Antennal Binding Protein Research Articles

Antennal Transcriptome of the Fruit-Sucking Moth Eudocima materna: Identification of Olfactory Genes and Preliminary Evidence for RNA-Editing Events in Odorant Receptors.

Unappealing shriveled fruits are a characteristic of one of the most elusive fruit pests. The perpetrator, Eudocima materna, attacks the fruit at a fully formed stage and, therefore, the antennal transcriptome for this insect was deduced to identify the molecular elicitors involved in the attraction to its host plants. A total of 260 olfactory genes, including 16 odorant-binding proteins (OBPs), four pheromone-binding proteins (PBPs), 40 antennal-binding proteins (ABPs), 178 odorant receptors (ORs), 17 chemosensory proteins (CSPs) and five sensory neuron membrane proteins (SNMPs) were identified. Phylogenetic analysis shows the divergence of E. materna proteins from closely related lepidopterans and provides insights on genes that have exclusively evolved in this insect. STRING network analysis revealed interactions of olfactory proteins among themselves and the proteins of other groups. Interestingly, online tools predicted RNA-editing events in the odorant receptor sequences, suggesting the possibility of multiple protein forms. Transcripts matching transposable element sequences were also detected in the dataset. Thus, the work reported here provides a valuable resource to design molecular methods for pest control.

Identification and characterization of chemosensory gene families in the bark beetle, Tomicus yunnanensis

The bark beetle, Tomicus yunnanensis (Coleoptera: Scolytinae), is a seriously destructive pest of Yunnan pine (Pinus yunnanensis) and is distributed solely in Southwestern China. It has been a challenge to control this pest owing to its resistance to chemical pesticides, which have been used as the main control strategy of this species in recent years. Since this approach will continue until an alternative mitigation strategy is implemented, it is essential to develop novel or improved biocontrol approaches. In the current study, we aimed to identify most, if not all, of the bark beetle's chemosensory genes, and to address their respective phylogenetic relationships and expression characteristics. Digital gene expression (DGE) profiling and a comparison of the profiles at three developmental stages yielded 40,287,265 clean reads and a large number of differentially expressed genes (DEGs), with 21 up- and 20 down-regulated DEGs involved in chemoreception. Transcriptome of the three mixed stages revealed a total of 80 transcripts encoding chemosensory-related proteins comprising 45 odorant-binding proteins (OBPs), 12 chemosensory proteins (CSPs), 20 receptor proteins [9 odorant receptors (ORs), 8 gustatory receptors (GRs) and 3 ionotropic receptors (IRs)] and 3 sensory neuron membrane proteins (SNMPs). As many as 38 full-length sequences were acquired with a combination of transcriptomic analysis and rapid amplification of cDNA ends (RACE) strategy. Phylogenetic analysis showed that T. yunnanensis OBPs were clustered into four sub-groups: 27 Minus-C OBPs, 5 antennal binding proteins (ABPIIs), 10 Classic OBPs and one Plus-C OBP; meanwhile, the ORs were grouped into four clades (1, 2, 7b and Orco). Expression profiles revealed that 66 of 80 genes were detected in the three DGE libraries, and 15 soluble olfactory proteins were antennae-predominant, possibly guiding olfactory-associated behaviors of this beetle. Taken together, our study has provided valuable data for further functional studies of this beetle and will facilitate the identification of potential molecular targets associated with chemosensory reception for use in biocontrol strategies.

Shotgun proteomic analysis on the embryos of silkworm Bombyx mori at the end of organogenesis

Embryonic development of silkworm, Bombyx mori is a process of systematical expression of genes and proteins which is dominated by complex regulatory networks. To gain comprehensive insight into the molecular basis of embryonic development and its regulation mechanisms, the proteome profile of the B. mori embryos at the end of organogenesis (tubercle appearance stage, TA) was characterized using LTQ-Orbitrap mass spectrometer. Totally 963 proteins were identified with a false discovery rate (FDR) of 0.12%. They were involved in embryonic development, chemoreception, and stimuli response and so forth. The proteins with the largest number of identified unique peptides, implying their possibly higher abundance, were involved in heat shock response, lipid transport and metabolism, and apoptosis. It was consistent with the physiological status of embryo at the end of organogenesis. Many functionally important proteins were identified for the first time in B. mori embryo such as the progesterone receptor membrane component 2, antennal binding protein, sericotropin, and molting fluid carboxypeptidase A (MF-CPA). 253 (26.27%) specific proteins in TA versus labrum appearance stage (LA, four days before TA) embryos were identified, which were mainly associated with musculature, nervous system, and chemoreception system. They disclosed the differential temporal and spatial expression of proteins in the process of organogenesis. The relative mRNA levels of fifteen identified proteins in the two experimented stages were also compared using quantitative reverse transcription PCR (qRT-PCR) and showed some inconsistencies with protein expression. Gene Ontology (GO) annotation of the identified proteins showed that the most proteome representations were in the categories of “binding” and “catalytic” in molecular function, and “cellular process” and “metabolic process” in biological process.

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth, Amyelois transitella

BackgroundThe navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.MethodologyBy analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.ConclusionWe have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

Global transcriptional analysis of pheromone biosynthesis-related genes in the female turnip moth, Agrotis segetum (Noctuidae) using a custom-made cDNA microarray

Using a custom-made cDNA microarray, global transcriptional analyses were conducted to identify genes differentially regulated in the pheromone gland as compared to the remaining insect tissue of the moth Agrotis segetum (Noctuidae). A two-fold or larger difference in relative expression levels was found for 227 of 864 genes investigated comparing the two tissues. Unexpectedly, an antennal binding protein homologue, containing a pheromone-binding/general odorant-binding protein PFAM domain, was expressed at a 56-fold higher level in the pheromone gland. Relatively higher expression levels in the pheromone gland were also found for other gene representatives putatively encoding odorant-binding proteins and chemosensory proteins, as well as a number of gene representatives putatively encoding proteins involved in juvenile hormone interactions. The largest relative up-regulation (84-fold) in the pheromone gland was found for a gene encoding a Δ11-desaturase homologue implicated in desaturation of pheromone precursors. For three gene representatives, the expression patterns were independently verified by quantitative real-time PCR (qPCR). Additionally the expression pattern in the pheromone gland for the Δ11-desaturase homologue was shown by qPCR to follow the previously known pattern of pheromone production in female A. segetum, both with respect to age and circadian rhythm, whereas the expression of a Δ9-desaturase and a chemosensory protein homologue did not share this pattern.

Characterisation of Bombyx mori Odorant-binding Proteins Reveals that a General Odorant-binding Protein Discriminates Between Sex Pheromone Components

In many insect species, odorant-binding proteins (OBPs) are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors (ORs) within the antennal sensilla. In the silkworm Bombyx mori, the OBPs are subdivided into three main subfamilies; pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal-binding proteins (ABPs). We used the MotifSearch algorithm to search for genes encoding putative OBPs in B. mori and found 13, many fewer than are found in the genomes of fruit flies and mosquitoes. The 13 genes include seven new ABP-like OBPs as well as the previously identified PBPs (three), GOBPs (two) and ABPx. Quantitative examination of transcript levels showed that BmorPBP1, BmorGOBP1, BmorGOBP2 and BmorABPx are expressed at very high levels in the antennae and so could be involved in olfaction. A new two-phase binding assay, along with other established assays, showed that BmorPBP1, BmorPBP2, BmorGOBP2 and BmorABPx all bind to the B. mori sex pheromone component (10E,12Z)-hexadecadien-1-ol (bombykol). BmorPBP1, BmorPBP2 and BmorABPx also bind the pheromone component (10E,12Z)-hexadecadienal (bombykal) equally well, whereas BmorGOBP2 can discriminate between bombykol and bombykal. X-ray structures show that when bombykol is bound to BmorGOBP2 it adopts a different conformation from that found when it binds to BmorPBP1. Binding to BmorGOBP2 involves hydrogen bonding to Arg110 rather than to Ser56 as found for BmorPBP1.

Identification of up-regulated proteins in the hemolymph of immunized Bombyx mori larvae

Insects defend themselves against foreign invaders via both a cellular response and a humoral response. The objective of this study was to identify proteins which were differently regulated in the immunized Bombyx mori larvae. Heat-inactivated bacteria ( Bacillus megaterium) were injected into B. mori larvae, 4 days after final ecdysis. After 6 h, we identified the immune proteins in the hemolymph which had been differentially regulated in the immune-challenged larvae, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS). After the bacterial injection, more than 30 spots determined to have been up-regulated, and 11 spots were down-regulated. The heat shock 70 kDa protein cognate was one of the up-regulated hemocytic proteins, and peptidoglycan recognition protein, antichymotrypsin precursor, and gloverin-like protein 1∼4 were newly synthesized in the plasma. Antennal binding protein 7 was up-regulated in the plasma. Our results indicated that these immune response proteins were involved with the carrying out of innate immune responses.

The expression pattern of four odorant-binding proteins in male and female silk moths, Bombyx mori

Four recombinant odorant-binding proteins of Bombyx mori, pheromone-binding protein (PBP), general odorant-binding protein 1 (GOBP1), general odorant-binding protein 2 (GOBP2) and antennal binding protein X (ABPX), were expressed in E. coli and used to raise polyclonal antisera. Immunoblots of antennal homogenates showed that these antisera were specific. In Western blot analysis and immunocytochemical labelling experiments, the sera against recombinant PBP and GOBP2 of B. mori gave identical results as sera against native PBP and GOBP2 of Antheraea polyphemus, respectively, thus confirming earlier results obtained with the latter. Labelling consecutive cross sections of various sensillum types with all four antisera revealed different labelling patterns in male and female sensilla (s.) trichodea and s. basiconica. Long s. trichodea in males and females represented uniform labelling types, whereas for short s. trichodea, s. intermedia, and s. basiconica a great variety of labelling patterns was observed, some being more common than others. Long s. trichodea, which in males are uniformly tuned to the pheromone components bombykol and bombykal, all strongly expressed PBP; labelling with antisera against the other three odorant-binding proteins hardly was above background, only in some hairs GOBP1 was expressed somewhat more strongly. Long s. trichodea of females, which respond specifically to linalool and benzoic acid, showed a different labelling pattern. Here, we observed strong labelling with antibodies against GOBP2 and medium labelling with anti-GOBP1, sometimes with anti-ABPX. S. basiconica in both sexes most commonly co-expressed GOBP1 and GOBP2, but other patterns were occasionally found, with some of them showing PBP expression, also in females. The great variety of labelling types in short s. trichodea, s. intermedia, and s. basiconica suggests a similar variety of functional subtypes as observed in plant odour-sensitive sensilla of other moth species.

Odorant-binding proteins from a primitive termite.

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.

Binding proteins from the antennae of Bombyx mori

From an antennal library of Bombyx mori cDNA clones encoding different binding proteins have been isolated. The deduced amino acid sequences showed only moderate homology to each other but shared several common structural features. Based on a sequence comparison with the antennal binding proteins from different moth species, one of the clones appears to encode a pheromone binding protein, whereas two others represent new members of the two general odorant binding protein families. A fourth clone encodes a protein which is related to antennal binding proteins so far found only in Drosophila melanogaster.

Odorant binding proteins of Heliothis virescens

cDNA clones coding for three different binding proteins were isolated from an antennal library of Heliothis virescens. The deduced amino acid sequences showed only moderate homology to each other but shared several common structural features. Based on a comparison with the predicted primary structures of antennal binding proteins from different moth species, one of the clones (Hel-1) was found to encode a pheromone binding protein, whereas the two others (Hel-10 and -11) encode general odorant binding proteins.