Peter W.K. Ma

Characterization of PBAN and PBAN-encoding gene neuropeptides in the central nervous system of the corn earworm moth, Helicoverpa zea

Both female and male adult corn earworm moths, Helicoverpa zea, were utilized to demonstrate, by immunochemical techniques, the presence and localization of the pheromone biosynthesis activating neuropeptide (Hez-PBAN). A polyclonal antibody was used and was shown to be specific to the C-terminal end of Hez-PBAN. Several other peptides with a similar five amino acid C-terminal ending also cross-reacted in competitive enzyme-linked immunosorbent assays (ELISAs). Immunocytochemical methods determined that in both adult male and female moths immunoreactive material was found in three clusters of cells in the subesophageal ganglion (SEG) and that axons projected from these cell bodies to the corpora cardiaca (CC) and down the ventral nerve cord (VNC). The CC also contained immunoreactive material. Each thoracic and abdominal ganglion also contained a pair of cell bodies with immunoreactivity. In addition, two pairs of axons originating from cell bodies in the SEG extended the entire length of the VNC and terminated in the last abdominal ganglion. ELISAs indicated that the brain-SEG complex contained the highest levels of PBAN-like material followed by the CC and thoracic ganglia respectively. The abdominal ganglia contained low levels of this material. Bioassays of nervous tissue also indicated the same relative levels of PBAN-like material in each part of the nervous system. HPLC fractionation of nervous tissue followed by ELISAs indicated the presence of several other PBAN-related peptides in the brain-SEG complex and CC. The thoracic ganglia had a different profile and contained lower levels of the peptides. These results indicate that male and female adult moths have similar localization of PBAN-related peptides and that the five PBAN-related peptides previously deduced from the gene sequence are probably present in the SEG and CC.

Cloning and functional expression of a cDNA encoding a metabolic acyl-CoA Δ9-desaturase of the cabbage looper moth, Trichoplusia ni

Acyl-CoA delta 9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity. In this research, a cDNA sequence was obtained from Trichoplusia ni adult fat body mRNA by using RT-PCR with degenerate primers based on other characterized delta 9-desaturase sequences. The remainder of the sequence was amplified using 3- and 5-RACE. A 1439 bp cDNA reconstructed from three overlapping PCR products contains an ORF encoding a 353-amino acids (aa) protein that shows clear homology (greater than 50% aa identity and greater than 65% aa similarity to characterized insect and vertebrate desaturases). The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transformation. The newly characterized desaturase from T. ni produced fatty acids delta 9-16 and delta 9-18 in a 1:6 ratio, compared to a 5:1 ratio, respectively, with the yeast delta 9 desaturase. A Northern blot hybridization and a RT-PCR experiment showed that temporal and tissue-specific patterns of expression of the corresponding mRNA are distinct from those of the delta 11-desaturase mRNA present in the pheromone glands of adult females. Based on its homology to other desaturases, the widespread distribution of its corresponding mRNA in various tissues, and its functional assay, we conclude that this cDNA encodes the apoprotein corresponding to the desaturase component of the metabolic delta 9-desaturase complex of T. ni.

Calcium involvement in the stimulation of sex pheromone production by PBAN in the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae)

Abstract An in vitro assay was used to study the stimulation of sex pheromone production by pheromone biosynthesis activating neuropeptide (PBAN) in female European corn borer (ECB), Ostrinia nubilalis . Synthetic Bom-PBAN was active at 0.25 nM and maximally stimulated in vitro pheromone production at 2.5 nM. With 2.5 nM Bom-PBAN, maximum pheromone production was reached within 90 min of incubation. The calcium ionophore, A23187, stimulated pheromone production independent of PBAN. Both Bom-PBAN-stimulated and ionophore-stimulated pheromone production are dependent on the presence of calcium in the incubation medium. A calcium-free incubation medium and a medium with calcium replaced by 5 mM magnesium did not support Bom-PBAN-stimulated and ionophore-stimulated pheromone production. Furthermore, a potent calcium blocker, lanthanum, inhibits Bom-PBAN stimulated pheromone production at 0.5 mM. These results suggest that the activation of sex pheromone production by PBAN in ECB is mediated, in part, by calcium ion.

Sites of synthesis and release of PBAN-like factor in the female European corn borer, Ostrinia nubilalis

Abstract Sex-pheromone production in female European corn borer moths, Ostrinia nubilalis, is regulated by PBAN(pheromone biosynthesis activating neuropeptide)-like factors. Using a decapitated-moth bioassay, three discrete sets of neurosecretory cells were identified in the subesophageal ganglion of female O. nubilalis that contained PBAN-like biological activity. Immunocytochemical studies with a polyclonal antiserum raised against a synthetic-truncated Hez-PBAN revealed the presence of PBAN-like immunoreactivity throughout the entire ventral nervous system. The corpora cardiaca also exhibited PBAN-like biological activity and immunoreactivity. Corpora cardiacectomized-allatectomized females produced significantly less pheromone than sham-operated females. Cobalt anterograde/retrograde filling studies did not show direct neural connections between the terminal abdominal ganglion and the sex-pheromone gland. Transection of the ventral nerve cord did not impair pheromone production. Removal of the entire abdominal ventral nerve cord did not affect the response of the operated females to exogenous PBAN. Results of the present investigation show that PBAN-like factors in female O. nubilalis moth are synthesized in three sets of neurosecretory cells in the subesophageal ganglion, and that release of these factors from the corpora cardiaca plays a more important role in pheromone production than does the ventral nerve cord.

Expression of a gene that encodes pheromone biosynthesis activating neuropeptide in the central nervous system of corn earworm, Helicoverpa zea

Abstract Expression of the pheromone biosynthesis activating neuropeptide (Hez-PBAN) gene in the central nervous system of larval, pupal and adult Helicoverpa zea was studied using Northern hybridization analyses, reverse-transcriptase–polymerase chain reaction (RT-PCR), in situ hybridization histochemistry, and whole-mount immunocytochemistry. Northern hybridization experiments demonstrated the presence of a 0.8-Kb Hez-PBAN transcript in the subesophageal ganglion in adults, pupae and day 0–1 final instar larvae. In the subesophageal ganglion, Hez-PBAN mRNA was localized by in situ hybridization histochemistry to the mandibular, maxillary and labial cell clusters. Whole-mount immunocytochemical studies showed that these three cell clusters also possess PBAN-like immunoreactivity. Low levels of Hez-PBAN mRNA were revealed in other parts of the central nervous system, including the brain, thoracic ganglia and abdominal ganglia by RT-PCR. In day 0–1 final instar larvae, these low levels of Hez-PBAN mRNA were localized by in situ hybridization histochemistry to a pair of ventral midline neurons in each thoracic ganglion and some abdominal ganglia. PBAN-like immunoreactivity was also detected in these neurons. This study shows that in H. zea, the Hez-PBAN gene is expressed predominantly in the subesophageal ganglion, and at relatively low levels in other parts of the central nervous system.

Sex Pheromone Gland of the Female European Corn Borer Moth, Ostrinia nubilalis (Lepidoptera, Pyralidae): Ultrastructural and Biochemical Evidences

Abstract The sex pheromone gland of the female European corn borer moth, Ostrinia nubilalis was studied using light and electron microscopy. The pheromone gland is formed by hypertrophied epidermal cells at the mid-dorsal region of the intersegmental membrane between abdominal segments 8 and 9/10. Active glandular cells contain extensive apical membrane foldings, a single nucleus, many free ribosomes, numerous mitochondria, microtubules and lipid droplets. Smooth endoplasmic reticulum is scanty. In young moths, the glandular cells are smaller in size, the microvilli at the apical membrane are poorly developed and the cytoplasm contains fewer mitochondria, microtubules, and no lipid droplets. The surrounding unmodified epidermal cells are small cuboidal or squamous cells. These cells have ill-defined apical membrane foldings and do not contain lipid droplets in the cytoplasm and the overlying cuticle. Fatty acids analyses revealed the presence of the sex pheromone components, (E)-11-tetradecenyl acetate, and their immediate precursors, methyl (E)-11- and methyl (Z)-11-tetradecenoate, only in the dorsal portion of the cylindrical intersegmental membrane. Results of the present study show that the sex pheromone gland of O. nubilalis is restricted to the dorsal aspect of the intersegmental membrane between segments 8–9/10 and is not a ring-gland.

BACULOVIRUS EXPRESSION OF AN INSECT GENE THAT ENCODES MULTIPLE NEUROPEPTIDES

Sex pheromone production in the corn earworm, Helicoverpa zea, is regulated by a 33-amino-acid neuropeptide named Hez-PBAN (pheromone biosynthesis activating neuropeptide). Hez-PBAN is encoded in a preprohormone that also contains four other structurally related peptides. Two recombinant baculoviruses that contain two different sequences of Hez-PBAN cDNA under the control of a strong polyhedrin promotor were constructed. The first virus, AcWT-PBAN, contains the entire prepro-Hez-PBAN coding sequence. The second virus, AcBX-PBAN, contains a synthetic chimera gene encoding a bombyxin signal peptide sequence fused to a pro-Hez-PBAN sequence. Cell extracts, culture medium of BTI-TN-5B1-4 cells, and hemolymph from 4th instar Trichoplusia ni larvae, all infected with AcBX-PBAN, showed a high level of pheromonotropic activity. Pheromonotropic activity was not detected in the cells infected with AcWT-PBAN. Results of chromatographic and immunochemical studies showed that some of the potential processing sites in the expressed pro-Hez-PBAN sequence were not used during posttranslational processing in the AcBX-PBAN-4-infected BTI-TN-5B1-4 cells and 4th instar T. ni larvae. However, the processing pattern of the recombinant pro-Hez-PBAN in AcBX-PBAN-infected 4th instar T. ni larvae was similar to that exhibited in the central nervous system of H. zea adult females, since a PBAN-like immunoreactive-peptide-band was found in the hemolymph of Ac-BX-PBAN-4-infected 4th instar T. ni larvae. In a droplet feeding assay, neonate and 3rd instar T. ni larvae infected with AcBX-PBAN-4 showed a significant reduction in survival time (26% and 19%, respectively) when compared to control larvae that were infected with a polyhedrin-deficient virus, Ac-E10.

Anatomy of the neurosecretory cells in the cerebral and subesophageal ganglia of the female European corn borer moth, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)

Abstract The anatomy of the neurosecretory cells in the brain-subesophageal ganglion complex of female European corn borer moth Ostrinia nubilalis (Lepidoptera: Pyralidae) was studied using histological and cobalt backfilling techniques. Histological staining revealed the presence of 2 median and one lateral neurosecretory cell groups in the brain. These brain neurosecretory cells are made up of mainly type A cells with a few type B cells in the median group. Three type C neurosecretory cell clusters occupy the apparent mandibular, maxillary, and labial neuromeres at the ventral median aspect of the subesophageal ganglion. Axonal pathways of the neurosecretory cell groups were delineated by retrograde cobalt filling from the corpora cardiaca. Fibers of the 3 brain neurosecretory cell groups merged to form a distinct axonal tract that exits the brain via the fused nervi corporis cardiaci-1 + 2. Cobalt backfilling from the corpora cardiaca filled 4 groups of cell bodies in the subesophageal ganglion. The presence in the subesophageal ganglion of extensive dendritic arborizations derived from the brain suggests interactions between neurosecretory cell groups in the 2 head ganglia.

Involvement of the Nervous System with PBAN

It is generally accepted that PBAN (pheromone biosynthesis activating neuropeptide) and PBAN-like compounds possessing pheromonotropic activity are biosynthesized in the subesophageal ganglion (SEG). Immunocytochemical studies in several species have revealed discrete groups of PBAN-like immunoreactive cells in the SEG to support this conclusion (Davis et al. 1993; Kingan et al. 1992; Tips et al. 1993). In two lepidopterous speciesHelicoverpa zeaandBombyx moria gene has been characterized from the SEG and found to encode PBAN and four other related peptides (Kawano et al. 1992; Ma et al. 1994). PBAN immunoreactivity also was found by these investigators in the corpora cardiaca (CC), as well as along the ventral nerve cord to the terminal abdominal ganglion. These data support a multiplicity of functions and means of transportation for these peptides, including PBAN. This chapter, however, is not intended as a review of all data that support either a mode of action for PBAN involving release from the CC into the hemolymph or involving transportation down the ventral nerve cord. The focus will be on research from our laboratory on several species and how some aspects of the mode of action of PBAN may or may not involve the nervous system in those species.