Studies on Biosynthesis of a Type II Sex Pheromone, Transportation of the Hydrophobic Precursor in a Body, and Mechanism of Their Hormonal Regulation

尺蛾科日本巨环虫（Ascotis selenaria cretaceous, Assc）的处女雌虫分泌C_19 3,4-环氧-6,9-二烯来吸引雄虫。先前的实验表明，环氧烯基信息素是由信息素腺体外产生的相应的3,6,9-三烯生物合成的，并与脂蛋白结合后通过血淋巴运输到它。为了阐明这种转运，我们用KBr平衡密度梯度超离心纯化了在SDS-PAGE上显示出两个条带（apoLp I和apoLp II，分别为约250 kDa和约80 kDa）的雌性飞蛾高密度脂蛋白（HDLp），并通过溶剂提取物的气相色谱-质谱分析证实了其与三烯的关联。接下来，HDLp的作用揭示了局部应用氘化三烯基前体的腹部的女性。三烯基前体与HDLp有关。在它们的信息素腺体中，我们检测到更多的三烯和氘化环氧信息素，表明三烯通过血淋巴运动。实验与男性飞蛾A.s.cretacea和雌性飞蛾的完全混乱显示相同的HDLp协会三烯局部应用。这一结果表明,成年女性A.s.cretacea没有开发HDLp专业三烯运输。为了调节其在信息素腺中的生物合成，Assc雌性在食道下神经节（SG）中产生信息素生物合成激活神经肽（PBAN），许多鳞翅目的雌性也是如此。我们已经分离出Assc-PBAN cDNA，该cDNA编码181个氨基酸，包括PBAN同源物和其他四个假定的肽：滞育激素同源物，α-SG神经肽（SGNP）， β-SGNP和γ-SGNP，它们的c端都有一个FXPR(K)L基序。虽然从其他15个物种中已经鉴定出含有30-35个氨基酸的pban，但Assc-PBAN同源物由28个氨基酸组成，与其他物种相比同源性较低（< 46%）。含有8个氨基酸的Assc- β-SGNPs也比其他16-22个氨基酸的β-SGNPs短。此外，所有已知的PBAN cDNA在β-SGNP和PBAN之间都有一个GRR序列作为切割位点，但Assc-PBAN cDNA在相应位置显示了一个不寻常的GR序列，表明β-SGNP和PBAN之间可能存在非切割。天然信息素异位肽的色谱行为揭示了Assc-PBAN的独特结构，包括βSGNP，即SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH_2，这是首次从产生环氧烯基性信息素的昆虫中检测到PBAN。少

Virgin females of the Japanese giant looper (Ascotis selenaria cretacea, Assc) in the family of Geometridae secrete C_19 3,4-epoxy-6,9-diene to attract males. Previous experiments have suggested that the epoxyalkenyl pheromone is biosynthesized from the corresponding 3,6,9-triene produced outside of a pheromone gland and transported to it via hemolymph after association with lipophorin. In order to clarify this transport, high-density lipophorin (HDLp) in the female moths showing two bands (apoLp I with ca.250 kDa and apoLp II with ca.80 kDa) on an SDS-PAGE was purified by KBr equilibrium density-gradient ultracentrifugation, and the association of the triene was confirmed by GC-MS analysis of a solvent extract from the isolated protein. Next, the role of HDLp was revealed by a topical application of the deuterated trienyl precursor to the abdomens of the females. The trienyl precursor was associated with HDLp. In their pheromone glands, the triene and the deuterated epoxy pheromone we … More re detected, indicating movement of the triene via the hemolymph. Experiments with male moths of A.s.cretacea and female moths of Bombyx moil showed the same association of HDLp with the triene topically applied. This result suggested that the adult females of A.s.cretacea did not develop HDLp specialized in the triene transport.To regulate its biosynthesis in the pheromone gland, Assc females produce a pheromone biosynthesis-activating neuropeptide (PBAN) in the suboesophageal ganglion (SG), as do females in many lepidopteran species. We have isolated Assc-PBAN cDNA, which encodes 181 amino acids, including a PBAN homologue and four other putative peptides : a diapause hormone homologue, α-SG neuropeptide (SGNP), β-SGNP, and γ-SGNP, all of which shared an FXPR(K)L motif on their C-termini. Although PBANs with 30-35 amino acids have been characterized from 15 other species, the Assc-PBAN homologue consisted of 28 amino acids and showed low homology (< 46 %) compared with the others. Assc-β-SGNP with 8 amino acids was also shorter than the other β-SGNPs (16-22 amino acids). Furthermore, all of the known PBAN cDNAs have a GRR sequence between β-SGNP and PBAN as a cleavage site, but the Assc-PBAN cDNA showed an unusual GR sequence at the corresponding position, indicating the possibility of non-cleavage between the β-SGNP and PBAN. The chromatographic behavior of the natural pheromonotoropic peptide revealed the unique structure of Assc-PBAN including βSGNP, i.e., SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH_2, as the first determination of PBAN from the insects producing an epoxyalkenyl sex pheromone. Less