Juvenile hormone III produced in male accessory glands of the longhorned beetle, Apriona germari, is transferred to female ovaries during copulation

Abstract

We report on juvenile hormone (JH) biosynthesis in vitro by male accessory glands (MAGs) in the longhorned beetle, Aprionona germari, accompanied by the transfer of JH from males to females during copulation. JH was extracted from the MAGs and separated by reversed-phase high-performance liquid chromatography. JH III was identified as the major JH by gas chromatography-mass spectrometry. A radiochemical assay and a non-radioactive method were used to measure the in vitro rate of JH biosynthesis by the MAGs. After 4 h of incubation with 3H-methionine in the medium, the radioactivity in the MAGs substantially increased. In a separate assay, incubation of the MAGs with non-radioactive methionine for 4 h resulted in a 39% increase in JH III. Seven-day-old males were injected with medium 199 containing 3H-methionine and 24 h later they were mated with virgin females. Hemolymph and the MAGs were collected from the mated males and hemolymph, ovaries and eggs were collected from the mated females for assaying radioactive JH. The radioactivity incorporated into JH in the MAGs was transferred to the females during copulation and later transferred into their eggs. Assayed 1 h after copulation, JH III level in the MAGs decreased 42% and the content of JH III in the male hemolymph did not change, whereas the content of JH III in the female hemolymph and ovaries both increased.

JH biosynthesis by reproductive tissues and corpora allata in adult longhorned beetles, Apriona germari

“…Identification of FA was based on ion intensities of six diagnostic ions m/z 5 81, 109, 121, 135, 149, and 205 (Burns et al, 2002 (Tian et al, 2010). After 4 h incubations in the presence of radioactive methionine, there were significant increases in radioactive JH III in the testes and ovaries, but not in the CA of males or females (Fig.…”

“…The virgin male and female adult bodies are covered with yellow bristle, which would be rubbed to form black blots on the sternum of mated male and the tergum of mated females during copulation; these are called ''mating blots '' (Ji et al, 1998). As the female adults produce eggs after emergence regardless of copulation, the virgin status of females was confirmed because their eggs did not hatch after normal incubation times (Tian et al, 2010).…”

“…In another variation on the classical view, we found a JHAMT activity that transferred a methyl group from methionine to produce JH III in MAGs; JH III was transferred to females during copulation. The transferred JH was ultimately taken up from hemolymph by ovaries and transferred into eggs (Tian et al, 2010). These findings provoked the hypothesis that testes, ovaries, and MAGs can biosynthesize JH III from long-chain intermediates in the JH biosynthetic pathway, beginning with farnesol.…”

“…After removing external water by blotting, we cultured half-tissues, separately, in Medium 199 (final methionine 250 mM, CaCl 2 10 mM, 1% Ficoll 400, 0.1% BSA, pH 7.0) for 4 h with unlabeled methionine and the other half-tissues were quick-frozen as controls. We processed sets of 10 tissues for JH III extraction in hexane and determined JH III content by RP-HPLC using external standard JH III (Tian et al, 2010) and performed three independent biological replicates.…”

“…After the incubations, the reactions were stopped by adding of 500 ml methanol: ethyl ether (1:1, V:V). MAGs were then homogenized in the medium and were extracted for three times with 1 ml of isooctane (Tian et al, 2010). We estimated radioactivity in 1 ml of total isooctane extract in a liquid scintillation counter (LS 6500; Beckman) and conducted three independent biological replicates.…”