Search

Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm ( Spodoptera frugiperda ) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae.

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens ) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125 -iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens . Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.

Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ∼1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ∼30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.

Photorhabdus luminescens produces several types of protein toxins, which are essential for participation in a trilateral symbiosis with nematodes and insects. The nematodes, carrying the bacteria, invade insect larvae and release the bacteria, which kill the insects with their toxins. Recently, the molecular mechanisms of the toxin complexes PTC3 and PTC5 have been elucidated. The biologically active components of the toxin complexes are ADP-ribosyltransferases, which modify actin and Rho GTPases, respectively. The actions of the toxins are described and compared with other bacterial protein toxins acting on the cytoskeleton.

Topical laboratory selection of tobacco budworm larvae, Heliothis virescens , with technical spinosad for multiple generations resulted in larvae 1068-fold resistant to topical applications of the insecticide and 316.6-fold resistant to insecticide treated diet as compared to the parental strain. The penetration of 2′-O-methyl[ 14 C]spinosyn A across the cuticle of the susceptible (parental) and selected (resistant) tobacco budworms increased with time 3–12 h after application. A trend of reduced penetration in the resistant strain was found but the differences were not statistically significant. 2′-O-methyl[ 14 C]spinosyn A when injected into the hemocoel was not metabolized 96 h after treatment in both the susceptible and resistant strain, suggesting that a change in metabolism was not the mechanism of resistance. Electrophysiological studies indicated that dose-dependent spinosyn A-induced currents occurred in neurons from spinosyn resistant and susceptible (adult) tobacco budworms. At both 10 and 100 nM spinosyn A, however, the amplitude of these currents in the resistant insects was significantly smaller than the amplitude of currents observed from neurons from susceptible tobacco budworm adults. This suggests that neurons from resistant insects have decreased sensitivity to spinosyn A. However, the reduced inward currents in the resistant strain may or may not be related to the mode of action of the spinosyns. No statistically significant cross-resistance was noted for the spinosad resistant tobacco budworms for topical applications of permethrin (Pounce ® ), profenofos (Curacron ® ), emamectin benzoate (Denim ® ), or indoxacarb (Steward ® ). A statistically significant reduction in susceptibility to acetamiprid (Mospilan ® ) in artificial diet as determined from a resistance ratio of 0.482 was found.

A benzoylphenylurea insect growth regulator with the common name noviflumuron was evaluated for use as a baiting toxicant against the eastern subterranean termite, Reticulitermes flavipes (Kollar). Noviflumuron demonstrated significantly greater potency and faster speed of action compared with the commercial standard hexaflumuron. In addition, noviflumuron was not a feeding deterrent on filter paper at concentrations of up to 10,000 ppm. The rates of uptake, clearance, and insect-to-insect transfer of [14C] noviflumuron were measured in R. flavipes in laboratory assays and compared with those previously reported for [14C]hexaflumuron. Under a continuous exposure regime, the uptake profile for noviflumuron was similar to that for hexaflumuron, although the time period of maximal uptake was shorter for noviflumuron. Noviflumuron was cleared from termites in a first order process with a half-life of approximately 29 d, whereas the half-life of hexaflumuron was much shorter (8-9 d). Noviflumuron was efficiently transferred from treated to untreated termites by trophallaxis via kinetics similar to those reported for hexaflumuron; however, the systemic dose of noviflumuron required to result in toxicity of R. flavipes was found to be at least two- to three-fold less than that of hexaflumuron. The faster activity of noviflumuron compared with hexaflumuron in R. flavipes can be at least partially explained by the combination of slower clearance and greater intrinsic activity.

Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis -resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis -resistant strains of Plutella xylostella and Spodoptera frugiperda . Cry1Bh1 is active against susceptible and B. thuringiensis -resistant colonies of O. nubilalis , P. xylostella , and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda . Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.

The rates of uptake, clearance, insect-to-insect transfer, and metabolism of [14C] hexaflumuron [N-(((3,5-dichloro-4-(1,1,2,2-tetrafluroethoxy)phenyl)- amino)carbonyl)-2,6-diflurobenzamide] were measured in eastern subterranean termite workers, Reticulitermes flavipes (Kollar), fed cellulose diets containing either 0.1 or 0.5% (wt:wt) hexaflumuron. The rate of uptake, level of maximum uptake, and amount of insect-to-insect transfer were concentration dependent. The clearance rate constant for hexaflumuron was independent of concentration, with a mean value of 3.2 x 10(-3)/h. This corresponds to a mean half-life for hexaflumuron inside termites of 9 d. No evidence of metabolism of hexaflumuron to additional products was detected when extracting and examining the radioactivity contained in the fecal and regurgitated material within the termite holding apparatus 40 d after exposure to the chemical. Hexaflumuron was efficiently transferred from treated to untreated termites, through trophallaxis, resulting in spread of the toxicant throughout the insect population. The combination of uptake and efficient transfer of hexaflumuron between treated and untreated termites ensures broad distribution of the material even to insects not directly exposed to the toxicant. The distribution of hexaflumuron by termite workers, along with their minimal ability to metabolize the compound to other metabolites, and their slow ability to clear the material from the termite population results in death of the entire group of termites contained within the holding apparatus.

This is the second in a series of reports that define the mode of action of spinosyns, a novel class of naturally derived insecticidal macrocyclic lactones. The concentration dependence of central nervous system (CNS) stimulation was determined using ganglia isolated from cockroaches, house fly larvae, and tobacco hornworm larvae. The tobacco hornworm nervous system was most sensitive, with a steep dose-response relation and an ED50of 5 nM for spinosyn A. The response of the housefly CNS was comparable, showing no clear effect at 3 nM and near-maximal stimulation at 10 nM spinosyn A. The cockroach CNS also displayed a steep dose-response relation, with a threshold of 10 nM and an EC50of 32 nM. That the nerve cords of cockroaches are exposed to comparable levels of spinosyn A during poisoning was established by comparison of the concentration of spinosyn A taken up by cockroach nerve cords during exposurein vivo,in cockroaches treated with a threshold dose, with that in isolated nerve cords equilibrated with a solution of spinosyn A in saline. A saline concentration of 21 nM was estimated to give the same nerve cord concentration as found at the threshold dose for prostration. We can conclude that during poisoning, spinosyn A reaches a concentration inside the insect that is sufficient to directly excite the central nervous system.

In vitro studies have demonstrated that N-(4-chloro-3-methyl-5-isothiazolyl)-N-methyl-2-[p-((α,α,α-trifluoro-p-tolyl)oxy]phenyl]acetamide (2a) undergoes NADPH-dependent metabolism, which is catalyzed by monooxygenase enzymes, in rat liver microsomes. The primary metabolite in rat was found to arise from ring-methyl hydroxylation, while N-demethylation to give N-(4-chloro-3-methyl-5-isothiazolyl)-2-[p-[(a,a,a-trifluoro-p-tolyl)oxy]phenyl]acetamide (1) was also observed to occur, but at a slower rate. In microsomal proteins prepared from tobacco budworm midgut tissues, the reverse was observed, as 1 is the predominant metabolite, while ring-methyl hydroxylation occurs at a slower rate. The overall rate of metabolism in trout liver microsomes was found to be 50-fold slower than in rat and afforded 1 as the predominant metabolite. Metabolism studies conducted on the N-alkyl-N-(5-isothiazolyl)- and N-(alkyl-isothiazolin-5-ylidene)phenylacetamides (2 and 3) have shown that the ring-alkylated isomers 3 were converted to 1 more rapidly than isomers 2 in all three species. In general, the rate of conversion to 1, or bioactivation, increased with increasing radical or carbocation stability of the alkyl group in rat and trout liver. In tobacco budworm, however, bioactivation was highest in the ethyl and n-propyl analogues. The ratio of bioactivation in tobacco budworm to that in trout, used as a predictor of selectivity, was observed to be highest with the methyl group.

Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ∼1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ∼30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.

Ronald Estabrook made his initial impact studying cytochrome P450 by demonstrating the oxidative metabolism function of this unique class of enzymes, which had an unusual spectral peak at 450 nm when reduced and in the presence of carbon monoxide. Utilizing a photochemical action spectrum, he demonstrated that a cytochrome P450 was responsible for steroid 21 hydroxylation catalyzed by microsomes prepared from adrenal cortex tissue. As a young postdoctoral student, I was given the unique opportunity to learn from a true pioneer in this field. Ron had a surprisingly small laboratory at that time that allowed me to closely interact with a great scientist to learn about the important role cytochrome P450 proteins play in a wide variety of different organisms catalyzing oxidative metabolism reactions essential to life and to provide organisms, with the means to defend against xenobiotics.

Chlorpyrifos (O,O-diethyl O-(3,5, 6-trichloro-2-pyridinyl)- phosphorothioate) is an organophosphorus (OP) insecticide used for controlling insect pests. Currently, the reference dose (RfD) used by the Environmental Protection Agency (EPA) to establish acceptable human exposure tolerances for chlorpyrifos is based upon inhibition of blood butyrylcholinesterase (BuChE), which is not the target enzyme of chlorpyrifos, and does not play any role in cholinergic transmission. Data are presented showing that inhibition of acetylcholinesterase (AChE) associated with red blood cells (RBC), an enzyme similar to or identical with that in the nervous system, is a more appropriate endpoint on which to base the RfD. Basing an acceptable level of human exposure (e.g., RfD) on inhibition of RBC AChE provides a significant margin of safety, since it is 12- to 14-fold more sensitive as an indicator of chlorpyrifos exposure than the AChE in the most sensitive relevant neurological tissues (brain or retina). Inhibition of RBC AChE activity is consistently exhibited at lower dosages of chlorpyrifos than those required to result in clinical symptoms of OP toxicity, or alterations in cognitive functional responses. There is no unique sensitivity of the fetus or neonates to chlorpyrifos when administered by an appropriate oral dose. Thus, inhibition of RBC AChE activity is an appropriate surrogate measurement of chlorpyrifos exposure and provides a conservative endpoint for establishing appropriate margins of safety for both adults and infants.