Man-Qun Wang

Molecular Characterization and Expression Pattern of Two General Odorant Binding Proteins from the Diamondback Moth, Plutella xylostella

In the Lepidoptera, odorant signals are thought to be mediated by general odorant binding proteins (GOBPs) in the sensillar lymph surrounding the olfactory receptors. We describe the identification and characterization of two new cDNAs encoding GOBPs from the antennae of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), a species for which no GOBPs have been identified to date. We focused our investigation on this olfactory protein family by using reverse transcription–polymerase chain reaction strategies. The deduced amino acid sequences of PxylGOBP1 and PxylGOBP2 revealed open reading frames of 168 and 163 amino acids, respectively, with six cysteine residues in conserved positions relative to other known GOBPs. The alignment of the mature PxylGOBPs with other Lepidoptera GOBPs showed high sequence identity (70–80%) with other full-length sequences from GenBank. Sequence identity between PxylGOBP1and PxylGOBP2 was only 50%, suggesting that the two proteins belong to different classes of lepidopteran GOBPs. The expression patterns of the two PxylGOBP genes, with respect to tissue distribution and sex, were further investigated by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Although the two GOBP genes were expressed only in the antennae of both sexes, reflecting the antennal specificity of GOBPs, the transcription levels of these genes depended on the sex, the age, the mating status, and the genes.

Structure-Based Analysis of the Ligand-Binding Mechanism for DhelOBP21, a C-minus Odorant Binding Protein, from Dastarcus helophoroides (Fairmaire; Coleoptera: Bothrideridae).

Odorant binding proteins (OBPs) transport hydrophobic odor molecules across the sensillar lymph to trigger a neuronal response. Herein, the Minus-C OBP (DhelOBP21) was characterized from Dastarcus helophoroides, the most important natural parasitic enemy insect that targets Monochamus alternatus. Homology modeling and molecular docking were conducted on the interaction between DhelOBP21 and 17 volatile molecules (including volatiles from pine bark, the larva of M. alternatus, and the faeces of the larva). The predicted three-dimensional structure showed only two disulfide bridges and a hydrophobic binding cavity with a short C-terminus. Ligand-binding experiments using N-phenylnaphthylamine (1-NPN) as a fluorescent probe showed that DhelOBP21 exhibited better binding affinities against those ligands with a molecular volume between 100 and 125 ų compared with ligands with a molecular volume between 160 and 185 ų. Molecules that are too big or too small are not conducive for binding. We mutated the amino acid residues of the binding cavity to increase either hydrophobicity or hydrophilia. Ligand-binding experiments and cyber molecular docking assays indicated that hydrophobic interactions are more significant than hydrogen-bonding interactions. Although hydrogen-bond interactions could be predicted for some binding complexes, the hydrophobic interactions had more influence on binding following hydrophobic changes that affected the cavity. The orientation of ligands affects binding by influencing hydrophobic interactions. The binding process is controlled by multiple factors. This study provides a basis to explore the ligand-binding mechanisms of Minus-C OBP.

Construction and analysis of cDNA libraries from the antennae of Batocera horsfieldi and expression pattern of putative odorant binding proteins.

Abstract A high-quality cDNA library was constructed from female and male antenna of the longhorned beetle, Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae), a serious pest of Populus (Salicales: Salicaceae). The titer was approximately 2.37 × 106 pfu/mL, and this complies with the test requirement. From the libraries, 692 clones were selected randomly, sequenced, and further analyzed, and the recombinational efficiency reached 93.85%. By alignment and cluster analysis, we identified four odorant binding proteins, two pheromone-binding proteins (have the characteristic six conserved cysteine residues), four Minus-C odorant binding proteins (lost two conserved cysteines), and three chemosensory proteins. In this study, we describe the identification and characterization of four new cDNAs that encode Minus-C odorant binding proteins (Minus-C OBPs) from B. horsfieldi antennal cDNA libraries. Our investigation focused on the expression pattern of the Minus-C OBP genes in various tissues in both sexes at different developmental stages, using reverse transcription PCR (RT-PCR) and realtime PCR (qPCR) strategies. Minus-C OBP1, 2, and 3 were expressed in all tested tissues, with the exception of the head (without antenna, labial palps, and maxillary palps). Minus-C OBP4 was expressed in the antenna, legs, and abdomen, but not in the labial palps, maxillary palps, or head. The qPCR results revealed MinusC OBPs were expressed in the antenna throughout the adult life, and that the transcript levels of these genes depended on the sex, age, and mating status of adults.

Identification and Comparative Expression Profiles of Chemoreception Genes Revealed from Major Chemoreception Organs of the Rice Leaf Folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae).

To better understand the olfactory mechanisms in the rice leaf folder, Cnaphalocrocis medinalis (Guenée), a serious pest of rice in Asia, we established six partial transcriptomes from antennae, protarsus, and reproductive organs of male and female adults. A total of 102 transcripts were identified, including 29 odorant receptors (ORs), 15 ionotropic receptors (IRs), 30 odorant-binding proteins (OBPs), 26 chemosensory proteins (CSPs), and 2 sensory neuron membrane proteins (SNMPs). The expression patterns of these genes were calculated by fragments per kilobase of exon per million fragments mapped (FPKM) and validated by real-time quantitative PCR (RT-qPCR). Some transcripts were exclusively expressed in specific organs, such as female protarsus, whereas others were universally expressed, this varied expression profile may provide insights into the specific functions mediated by chemoreception proteins in insects. To the best of our knowledge, among the 102 identified transcripts, 81 are novel and have never been reported before. In addition, it also is the first time that ORs and IRs are identified in C. medinalis. Our findings significantly enhance the currently limited understanding olfactory mechanisms of the olfactory mechanisms underlying the chemoreception system in C. medinalis.

Insight Into the Ultrastructure of Antennal Sensilla of Mythimna separata (Lepidoptera: Noctuidae)

The oriental armyworm, Mythimna separata (Walk), is one of the most serious pests of cereals in Asia and Australasia. The structure and distribution of the antennal sensilla of M. separata were studied by scanning electron microscopy and transmission electron microscopy. The results showed that antennae of both female and male M. separata are filiform in shape. Three groups and seven morphological sensillum types were recorded in both sexes, including uniporous sensilla (sensilla chaetica), multiporous sensilla (sensilla trichodea, basiconica, coeloconica, and styloconica), and aporous sensilla (sensilla squamiformia and Böhm bristles). S. trichodea, which were the most abundant sensilla, was made of three subtypes (ST I, ST II, and ST III) according to external features and two subtypes of s. basiconica (SB I and SB II) and s. coeloconica (SCo I and SCo II) were identified, respectively. Sexual dimorphisms in sensilla of M. separata were mainly perceived as the variations in the numbers of several sensilla subtypes. Also, the possible functions of the antennal sensilla were discussed. These results contribute to our understanding of the function of antennae in the behavior of M. separata.

Molecular cloning and expression of pheromone‐binding protein1 from the diamondback moth, Plutella xylostella

We report on the identification and characterization of a new cDNA encoding the pheromone‐binding protein1 (PxylPBP1) from male antennae of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). The deduced amino acid sequences of PxylPBP1 revealed a mature protein of 164 amino acids, with six cysteine residues in conserved positions relative to other known PBP. The alignment of the mature PxylPBP1 with other Lepidoptera PBP showed high sequence similarity (70–80%) with other full‐length sequences from GenBank. Reverse transcription polymerase chain reaction (PCR) analysis revealed that the PxylPBP1 gene is only expressed in antennae. Real‐time PCR further indicated that the expression of PxylPBP1 gene is higher in male moths, and higher in mated moths regardless of gender. The transcription levels of PxylPBP1 gene of unmated male moths reduced with age, and peaked in 4‐h‐old female moths.

Effects of polyamines and polyamine synthesis inhibitor on antennal electrophysiological responses of diamondback moths, Plutella xylostella

Electroantennography showed that treatment with polyamines and α‐difluoromethyl‐ornithine (DFMO) significantly affected the sensitivity of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), antennae to odors. Response to a female pheromone extract was significantly higher in antennae from spermine‐treated males than in those from acetone‐treated males, and significantly lower in spermidine‐treated males, whereas the DFMO‐treatment had no significant effect. The effect of polyamine and DFMO treatment on olfactory sensitivity differed between the sexes, and correlated with the mating status of adults. Amine putrescine, spermidine, and DFMO treatments enhanced the sensitivity of unmated male antennae to most of the odors tested, but inhibited the sensitivity of virgin female antennae. Spermine treatment enhanced the sensitivity of virgin female antennae to all tested odors, but inhibited the sensitivity of mated female antennae to cabbage juice. These results suggest that polyamines play a regulatory role in the detection of odors and that their effects depend on the odor source, and the gender and mating status of diamondback moths.

Electrophysiological Responses of the Rice Leaffolder, Cnaphalocrocis medinalis, to Rice plant Volatiles

Abstract The rice leaffolder, Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae), is one of the most destructive pests of rice. Electrophysiological responses of this species to 38 synthetic volatiles known to be released from rice plants (Poaceae: Oryza spp.) were studied using the electroantennogram (EAG) method. Compounds that elicited the strongest EAG responses for each physiological condition were selected for EAG dose-response tests at five concentrations. These compounds included: methyl salicylate, heptanol, linalool, cyclohexanol, and 2-heptanone for one-day-old male moths; heptanol, hexanal, (Z)-2-hexen-1-ol, and nonadecane for one-day- old females; methyl salicylate, heptanol, (E)-2-hexen-1-ol, and (Z)-2-hexen-1-ol for three-day- old males; linalool, heptanol, (E)-2-hexen-1-ol, 2-heptanone, and hexanal for three-day-old females; 2-heptanone, cyclohexanol, linalool, heptanol, and methyl salicylate for five-day-old virgin females; and methyl benzoate, (Z)-2-hexen-1-ol, heptanol, linalool, and hexanal for five- day-old mated females. Female and male C. medinalis exhibited broad overlap in their EAG responses, and there was no clear difference between male and female EAG responses to different compounds. Statistical analyses revealed that both volatile compound chemical structure and C. medinalis physiological condition (age, sex, and mating condition) had an effect on EAG response.

Chemosensory protein genes of batocera horsfieldi (hope): identification and expression pattern

Chemoreception is an essential feature for selection of host plants by insects. Chemosensory proteins (CSPs) are a group of small proteins (approximately 13 kDa) that are expressed widely in various insect sensory appendages. Batocera horsfieldi (Hope) is a major pest of Popolus and utilizes a variety of semiochemicals for its mating and oviposition. However, no CSP gene has been identified in B. horsfieldi. Here, to obtain the DNA sequences of B. horsfieldi CSPs, we used both bioinformatics and experimental approaches to analyse the antennal expressed sequence tags (ESTs). Among 649 EST clones, three unique CSP sequences (BhorCSP1, 2 and 3) were identified and characterized from B. horsfieldi (Hope) antennal cDNA libraries based on their four conserved cysteine residues, which have a characteristic spacing pattern. A phylogenetic tree of BhorCSPs and other insect CSPs showed that BhorCSP1 and BhorCSP2 are more similar to CSPs of Tribolium castaneum than BhorCSP3. BhorCSP3 is closely related to CSPs of the Dipteran, Culex quinquefasciatus. Using reverse transcription polymerase chain reaction (RT‐PCR) and real‐time Q‐PCR strategies, we examined the expression patterns of these three putative CSP genes in different tissues, sexes and developmental stages. Our analysis shows that all three genes were expressed in all tissues examined, including the antennae, labial palps, maxillary palps, legs, abdomen and wings but not in the head. Real‐time Q‐PCR results revealed that these CSP genes were expressed throughout the life of the adults, and the transcription levels of these genes depended on the sex, age and mating status of adults.

Effects of putrescine on diapause induction and intensity, and post-diapause development of Helicoverpa armigera

Effects of putrescine on diapause induction and intensity, and post‐diapause development in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) from Wuhan, Hubei, China were studied in the laboratory at different temperatures and photoperiod conditions. The results showed a generally higher incidence of diapause in putrescine‐feeding groups. Putrescine feeding distinctly restricted the development of non‐diapause‐destined and diapause‐destined larvae, except at 0.01% putrescine concentration, which accelerated the development of larvae at 21 °C. Putrescine had no significant effect on the duration of diapause, although it inhibited the post‐diapause development of pupae. Photoperiod played a different role in diapause induction and post‐diapause development between 21 and 23 °C. Putrescine played a definite role in diapause induction and intensity, and post‐diapause development in H. armigera, and a significant interaction between putrescine, photophase, and temperature is to be expected.