Tiantao Zhang

Morphology and ultrastructure of antennal sensilla of Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) and their probable functions

Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is a polyembryonic endoparasitoid of the Asian corn borer, Ostrinia furnacalis and the European corn borer, Ostrinia nubilalis. To better understand the host location mechanism, we examined the external morphology and ultrastructure of the antennal sensilla of this parasitoid by using scanning and transmission electron microscopy. Antennae of male and female of the M. cingulum are filiform in shape, 5.90-6.64mm in length and consist of scape, pedicel, and flagellum with 39 and 40 flagellomeres, respectively. Cuticular pore and nine types of morphologically distinct sensilla were identified in both sexes, including two types of sensilla chaetica (nonporous), s. trichodea (nonporous), s. basiconica I (nonporous blunt tip), s. basiconica II (porous wall) and basiconica III (nonporous wall) with branched blunt tip, s. coeloconica with finger-like projections, protruded s. campaniform with central tip pore, and plate-like s. placodea (porous). We compared number, morphology, and distribution of sensilla between sexes. S. campaniform and non-porous basiconica type I may play a role in gustatory functions whereas type II, and s. placodea may play a function in detecting odor stimuli due to their pores wall. The sensilla chaetica and s. trichodea may be involved in mechnosensation. S. coeloconica probably plays a role as thermo-hygro receptor, whilst cuticular pores may detect odor stimuli. No differences in antenna shape and basic structure in the males and females, but male antennae length and width were significantly greater than those of females. Furthermore, males had more placodea than females. The sensilla types, morphology, and structure of both sexes were compared to those found in other parasitic hymenoptera, especially braconid wasps.

Field trials to evaluate the effects of transgenic cry1Ie maize on the community characteristics of arthropod natural enemies

Possible non-target effect of transgenic cry1Ie maize exerts on natural enemy community biodiversity in the field is unresolved. In the present study, a 2-yr comparison of transgenic cry1Ie maize (Event IE09S034, Bt maize) and its near isoline (Zong 31, non-Bt maize) on natural enemy community biodiversity were compared with whole plant inspections, pitfall traps and suction sampler. Natural enemy diversity indices (Shannon-Wiener’, Simpson’s and Pielou’s index) and abundance suggested there were no significant differences between the two types of maize. The only exceptions were the Pielou’s index for whole plant inspections in 2013 and abundance for pitfall traps in 2012, which were significantly higher in Bt maize than those of non-Bt maize. The main species of natural enemies were identical in Bt and non-Bt maize plots for each method and the three methods combined. For whole plant inspections, Bt maize had no time-dependent effect on the entire arthropod natural enemy community, and also no effect on community dissimilarities between Bt and non-Bt maize plots. These results suggested that despite the presence of a relatively minor difference in natural enemy communities between Bt and non-Bt maize, transgenic cry1Ie maize had little, if any, effect on natural enemy community biodiversity.

Identification of putative chemosensory receptor genes from yellow peach moth Conogethes punctiferalis (Guenée) antennae transcriptome

The yellow peach moth, Conogethes punctiferalis, is an extremely important polyphagous insect in Asia. The chemosensory systems of moth play an important role in detecting food, oviposition sites and mate attraction. Several antennal chemosensory receptors are involved in odor detection. Our study aims to identify chemosensory receptor genes for potential applications in behavioral responses of yellow peach moth. By transcriptomic analysis of male and female antennae, 83 candidate chemosensory receptors, including 62 odorant receptors, 11 ionotropic receptors and 10 gustatory receptors were identified. Through Blast and sequence alignment, the highly conserved co-receptor Orco was annotated, eight unigenes clustered into pheromone receptors, and two clustered as sugar receptor. Among the IRs, one unigenes was similar with co-receptors IR25a. Expression levels of 50 odorant receptors were further evaluated by quantitative real-time PCR in antennae. All the ORs tested were detected in antennae and some of which were associated with sex-biased expression. The chemosensory receptors identified in C. punctiferalis provide a foundational resource for further analysis on olfaction for behavior. The expression profiles of ORs in antennae indicated variant functions in olfactory recognition, and our results provided the possibility for the potential application of semiochemical to control this pest moth.

Three amino acid residues bind corn odorants to McinOBP1 in the polyembryonic endoparasitoid of Macrocentrus cingulum Brischke.

Odorant binding proteins (OBPs) play a central role in transporting odorant molecules from the sensillum lymph to olfactory receptors to initiate behavioral responses. In this study, the OBP of Macrocentrus cingulum McinOBP1 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR experiments indicate that the McinOBP1 is expressed mainly in adult antennae, with expression levels differing by sex. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the McinOBP1 can bind green-leaf volatiles, including aldehydes and terpenoids, but also can bind aliphatic alcohols with good affinity, in the order trans-2-nonenal>cis-3-hexen-1-ol>trans-caryophelle, suggesting a role of McinOBP1 in general odorant chemoreception. We chose those three odorants for further homology modeling and ligand docking based on their binding affinity. The Val58, Leu62 and Glu130 are the key amino acids in the binding pockets that bind with these three odorants. The three mutants, Val58, Leu62 and Glu130, where the valine, leucine and glutamic residues were replaced by alanine, proline and alanine, respectively; showed reduced affinity to these odorants. This information suggests, Val58, Leu62 and Glu130 are involved in the binding of these compounds, possibly through the specific recognition of ligands that forms hydrogen bonds with the ligands functional groups.

Transcriptome Comparison Analysis of Ostrinia furnacalis in Four Developmental Stages

The Asian corn borer, Ostrinia furnacalis, is one of the most destructive pests of maize and causes huge losses in maize yield each year. In order to characterize the different developmental stages, a high-throughput sequencing platform was employed to perform de novo transcriptome assembly and gene expression analysis for the egg, larva, pupa and adult stages. Approximately 185 million reads were obtained, trimmed, and assembled into 42,638 unigenes with an average length of 801.94 bp and an N50 length of 1,152 bp. These unigene sequences were annotated and classified by performing Gene Ontology (GO), Cluster of Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classifications. Comparison of the gene expression profiles of the two transitional stages revealed dramatic differences. Some differentially expressed genes are associated with digestion, cuticularization olfactory recognition and wing formation as well as growth and development. In total, 12 putative insect development-related genes were identified. Real-time quantitative PCR (RT-qPCR) results and sequencing based on relative expression levels of randomly selected genes confirmed these expression patterns. These data represent the most comprehensive transcriptomic resource currently available for O. furnacalis and will facilitate the study of developmental pathways, cuticularization, wing formation and olfactory recognition.

Gene set of chemosensory receptors in the polyembryonic endoparasitoid Macrocentrus cingulum

Insects are extremely successful animals whose odor perception is very prominent due to their sophisticated olfactory system. The main chemosensory organ, antennae play a critical role in detecting odor in ambient environment before initiating appropriate behavioral responses. The antennal chemosensory receptor genes families have been suggested to be involved in olfactory signal transduction pathway as a sensory neuron response. The Macrocentrus cingulum is deployed successfully as a biological control agent for corn pest insects from the Lepidopteran genus Ostrinia. In this research, we assembled antennal transcriptomes of M. cingulum by using next generation sequencing to identify the major chemosensory receptors gene families. In total, 112 olfactory receptors candidates (79 odorant receptors, 20 gustatory receptors, and 13 ionotropic receptors) have been identified from the male and female antennal transcriptome. The sequences of all of these transcripts were confirmed by RT-PCR, and direct DNA sequencing. Expression profiles of gustatory receptors in olfactory and non-olfactory tissues were measured by RT-qPCR. The sex-specific and sex-biased chemoreceptors expression patterns suggested that they may have important functions in sense detection which behaviorally relevant to odor molecules. This reported result provides a comprehensive resource of the foundation in semiochemicals driven behaviors at molecular level in polyembryonic endoparasitoid.

Identification and expression pattern analysis of chemosensory receptor genes in the Macrocentrus cingulum (Hymenoptera: Braconidae) antennae

Macrocentrus cingulum is an important polyembryonic endoparasitic wasp that attacks larvae of the Asian corn borer, Ostrinia furnacalis (Guenee) and the European corn borer, O. nubilalis (Hubner). Parasitoids use antennae as the main sensory organ to recognize herbivore-induced plant volatiles as host searching cues. The antennal olfaction proteins, odorant receptors (ORs) and ionotropic receptors (IRs) are involved in olfactory signal transduction pathway as a sensory neuron response. In the present study, we constructed a cDNA library from the male and female antennae for identifying the olfaction-related genes in M. cingulum. For that, we sequenced 3160 unique gene sequences and annotated them with gene ontology (GO), cluster of ortholo- gous groups of proteins (COG), and KEGG ontology (KO). Through the homology search, we identifi ed 9 odorant receptors (ORs), 3 ionotropic receptors (IRs) and 1 odorant binding protein (OBP) genes from the cDNA library sequences. Additionally, the expres- sion patterns of these ORs and IRs in different tissues (antennae, heads, thoraxes, abdomens, and legs) were demonstrated by RT-PCR. The qualitative gene expression analyses showed that most of the OR genes were more highly expressed in female than male antennae; whereas IRs, unlike ORs, were more expressed in various male than females tissues. We are the fi rst to report ORs and IRs in M. cingulum, which should help in deciphering the molecular basis of olfaction system in this wasp.

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.

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.

Binding specificity of two PBPs in the yellow peach moth Conogethes punctiferalis (Guenée)

Pheromone binding proteins (PBPs) play an important role in olfaction of insects by transporting sex pheromones across the sensillum lymph to odorant receptors. To obtain a better understanding of the molecular basis between PBPs and semiochemicals, we have cloned, expressed, and purified two PBPs (CpunPBP2 and CpunPBP5) from the antennae of Conogethes punctiferalis. Fluorescence competitive binding assays were used to investigate binding affinities of CpunPBP2 and CpunPBP5 to sex pheromone and volatiles. Results indicate both CpunPBP2 and CpunPBP5 bind sex pheromones E10-16:Ald, Z10-16:Ald and hexadecanal with higher affinities. In addition, CpunPBP2 and CpunPBP5 also could bind some odorants, such as 1-tetradecanol, trans-caryopyllene, farnesene, and β-farnesene. Homology modeling to predict 3D structure and molecular docking to predict key binding sites were used, to better understand interactions of CpunPBP2 and CpunPBP5 with sex pheromones E10-16:Ald and Z10-16:Ald. According to the results, Phe9, Phe33, Ser53, and Phe115 were key binding sites predicted for CpunPBP2, as were Ser9, Phe12, Val115, and Arg120 for CpunPBP5. Binding affinities of four mutants of CpunPBP2 and four mutants of CpunPBP5 with the two sex pheromones were investigated by fluorescence competitive binding assays. Results indicate that single nucleotides mutation may affect interactions between PBPs and sex pheromones. Expression levels of CpunPBP2 and CpunPBP5 in different tissues were evaluated using qPCR. Results show that CpunPBP2 and CpunPBP5 were largely amplified in the antennae, with low expression levels in other tissues. CpunPBP2 was expressed mainly in male antennae, whereas CpunPBP5 was expressed mainly in female antennae. These results provide new insights into understanding the recognition between PBPs and ligands.