheritance of introduced genes followed the Mendelian rules.

PnTx4-3, a new insect toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic towards insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a class of peptides that are able to inhibit glutamate uptake in preparations of mammalian central nervous systems (CNS). In this work a new toxin called PnTx4-3 was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Edman sequencing of PnTx4-3 revealed that it was a polypeptide of 48 amino acid residues, containing 10 cysteines cross-linked by five disulfide bridges. The molecular mass measured by ES-Q-TOF mass spectrometry was 5199.49±0.64 Da, which is very close to the calculated mass from amino acid sequence (5199.99 Da). This toxin induces immediate excitatory effects when injected intrathoracically in house flies and cockroaches. Intracerebroventricular injections of 30 μg of PnTx4-3 in mice resulted in no apparent signs of intoxication. In order to make an orthologous comparison, pharmacological characterisation were carried out in rat brain synaptosomes by using [ 3H]- l-glutamate, showed that the whole PhTx4 fraction as well as the pure toxins PnTx4-3, Tx4(6-1) and Tx4(5-5) obtained of this fraction, were able to inhibit the glutamate uptake in the micromolar concentration range. PnTx4-3 inhibits the glutamate uptake in a dose dependent manner, with an IC 50 of approximately 1 μM. PnTx4-3 is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.

An improved baculovirus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin

Baculovirus occlusion bodies, large proteinaceous structures which contain virions, have recently been engineered to incorporate foreign proteins. The major constituent protein of occlusion bodies from the baculovirus Autographa californica nucleopolyhedrovirus is polyhedrin, and assembly of recombinant occlusion bodies which incorporate a foreign protein depends on an interaction between native polyhedrin and a polyhedrin–foreign protein fusion. This technology has now been applied to the generation of a recombinant baculovirus (ColorBtrus) that produces occlusion bodies incorporating the Bacillus thuringiensis (Bt) insecticidal Cry1Ac toxin protein. ColorBtrus coexpresses native polyhedrin and a fusion protein in which polyhedrin is fused to the Bt toxin, which is in turn fused to green fluorescent protein (GFP). Analysis of ColorBtrus occlusion bodies confirmed that they include both Bt toxin and GFP, yet still incorporate virions. Bioassay of ColorBtrus demonstrated that its speed of action and pathogenicity are strikingly enhanced compared to wild-type virus. ColorBtrus represents a novel, powerful biological insecticide that combines positive attributes of both Bt toxin and baculovirus based systems.

Insect toxins from spruce endophytes

Extracts of fermentation cultures of a fungal endophyte (DAOM 221611) from spruce needles have afforded the known macrocyclic antibiotic vermiculin (1), 7α,8β,11-trihydroxydrimane (2), and eight novel 13-carbon γ-lactones, namely trans-3-methyldodec-cis-6-en-4-olide (3), trans-8-hydroxy-3-methyldodec-cis-6-en-4-olide (4), trans-8-acetoxy-3-methyldodec-cis-6-en-4-olide (5), trans-9-hydroxy-3-methyl-8-oxo-dodec-trans-6-en-4-olide (6), trans-8,9-dihydroxy-3-methyldodec-cis-6-en-4-olide (7), trans-9-hydroxy-8-oxo-3-methyldodecan-4-olide (8), trans-7,9-dihydroxy-3-methyl-8-oxo-dodecan-4-olide (9), and trans-6-hydroxymethyl-3-methyl-7-oxo-undecan-4-olide (10). A known JH III metabolism product, 10,11-dihydroxyfarnesenic acid (11), was also isolated and synthesized from farnesol. Other endophyte cultures from black spruce and white spruce afforded the novel 6,7-dihydroxy-2-propyl-2,4-octadien-4-olide (16), 5,6,8-trihydroxy-4-(1'-hydroxyethyl) isocoumarin (17) plus the known sescandelin (18), sescandelin B (19), and 4-hydroxy-2-methoxyacetanilide (20). Several of the γ-lactones showed toxicity to spruce budworm (Choristoneura fumiferana Clem.) larvae and vermiculin 1 and compound 16 were toxic to spruce budworm cells.Key words: toxigenic endophytes, insect toxins, γ-lactones, isocoumarins.

Effect of signal sequence and promoter on the speed of action of a genetically modified Autographa californica nucleopolyhedrovirus expressing the scorpion toxin LqhIT2

Abstract We report on the construction and optimization of recombinant Autographa californica nucleopolyhedrovirus engineered to express the insect-selective toxin IT2 from the scorpion Leiurus quinquestriatus hebraeus . We constructed a series of viruses expressing the synthetic Lqhit2 gene with different signal sequences or controlled by different promoters. The effect of the various viruses on speed of response was assayed in Heliothis virescens larvae. In addition, the performance of the optimum recombinant viral construct was compared with similar constructs carrying the Androctonus australis hector insect toxin controlled by two different promoters. There were no significant differences in speed of response of viruses with the early hr5/ie1 the early hr5/lef3 , or the early/late hr5/39K promoter driving toxin expression. However, the choice of signal sequence resulted in significant effects. The signal sequence from the bombyxin gene of the silkworm Bombyx mori , proved nominally the best. When the signal sequences were used from the following genes, the viruses acted significantly slower: AcMNPV gp67 , a lepidopteran adipokinetic hormone, a dipteran chymotrypsin, and the homologous Lqhit2 . Finally, the signal sequences of the genes for cuticle protein II of Drosophila melanogaster and of the insect toxins of the scorpions A. australis hector and Hottentota judaicus performed very poorly. The speed of action of AcMNPV, carrying the synthetic Lqhit2 gene with the bombyxin secretion signal and driven by the hr5/ie1 promoter [Ac.LqhIT2(hr5/ie1)], was compared to that of the same virus carrying the AaIT gene under the control of the p10 promoter, or the hr5/ie1 promoter in H. virescens , Trichoplusia ni , and Spodoptera exigua larvae. All recombinant viruses elicited the response significantly faster than the common progenitor wild-type virus in all tests. The response elicited by Ac.LqhIT2(hr5/ie1) was nominally faster than that of both viruses expressing AaIT in all insects tested.

Purification of two depressant insect neurotoxins and their gene cloning from the scorpion Buthus martensi Karsch.

Insect-specific neurotoxins are important components of scorpion venoms. In this study, two toxins from the scorpion Buthus martensi Karsch (BmK) were purified. They shared high sequence homology with other depressant insect toxins and were designated BmK ITa and BmK ITb, respectively. They were able to suppress the action potential of cockroach isolated axon, which is due to a decrease in the peak sodium current. Furthermore, the effect of BmK ITb was lower than that of BmK ITa, and some of the electrophysiological characteristics of BmK ITb even resemble that of excitatory insect toxins. Their primary structures were determined by N-terminal partial sequence determination and cDNA cloning. The differences in their structures, especially the 31st residues, may result in the unique activity of BmK ITb.