Yu Cheng Zhu

Oxidative conjugation of catechols with proteins in insect skeletal systems

Abstract Cuticle sclerotization or tanning is a vital process that occurs during each stage of insect development to harden and stabilize the newly secreted exoskeleton. The structural polymers protein and chitin make up the bulk of the cuticle, and chemical interactions between these biopolymers with quinonoid tanning agents are largely responsible for the physical properties of the mature exoskeleton. The oxidative conjugation of catechols with cuticular proteins plays an important role in this metabolism. The main hypothesis for cuticle sclerotization involves the formation of adducts and cross-links between nucleophilic imidazole nitrogens of histidyl residues in the proteins and electrophilic ring or side-chain carbons of ortho-quinones and para-quinone methides derived from the catechols, N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenylethanol. C–N and C–O linkages between these quinone tanning agents and proteins in cuticles from a variety of insects from several orders have been elucidated. cDNAs for both the tyrosinase and laccase types of phenoloxidases that catalyze the cross-linking reactions have been isolated and sequenced. The sequences of laccase cDNAs from two insect species were more similar to fungal laccases than to those from plants. These results provide insights into how insects use structural proteins, catechols, and oxidative enzymes to form catechol–amino acid adducts during sclerotization.

Evidence of field-evolved resistance to Cry1Ac-expressing Bt cotton in Helicoverpa armigera (Lepidoptera: Noctuidae) in northern China

BACKGROUND Evolution of resistance threatens the continued success of transgenic crops expressing insecticidal proteins. One of the key factors for successful resistance management is the timely implementation of monitoring programmes to detect early changes of resistance allele frequency in field populations. F(1)/F(2) screen, dose-response bioassays and field survey were used to monitor resistance to the Cry1Ac-expressing cotton in a field population of Helicoverpa armigera (Hübner), the primary target of transgenic Bt cotton in China. RESULTS Field survey showed an increased trend of egg populations of H. armigera on Bt cotton in the Qiuxian area from 2003 to 2007. By using the F(2) screening procedure, the resistance allele frequency in the Qiuxian (Hebei, China) population of H. armigera collected during 2007 was estimated to be 0.075 (95% CI: 0.053-0.100), which was 12 times greater than that estimated 9 years ago. Dose-response bioassay with the field population collected from the same area showed a significant resistance level (11-fold) to Cry1Ac toxin compared to a laboratory susceptible strain. CONCLUSION This study documented a case of field-evolved resistance in H. armigera after several years of intensive planting of Bt cotton. Proactive tactics must be adopted to prevent further increase of resistance gene frequency in the Qiuxian region.

Molecular cloning of trypsin-like cDNAs and comparison of proteinase activities in the salivary glands and gut of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae)

Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseinase and 90% of BApNAse activities in the salivary glands were inhibited by aprotinin, benzamidine, and PMSF, whereas only 46% azocaseinase and 60% BApNAse activities in the gut were suppressed by benzamidine, leupeptin, and TLCK. The pH optima for azocaseinase activity in salivary glands ranged from 6.2 to 10.6, whereas the pH optima for gut proteinases was acidic for general and alkaline for tryptic proteinases. Zymogram analysis demonstrated that approximately 26-kDa proteinases from salivary glands were active against both gelatin and casein substrates. Three trypsin-like cDNAs, LlSgP2-4, and one trypsin-like cDNA, L1GtP1, were cloned from salivary glands and gut, respectively. Putative trypsin precursors from all cloned cDNAs contained a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included His, Asp, and Ser residues for the catalytic amino acid triad of serine proteinase active sites, residues for the binding pocket, and four pairs of cysteine residues for disulfide bridges. Deduced trypsin-like proteins from LlSgP2, LlSgP3, and LlGtP1 cDNAs shared 98-99% sequence identity with a previously reported trypsin-like precursor, whereas the trypsin-like protein of LlSgP4 shared only 44% sequence identity with all other trypsin-like proteins, indicating multi-trypsin forms are present in L. lineolaris.

Expressed sequence tags from larval gut of the European corn borer (Ostrinia nubilalis): Exploring candidate genes potentially involved in Bacillus thuringiensis toxicity and resistance

BackgroundLepidoptera 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.ResultsWe 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.ConclusionWe 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.

Imidacloprid Susceptibility Survey and Selection Risk Assessment in Field Populations of Nilaparvata lugens (Homoptera: Delphacidae)

Abstract Imidacloprid has been used for many years to control planthopper Nilaparvata lugens (Stål) (Homoptera: Delphacidae) in China. To provide resistance assessment for the national insecticide resistance management program, we collected a total of 42 samples of the planthoppers from 27 locations covering eight provinces to monitor their dose responses and susceptibility changes to imidacloprid over an 11-yr period (1996–2006). Results showed that most field populations maintained susceptibility from 1996 to 2003 except for a population from Guilin, Guangxi, in 1997, which showed a low level of resistance to imidacloprid. However, surveys conducted in 2005 indicated that 16 populations from six provinces quickly developed resistance with resistance ratios ranging from 79 to 811. The data collected in 2006 revealed that the resistance levels in 12 populations collected from seven different provinces decreased slightly (RR = 107–316), except the Tongzhou population (Jiangsu Province), which developed 625-fold resistance. Dominant and intensive use of imidacloprid in a wide range of rice, Oryza savita L., growing areas might be a driving force for the resistance development. Migration of the insect also significantly boosted the resistance levels due to extensive and intensive use of imidacloprid in emigrating areas and continuous postmigration sprays of the chemical. In addition, laboratory resistance selection using imidacloprid showed that resistance ratio increased to 14-fold after 27 generations, suggesting that quick resistance development might be associated with more frequent applications of the insecticide in recent years.

Using an F2 screen to monitor frequency of resistance alleles to Bt cotton in field populations of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

BACKGROUND In an effort to control the most devastating cotton pest, Helicoverpa armigera (Hübner), in Qiuxian County (Hebei, China), Bt cotton has been planted almost exclusively since 1998 in this area. Owing to the high insecticidal selection pressures in this region, monitoring of Bt resistance in H. armigera is necessary so that proactive actions can be implemented before field control measures fail. From 2003 to 2005, an F(2) screen was conducted in order to monitor Bt resistance in H. armigera populations collected from this area. RESULTS The F(2) screen showed that 15 out of 278 isofemale lines carried resistance alleles to Bt cotton. The resistance allele frequency in field populations of H. armigera ranged from 0.0119 to 0.0297, with an overall frequency of 0.0146 and a 95% confidence interval of 0.0084-0.0225 for the 3 year period. This value is greater than the value reported from 1999 (P < 0.05). CONCLUSION A fluctuating but overall increase in resistance allele frequency was detected in the field populations of H. armigera in Qiuxian County from 1999 to 2005. To prevent further increases in Bt resistance frequency in this pest, it is necessary to introduce Bt cotton expressing multiple Bt toxins and integrate this technology with other tactics for management of this key pest.

Major putative pesticide receptors, detoxification enzymes, and transcriptional profile of the midgut of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae).

Insecticide resistance mechanisms, including those for Cry proteins (Bt), in Heliothis virescens are not well understood. Sequencing of midgut transcriptomes may facilitate the discovery of the genes responsible for resistance development. In this study, a total of 5856 Sanger sequences were obtained and assembled to 1687 contigs (464) and singletons (1233) with average length of 507 bp. Blast similarity search showed that 1372 cDNAs from this study matched different genes or cDNAs in the GenBank and other sequence databases. Blast2go annotation identified 611 highly similar proteins with metabolic and cellular processes as major biological functions and catalytic activity and binding as major molecular functions. At least 143 contigs and singletons were associated with pesticide activation, detoxification, and resistance development. These cDNAs, with average length of 601 bp, matched nine groups of pesticide resistance related genes. At least 80 cDNAs coded for Bt resistance related enzymes and potential receptors, including 58 proteinases, 4 cadherins, 13 aminopeptidase, and 5 alkaline phosphatases. Other putative detoxification enzymes included 20 cytochrome P450 oxidases, 11 glutathione S-transferases, 9 esterases, 8 sodium channels, and 15 cytochrome oxidases. Of the 143 contigs and singletons, 111 cDNA sequences seemed to be new resistance candidate gene transcripts in GenBank because they either priorly matched resistance candidate cDNAs of other species, or had low sequence identity with those previously sequenced from H. virescens. This study provides a foundation for future research to develop a gut-specific DNA microarray for analysis of the global changes of gene expression in response to biological and chemical pesticides. Future development resistance management strategies could benefit from this study and help continue research to identify key genes targetable by classic and novel approaches.

Spray Toxicity and Risk Potential of 42 Commonly Used Formulations of Row Crop Pesticides to Adult Honey Bees (Hymenoptera: Apidae)

ABSTRACT To combat an increasing abundance of sucking insect pests, >40 pesticides are currently recommended and frequently used as foliar sprays on row crops, especially cotton. Foraging honey bees may be killed when they are directly exposed to foliar sprays, or they may take contaminated pollen back to hives that maybe toxic to other adult bees and larvae. To assess acute toxicity against the honey bee, we used a modified spray tower to simulate field spray conditions to include direct whole-body exposure, inhalation, and continuing tarsal contact and oral licking after a field spray. A total of 42 formulated pesticides, including one herbicide and one fungicide, were assayed for acute spray toxicity to 4-6-d-old workers. Results showed significantly variable toxicities among pesticides, with LC50s ranging from 25 to thousands of mg/liter. Further risk assessment using the field application concentration to LC1 or LC99 ratios revealed the risk potential of the 42 pesticides. Three pesticides killed less than 1% of the worker bees, including the herbicide, a miticide, and a neonicotinoid. Twenty-six insecticides killed more than 99% of the bees, including commonly used organophosphates and neonicotinoids. The remainder of the 13 chemicals killed from 1–99% of the bees at field application rates. This study reveals a realistic acute toxicity of 42 commonly used foliar pesticides. The information is valuable for guiding insecticide selection to minimize direct killing of foraging honey bees, while maintaining effective control of field crop pests.

Survey of Susceptibilities to Monosultap, Triazophos, Fipronil, and Abamectin in Chilo suppressalis (Lepidoptera: Crambidae)

Abstract To provide a foundation for national resistance management of the Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), a study was carried out to determine dose-response and susceptibility changes over a 5-yr period in the insect from representative rice, Oryza sativa L., production regions. In total, 11 populations were collected from 2002 to 2006 in seven rice-growing provinces in China, and they were used to examine their susceptibility levels to monosultap, triazophos, fipronil, and abamectin. Results indicated that most populations had increased tolerance to monosultap. Several field populations, especially those in the southeastern Zhejiang Province, were highly or extremely highly resistant to triazophos (resistance ratio [RR] = 52.57–899.93-fold), and some populations in Anhui, Jiangsu, Shanghai, and the northern rice regions were susceptible or had a low level of resistance to triazophos (RR = 1.00–10.69). Results also showed that most field populations were susceptible to fipronil (RR < 3), but the populations from Ruian and Cangnan, Zhejiang, in 2006 showed moderate levels of resistance to fipronil (RR = 20.99–25.35). All 11 field populations collected in 2002–2006 were susceptible to abamectin (RR < 5). The tolerance levels in the rice stem borer exhibited an increasing trend (or with fluctuation) over a 5-yr period for different insecticides, and they reached a maximal level in 2006 for all four insecticides. Analysis of regional resistance ratios indicated that the history and intensity of insecticide application are the major driving forces for the resistance evolution in C. suppressalis. Strategic development of insecticide resistance management also is proposed.

Electrical penetration graph evidence that pymetrozine toxicity to the rice brown planthopper is by inhibition of phloem feeding.

BACKGROUND Pymetrozine is a valuable novel insecticide for control of sucking insects, including the brown planthopper Nilaparvata lugens (Stål), one of the most serious pests on rice. This study was conducted to elucidate the action mechanisms of pymetrozine on the feeding behaviour of the planthopper. RESULTS The activity test showed that pymetrozine primarily functioned as an antifeedant that caused starvation and death in N. lugens, rather than having neurotoxicity. Pymetrozine-treated insects died at a significantly slower speed than insects treated with starvation. Electrical penetration graph (EPG) data indicated that pymetrozine significantly increased the duration of non-probing periods and had a strong inhibition to phloem ingestion. The inhibition was strongly dose dependent, resulting in a complete suppression of the activity in the phloem region when the pymetrozine concentration was increased to 400 mg L(-1) . Starvation caused by inhibition of phloem ingestion might be a major toxicity mechanism of pymetrozine. EPG data also showed that pymetrozine had no significant effect on stylet movement and duration of xylem sap ingestion. CONCLUSION The study revealed that pymetrozine disturbed the feeding behaviour of N. lugens mainly by increasing the non-probe period and inhibiting phloem ingestion. The inhibition resulted in a slow death similar to starvation.