Shuxiong Bai

Male- and female-biased gene expression of olfactory-related genes in the antennae of Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae)

The Asian corn borer (ACB), Ostrinia furnacalis (Guenée), is a destructive pest insect of cultivated corn crops, for which antennal-expressed receptors are important to detect olfactory cues for mate attraction and oviposition. Few olfactory related genes were reported in ACB, so we sequenced and characterized the transcriptome of male and female O. furnacalis antennae. Non-normalized male and female O. furnacalis antennal cDNA libraries were sequenced on the Illumina HiSeq 2000 and assembled into a reference transcriptome. Functional gene annotations identified putative olfactory-related genes; 56 odorant receptors (ORs), 23 odorant binding proteins (OBPs), and 10 CSPs. RNA-seq estimates of gene expression respectively showed up- and down-regulation of 79 and 30 genes in female compared to male antennae, which included up-regulation of 8 ORs and 1 PBP gene in male antennae as well as 3 ORs in female antennae. Quantitative real-time RT-PCR analyses validated strong male antennal-biased expression of OfurOR3, 4, 6, 7, 8, 11, 12, 13 and 14 transcripts, whereas OfurOR17 and 18 were specially expressed in female antennae. Sex-biases gene expression described here provides important insight in gene functionalization, and provides candidate genes putatively involved in environmental perception, host plant attraction, and mate recognition.

Physiological Responses Induced by Ostrinia furnacalis (Lepidoptera: Crambidae) Feeding in Maize and Their Effects on O. furnacalis Performance

Abstract Plants damaged by herbivorous insects often respond by mounting a series of defense responses that can inhibit the insects fitness. Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae) is a major insect pest in maize throughout much of Asia, Australia, and the western Pacific islands. We examined the effects of O. furnacalis-induced maize defenses on O. furnacalis fitness, and explained the effects from biochemical changes that occur in maize leaves in response to O. furnacalis feeding. The results of the age-stage, two-sex life table showed that significantly longer larval and pupal life spans, and total preoviposition period (TPOP) occurred. A decrease in the longevity and fecundity of female adults was observed in O. furnacalis fed on O. furnacalis-damaged leaves. The mean generation time (T), finite rate of increase (λ), net reproductive rate (R0), and intrinsic rate of increase (r) were also correspondingly affected. Biochemical assays indicated that 24 h of O. furnacalis herbivory resulted in decreased levels of the benzoxazinoids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-β-D-glucopyranose (DIMBOA-Glc), and a corresponding increase in 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-β-D-glucopyranose (HDMBOA-Glc). Maize also exhibited higher activities of the defensive enzymes—peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO)—after 24 h of herbivory. We concluded that exposure to O. furnacalis-damaged leaves had an inhibitory impact on the fitness of the neonate to pupa stages of O. furnacalis. The observed higher level of HDMBOA-Glc and higher enzymatic activities of POD, SOD, CAT, and PPO may account, in part, for the observed inhibitory effects on O. furnacalis fitness.

Down-regulation of aminopeptidase N and ABC transporter subfamily G transcripts in Cry1Ab and Cry1Ac resistant Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae)

The Asian corn borer (ACB), Ostrinia furnacalis (Lepidoptera: Crambidae), is a highly destructive pest of cultivated maize throughout East Asia. Bacillus thuringiensis (Bt) crystalline protein (Cry) toxins cause mortality by a mechanism involving pore formation or signal transduction following toxin binding to receptors along the midgut lumen of susceptible insects, but this mechanism and mutations therein that lead to resistance are not fully understood. In the current study, quantitative comparisons were made among midgut expressed transcripts from O. furnacalis susceptible (ACB-BtS) and laboratory selected strains resistant to Cry1Ab (ACB-AbR) and Cry1Ac toxins (ACB-AcR) when feeding on non-Bt diet. From a combined de novo transcriptome assembly of 83,370 transcripts, ORFs of ≥ 100 amino acids were predicted and annotated for 28,940 unique isoforms derived from 12,288 transcripts. Transcriptome-wide expression estimated from RNA-seq read depths predicted significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as a putative ATP binding cassette transporter group G (abcg) gene in both ACB-AbR and -AcR (log2[fold-change] ≥ 1.36; P < 0.0001). The transcripts that were most highly differentially regulated in both ACB-AbR and -AcR compared to ACB-BtS (log2[fold-change] ≥ 2.0; P < 0.0001) included up- and down-regulation of serine proteases, storage proteins and cytochrome P450 monooxygenases, as well as up-regulation of genes with predicted transport function. This study predicted the significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as abccg gene in both ACB-AbR and -AcR. These data are important for the understanding of systemic differences between Bt resistant and susceptible genotypes.

Field efficacy of transgenic cotton containing single and double toxin genes against the Asian corn borer (Lep., Pyralidae)

Abstract:u2002 Insect resistant transgenic cotton (Gossypium hirsutum L.) is expected to provide satisfactory control of lepidopteran species in the cotton field. The Asian corn borer, Ostrinia furnacalis (Guenée) (Lep., Pyralidae), is an important component of the lepidopteran pest complex of cotton in China. Insect resistant transgenic cotton cultivars GK2, carrying cry1A gene, and SGK321, carrying both cry1A and CpTI genes, were evaluated for resistance to Asian corn borer. Field trials were conducted with artificial infestation of Asian corn borer at squaring, flowering and flowering‐boll cotton plants, which coincided with the generations of natural Asian corn borer occurrence. Damage ratings were significantly reduced in transgenic cotton cultivars both GK2 and SGK321 compared with their parental non‐transgenic cotton cultivars Simian3 and Shiyuan321, respectively. In addition, percentage of plants stem bored and number of tunnels per plant were significantly higher on GK2 than on SGK321 in the second generation. Laboratory bioassays were carried out by exposing neonates to plant tissues collected from the field. Tissues assayed included the new leaves, match‐head squares and white flowers, which are the tissues initially attacked by the neonates in the field. Low larval survival rates were observed on SGK321 and GK2, contrasting greatly to the high number of survivors found on their non‐transgenic cotton tissue isolated throughout the season. However, larval survival was higher on new leaves isolated from late‐season transgenic cotton plants and fruit tissues than on early‐season. In addition, higher larval survival was observed on GK2 than SGK321 in assays with the late season tissues. This may be associated with reduced levels of available toxin in plant tissues as they age. Both laboratory and field data indicated that SGK321 and GK2 were highly resistant to Asian corn borer. The high level of efficacy for insect resistant transgenic cotton against Asian corn borer offers the potential for season‐long control.

Does Bt maize expressing Cry1Ac protein have adverse effects on the parasitoid Macrocentrus cingulum (Hymenoptera: Braconidae)?

The potential effects of insect‐resistant, genetically engineered (GE) crops on non‐target organisms, especially on predators and parasitoids, must be evaluated before their commercial cultivation. The effects of GE maize that produces Cry1Ac toxin on the parasitoid Macrocentrus cingulum were assessed by direct bioassay and indirect bioassay. In the indirect bioassay, parasitism rate, cocoon weight and the number of M. cingulum progeny produced per host were significantly reduced when M. cingulum‐parasitized Cry1Ac‐susceptible Ostrinia furnacalis were fed a diet containing purified Cry1Ac; however, life‐table parameters of M. cingulum were not adversely affected when the same assay was performed with Cry1Ac‐resistant O. furnacalis. These results indicated that the detrimental effects detected with a Cry1Ac‐susceptible host were mediated by poor host quality. In a direct bioassay, no difference in life‐table parameters were detected when M. cingulum adults were directly fed a 20% honey solution with or without Cry1Ac; however, survival and longevity were significantly reduced when M. cingulum adults were fed a honey solution containing potassium arsenate, which was used as a positive control. The stability and bioactivity of Cry1Ac toxin in the food sources and Cry1Ac toxin uptake by the host insect and parasitoid were confirmed by enzyme‐linked immunosorbent assay and sensitive‐insect bioassays. Our results demonstrate that M. cingulum is not sensitive to Cry1Ac toxin at concentrations exceeding those encountered in Bacillus thuringiensis maize fields. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non‐target organisms and will be useful for assessing other non‐target impacts.

Molecular cloning, expression profile, odorant affinity, and stability of two odorant-binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant-binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant-binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real-time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant-binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans-caryophelle, farnesene, and cis-3-Hexen-1-ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild-type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α-helical content.

Evaluation of Bt Corn with Pyramided Genes on Efficacy and Insect Resistance Management for the Asian Corn Borer in China

A Bt corn hybrid (AcIe) with two Bt genes (cry1Ie and cry1Ac) was derived by breeding stack from line expressing Cry1Ie and a line expressing Cry1Ac. Efficacy of this pyramided Bt corn hybrid against the Asian corn borer (ACB), Ostrinia furnacalis, was evaluated. We conducted laboratory bioassays using susceptible and resistant ACB strains fed on artificial diet or fresh plant tissues. We also conducted field trials with artificial infestations of ACB neonates at the V6 and silk stages. The toxin-diet bioassay data indicated that mixtures of Cry1Ac and Cry1Ie proteins had synergistic insecticidal efficacy. The plant tissue bioassay data indicated that Bt corn hybrids expressing either a single toxin (Cry1Ac or Cry1Ie) or two toxins had high efficacy against susceptible ACB. Damage ratings in the field trials indicated that the Bt corn hybrids could effectively protect against 1st and the 2nd generation ACB in China. The hybrid line with two Bt genes showed a higher efficacy against ACB larvae resistant to Cry1Ac or CryIe than the hybrid containing one Bt gene, and the two gene hybrid would have increased potential for managing or delaying the evolution of ACB resistance to Bt corn plants.

C-terminus Methionene Specifically Involved in Binding Corn Odorants to Odorant Binding Protein4 in Macrocentrus cingulum

The soluble carrier proteins, OBPs carry odor components through sensilium lymph to specific receptors within the antennal sensilla to trigger behavioral responses. Herein, McinOBP4 was characterized from the Macrocentrus cingulum, which is the specialist parasitic insect of Ostrinia furnacalis for better understanding of olfactory recognition mechanism of this wasp. The classical odorant binding protein McinOBP4 showed good binding affinity to corn green leaf volatiles. RT-qPCR results showed that the McinOBP4 was primarily expressed in male and female wasp antennae, with transcripts levels differing by sex. Fluorescence assays indicate that, McinOBP4 binds corn green leaf volatiles including terpenoides and aliphatic alcohols as well as aldehydes with good affinity. We have also conducted series of binding assay with first mutant (M1), which lacked the last 8 residues and a second mutant (M2), with Met119 replaced by Leucine (Leu119). In the acidic conditions, affinity N-phenylnaphthylamine (1-NPN) to McinOBP4 and M1 were substantially decreased, but increase in basic condition with no significant differences. The lack of C-terminus showed reduced affinity to terpenoides and aliphatic alcohols as well as aldehydes compounds of corn odorants. The mutant M2 with Met119 showed significant reduction in binding affinity to tested odorants, it indicating that Met119 forming hydrophobic chain with the odorants functional group to binding. This finding provides detailed insight of chemosensory function of McinOBP4 in M. cingulum and help to develop low release agents that attract of this wasp to improve ecologically-friendly pest management strategy.

EXPRESSION PATTERNS OF THE GLYCOGEN PHOSPHORYLASE GENE RELATED TO LARVAL DIAPAUSE IN Ostrinia furnacalis.

Glycogen phosphorylase (GP) acts in the first step in release of glucose from glycogen, a form of energy storage for most organisms. To investigate the characteristics and expression pattern of GP gene (Ofgp) in the Asian corn borer, Ostrinia furnacalis (Guenée), larvae, we cloned and analyzed tissue transcription of Ofgp. The results indicate that the open reading frame (ORF) is 2,526 bp, encoding 841 amino acid. The calculated three-dimensional structure shows 33 α-helices and 24 β-sheets. Ofgp transcription levels varied significantly during the second to fifth instars under long-day (28 °C, 16:8 L:D photoperiod, and 70-80% relative humidity (RH)) and short-day (24.5 °C, 11:13 L:D photoperiod, and 70-80% RH) conditions, remained low during the prediapause phase, and then increased after about 36 d under short-day photoperiod. In the larvae reared under long-day condition, hemolymph ranked the highest in the transcript level of Ofgp. The highest transcription was recorded in the fat body and was lower in the other tissues in larvae reared under short-day condition. We found that Ofgp transcription increased linearly from October 2012 to January 2013. The transcript level was negatively correlated with environmental temperature. We infer the higher Ofgp transcription may enhance the cold hardiness of the diapause larvae.

Effects of host plants on the fitness of Athetis lepigone (Möschler)

Athetis lepigone (Möschler) is a new insect pest that feeds on corn seedlings in summer corn region of China. It is an omnivorous insect, which not only infests corn, soya bean, peanut and other crops, but also feeds on fallen and decaying leaves of some crops and weeds. Effects of host plants, corn, cotton, peanut, wheat and soya bean, on larval and pupal development and survival, and the fecundity of adults of A. lepigone were studied under laboratory conditions. The developmental time from egg to adult was the shortest (30.78 days) on germinating wheat kernels and the longest (40.13 days) on corn leaves. Larval survival was the highest (80%) on germinating wheat kernels and lowest (55%) on soya bean leaves. Hatching rate was the highest (92.98%) where eggs were oviposited by the females developed from larvae that were fed on germinating wheat kernels. Similarly, fecundity was the greatest on germinating wheat kernels (337), while the lowest fecundity (145) was observed on cotton leaves. The highest intrinsic rate of increase (0.22) and net reproduction rate (181.39) were found on germinating wheat kernels. Germinating wheat kernels were recognized as the most suitable host plant for A. lepigone because of the shortest developmental period, highest survival and highest fecundity. The results correspond with the field habits of A. lepigone and clearly show differences in development and adaptability on the five tested host plants. The findings will serve as baseline information to better understand the biology of the pest, which can be utilized in an effective management programme.