Copyright (c) 2009 University of Nebraska - Lincoln All rights reserved. http://digitalcommons.unl.edu/entomologyfacpub Recent documents in Faculty Publications: Department of Entomology en-us Wed, 21 Oct 2009 23:18:43 PDT 3600 http://digitalcommons.unl.edu/entomologyfacpub/169 http://digitalcommons.unl.edu/entomologyfacpub/169 Tue, 20 Oct 2009 11:39:38 PDT BACKGROUND: The high dose plus refuge is one of the major components of the resistance management plan mandated for transgenic corn expressing Cry toxins from Bacillus thuringiensis Berliner (Bt) that targets the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae). This strategy was based on assumptions such as functional recessive inheritance, which has not been previously tested for O. nubilalis. The authors used a field-derived resistant strain of O. nubilalis to define the nature of resistance to Cry1Ab toxin by examining the inheritance and on-plant survival of susceptible and resistant insects and their F1 progeny. RESULTS: The resistant strain exhibited >800-fold resistance to Cry1Ab. Resistance was primarily autosomal and controlled by more than one locus or multiple alleles at one locus. The degree of dominance D calculated on the basis of LC50 values was −0.45 (h′ = 0.27), indicating that resistance was incompletely recessive. No survivors were found on vegetative-stage Bt corn, although both resistant larvae and their F1 progeny were able to survive on reproductive corn 15 days after infestation. CONCLUSIONS: A field derived O. nubilalis strain exhibited high levels of resistance to Cry1Ab and survived on transgenic corn by feeding on tissues with low Cry1Ab expression. The Cry1Ab resistance was primarily autosomal, incompletely recessive and polygenic. Tissue and on-plant survival data indicated that dominance varies depending on plant stage. Andre L. B. Crespo http://digitalcommons.unl.edu/entomologyfacpub/168 http://digitalcommons.unl.edu/entomologyfacpub/168 Tue, 20 Oct 2009 11:19:12 PDT To examine how resistance to Bacillus thuringiensis (Bt) toxins influences movement and survival of European corn borer (Ostrinia nubilalis [Hübner]) neonates, the responses of Cry1Ab-resistant , -susceptible, and hybrid (F1) larvae were examined using two different techniques. First, using an automated videotracking system, aspects of O. nubilalis movement were quantified in the presence of artificial diet incorporating 50% non-Bt or insect-resistant Cry1Ab maize tissue. Second, O. nubilalis dispersal and survival were measured 48-72 h after hatching on a Cry1Ab maize plant surrounded by two non-Bt maize plants. Video tracking indicated the presence of Cry1Ab tissue increased the total distance moved (m), time moving (%), and time away from the diet (%) for O. nubilalis while decreasing meander (degrees/cm). However, resistant larvae showed reduced movement and increased meander (≈localized searching) relative to susceptible or hybrid larvae on diet incorporating Cry1Ab tissue. Conversely, when placed onto Cry1Ab maize plants, resistant larvae were more likely than susceptible O. nubilalis to disperse onto adjacent non-Bt plants. The difference in on-plant dispersal seems to reflect greater survival after toxin exposure for resistant larvae rather than increased activity. These results suggest that simplified 'Petri dish' tests may not be predictive of larval movement among non-Bt and insect-resistant Bt maize plants. Because models of O. nubilalis resistance evolution incorporate various movement and survival parameters, improved data for on-plant behavior and survival of Bt- resistant , -susceptible, and hybrid larvae should help preserve the efficacy of transgenic insect-resistant maize. Jarrad R. Prasifka http://digitalcommons.unl.edu/entomologyfacpub/167 http://digitalcommons.unl.edu/entomologyfacpub/167 Thu, 24 Sep 2009 13:42:34 PDT Background: Lepidoptera represents more than 160,000 insect species which include some of the most devastating pests of crops, forests, and stored products. However, the genomic information on lepidopteran insects is very limited. Only a few studies have focused on developing expressed sequence tag (EST) libraries from the guts of lepidopteran larvae. Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt) toxins, and for discovering new targets for novel toxins for use in pest management. This study analyzed the ESTs generated from the larval gut of the European corn borer (ECB, Ostrinia nubilalis), one of the most destructive pests of corn in North America and the western world. Our goals were to establish an ECB larval gut-specific EST database as a genomic resource for future research and to explore candidate genes potentially involved in insect-Bt interactions and Bt resistance in ECB. Results: We constructed two cDNA libraries from the guts of the fifth-instar larvae of ECB and sequenced a total of 15,000 ESTs from these libraries. A total of 12,519 ESTs (83.4%) appeared to be high quality with an average length of 656 bp. These ESTs represented 2,895 unique sequences, including 1,738 singletons and 1,157 contigs. Among the unique sequences, 62.7% encoded putative proteins that shared significant sequence similarities (E-value ≤ 10-3)with the sequences available in GenBank. Our EST analysis revealed 52 candidate genes that potentially have roles in Bt toxicity and resistance. These genes encode 18 trypsin-like proteases, 18 chymotrypsin-like proteases, 13 aminopeptidases, 2 alkaline phosphatases and 1 cadherin-like protein. Comparisons of expression profiles of 41 selected candidate genes between Cry1Ab-susceptible and resistant strains of ECB by RT-PCR showed apparently decreased expressions in 2 trypsin-like and 2 chymotrypsin-like protease genes, and 1 aminopeptidase genes in the resistant strain as compared with the susceptible strain. In contrast, the expression of 3 trypsin- like and 3 chymotrypsin-like protease genes, 2 aminopeptidase genes, and 2 alkaline phosphatase genes were increased in the resistant strain. Such differential expressions of the candidate genes may suggest their involvement in Cry1Ab resistance. Indeed, certain trypsin-like and chymotrypsin-like proteases have previously been found to activate or degrade Bt protoxins and toxins, whereas several aminopeptidases, cadherin-like proteins and alkaline phosphatases have been demonstrated to serve as Bt receptor proteins in other insect species. Conclusion: We developed a relatively large EST database consisting of 12,519 high-quality sequences from a total of 15,000 cDNAs from the larval gut of ECB. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Our work provides a foundation for future research to develop an ECB gut-specific DNA microarray which can be used to analyze the global changes of gene expression in response to Bt protoxins/toxins and the genetic difference(s) between Bt- resistant and susceptible strains. Furthermore, we identified 52 candidate genes that may potentially be involved in Bt toxicity and resistance. Differential expressions of 15 out of the 41 selected candidate genes examined by RT-PCR, including 5 genes with apparently decreased expression and 10 with increased expression in Cry1Ab-resistant strain, may help us conclusively identify the candidate genes involved in Bt resistance and provide us with new insights into the mechanism of Cry1Ab resistance in ECB. Chitvan Khajuria http://digitalcommons.unl.edu/entomologyfacpub/166 http://digitalcommons.unl.edu/entomologyfacpub/166 Mon, 14 Sep 2009 11:01:47 PDT A major assumption of the high-dose/refuge strategy proposed for insect resistance management strategies for transgenic crop plants that express toxins from Bacillus thuringiensis is that resistance traits that evolve in pest species will be recessive. The inheritance of Cry1F resistance and larval survival on commercially available Cry1F corn hybrids were determined in a laboratory-selected strain of European corn borer, Ostrinia nubilalis (Hu¨ bner), displaying more than 3000-fold resistance to Cry1F. Concentration-response bioassays of reciprocal parental crosses indicated that the resistance is autosomal and recessive. Bioassays of the backcross of the F1 generation with the selected strain were consistent with the hypothesis that a single locus, or a set of tightly linked loci, is responsible for the resistance. Greenhouse experiments with Cry1F-expressing corn hybrids indicated that some resistant larvae survived the high dose of toxin delivered by Cry1F-expressing plants although F1 progeny of susceptible by resistant crosses had fitness close to zero. These results provide the first direct evidence that the high dose/refuge strategy currently in place to manage resistance in Cry1F-expressing corn is appropriate. E. J. G. Pereira http://digitalcommons.unl.edu/entomologyfacpub/165 http://digitalcommons.unl.edu/entomologyfacpub/165 Mon, 14 Sep 2009 10:54:57 PDT Agricultural research in the 21st century has become a collaborative effort. Research on crop pests like Spodoptera frugiperda (J.E. Smith), commonly known as the fall armyworm (FAW), can involve international collaboration because it is a pest not only in the southern United States, but also in Latin and South America. Our interest to study the genetic variation of 24 subpopulations of FAW from the southern United States, Mexico, Puerto Rico, Brazil and Argentina required insect collection procedures that preserve the integrity of DNA for molecular genetic analysis. The samples were collected primarily from maize (Zea mays L.), but also included outliers collected from pigweed (Amaranthus sp.), Royal Paulownia (Paulownia tomentosa (Thunb.) Sieb. and Zucc. ex Steud.), lemon tree (Citrus limon (L.) Burm) and Bermuda grass (Cynodon dactylon (L.) Pers.). A common insect preservation technique is to place individual insects in 95% ethanol (ETOH). However, various regulations for shipping and the size of this insect often prevent large sample sizes stored in ETOH from being imported. Genomic DNA from samples preserved in 95% ETOH, lyophilized and fresh insects was extracted and evaluated using DNA quantification and polymerase chain reaction-amplified fragment length polymorphism (PCR-AFLP). All three treatments yielded high-quality/high molecular weight (c. 70- 150 µg) DNA. No differences in quality of genomic DNA for AFLP analysis were observed. Lyophilization is a reliable tool to preserve FAW samples, which yields highquality DNA for use in AFLP genetic analysis. Pete L. Clark http://digitalcommons.unl.edu/entomologyfacpub/164 http://digitalcommons.unl.edu/entomologyfacpub/164 Mon, 14 Sep 2009 10:44:59 PDT The purpose of this research was to evaluate the genetic similarity and structure of the fall armyworm, Spodoptera frugiperda (J.E. Smith), populations associated with maize and cotton crops in Brazil using amplified fragment length polymorphisms. Mean genetic similarity among populations was 0.45. The unweighted pair group method with arithmetic mean analysis dendrograms did not separate populations of S. frugiperda into clusters related to the host plant in which the insects were collected. No genetic variation was observed among maize and cotton populations of S. frugiperda, suggesting that the same populations are injuring both crops in Brazil. This research validates the need for stewardship of crop-protection methods for managing S. frugiperda to reduce the incidence of pesticide resistance, due to the spatial and temporal overlapping of maize and cotton crops in some regions in Brazil. S. Martinelli http://digitalcommons.unl.edu/entomologyfacpub/163 http://digitalcommons.unl.edu/entomologyfacpub/163 Wed, 29 Jul 2009 11:55:36 PDT Between 2005 and 2007, 656 new county records and eight new state records were obtained for Nebraska's short-horned grasshoppers (Acrididae and Romaleidae). This represents an increase of 30% in the known grasshopper county records in Nebraska. Based on the large number of new distribution records, it appears that the previous distributions of Nebraska's grasshoppers greatly underestimated the actual distributions. The new grasshopper state records were: Boopedon gracile, Chloealtis abdominalis, Encoptolophus subgracilis, Melanoplus bispinosus, Melanoplus borealis, Melanoplus punctulatus, Paratylotropidia brunneri, and Psinidia fenestralis were obtained. These new records will aid in both grasshopper monitoring for pest outbreaks and in conservation of rare and declining species which are often habitat specialists associated with prairie ecosystems. Mathew Brust http://digitalcommons.unl.edu/entomologyfacpub/162 http://digitalcommons.unl.edu/entomologyfacpub/162 Thu, 09 Jul 2009 08:22:02 PDT The susceptibility of the egg, pupa, and adult of Haematobia irritans (L.) (Diptera: Muscidae) to isolates of the fungi Metarhizium anisopliae (Metsch.) Sor., Beauveria bassiana (Bals.) Vuill., and Paecilomyces fumosoroseus (Wize) Brown and Smith, was evaluated under laboratory conditions. Groups of 20 eggs than 4 h old, pupae less than 48h old and adults were sprayed with a conidial suspension of each isolate. Eggs, pupae and adults of horn fly were susceptible to these entomopathogenic fungi. For treated eggs, the isolates Ma3, Ma 15, Ma25, Pfr1, and Pfr8 reduced adult emergence to 3.8% to 6.3% in comparison with the control (72%). The mortality of pupae infected by the isolates Ma2, Ma25, and Pfr10 ranged between 50% and 71.3%. Mortality of adults after treatment with the isolates Ma6, Ma 10, Ma 14, Ma 15, Pfr 1, Pfr 9, Pfr 10, Pfr 11, and Pfr12 were higher than 90%. The isolate Ma6 produced the lowest LC50 against adult horn flies (8.08 x102conidia/ml). These findings supported the hypotheses that isolates of M. anisopliae, and P. fumosoroseus are pathogenic against the different biological stages of horn flies by reducing adult emergence when applied on groups of eggs and pupae, and producing mortality when applied to adults. C. A. Angel-Sahagún http://digitalcommons.unl.edu/entomologyfacpub/161 http://digitalcommons.unl.edu/entomologyfacpub/161 Thu, 09 Jul 2009 08:22:01 PDT Cry1Ab toxin binding analysis was performed to determine whether resistance in laboratory-selected Ostrinia nubilalis strains is associated with target site alteration. Brush border membrane vesicles were prepared using dissected midguts from late instars of susceptible and resistant strains (Europe-R and RSTT) of O. nubilalis. Immunoblot analysis indicated that three different proteins bound to Cry1Ab toxin and were recognized by an anticadherin serum. In a comparison of resistant and susceptible strains, reduced Cry1Ab binding was apparent for all three bands corresponding to cadherin-like proteins in the Europe-R strain, while reduced binding was apparent in only one band for the RSTT strain. Real-time analysis of Cry1Ab binding to gut receptors using surface plasmon resonance suggested slight differences in affinity in both resistant strains. Additional binding analysis was conducted using 125I-labeled Cry1Ab, Cry1Ac, and Cry1Aa. Slight differences were again observed between the resistant and susceptible strains for Cry1Ab binding. However, when binding of 125I-labeled Cry1Aa was tested, a 10-fold reduction in the concentration of binding sites was observed in the Europe-R strain. Expression of the O. nubilalis cadherin gene was similar in both the resistant and susceptible strains and did not account for differences in binding. In combination, the results of the present work suggest that differences in susceptibility to Cry1A toxins in the Europe-R strain of O. nubilalis are associated with altered receptor binding, although the precise nature of this mechanism is still uncertain. Herbert A. A. Siqueira http://digitalcommons.unl.edu/entomologyfacpub/160 http://digitalcommons.unl.edu/entomologyfacpub/160 Tue, 16 Jun 2009 09:08:55 PDT To delay evolution of insect resistance to transgenic crops producing Bacillus thuringiensis (Bt) toxins, nearby ''refuges'' of host plants not producing Bt toxins are required in many regions. Such refuges are expected to be most effective in slowing resistance when the toxin concentration in Bt crops is high enough to kill all or nearly all insects heterozygous for resistance. However, Bt corn, Zea mays, introduced recently does not meet this ''high-dose'' criterion for control of western corn rootworm (WCR), Diabrotica virgifera virgifera. A greenhouse method of rearing WCR on transgenic corn expressing the Cry3Bb1 protein was used in which approximately 25% of previously unexposed larvae survived relative to isoline survival (compared to 1-4% in the field). After three generations of full larval rearing on Bt corn (Constant-exposure colony), WCR larval survival was equivalent on Bt corn and isoline corn in greenhouse trials, and the LC50 was 22-fold greater for the Constant-exposure colony than for the Control colony in diet bioassays with Cry3Bb1 protein on artificial diet. After six generations of greenhouse selection, the ratio of larval recovery on Bt corn to isoline corn in the field was 11.7-fold greater for the Constant-exposure colony than the Control colony. Removal from selection for six generations did not decrease survival on Bt corn in the greenhouse. The results suggest that rapid response to selection is possible in the absence of mating with unexposed beetles, emphasizing the importance of effective refuges for resistance management. Lisa N. Meihls