Isolation, endocrine regulation and transcript distribution of a putative primary JH-responsive gene from the pine engraver, Ips pini (Coleoptera: Scolytidae).

We isolated a cDNA of unknown function from a juvenile hormone III (JH III)-treated male midgut cDNA library prepared from the pine engraver beetle, Ips pini, and examined its genomic structure. The gene, tentatively named "Ipi10G08", encoded a 410 amino acid translation product that shared 26-37% identity with unannotated matches from several insects. Semi-quantitative RT-PCR analysis of Ipi10G08 following application of a 10 microg dose of JH III demonstrated an early induction for both male and female beetles, with transcripts being detectable after 45 min. An expression profile of male midgut tissue indicated Ipi10G08 transcript levels reach a maximum induction of approximately 22.5-fold control levels at 4h post-treatment. Tissue distribution studies displayed a large induction of Ipi10G08 mRNA in the alimentary canal of JH III-treated beetles, especially in males. A dose curve from both sexes suggested there may be a difference in the ability to respond to lower levels of JH III and immunoblot analysis indicated that although JH III highly induces transcript levels in females, protein levels are not similarly induced, while protein levels are induced in males. Ipi10G08 is likely a primary JH response gene and may provide insight into how this hormone exerts its actions.

Isolation, endocrine regulation and mRNA distribution of the 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) gene from the pine engraver, Ips pini (Coleoptera: Scolytidae).

We isolated a full-length cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) from the pine engraver beetle, Ips pini (Say), and examined its genomic structure. The intron-less gene has a predicted 460 amino acid cytosolic protein product with 73% identity to HMG-S from Dendroctonus jeffreyi, and high identity (58-64%) with other insect HMG-Ss. Topically applied juvenile hormone (JH) III induced HMG-S mRNA levels up to 6.5-fold in both sexes, mostly in the anterior midgut, though there were differences between males and females in the timing, sensitivity to JH III dose and tissue distribution of HMG-S mRNA. These data further validate the coordinate regulation of mevalonate pathway genes for de novo isoprenoid pheromone production in bark beetles.

Effects of juvenile hormone on gene expression in the pheromone-producing midgut of the pine engraver beetle, Ips pini.

Juvenile hormone III (JH III) stimulates biosynthesis of the monoterpenoid aggregation pheromone component, ipsdienol, in the anterior midgut of the male pine engraver beetle, Ips pini (Say). To understand better the hormonal regulation of pheromone biosynthesis in this forest pest, and identify JH III-responsive genes, microarrays were prepared and hybridized to cDNA from midguts of JH III-treated beetles. Expression patterns were confirmed by quantitative real-time RT-PCR. JH III co-ordinately regulated mevalonate pathway genes and many other genes implicated in pheromone biosynthesis. Sex differences in basal levels of mevalonate pathway genes were consistent with their role in male-specific pheromone biosynthesis. This is the first microarray-based study of the developmental and hormonal regulation of insect pheromone biosynthesis.

Preparative chiral liquid chromatography for enantiomeric separation of pheromones.

Cellulose triacetate was investigated as a chiral stationary phase for preparatively separating the enantiomers of lineatin, frontalin, exo-brevicomin, endo-brevicomin, verbenone, (E)-conophthorin, and grandisol. Tens of milligrams of both enantiomers were efficiently prepared in high percentage enantiomeric excess from one injection of each compound except grandisol. We prepared grandisyl acetate, benzoate, and 4-bromobenzoate to determine if derivatization of the free alcohol might improve separation. Of these, grandisyl 4-bromobenzoate provided the best separation but was still not very well resolved. Preparative separation of enantiomers on cellulose triacetate is a viable alternative to stereoselective synthesis when semiochemicals of very high enantiomeric purity are required for biological testing.

Changes in the intramolecular stable carbon isotope ratios with age of the European cave bear (Ursus spelaeus).

Changes with age in the diet and metabolism of the extinct European cave bear (Ursus spelaeus) from the Divje Babe archaeological site in northwestern Slovenia were investigated by comparing the stable carbon isotope ratio of whole bone collagen with that of the peptide-bonded carboxyl carbons in collagen. These carboxyl carbons were selectively released by decarboxylation of the collagen hydrolysate with ninhydrin (2,2-dihydroxy-1,3-indanedione). The stable carbon isotope ratio increased with age for both the peptide-bonded and other carbons. Although only one-third of the carbon atoms in collagen are peptide-bonded, they account for most of the observed change with age of the stable carbon isotope ratio of the whole collagen. Stable nitrogen isotope ratios decreased with age and were negatively correlated with changes in the stable carbon isotope ratios. These observations support previous investigations on the unique metabolism of hibernating bears.