Antennal-specific pheromone-degrading aldehyde oxidases from the moths Antheraea polyphemus and Bombyx mori

Female moths produce blends of odorant chemicals, called pheromones. These precise chemical mixtures both attract males and elicit appropriate mating behaviors. To locate females, male moths must rapidly detect changes in environmental pheromone concentration. Therefore, the regulation of pheromone concentration within antennae, their chief organ of smell, is important. We describe antennal-specific aldehyde oxidases from the moths Antheraea polyphemus and Bombyx mori that are capable of catabolizing long chain, unsaturated aldehydes such as their aldehyde pheromones. These soluble enzymes are associated uniquely with male and female antennae and have molecular masses of 175 and 130 kDa, respectively. The A. polyphemus aldehyde oxidase has been localized to the olfactory sensilla which contain the pheromone receptor cell dendrites. These same sensilla contain a previously described sensilla-specific esterase that degrades the acetate ester component of A. polyphemus pheromone. We propose that sensillar pheromone-degrading enzymes modulate pheromone concentration in the receptor space and hence play a dynamic role in the pheromone-mediated reproductive behaviors of these animals.     

Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori

Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, followed by digestion with endoproteinase Lys-C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and protein sequencing supported the assignment of disulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.     

Identification of the Z-W bivalent in the silkworm,Bombyx mori

None of the 56 chromosomes including sex chromosomes have been identified in the silkworm so far, though the 28 linkage groups have been determined (Doira, 1986). The present study aims to demonstrate the sex chromosome bivalent in the oocyte by using a particular strain, the sex-limited yellow cocoon (Sy), in which a large fragment of the second chromosome was translocated onto the W chromosome. Among 28 bivalents in the oocyte of the Sy strain, an asymmetrical synaptonemal complex was observed, while in the oocyte of the control strains no such complex was found. We consider this complex as the Z-W bivalent in the silkworm.     

Immunocytochemical identification of neuroactive substances in the antennal lobe of the male silkworm moth Bombyx mori

As a first step towards understanding the functional role of neuroactive substances in the first olfactory center of the male silkworm moth Bombyx mori, we carried out an immunocytochemical identification of antennal lobe neurons. Antibodies against gamma-aminobutyric acid (GABA), FMRFamide, serotonin, tyramine and histamine were applied to detect their existence in the antennal lobe. In the present immunocytochemical study, we clarified four antenno-cerebral tracts from their origin and projection pathways to the protocerebrum, and revealed the following immunoreactive cellular organization in the antennal lobe. 1) Local interneurons with cell bodies in the lateral cell cluster showed GABA, FMRFamide and tyramine immunoreactivity. 2) Projection neurons passing through the middle antenno-cerebral tract with cell bodies in the lateral cell cluster showed GABA and FMRFamide immunoreactivity. Projection neurons passing through the outer antenno-cerebral tract with cell bodies in the lateral cell cluster showed FMRFamide immunoreactivity. 3) Centrifugal neurons passing through the inner antenno-cerebral tract b with cell bodies located outside the antennal lobe showed serotonin and tyramine immunoreactivity. Our results revealed basic distribution patterns of neuroactive substances in the antennal lobe and indicated that each projection pathway from the antennal lobe to the protocerebrum contains specific combination of neuroactive substances.     

Combinative stimulation inactivates sex pheromone production in the silkworm moth, Bombyx mori.

Mating results in a strong suppression of sex pheromone (bombykol) production in the female silkworm moth, Bombyx mori. The mechanical stimulation from the insertion of a penis, inflation of the bursa copulatrix (BC), or copulation with the sterile male whose penis was removed in order to prevent ejaculation (pr-male) induced only a partial decline in bombykol production. Artificial insemination stimulates oviposition of fertilized eggs as does normal mating. However, bombykol production did not decline in artificially inseminated females. When females were artificially inseminated before or after mating with pr-males, some females had a small amount of bombykol, similar to females mated with normal males, while other females had a large amount of bombykol similar to virgin females. The former usually laid fertilized eggs, while the latter laid only unfertilized eggs though semen filled their spermatophores and spermathecae. The mechanical stimulation caused by mating with a pr-male could be replaced by covering the abdominal tip with melted paraffin. Neither implantation of the BC obtained from mated females, nor injection of the spermatophore extract, into a female mated with a pr-male could inactivate bombykol production. Injection of hemolymph from a mated female into a virgin also failed to affect bombykol production These results indicate that a combination of both the tactile stimulation of the abdominal tip and the arrival of fertile spermatozoa in the vestibulum trigger a neural inactivation mechanism of bombykol production after mating.     

Identification of plasmalogen in the gut of silkworm (Bombyx mori)

Herbivorous insect species are constantly challenged with endogenous and exogenous oxidative stress. Consequently, they possess an array of antioxidant enzymes and small molecular weight antioxidants. Lipid-soluble small molecular antioxidants, such as tocopherols, have not been well studied in insects but may play important antioxidant roles. In this study, we identified plasmalogen phosphatidylethanolamines (pPEs) as well as α-, β/γ-, δ-tocopherol in the larvae of the silkworm Bombyx mori by LCMS analyses and examined their distribution. Plasmalogen are reported to inhibit the metal ion induced oxidation. The composition of tocopherols was the same among gut contents, gut tissues, and the other tissues. However, plasmalogens, a unique class of glycerophospholipids rich in polyunsaturated fatty acids and containing a vinyl ether bond at the sn-1 position, were mainly distributed in gut tissues. Plasmalogens might protect gut tissues from oxidation stress.     

Identification of an aldehyde oxidase involved in indole-3-acetic acid synthesis in Bombyx mori silk gland

Auxin is thought to be an important factor in the induction of galls by galling insects. We have previously shown that both galling and nongalling insects synthesize indole-3-acetic acid (IAA) from tryptophan (Trp) via two intermediates, indole-3-acetaldoxime (IAOx) and indole-3-acetaldehyde (IAAld). In this study, we isolated an enzyme that catalyzes the last step “IAAld → IAA” from a silk-gland extract of Bombyx mori. The enzyme, designated “BmIAO1”, contains two 2Fe–2S iron–sulfur-cluster-binding domains, an FAD-binding domain, and a molybdopterin-binding domain, which are conserved in aldehyde oxidases. BmIAO1 causes the nonenzymatic conversion of Trp to IAAld and the enzymatic conversion of IAOx to IAA, suggesting that BmIAO1 alone is responsible for IAA production in B. mori. However, a detailed comparison of pure BmIAO1 and the crude silk-gland extract suggested the presence of other enzymes involved in IAA production from Trp.

Abbreviations: BA: benzoic acid; CE: collision energy; CXP: collision cell exit potential; DP: declustering potential; IAA: indole-3-acetic acid; IBI1: IAA biosynthetic inhibitor-1; IAAld: indole-3-acetaldehyde; ICA: indole-3-carboxylic acid; IAOx: indole-3-acetaldoxime; IEtOH: indole-3-ethanol; LC–MS/MS: liquid chromatography–tandem mass spectrometry; Trp: tryptophan     

Identification of lipases involved in PBAN stimulated pheromone production in Bombyx mori using the DGE and RNAi approaches

Background

Pheromone biosynthesis activating neuropeptide (PBAN) is a neurohormone that regulates sex pheromone synthesis in female moths. Bombyx mori is a model organism that has been used to explore the signal transduction pattern of PBAN, which is mediated by a G-protein coupled receptor (GPCR). Although significant progress has been made in elucidating PBAN-regulated lipolysis that releases the precursor of the sex pheromone, little is known about the molecular components involved in this step. To better elucidate the molecular mechanisms of PBAN-stimulated lipolysis of cytoplasmic lipid droplets (LDs), the associated lipase genes involved in PBAN- regulated sex pheromone biosynthesis were identified using digital gene expression (DGE) and subsequent RNA interference (RNAi).

Results

Three DGE libraries were constructed from pheromone glands (PGs) at different developed stages, namely, 72 hours before eclosion (−72 h), new emergence (0 h) and 72 h after eclosion (72 h), to investigate the gene expression profiles during PG development. The DGE evaluated over 5.6 million clean tags in each PG sample and revealed numerous genes that were differentially expressed at these stages. Most importantly, seven lipases were found to be richly expressed during the key stage of sex pheromone synthesis and release (new emergence). RNAi-mediated knockdown confirmed for the first time that four of these seven lipases play important roles in sex pheromone synthesis.

Conclusion

This study has identified four lipases directly involved in PBAN-stimulated sex pheromone biosynthesis, which improve our understanding of the lipases involved in releasing bombykol precursors from triacylglycerols (TAGs) within the cytoplasmic LDs.     

Interneurons sensitive to female pheromone in the deutocerebrum of the male silkworm moth, Bombyx mori

ABSTRACT. In response to minute quantities of female sex pheromone, the male silkworm moth, Bombyx mori L., walks upwind to locate the odour source. The axons of antennal receptors specific for the two known components of the pheromone terminate in the deutocerebrum. In this study, single interneurons were recorded extracellularly in the deutocerebrum of the male silkworm moth. Responses were characterized as the antennae were presented with puffs of clean air, or air containing either or both components of the female pheromone, bombykol and bombykal. An apparatus is described which added bombykol or bombykal to a constant air stream flowing over the antenna. Most units (87%) showed qualitatively different responses to bombykol and bombykal. A majority of the pheromone-sensitive units (65%) also showed mechanosensory responses to air puffs. Two units were recorded which were slightly inhibited by either bombykol or bombykal alone, but were excited by a mixture of the two.     

Purification and characterization of cocoonase from the silkworm Bombyx mori

Cocoonase (CCN) which facilitates the degradation of a cocoon is recognized as a trypsin-like serine protease. In this study, CCN from the silkworm Bombyx mori was purified and comprehensively characterized. Its activity was maximal at about pH 9.8. It was stable above pH 3.4 at 4?°C and below 50?°C at pH 7.5. CuSO₄, FeSO₄, and ZnSO₄ showed inhibitory effects on CCN, but other salts improved activity. Typical trypsin inhibitors inhibited CCN, but the relative inhibitory activities were much lower than those against bovine trypsin. An extract of cocoon shells inhibited trypsin, but it was only slightly inhibitory against CCN. There were significant differences in catalytic efficiencies and substrate specificities as between CCN and bovine trypsin.