Tarlochan S. Dhadialla

Bisacylhydrazine Insecticides for Selective Pest Control

In this chapter, we review five members of a novel class of chemistry, the non-steroidal bisacylhydrazine (BAH) compounds that are true agonists of the steroidal insect moulting hormone, 20-hydroxyecdysone. Also referred to as ecdysone agonists (EAs), the five BAH compounds have been commercialized for the control of lepidopteran and coleopteran larvae. Of these, four compounds (methoxyfenozide, tebufenozide, chromafenozide, and fufenozide) are predominantly toxic to lepidopteran larvae, while the fifth compound, halofenozide, is active on both lepidopteran and coleopteran larval pests in turf. The evidence for the basis of this insect selective toxicity is reviewed. The non-steroidal EA BAH insecticidal compounds are important tools in integrated pest management and insect resistance management programmes because of their selective insect toxicity, novel mode of action, and reduced risk for eco- and mammalian toxicology. In reviewing these BAH insecticides, there is greater emphasis on methoxyfenozide, the most widely used insecticide in this class of chemistry.

An Overview of Insect Growth Disruptors; Applied Aspects

Since the initial suggestion of using insect hormones or their mimics as the third-generation insecticides, great strides have been made in the discovery of three classes of such insect growth-disrupting insecticides. We propose the use of the term “insect growth disruptors” instead of the mis-named term “insect growth regulators”. We review the commercial insecticides that mimic the mode of action of the insect growth and development hormones, the sesquiterpenoid juvenile hormones and the steroidal 20-hydroxyecdysone and the third class of insecticides that disrupt growth and development by interfering with chitin synthesis in target insect pests. In this chapter, the emphasis is on the application of these insecticides to control insect pests and development of resistance. Since the non-steroidal ecdysone agonist insecticides have been extensively reviewed in other chapters of this volume and elsewhere, only salient features of these novel insecticides are discussed.

16th International Ecdysone Workshop: July 10–14, 2006, Ghent University, Belgium

Chitin synthesis and degradation are recurrent, fundamental events of insect development. The final step of chitin anabolism is the polymerization of UDP-N-acetylglucosamine units which requires chitin synthase activity. We present here results on the cloning and characterization of the chitin synthase 1 (CfChS1) gene from the spruce budworm (SBW; Choristoneura fumiferana), an important North American forest pest insect. Using degenerate primers from conserved regions of other insect ChS, a CfChS1 fragment was isolated by PCR on a cDNA library made from freshly ecdysed, L6 SBW larvae. The full length of the CfChS1 cDNA was determined to be 5.3 kb by the sequencing of overlapping cDNA clones and 5-RACE experiments. The encoded enzyme is 1565 amino acid long and possesses the two catalytic domain signature sequences found in all insect ChS (EDR, position 858 to 860 and QRRW, position 895 to 898). Computer-assisted analysis also predicts the existence of 16 transmembrane helices in the amino acid sequence, implying that CfChS1 N-and C- termini share the same (extracellular) topological space. The expression of CfChS1 mRNA was closely associated with larval-larval and larval-pupal molts as well as with the formation of adult cuticle. In larvae, mRNA was generally absent during intermolt periods, but accumulated to high levels immediately after ecdysis, consistent with renewed chitin synthesis. Accumulation was also observed 24h before and after this event, and even up to 48h after ecdysis to the 6th larval instar. CfChS1 expression was restricted to the epidermis and did not accumulate in fat body or midgut tissues. Treatment of larvae with ecdysone and with the non-steroidal ecdysone agonist tebufenozide, repressed the transcription of CfChS1, within 6 to 12h of application. These results indicate that CfChS1 expression could be stimulated by the falling 20-hydroxyecdysone titers that characterize the late phase of the molting process. We are currently investigating the interplay between CfChS1 and other genes expressed during molting in the SBW.

Protein synthesis by the milk gland and fat body of the tsetse fly, Glossina pallidipes

Abstract The patterns of protein synthesis by the milk gland and the fat body of female Glossina pallidipes during the pregnancy cycle were studied by incubation with [ 35 S]methionine both in vivo and in vitro . The pattern of protein synthesis by the milk gland changed with the stage of the larva in the uterus. Very little synthesis occurred in the milk gland until the first instar larva hatched. Then four proteins (13, 16, 24 and 72 kDa) were prominently synthesized. As the larva matured, the synthesis of 19, 38, 40 and 72 kDa proteins increased, whereas that of the 13 and 24 kDa proteins decreased. Just before larviposition, only the 16 and 72 kDa proteins were still being synthesized. The milk gland secreted into the medium primarily the 13, 16, 19 and 72 kDa proteins, all of which were found in the larval gut after a 5 hr pulse of labeled methionine in vivo . During most of the pregnancy cycle protein synthesis in the fat body was low compared to that of the milk gland and only small amounts of several low molecular weight proteins (less than or equal to 16 kDa) were released into the medium. But when a large third instar larva was present in the uterus, the fat body synthesized and secreted a 72 kDa and a 15–17 kDa complex of proteins.

CHAPTER 6:The Bisacylhydrazine Insecticides

The bisacylhydrazine (BAH) insecticides are a class of green chemistry compounds very specific to the target insect pests that induce a premature molting process in the affected insects. The BAH insecticides bind to the ecdysone receptors in the target pests with a greater affinity than that observed for the natural hormone, 20-hydroxyecdysone. This confers a greater level of selectivity towards most non-target organisms present in the ecosystems where the BAH insecticides are used. The BAH insecticides act directly on immature stages of the target insects (eggs and larvae) and cause some sublethal effects, such as delayed developmental rates and reduced fecundity and fertility on the adult stage of the pests. These insecticides have very good ecotoxicological profile, having virtually no impact on most non target organisms including beneficial insects and pollinators, (bees, predators and parasitoids), birds, fish and terrestrial invertebrates. The BAH insecticides also have low-toxicity to mammals making them reduced risk materials for humans handling the products. The most widely registered BAH is methoxyfenozide, which has registrations in more than 50 countries in a variety of crops ranging from vegetables to specialty uses such as forestry and tea. Due to their characteristics, the BAH insecticides can be incorporated in insecticide resistance and integrated pest management programs. Data related to these aspects of BAH insecticides are presented.

Development of stable isotope and selenomethionine labeling methods for proteins expressed in Pseudomonas fluorescens

Pseudomonas fluorescens is a robust protein expression system that is very well suited for high throughput protein expression for structural genomics studies. Since NMR spectroscopy and X-ray crystallography are both used by various investigators in structure elucidation studies, the availability of target proteins labeled with stable isotopes or selenomethionine is essential for the determination of protein structures. A completely defined medium for the expression and stable isotope labeling of proteins in P. fluorescens has been developed. The expression level of Bacillus thuringiensis Cry34 in the modified medium is comparable to that obtained in the original medium. In addition, more than 95% incorporation of 15N was obtained in Cry34 using 15N ammonium sulfate and the quality of the protein, as assessed by NMR analysis, is comparable to that made using commercial medium. High levels of selenomethionine (SeMet) incorporation in the Xenorhabdus nematophilus insecticidal protein XptA2 were also obtained in P. fluorescens using the defined medium, allowing development of a method for obtaining highly purified XptA2. The following observations were made when inhibitors of endogenous methionine biosynthesis were used in P. fluorescens culture when SeMet was substituted in XptA2: (I) there is little inhibition of cell growth or recombinant XptA2 expression in the presence of SeMet concentrations up to 300 mg/L in cell culture, (II) there was greater than 95% SeMet incorporation ratio in recombinant SeMet-labeled XptA2 (SeMet-XptA2) and the incorporation ratio is consistent and reproducible and (III) finally, purified SeMet-XptA2 possesses similar protein structure and insecticidal activity relative to the unlabeled counterpart XptA2 as shown by bioassay and differential scanning calorimetric analysis. The high SeMet incorporation should provide high accuracy and resolution in XptA2 phase determination by multiwavelength anomalous diffraction (MAD), indicating that P. fluorescens is an excellent expression host to produce SeMet-labeled proteins for structural study.