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In JoVE (1)
Other Publications (27)
- Journal of Medical Entomology
- The Journal of Biological Chemistry
- Insect Biochemistry and Molecular Biology
- Journal of Insect Science (Online)
- Journal of Economic Entomology
- Journal of Economic Entomology
- Journal of Economic Entomology
- Nature Biotechnology
- Journal of Economic Entomology
- Insect Biochemistry and Molecular Biology
- Pest Management Science
- Insect Molecular Biology
- PloS One
- GM Crops & Food
- PloS One
- PloS One
- Insect Biochemistry and Molecular Biology
- PloS One
- PloS One
- Journal of Invertebrate Pathology
- Scientific Reports
- Scientific Reports
- Trends in Biotechnology
- Insect Biochemistry and Molecular Biology
Articles by Jeffrey A. Fabrick in JoVE
Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells
Jeffrey A. Fabrick1, J. Joe Hull1
1U.S. Arid Land Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture
Other articles by Jeffrey A. Fabrick on PubMed
Journal of Medical Entomology. May, 2002 | Pubmed ID: 12061447
The sensitivity and accuracy of near-infrared spectroscopy (NIRS) was compared with that of the pteridine fluorescence technique for estimating the chronological age of house flies, Musca domestica (L.). Although results with both techniques were significantly correlated with fly age, confidence limits on predicted ages generally were smaller with NIRS. Young flies could be readily differentiated from old flies by using NIRS. Age predictions using the pteridine method are dependent upon size, sex, and temperature at which adult flies are exposed. In contrast, those factors do not need to be determined for age-grading using NIRS. Classification accuracy using the NIRS method was similar for whole flies, fresh heads, dried heads, and ethanol-preserved heads. The NIRS method was also suitable for predicting age of stable flies, Stomoxys calcitrans (L.), and face flies, Musca autumnalis De Geer. NIRS has several advantages over the measurement of pteridine levels for age-grading field-collected flies, including speed and portability of instrumentation, and not needing to determine sex, size, and temperatures to which adult flies were exposed.
Innate Immunity in a Pyralid Moth: Functional Evaluation of Domains from a Beta-1,3-glucan Recognition Protein
The Journal of Biological Chemistry. Jun, 2004 | Pubmed ID: 15084591
Invertebrates, like vertebrates, utilize pattern recognition proteins for detection of microbes and subsequent activation of innate immune responses. We report structural and functional properties of two domains from a beta-1,3-glucan recognition protein present in the hemolymph of a pyralid moth, Plodia interpunctella. A recombinant protein corresponding to the first 181 amino-terminal residues bound to beta-1,3-glucan, lipopolysaccharide, and lipoteichoic acid, polysaccharides found on cell surfaces of microorganisms, and also activated the prophenoloxidase-activating system, an immune response pathway in insects. The amino-terminal domain consists primarily of an alpha-helical secondary structure with a minor beta-structure. This domain was thermally stable and resisted proteolytic degradation. The 290 residue carboxyl-terminal domain, which is similar in sequence to glucanases, had less affinity for the polysaccharides, did not activate the prophenoloxidase cascade, had a more complicated CD spectrum, and was heat-labile and susceptible to proteinase digestion. The carboxyl-terminal domain bound to laminarin, a beta-1,3-glucan with beta-1,6 branches, but not to curdlan, a beta-1,3-glucan that lacks branching. These results indicate that the two domains of Plodia beta-1,3-glucan recognition protein, separated by a putative linker region, bind microbial polysaccharides with differing specificities and that the amino-terminal domain, which is unique to this class of pattern recognition receptors from invertebrates, is responsible for stimulating prophenoloxidase activation.
Insect Biochemistry and Molecular Biology. Nov, 2004 | Pubmed ID: 15522618
Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins. We previously identified three mutant alleles (r1, r2, r3) of a cadherin gene in pink bollworm (Pectinophora gossypiella) linked with recessive resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton. Here we describe a polymerase chain reaction (PCR)-based method that detects the mutation in genomic DNA of each of the three resistant alleles. Using primers that distinguish between resistant and susceptible (s) alleles, this method enables identification of 10 genotypes (r1r1, r1r2, r1r3, r2r2, r2r3, r3r3, r1s, r2s, r3s, and ss) at the cadherin locus. For each of the three resistant alleles, the method detected the resistance allele in a single heterozygote (r1s, r2s, or r3s) pooled with DNA from the equivalent of 19 susceptible (ss) individuals. The results suggest that the DNA-based detection method described here could greatly increase the efficiency of monitoring for resistance to Cry1Ac compared to bioassays that detect rare individuals with homozygous resistance.
Journal of Insect Science (Online). 2004 | Pubmed ID: 15861231
Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database.
Journal of Economic Entomology. Oct, 2006 | Pubmed ID: 17066779
Transgenic crops producing toxins from the bacterium Bacillus thuringiensis (Bt) kill insect pests and can reduce reliance on insecticide sprays. Although Bt cotton (Gossypium hirsutum L.) and Bt corn (Zea mays L.) covered 26 million ha worldwide in 2005, their success could be cut short by evolution of pest resistance. Monitoring the early phases of pest resistance to Bt crops is crucial, but it has been extremely difficult because bioassays usually cannot detect heterozygotes harboring one allele for resistance. We report here monitoring of resistance to Bt cotton with DNA-based screening, which detects single resistance alleles in heterozygotes. We used polymerase chain reaction primers that specifically amplify three mutant alleles of a cadherin gene linked with resistance to Bt cotton in pink bollworm, Pectinophora gossypiella (Saunders), a major pest. We screened DNA of 5,571 insects derived from 59 cotton fields in Arizona, California, and Texas during 2001-2005. No resistance alleles were detected despite a decade of exposure to Bt cotton. In conjunction with data from bioassays and field efficacy tests, the results reported here contradict predictions of rapid pest resistance to Bt crops.
Journal of Economic Entomology. Dec, 2006 | Pubmed ID: 17195682
Resistance to transgenic cotton, Gossypium hirsutum L., producing Bacillus thuringiensis (Bt) toxin Cry1Ac is linked with three recessive alleles of a cadherin gene in laboratory-selected strains of pink bollworm, Pectinophora gossypiella (Saunders), a major cotton pest. Here, we analyzed a strain (MOV97-R) with a high frequency of cadherin resistance alleles, a high frequency of resistance to 10 microg of Cry1Ac per milliliter of diet, and an intermediate frequency of resistance to 1000 microg of Cry1Ac per ml of diet. We selected two strains for increased resistance by exposing larvae from MOV97-R to diet with 1000 microg of Cry1Ac per ml of diet. In both selected strains, two to three rounds of selection increased survival at 1000 microg of CrylAc per ml of diet to at least 76%, indicating genetic variation in survival at this high concentration and yielding >4300-fold resistance relative to a susceptible strain. Variation in cadherin genotype did not explain variation in survival at 1000 microg of Cry1Ac per ml of diet, implying that one or more other loci affected survival at this concentration. This conclusion was confirmed with results showing that when exposure to Cry1Ac stopped, survival at 1000 microg of Cry1Ac per ml of diet dropped substantially, but survival at 10 microg Cry1Ac per ml of diet remained close to 100% and all survivors had two cadherin resistance alleles. Although survival at 1000 microg of Cry1Ac per ml of diet is not required for resistance to Bt cotton, understanding how genes other than cadherin confer increased survival at this high concentration may reveal novel mechanisms of resistance.
Effects of Pink Bollworm Resistance to Bacillus Thuringiensis on Phenoloxidase Activity and Susceptibility to Entomopathogenic Nematodes
Journal of Economic Entomology. Jun, 2009 | Pubmed ID: 19610442
Widespread planting of crops genetically engineered to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) imposes selection on many key agricultural pests to evolve resistance to Bt. Fitness costs can slow the evolution of Bt resistance. We examined effects of entomopathogenic nematodes on fitness costs of Bt resistance in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), a major pest of cotton, Gossypium hirsutum L., in the southwestern United States that is currently controlled by transgenic cotton that produces Bt toxin Cry1Ac. We tested whether the entomopathogenic nematodes Steinernema riobrave Cabanillas, Poinar, and Raulston (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) affected fitness costs of resistance to Cry1Ac in two laboratory-selected hybrid strains of pink bollworm reared on non-Bt cotton bolls. The nematode S. riobrave imposed a recessive fitness cost for one strain, and H. bacteriophora imposed a fitness cost affecting heterozygous resistant individuals for the other strain. Activity of phenoloxidase, an important component of insects' immune response, did not differ between Bt-resistant and Bt-susceptible families. This suggests phenoloxidase does not affect susceptibility to entomopathogenic nematodes in Bt-resistant pink bollworm. Additionally, phenoloxidase activity does not contribute to Bt resistance, as has been found in some species. We conclude that other mechanisms cause higher nematode-imposed mortality for pink bollworm with Bt resistance genes. Incorporation of nematode-imposed fitness costs into a spatially explicit simulation model suggests that entomopathogenic nematodes in non-Bt refuges could delay resistance by pink bollworm to Bt cotton.
Nature Biotechnology. Dec, 2010 | Pubmed ID: 21057498
Genetically engineered crops that produce insecticidal toxins from Bacillus thuringiensis (Bt) are grown widely for pest control. However, insect adaptation can reduce the toxins' efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to provide susceptible insects to mate with resistant insects. Variable farmer compliance is one of the limitations of this approach. Here we report the benefits of an alternative strategy where sterile insects are released to mate with resistant insects and refuges are scarce or absent. Computer simulations show that this approach works in principle against pests with recessive or dominant inheritance of resistance. During a large-scale, four-year field deployment of this strategy in Arizona, resistance of pink bollworm (Pectinophora gossypiella) to Bt cotton did not increase. A multitactic eradication program that included the release of sterile moths reduced pink bollworm abundance by >99%, while eliminating insecticide sprays against this key invasive pest.
Extraordinary Resistance to Insecticides Reveals Exotic Q Biotype of Bemisia Tabaci in the New World
Journal of Economic Entomology. Dec, 2010 | Pubmed ID: 21309242
A strain of the whitefly Bemisia tabaci (Gennadius) possessing unusually high levels of resistance to a wide range of insecticides was discovered in 2004 in the course of routine resistance monitoring in Arizona. The multiply resistant insects, collected from poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) plants purchased at a retail store in Tucson, were subjected to biotype analysis in three laboratories. Polyacrylamide gel electrophoresis of naphthyl esterases and sequencing of the mitochondrial cytochrome oxidase I gene (780 bp) confirmed the first detection of the Q biotype of B. tabaci in the New World. This U.S. Q biotype strain, referred to as Poinsettia'04, was highly resistant to two selective insect growth regulators, pyriproxyfen and buprofezin, and to mixtures of fenpropathrin and acephate. It was also unusually low in susceptibility to the neonicotinoid insecticides imidacloprid, acetamiprid, and thiamethoxam, relative to B biotype whiteflies. In 100 collections of whiteflies made in Arizona cotton (Gossypium spp.), vegetable, and melon (Cucumis melo L.) fields from 2001 to 2005, no Q biotypes were detected. Regions of the United States that were severely impacted by the introduction of the B biotype of B. tabaci in the 1980s would be well advised to promote measures that limit movement of the Q biotype from controlled environments into field systems and to formulate alternatives for managing this multiply-resistant biotype, in the event that it becomes more widely distributed.
Identification and Characterization of Functional Aquaporin Water Channel Protein from Alimentary Tract of Whitefly, Bemisia Tabaci
Insect Biochemistry and Molecular Biology. Mar, 2011 | Pubmed ID: 21146609
Some hemipteran xylem and phloem-feeding insects have evolved specialized alimentary structures or filter chambers that rapidly transport water for excretion or osmoregulation. In the whitefly, Bemisia tabaci, mass movement of water through opposing alimentary tract tissues within the filter chamber is likely facilitated by an aquaporin protein. B. tabaci aquaporin-1 (BtAQP1) possesses characteristic aquaporin topology and conserved pore-forming residues found in water-specific aquaporins. As predicted for an integral transmembrane protein, recombinant BtAQP1 expressed in cultured insect cells localized within the plasma membrane. BtAQP1 is primarily expressed in early instar nymphs and adults, where in adults it is localized in the filter chamber and hindgut. Xenopus oocytes expressing BtAQP1 were water permeable and mercury-sensitive, both characteristics of classical water-specific aquaporins. These data support the hypothesis that BtAQP1 is a water transport protein within the specialized filter chamber of the alimentary tract and functions to translocate water across tissues for maintenance of osmotic pressure and/or excretion of excess dietary fluid.
Increased Toxicity of Bacillus Thuringiensis Cry3Aa Against Crioceris Quatuordecimpunctata, Phaedon Brassicae and Colaphellus Bowringi by a Tenebrio Molitor Cadherin Fragment
Pest Management Science. Apr, 2011 | Pubmed ID: 21495115
BACKGROUND: Biopesticides containing Cry insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are effective against many lepidopteran pests, but there is a lack of Bt-based pesticides for efficient control of important coleopteran pests. Based on the reported increase in Bt toxin oligomerization by a polypeptide from the Cry3Aa receptor cadherin in Tenebrio molitor (Coleoptera: Tenebrionidae), it was hypothesized that this cadherin peptide, rTmCad1p, would enhance Cry3Aa toxicity towards coleopteran larvae. To test this hypothesis, the relative toxicity of Cry3Aa, with or without rTmCad1p, against damaging chrysomelid vegetable pests of China was evaluated. RESULTS: Cry3Aa toxicity was evaluated in the spotted asparagus beetle (Crioceris quatuordecimpunctata), cabbage leaf beetle (Colaphellus bowringi) and daikon leaf beetle (Phaedon brassicae). To assess the effect of rTmCad1p on Cry3Aa toxicity, neonate larvae were fed Cry3Aa toxin alone or in combination with increasing amounts of rTmCad1p. The data demonstrated that Cry3Aa toxicity was significantly increased in all three vegetable pests, resulting in as much as a 15.3-fold increase in larval mortality. CONCLUSION: The application of rTmCad1p to enhance Cry3Aa insecticidal activity has potential for use in increasing range and activity levels against coleopteran pests displaying low susceptibility to Bt-based biopesticides. Copyright Â© 2011 Society of Chemical Industry.
Insertion of an Intact CR1 Retrotransposon in a Cadherin Gene Linked with Bt Resistance in the Pink Bollworm, Pectinophora Gossypiella
Insect Molecular Biology. Oct, 2011 | Pubmed ID: 21815956
Three mutations in the Pectinophora gossypiella cadherin gene PgCad1 are linked with resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac. Here we show that the r3 mutation entails recent insertion into PgCad1 of an active chicken repeat (CR1) retrotransposon, designated CR1-1_Pg. Unlike most other CR1 elements, CR1-1_Pg is intact, transcribed by a flanking promoter, contains target site duplications and has a relatively low number of copies. Examination of transcripts from the PgCad1 locus revealed that CR1-1_Pg disrupts both the cadherin protein and a long noncoding RNA of unknown function. Together with previously reported data, these findings show that transposable elements disrupt eight of 12 cadherin alleles linked with resistance to Cry1Ac in three lepidopteran species, indicating that the cadherin locus is a common target for disruption by transposable elements.
Similar Genetic Basis of Resistance to Bt Toxin Cry1Ac in Boll-selected and Diet-selected Strains of Pink Bollworm
PloS One. 2012 | Pubmed ID: 22530065
Genetically engineered cotton and corn plants producing insecticidal Bacillus thuringiensis (Bt) toxins kill some key insect pests. Yet, evolution of resistance by pests threatens long-term insect control by these transgenic Bt crops. We compared the genetic basis of resistance to Bt toxin Cry1Ac in two independently derived, laboratory-selected strains of a major cotton pest, the pink bollworm (Pectinophora gossypiella [Saunders]). The Arizona pooled resistant strain (AZP-R) was started with pink bollworm from 10 field populations and selected with Cry1Ac in diet. The Bt4R resistant strain was started with a long-term susceptible laboratory strain and selected first with Bt cotton bolls and later with Cry1Ac in diet. Previous work showed that AZP-R had three recessive mutations (r1, r2, and r3) in the pink bollworm cadherin gene (PgCad1) linked with resistance to Cry1Ac and Bt cotton producing Cry1Ac. Here we report that inheritance of resistance to a diagnostic concentration of Cry1Ac was recessive in Bt4R. In interstrain complementation tests for allelism, F(1) progeny from crosses between AZP-R and Bt4R were resistant to Cry1Ac, indicating a shared resistance locus in the two strains. Molecular analysis of the Bt4R cadherin gene identified a novel 15-bp deletion (r4) predicted to cause the loss of five amino acids upstream of the Cry1Ac-binding region of the cadherin protein. Four recessive mutations in PgCad1 are now implicated in resistance in five different strains, showing that mutations in cadherin are the primary mechanism of resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona.
GM Crops & Food. Jul-Sep, 2012 | Pubmed ID: 22572905
Evolution of resistance by pests can reduce the benefits of transgenic crops that produce toxins from Bacillus thuringiensis (Bt) for insect control. One of the world's most important cotton pests, pink bollworm (Pectinophora gossypiella), has been targeted for control by transgenic cotton producing Bt toxin Cry1Ac in several countries for more than a decade. In China, the frequency of resistance to Cry1Ac has increased, but control failures have not been reported. In western India, pink bollworm resistance to Cry1Ac has caused widespread control failures of Bt cotton. By contrast, in the state of Arizona in the southwestern United States, monitoring data from bioassays and DNA screening demonstrate sustained susceptibility to Cry1Ac for 16 y. From 1996-2005, the main factors that delayed resistance in Arizona appear to be abundant refuges of non-Bt cotton, recessive inheritance of resistance, fitness costs associated with resistance and incomplete resistance. From 2006-2011, refuge abundance was greatly reduced in Arizona, while mass releases of sterile pink bollworm moths were made to delay resistance as part of a multi-tactic eradication program. Sustained susceptibility of pink bollworm to Bt cotton in Arizona has provided a cornerstone for the pink bollworm eradication program and for integrated pest management in cotton. Reduced insecticide use against pink bollworm and other cotton pests has yielded economic benefits for growers, as well as broad environmental and health benefits. We encourage increased efforts to combine Bt crops with other tactics in integrated pest management programs.
An Initial Event in the Insect Innate Immune Response: Structural and Biological Studies of Interactions Between β-1,3-glucan and the N-terminal Domain of β-1,3-glucan Recognition Protein
Biochemistry. Jan, 2013 | Pubmed ID: 23237493
In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the nuclear magnetic resonance (NMR) solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone (15)N-(1)H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-βGRP-laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-βGRP-laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of an X-ray crystallographic structure of the N-βGRP-laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements conducted with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of the N-βGRP-laminarin macro complex and that a decreased stability is accompanied by a reduced level of activation of the proPO pathway. An increased level of β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of the βGRP-β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway.
PloS One. 2013 | Pubmed ID: 23357950
Mirid plant bugs (Hemiptera: Miridae) are economically important insect pests of many crops worldwide. The western tarnished plant bug Lygus hesperus Knight is a pest of cotton, alfalfa, fruit and vegetable crops, and potentially of several emerging biofuel and natural product feedstocks in the western US. However, little is known about the underlying molecular genetics, biochemistry, or physiology of L. hesperus, including their ability to survive extreme environmental conditions.
Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab
PloS One. 2013 | Pubmed ID: 24244692
Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.
Molecular and Functional Characterization of Multiple Aquaporin Water Channel Proteins from the Western Tarnished Plant Bug, Lygus Hesperus
Insect Biochemistry and Molecular Biology. Feb, 2014 | Pubmed ID: 24333473
Aquaporins (AQPs) are integral membrane channel proteins that facilitate the bidirectional transfer of water or other small solutes across biological membranes involved in numerous essential physiological processes. In arthropods, AQPs belong to several subfamilies, which contribute to osmoregulation, respiration, cryoprotection, anhydrobiosis, and excretion. We cloned and characterized five novel AQPs from the western tarnished plant bug, Lygus hesperus, a polyphagous insect pest of food and fiber crops throughout western North America. The L. hesperus AQPs (LhAQP1-5) belong to different phylogenetic subfamilies, have unique transcription profiles and cellular localizations, and all transport water (but not glycerol) when heterologously expressed in Xenopus laevis oocytes. Our results demonstrate that multiple AQPs with possible compensatory functions are produced in L. hesperus that likely play important roles in maintaining water homeostasis in this important insect pest.
Alternative Splicing and Highly Variable Cadherin Transcripts Associated with Field-evolved Resistance of Pink Bollworm to Bt Cotton in India
PloS One. 2014 | Pubmed ID: 24840729
Evolution of resistance by insect pests can reduce the benefits of insecticidal proteins from Bacillus thuringiensis (Bt) that are used extensively in sprays and transgenic crops. Despite considerable knowledge of the genes conferring insect resistance to Bt toxins in laboratory-selected strains and in field populations exposed to Bt sprays, understanding of the genetic basis of field-evolved resistance to Bt crops remains limited. In particular, previous work has not identified the genes conferring resistance in any cases where field-evolved resistance has reduced the efficacy of a Bt crop. Here we report that mutations in a gene encoding a cadherin protein that binds Bt toxin Cry1Ac are associated with field-evolved resistance of pink bollworm (Pectinophora gossypiella) in India to Cry1Ac produced by transgenic cotton. We conducted laboratory bioassays that confirmed previously reported resistance to Cry1Ac in pink bollworm from the state of Gujarat, where Bt cotton producing Cry1Ac has been grown extensively. Analysis of DNA from 436 pink bollworm from seven populations in India detected none of the four cadherin resistance alleles previously reported to be linked with resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona. However, DNA sequencing of pink bollworm derived from resistant and susceptible field populations in India revealed eight novel, severely disrupted cadherin alleles associated with resistance to Cry1Ac. For these eight alleles, analysis of complementary DNA (cDNA) revealed a total of 19 transcript isoforms, each containing a premature stop codon, a deletion of at least 99 base pairs, or both. Seven of the eight disrupted alleles each produced two or more different transcript isoforms, which implicates alternative splicing of messenger RNA (mRNA). This represents the first example of alternative splicing associated with field-evolved resistance that reduced the efficacy of a Bt crop.
Transcriptome-based Identification of ABC Transporters in the Western Tarnished Plant Bug Lygus Hesperus
PloS One. 2014 | Pubmed ID: 25401762
ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate diverse physiological functions by coupling ATP hydrolysis with substrate transport across lipid membranes. In insects, these proteins play roles in metabolism, development, eye pigmentation, and xenobiotic clearance. While ABC transporters have been extensively studied in vertebrates, less is known concerning this superfamily in insects, particularly hemipteran pests. We used RNA-Seq transcriptome sequencing to identify 65 putative ABC transporter sequences (including 36 full-length sequences) from the eight ABC subfamilies in the western tarnished plant bug (Lygus hesperus), a polyphagous agricultural pest. Phylogenetic analyses revealed clear orthologous relationships with ABC transporters linked to insecticide/xenobiotic clearance and indicated lineage specific expansion of the L. hesperus ABCG and ABCH subfamilies. The transcriptional profile of 13 LhABCs representative of the ABCA, ABCB, ABCC, ABCG, and ABCH subfamilies was examined across L. hesperus development and within sex-specific adult tissues. All of the transcripts were amplified from both reproductively immature and mature adults and all but LhABCA8 were expressed to some degree in eggs. Expression of LhABCA8 was spatially localized to the testis and temporally timed with male reproductive development, suggesting a potential role in sexual maturation and/or spermatozoa protection. Elevated expression of LhABCC5 in Malpighian tubules suggests a possible role in xenobiotic clearance. Our results provide the first transcriptome-wide analysis of ABC transporters in an agriculturally important hemipteran pest and, because ABC transporters are known to be important mediators of insecticidal resistance, will provide the basis for future biochemical and toxicological studies on the role of this protein family in insecticide resistance in Lygus species.
Cadherin is Involved in the Action of Bacillus Thuringiensis Toxins Cry1Ac and Cry2Aa in the Beet Armyworm, Spodoptera Exigua
Journal of Invertebrate Pathology. May, 2015 | Pubmed ID: 25754522
Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins are effective against some insect pests in sprays and transgenic crops, although the evolution of resistance could threaten the long-term efficacy of such Bt use. One strategy to delay resistance to Bt crops is to "pyramid" two or more Bt proteins that bind to distinct receptor proteins within the insect midgut. The most common Bt pyramid in cotton (Gossypium hirsutum L.) employs Cry1Ac with Cry2Ab to target several key lepidopteran pests, including the beet armyworm, Spodoptera exigua (Hübner), which is a serious migratory pest of many vegetable crops and is increasingly important in cotton in China. While cadherin and aminopeptidase-N are key receptors of Cry1 toxins in many lepidopterans including S. exigua, the receptor for Cry2A toxins remains poorly characterized. Here, we show that a heterologous expressed peptide corresponding to cadherin repeat 7 to the membrane proximal extracellular domain (CR7-MPED) in the S. exigua cadherin 1b (SeCad1b) binds Cry1Ac and Cry2Aa. Moreover, SeCad1b transcription was suppressed in S. exigua larvae by oral RNA interference and susceptibility to Cry1Ac and Cry2Aa was significantly reduced. These results indicate that SeCad1b plays important functional roles of both Cry1Ac and Cry2Aa, having major implications for resistance management for S. exigua in Bt crops.
Scientific Reports. Oct, 2015 | Pubmed ID: 26455902
Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.
Scientific Reports. Nov, 2015 | Pubmed ID: 26559899
Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant "pyramids" producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus, and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India.
Trends in Biotechnology. Apr, 2016 | Pubmed ID: 26774592
The primary strategy for delaying the evolution of pest resistance to transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) entails refuges of plants that do not produce Bt toxins and thus allow survival of susceptible pests. Recent advances include using refuges together with Bt crop 'pyramids' that make two or more Bt toxins effective against the same pest, and planting seed mixtures yielding random distributions of pyramided Bt and non-Bt corn plants within fields. We conclude that conditions often deviate from those favoring the success of pyramids and seed mixtures, particularly against pests with low inherent susceptibility to Bt toxins. For these problematic pests, promising approaches include using larger refuges and integrating Bt crops with other pest management tactics.
De Novo Construction of an Expanded Transcriptome Assembly for the Western Tarnished Plant Bug, Lygus Hesperus
GigaScience. 2016 | Pubmed ID: 26823975
The plant bug Lygus hesperus Knight is a polyphagous pest of many economically important crops. Despite its pest status, little is known about the molecular mechanisms responsible for much of the biology of this species. Earlier Lygus transcriptome assemblies were limited by low read depth, or because they focused on specific conditions. To generate a more comprehensive transcriptome, we supplemented previous datasets with new reads corresponding to specific tissues (heads, antennae, and male reproductive tissues). This transcriptome augments current Lygus molecular resources and provides the foundational knowledge critical for future comparative studies.
GigaScience. Jun, 2016 | Pubmed ID: 27333791
The pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) is one of the world's most important pests of cotton. Insecticide sprays and transgenic cotton producing toxins of the bacterium Bacillus thuringiensis (Bt) are currently used to manage this pest. Bt toxins kill susceptible insects by specifically binding to and destroying midgut cells, but they are not toxic to most other organisms. Pink bollworm is useful as a model for understanding insect responses to Bt toxins, yet advances in understanding at the molecular level have been limited because basic genomic information is lacking for this cosmopolitan pest. Here, we have sequenced, de novo assembled and annotated a comprehensive larval midgut transcriptome from a susceptible strain of pink bollworm.
Molecular and Functional Characterization of Bemisia Tabaci Aquaporins Reveals the Water Channel Diversity of Hemipteran Insects
Insect Biochemistry and Molecular Biology. Oct, 2016 | Pubmed ID: 27491441
The Middle East-Asia Minor 1 (MEAM1) whitefly, Bemisia tabaci (Gennadius) is an economically important pest of food, fiber, and ornamental crops. This pest has evolved a number of adaptations to overcome physiological challenges, including 1) the ability to regulate osmotic stress between gut lumen and hemolymph after imbibing large quantities of a low nitrogen, sugar-rich liquid diet; 2) the ability to avoid or prevent dehydration and desiccation, particularly during egg hatching and molting; and 3) to be adapted for survival at elevated temperatures. One superfamily of proteins involved in the maintenance of fluid homeostasis in many organisms includes the aquaporins, which are integral membrane channel proteins that aid in the rapid flux of water and other small solutes across biological membranes. Here, we show that B. tabaci has eight aquaporins (BtAqps), of which seven belong to the classical aquaporin 4-related grade of channels, including Bib, Drip, Prip, and Eglps and one that belongs to the unorthodox grade of aquaporin 12-like channels. B. tabaci has further expanded its repertoire of water channels through the expression of three BtDrip2 amino-terminal splice variants, while other hemipteran species express amino- or carboxyl-terminal isoforms of Drip, Prip, and Eglps. Each BtAqp has unique transcript expression profiles, cellular localization, and/or substrate preference. Our phylogenetic and functional data reveal that hemipteran insects lost the classical glp genes, but have compensated for this by duplicating the eglp genes early in their evolution to comprise at least three separate clades of glycerol transporters.