Selection,effective dominance,and completeness of Cry1A.105/Cry2Ab2 dual-protein resistance in Helicoverpa zea(Boddie)(Lepidoptera:Noctuidae)

In the U.S.,Helicoverpa zea(Boddie)is a major pest targeted by both transgenic maize and cotton expressing Bacillus thuringiensis(Bt)proteins.Resistance of insect to Bt maize and cotton containing cry1A and cry2A genes has widely occurred in the U.S.In this study,two trials were performed to investigate larval survival and development of a Cry1A.105/Cry2Ab2 dual-protein resistant(VT2P-RR),a susceptible,and an F1 heterozygous(VT2P-RS)populations of H.zea on ears of nine Bt and three non-Bt maize hybrids.The Bt maize hybrids evaluated represent five common pyramided traits expressing two or three of the Cry1A.105,Cry1Ab,Cry1F,Cry2Ab2,and Vip3Aa20 proteins.In the laboratory,neonates of the three H.zea populations were inoculated on silks of ears collected from maize at R1-R2 plant stages;and larval survivorship was checked 10 d after neonate release.All three insect populations survived normally on non-Bt maize ears.Varied numbers of VT2P-RR and VT2P-RS survived on ears of Cry1A.105/Cry2Ab2 maize,while all larvae of the three populations died or could not develop on ears of Vip3Aa20-expressing maize.The results demonstrated that the dual-protein resistant H.zea was not cross-resistant to Vip3Aa20-expressing maize,and thus traits with vip3Aa20 gene should be effective to manage Cry1A.105/Cry2Ab2-resistant H.zea.The resistance in VT2P-RR was determined to be incomplete on Cry1A.105/Cry2Ab2 maize.The effective dominance levels varied greatly,from recessive to incompletely dominant,depending on maize hybrids and trials,suggesting that proper selection of maize hybrids could be important for mitigating the Cry1A.105/Cry2Ab2 resistance.The data generated should aid in modeling multiple-protein Bt resistance in H.zea.

Relative fitness of susceptible and Cry1A.105/Cry2Ab2-single-/dual-protein-resistant Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) on non-Bt diet and a diet containing a low concentration of two proteins

Helicoverpa zea(Boddie)is a destructive agricultural pest species that is targeted by both Bacillus thuringiensis(Bt)maize and cotton in the United States.Cry1A.105 and Cry2Ab2 are two Bt proteins expressed in a widely planted maize event MON 89034.In this study,two tests(Test-Ⅰ and Test-Ⅱ)were conducted to evaluate the relative fitness of Bt-susceptible and-resistant H.zea on non-Bt diet(Test-Ⅰ and Test-Ⅱ)and a diet containing a mix of Cry1A.105 and Cry2Ab2 at a low concentration(Test-Ⅱ only).Insect populations evaluated in Test-Ⅰ were two Bt-susceptible strains and three Bt-resistant strains(a single-protein Cry1A.105-,a single-protein Cry2Ab2-,and a dual-protein Cry1A.105/Cry2Ab2-resistant strains).Test-Ⅱ analyzed the same two susceptible strains,three backcrossed-and-reselected Cry1A.105/Cry2Ab2-single-/dual-protein-resistant strains,and three F1 heterozygous strains.Measurements of life table parameters showed that neither the single-nor dual-protein Cry1A.105/Cry2Ab2 resistance in H.zea was associated with fitness costs under the test conditions.The single Cry protein resistances at a concentration of a mix of Cry1A.105 and Cry2Ab2 that resulted in a zero net reproductive rate for the two susceptible strains were functionally incomplete recessive or codominant,and the dual-protein resistance was completely dominant.The lack of fitness costs could be a factor contributing to the rapid revolution of resistance to the Cry proteins in this species.Data generated from this study should aid our understanding of Cry protein resistance evolution and help in refining IRM programs for H.zea.

Resistance of the fall armyworm,Spodoptera frugiperda,to transgenic Bacillus thuringiensis Cry1 F corn in the Americas:lessons and implications for Bt corn IRM in China

The fall armyworm,Spodoptera frugiperda(J.E.Smith),is a major target pest of Bt crops(e.g.,corn,cotton,and soybean)in North and South America.This pest has recently invaded Africa and Asia including China and the invasion has placed a great threat to the food security in many countries of these two continents.Due to the extensive use of Bt crops,practical resistance of S.frugiperda to Cry IF corn(TC 1507)with field control problems has widely occurred in Puerto Rico,Brazil,Argentina,and the mainland United States.Analyzing data generated from decade-long studies showed that several factors might have contributed to the wide development of the resistance.These factors include(1)limited modes of action of Bt proteins used in Bt crops;(2)cross-resistance among Cryl proteins;(3)use of nonhigh dose Bt crop traits;(4)that the resistance is complete on Bt corn plants;(5)abundant in initial Cry IF resistance alleles;and(6)lack of fitness costs/recessive fitness costs of the resistance.The long-term use of Bt crop technology in the Americas suggests that Bt corn can be an effective tool for controlling S.frugiperda in China.IRM programs for Bt corn in China should be as simple as possible to be easily adopted by small-scale growers.The following aspects may be considered in its Bt corn IRM programs:(1)use of only"high dose^traits for both S.frugiperda and stalk borers;(2)developing and implementing a combined resistance monitoring program;(3)use“gene pyramiding”as a primary IRM strategy;and(4)if possible,Bt corn may not be planted in the areas where S.frugiperda overwinters.Lessons and experience gained from the global long-term use of Bt crops should have values in improving IRM programs in the Americas,as well as for a sustainable use of Bt corn technology in China.

Detection and monitoring of insect resistance to transgenic Bt crops

Transgenic crops expressing Bacillus thuringiensis （Bt） endotoxins havebecome one of the most important tools for managing corn and cotton insect pests in the US and other countries. The widespread adoption of transgenic Bt crops could place a high degree of selection pressure on the target insect populations and accelerate development of resistance, raising concerns about the long-term durability of Bt plants as an effective pest management tool. Conservation of Bt susceptibility in insects has become one of the most active research areas in modern agriculture. One of the key factors for a successful Bt resistance management plan is to have a cost-effective monitoring system that can provide information on. （i） the initial Bt resistance allele frequencies at low levels in field insect populations; and （ii） early shifts in Bt resistance allele frequencies so that proactive measures for managing resistance can be deployed well before field control failures. Developing such a monitoring program has been difficult because： （i） resistance traits that occur at very low frequencies are hard to detect; （ii） many factors affect the sensitivity and accuracy of a Bt resistance monitoring program; and （iii） monitoring resistance is costly. Several novel methods for detecting Bt resistance alleles developed during the last decade have made a cost-effective monitoring system possible. Future studies should focus on how to improve and standardize the methodologies for insect sampling and Bt resistance detection.

Effectiveness of spinosad against seven major storedgrain insects on corn

In January 2005, the United States Environmental Protection Agency registered spinosad as a stored grain protectant. No referenced data on the efficacy of spinosad on corn in suppressing major stored-grain insects have been published. In this paper, we evaluated the efficacy of spinosad against seven major stored-grain insects on shelled corn in the laboratory. Insect species tested were the red flour beetle, Tribolium castaneum （Jacquelin duVal）; rusty grain beetle, Cryptolestesferrugineus （Stephens）; lesser grain borer, Rhyzopertha dominica （F.）; sawtoothed grain beetle, Oryzaephilus surinamensis （L.）; rice weevil, Sitophilus oryzae （L.）; maize weevil, Sitophilus zeamais （Motschulsky）; and Indian meal moth, Plodia interpunctella （Htibner）. Corn kernels were treated with spinosad at 0, 0. 1, 0.5, 1, and 2 active ingredient （a.i.） mg/kg for controlling the seven species. Beetle adults or P. interpunctella eggs were introduced into each container holding 100 g of untreated or insecticide-treated corn. The seven insect species survived well on the control treatment, produced 28 to 336 progeny, and caused significant kernel damage after 49 days. On spinosad-treated corn, adult mortality of C. ferrugineus, R. dominica, 0. surinamensis, S. oryzae, and S. zeamais was 〉 98% at 1 and 2 mg/kg after 12 days. Spinosad at≥ 0.5 mg/kg completely suppressed egg-to-larval survival after 21 days and egg-to-adult emergence of P. interpunctella after 49 days, whereas 16% T. castaneum adults survived at 1 mg/kg after 12 days. Spinosad at 1 or 2 mg/kg provided complete or near complete suppression of progeny production and kernel damage of all species after 49 days. Our results indicate that spinosad at the current labeled rate of 1 mg/kg is effective against the seven stored-grain insect pests on corn.

Genomic and transcriptomic analysis unveils population evolution and development of pesticide resistance in fall armyworm Spodoptera frugiperda

The fall armyworm(FAW),Spodoptera frugiperda,is a destructive pest native to America and has recently become an invasive insect pest in China.Because of its rapid spread and great risks in China,understanding of FAW genetic background and pesticide resistance is urgent and essential to develop effective management strategies.Here,we assembled a chromosome-level genome of a male FAW(SFynMstLFR)and compared re-sequencing results of the populations from America,Africa,and China.Strain identification of 163 individuals collected from America,Africa and China showed that both C and R strains were found in the American pop-ulations,while only C strain was found in the Chinese and African populations.Moreover,population geno-mics analysis showed that populations from Africa and China have close relationship with significantly genetic differentiation from American populations.Taken toge-ther,FAWs invaded into China were most likely origi-nated from Africa.Comparative genomics analysis displayed that the cytochrome p450 gene family is extremely expanded to 425 members in FAW,of which 283 genes are specific to FAW.Treatments of Chinese populations with twenty-three pesticides showed the variant patterns of transcriptome profiles,and several detoxification genes such as AOX,UGT and GST spe-cially responded to the pesticides.These findings will be useful in developing effective strategies for manage-ment of FAW in China and other invaded areas.

Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in CrylAb-susceptible and CrylAb-resistant strains of sugarcane borer, Diatraea saccharalis

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is re- sponsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the CrylAb resistance in D. saccharalis is associ- ated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry 1 Ab-susceptible （Cry 1 Ab- S S） and Cry 1 Ab-resistant （Cry 1 Ab-RR） strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin- like proteinases were sequenced from CrylAb-SS and CrylAb-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all fimctional motifs, includ- ing signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between CrylAb-SS and CrylAb-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between CrylAb-SS and CrylAb-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry 1 Ab-SS and CrylAb-RR strains, but the difference was not statistically significant. Data suggest that the development of CrylAb resistance in D. saccharalis was not significantly as- sociated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.

Performance of Bt-susceptible and -heterozygous dual-gene resistant genotypes of Spodoptera frugiperda (J.E.Smith) (Lepidoptera:Noctuidae) in seed blends of non-Bt and pyramided Bt maize

A seed blend refuge has been implemented in the U.S.Corn Belt for Bt maize resistance management.The fall armyworm,Spodoptera frugiperda(J.E.Smith),is a target pest of Bt maize in the Americas.The larvae of this pest are mobile,which may affect the efficacy of seed blend refuges.In this study,field and greenhouse trials were conducted to determine the performance of Bt-susceptible(aabb)and-heterozygous dual-gene-resistant(AaBb)genotypes of S.frugiperda in seed blends of non-Bt and pyramided Bt maize.Three field trials evaluated larval survival,larval growth,and plant injury with aabb in seed blends of Bt maize expressing Cry1A.105/Cry2Ab2/Vip3A with 0–30%non-Bt seeds.Greenhouse tests investigated the performance of aabb and AaBb in seed blends of Cry1A.105/Cry2Ab2 with 0–30% non-Bt seeds.In pure non-Bt maize plots,after 9–13 d of neonates being released on the plants,0.39 and 0.65 larvae/plant survived with leaf injury ratings of 4.7 and 5.9(Davis's 1–9 scale)in the field and greenhouse,respectively.In contrast,live larvae and plant injury were virtually not observed on Bt plants across all planting patterns.Larval occurrence and plant injury by aabb on non-Bt plants were similar between seed blends and pure non-Bt plantings,suggesting that the blended refuges could provide an equivalent susceptible population as structured refuge under the test conditions.In the greenhouse,the two insect genotypes in seed blends performed similarly,indicating that the seed blends did not provide more favorable conditions for AaBb over aabb.The information generated from this study should be useful in managing S.frugiperda and evaluating if send blends could be suitable refuge options for Bt resistance management in the regions where the insect is a primary target pest.

Susceptibility of field populations of sugarcane borer from non-Bt and Bt maize plants to five individual Cry toxins

Sugarcane borer, Diatraea saccharalis （E）, is a major target of transgenic maize expressing Bacillus thuringiensis （Bt） proteins in South America and the US mid- south region. Resistance development in target pest populations is a major threat to the sustainable use of Bt crops. In our field trials in 2009, a significant number of live borers and plant injury fi＇om D. saccharalis were observed in an experimental SmartStaxTM maize line. The objective of this study was to assess the relative susceptibility of two field popula- tions ofD. saccharalis collected from non-Bt and Bt maize plants containing SmartStaxTM traits to five individual Cry proteins. The five Bt proteins included two proteins （Cry 1A. 105 and Cry2Ab2） that were expressed in SmartStaxTM maize plants and three other common Bt proteins （Cry 1Aa, Cry 1Ab and Cry l Ac） that were not produced in SmartStaxTM. Larval mortality and growth inhibition on Bt diet of the fourth generation after field collections were evaluated 7 days after release of neonates on the diet surface. The laboratory bioassays showed that 50% lethal concentration （LCs0） values for CrylA. 105 and Cry2Ab2 for the population originated from Bt plants were 3.55- and 1.34-fold greater, respectively, than those of the population collected from non-Bt plants. In contrast, relative to the popula- tion from non-Bt plants, the LCs0 of the population sampled from Bt plants were 3.85-, 2.5- and 1.64-fold more sensitive to CrylAa, CrylAb and CrylAc, respectively. The re- sults did not provide clear evidence to conclude that the observed field survival of D. saccharalis on Bt plants was associated with increased levels of resistance.

Lack of repellency of three commercial ultrasonic devices to the German cockroach （Blattodea： Blattellidae）

Three commercial ultrasonic devices （A, B, and C） were tested for their ability to repel the German cockroach, Blattella germanica （L.） （Blattodea： Blattellidae）, in Plexiglas enclosures. Device A generated peak frequencies at 26 kHz and 34 kHz, and produced a 95 ±1 dB sound pressure level （SPL） at 50 cm distance （0 dB = 20 log 10[20 μPa/ 20 μPa]）. Device B generated peak frequencies at 27 kHz and 35 kHz, and produced a 92 ± 4 dB SPL. Device C generated a wide range of frequencies between 28-42 kHz and produced an 88 ±2 dB SPL. Ultrasound from any of the three devices did not demonstrate sufficient repelling ability against the German cockroach in the tests. The result failed to provide evidence that ultrasonic technology could be used as an effective pest management tool to repel or eliminate the German cockroach.