Insecticidal activity of spore-free mutants of Bacillus thuringiensis against the Indian meal moth and almond moth

Three oligosporogenic mutants of Bacillus thuringiensis were assayed for toxicity against larvae of the Indian meal moth, Plodia interpunctella, and the almond moth, Ephestia cautella. The results were compared with insecticidal activity obtained from the parent strain (HD-1) and two standard B. thuringiensis formulations (HD-1-S-1971 and HD-1-S-1980) against the same insect species. The toxicity of the sporeless mutant preparations was significantly diminished against the Indian meal moth (10- to 26-fold increase in LC₅₀) but exceeded the toxicity of the standards against the almond moth. The toxicities of the B. thuringiensis preparations toward the Indian meal moth were consistent with the number of spores in the test samples, but spores did not contribute to toxicity to E. cautella larvae. A rationale for basing dosage on soluble protein was demonstrated for use in situations where spores are not a contributing factor in toxicity.     

Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12.

The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 protoxin and the corresponding HD-1 P1 protoxin. Comparing the toxicities of individual protoxins with that of sporulated cultures demonstrates that no individual protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.     

Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12

The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 protoxin and the corresponding HD-1 P1 protoxin. Comparing the toxicities of individual protoxins with that of sporulated cultures demonstrates that no individual protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.     

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.     

Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.     

Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA.

Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains.     

Differential Activity and Activation of Bacillus thuringiensis Insecticidal Proteins in Diamondback Moth, Plutella xylostella

Whole-crystal preparations from strains HD-1 and HD-133, activated Cry1Ab and Cry1C toxins as well as Cry1Aa, Cry1Ac, Cry1D, and Cry2Aa protoxins were tested for toxicity to 2nd-instar larvae of the diamondback moth, Plutella xylostella. Mortality data recorded after 2 and 5 days provided different results that were related to differential rates of solubilization, activation, and degradation of insecticidal crystal proteins. The two most active proteins are Cry1Ab and Cry1C, which are both present in HD-133. The Cry1Ab protoxin is activated within 2 days, whereas activation of the Cry1C protoxin occurs between 2 and 5 days. HD-133 is more active than HD-1 immediately after infection and remains toxic over 5 days owing to the sequential activation of its crystal components. Solubility properties of crystals and rates of activation of protoxins influence the overall toxicity of HD-1 and HD-133 to the diamondback moth. Received: 30 March 1999 / Accepted: 3 May 1999     

Contribution of Bacillus thuringiensis Spores to Toxicity of Purified Cry Proteins Towards Indianmeal Moth Larvae

The influence of Bacillus thuringiensis subsp. kurstaki HD-1 spores upon the toxicity of purified Cry1Ab and Cry1C crystal proteins toward susceptible and BT-resistant Indianmeal moth (IMM, Plodia interpunctella) larvae was investigated. With susceptible larvae, HD-1 spores were toxic in the absence of crystal protein and highly synergistic (approximately 35- to 50-fold) with either Cry1Ab or Cry1C protein. With BT-resistant IMM larvae, HD-1 spores were synergistic with Cry1Ab and Cry1C protein in all three resistant strains examined. Synergism was highest (approximately 25- to 44-fold) in insects with primary resistance toward Cry1C (IMM larvae with resistance to B. thuringiensis subsp. aizawai or entomocidus). However, HD-1 spores also synergized either Cry1Ab or Cry1C toxicity toward larvae resistant to B. thuringiensis subsp. kurstaki at a lower level (approximately five- to sixfold). With susceptible larvae, the presence of spores reduced the time of death when combined with each of the purified Cry proteins. Without spores, the speed of intoxication and eventual death for larvae treated with Cry1C and Cry1Ab proteins was much slower than for the HD-1 preparation containing both spores and crystals together. Neither spores nor toxin dose affected the mean time of death of resistant larvae treated with either Cry1Ab or Cry1C toxins. Both Cry1Ab and Cry1C toxins appeared to reduce feeding and consequently toxin consumption. Received: 1 December 1995 / Accepted: 3 January 1996     

The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing proteins was confirmed by their binding to pyruvate carboxylase, a biotin-containing enzyme, as well as to biotinylated ovalbumin and biotinylated bovine serum albumin but not to their nonbiotinylated counterparts. Activated HD-73 toxin also inhibited the enzymatic activity of pyruvate carboxylase. The biotin binding site is likely contained in domain III of the toxin. Two highly conserved regions within domain III are similar in sequence to the biotin binding sites of avidin, streptavidin, and a biotin-specific monoclonal antibody. In particular, block 4 of the B. thuringiensis toxin contains the YAS biotin-specific motif. On the basis of its N-terminal amino acid sequence, the 120-kDa biotin-containing protein is totally distinct from the 120-kDa aminopeptidase N reported to be a receptor for Cry1Ac toxin.     

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD₅₀]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD₅₀ of HD-1 with the FFD₅₀ expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.     

Activated insecticidal crystal proteins from Bacillus thuringiensis serovars killed adult house flies

Insecticidal crystal proteins (ICP) from Bacillus thuringiensis serovar kurstaki HD-1 and HD-73 were activated by immobilized trypsin or chymotrypsin. The activated toxins (10 μ g or more) as well as unactivated ICP killed adult house flies but not larvae. Bacillus thuringiensis strain son diego did not kill house flies. In this experimental system, the average life span of the adult house fly was 8 days and the activated toxins reduced it to 2 days. The unactivated insecticidal crystal protein also reduced it to 4 days at the same concentration as the activated toxin.     

Processing of delta-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juices of various insect larvae.

Midgut juices were prepared from Adoxophyes sp., smaller tea tortrix (STT); Bombyx mori, silkworm (SW); Spodoptera litura, common cutworm (CCW); Plutella xylostella, diamondback moth (DBM); and Musca domestica, housefly (HF) and immobilized onto Sepharose 4B. delta-Endotoxins (ICPs) from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 were digested by these immobilized gut juice proteases. All gut juices tested derived relatively proteolytic resistant cores from ICP. The molecular sizes of these cores, about 55 kDa in SDS-PAGE, were resulted. In the case of CCW, however, digestion was very strong and only 1/20 concentration of core protein remained relative to other digests. The N-terminal amino acid sequencing of the core proteins showed that they were truncated at the very end of the N-terminus of protoxin, CryIA, at different sites. Although housefly larvae were completely insensitive to active toxin, the gut juice produced the core, suggesting that the housefly may lack the binding sites for the core-active toxin.     

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.     

In vivo generation of hybrids between two Bacillus thuringiensis insect-toxin-encoding genes

The parasporal crystal of Bacillus thuringiensis is composed of polypeptides highly toxic to a number of insect larvae. The structural genes (cryIA) encoding the Lepidoptera-specific toxin from different bacterial strains diverge primarily in a single hypervariable region, whereas the N-terminal and C-terminal parts of the proteins are highly conserved. In this report, we describe the generation of hybrid genes between two cryIA genes. Two truncated cryIA genes were cloned in a plasmid vector in such way as to have only the hypervariable region in common. The two truncated cryIA genes were separated by the tetracycline-resistance determinant (or part of it). In vivo recombination between the hypervariable regions of the cryIA genes reconstituted an entire hybrid cryIA gene. Direct sequence analysis of 17 recombinant plasmids identified eleven different crossover regions which did not alter the reading frame and allowed the production of eight different hybrid proteins. The recombination events were independent from the RecA function of Escherichia coli. Some of the hybrid gene products were more specific in their insecticidal action and one had acquired a new biological activity.     

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.     

Binding and toxicity of Bacillus thuringiensis protein Cry1C to susceptible and resistant diamondback moth (Lepidoptera: Plutellidae)

We studied mechanisms of resistance to Bacillus thuringiensis insecticidal crystal protein Cry1C in the diamondback moth, Plutella xylostella (L.). Binding assays with midgut brush border membrane vesicles prepared from whole larvae showed no significant difference between resistant and susceptible strains in binding of radioactively-labeled Cry1C. These results indicate that reduced binding of Cry1C to midgut membrane target sites did not cause resistance to Cry1C. Thus, the mechanism of resistance to Cry1C differs from that observed in several previously reported cases of resistance to Cry1A toxins in diamondback moth. We tested Cry1C toxin and Cry1C crystalline protoxin against resistant and susceptible larvae using leaf disk bioassays. After adjusting for the size difference between Cry1C toxin and protoxin, we found that with resistant larvae, toxin was significantly more toxic than protoxin. In contrast, with susceptible larvae, no significant difference in toxicity occurred between Cry1C toxin and protoxin. The resistance ratios for Cry1C were 19 for toxin and 48 for protoxin. These results suggest that reduced conversion of Cry1C protoxin to toxin is a minor mechanism of resistance to Cry1C. Because neither reduced binding nor reduced conversion of protoxin to toxin appear to be major mechanisms, one or more other mechanisms are important in diamondback moth resistance to Cry1C.     

苏云金芽胞杆菌高效表达载体的构建

[目的]通过比较cry1A、cry3A、cry4A和cry8E四个基因的启动子转录活性,筛选出一个强启动子,利用强启动子构建一个苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)高效表达载体.[方法]利用启动子融合lacZ技术检测了4种启动子的转录活性.通过扫描电子显微镜观察晶体、SDS-PAGE、蛋白定量和生物活性测定等方法对新建高效表达载体进行功能验证.[结果]构建了Pcry1A、Pcry3A、Pcry4A和Pcry8E4个启动子融合报告基因lacZ的表达载体,经β-半乳糖苷酶活性分析得知,启动子活性从高到低依次为Pcry8E＞Pcry1A＞Pcry4A＞Pcry3A.选取cry8E启动子,以pHT315作为基础载体构建苏云金芽胞杆菌高效表达载体pHT315-8E21b,将cry1Ac基因连接到pHT315-8E21b和广泛应用的cry3A启动子指导的pSXY-422b上,分别转入无晶体突变株HD-73-,获得菌株HD-8E1Ac和HD-422-1Ac.扫描电子显微镜观察显示,HD-8E1Ac菌株可以形成菱形晶体,说明正确表达了cry1Ac基因.SDS-PAGE分析结合蛋白定量实验表明pHT315-8E21b表达效率高于pSXY-422b.对小菜蛾(Plutella xylostella)的生物活性测定表明HD-8E1Ac菌株对小菜蛾有生物活性,且菌株活性高于HD-422-1Ac.[结论]利用强启动子Pcry8E构建了一个能在Bt中高效表达的穿梭载体pHT315-8E21b,该载体可正确表达cry1Ac基因,其表达效率高于被广泛应用的pSXY422b.     

Cellular Fatty Acid Analysis of Isolates of Bacillus thuringiensis Serovar kurstaki,Strain HD-1

Whole-cell cellular fatty acid (CFA) composition was utilized to determine if Bacillus thuringiensis serovar kurstaki (HD-1) larvicides produced by different manufacturers could be distinguished from each other and to determine whether these larvicides were distinguishable from isolates deposited in public collections. This study analyzed Biobit, Dipel, Foray, Thuricide, and the non HD-1 larvicides Delfin and Javelin, as well as the 1971 and 1980 Standards of HD-1. Isolates of HD-1 deposited in the collections of the American Type Culture Collection, Bacillus Genetic Stock Center, Pasteur Institute, and United States Department of Agriculture (USDA) were also analyzed. The data were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages (UPGMA). Samples that linked at a Euclidean distance ≤2.0 units were considered to belong to the same fatty acid strain. Isolates of HD-1 from commercial products and deposits of HD-1 in the public collections and Standards were polytypic; 22 separate fatty acid strains were identified in the 1971 and 1980 Standards and 35 fatty acid strains were identified in the public collections. The type strain for Btk contained multiple fatty acid strains; three fatty acid strains were present in both the Bacillus Genetic Stock Center and the Pasteur Institute collections. In contrast, the type strain for serovar kurstaki in the USDA collection (HD-73) was monotypic and its fatty acid strain did not occur in other collections. We could distinguish between HD-1 and non-HD-1 larvicides using CFA composition. We conclude that CFA analysis may be used to identify commercial products.