JH BIOSYNTHESIS by REPRODUCTIVE TISSUES and CORPORA ALLATA in ADULT LONGHORNED BEETLES, Apriona Germari
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Article JH BIOSYNTHESIS BY REPRODUCTIVE TISSUES AND CORPORA ALLATA IN ADULT LONGHORNED BEETLES, Apriona germari Ling Tian College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China; Key Laboratory of Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Bao-Zhong Ji College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China Shu-Wen Liu Management Office of Sun Yatsen’s Mausoleum, Nanjing, China Chun-Ling He, Feng Jin, and Jie Gao College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China David Stanley USDA/Agricultural Research Service, Biological Control of Insects Research Laboratory, Columbia, Missouri Sheng Li Key Laboratory of Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Grant sponsor: Chinese National Science Foundation Program; Grant numbers: 30271086; 30471399; Grant sponsor: Natural Science Fund for Colleges and Universities in Jiangsu Province; Grant number: 04KJB180053. Correspondence to: Bao-Zhong Ji, Nanjing Forestry University, Longpan Road 159, Nanjing 210037, China. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 75, No. 4, 275–286 (2010) Published online in Wiley Online Library (wileyonlinelibrary.com). & 2010 Wiley Periodicals, Inc. DOI: 10.1002/arch.20395 276 Archives of Insect Biochemistry and Physiology, December 2010 We report on juvenile hormone (JH) biosynthesis from long-chain intermediates by specific reproductive tissues and the corpora allata (CA) prepared from adult longhorned beetles, Apriona germari. The testes, male accessory glands (MAGs), ovaries, and CA contained the long- chain intermediates in the JH biosynthetic pathway, farnesoic acid (FA), methyl farnesoate (MF), and JH III. The testes and ovaries, but not CA, produced radioactive JH III after the addition of 3H-methionine and, separately, unlabeled methionine, to the incubation medium. We inferred that endogenous FA is methylated to MF in the testes and ovaries. Addition of farnesol led to increased amounts of FA in the testes, MAGs, ovaries, and CA, indicating oxidation of farnesol to FA. Addition of FA to incubation medium yielded increased JH III, again indicating methylation of FA to MF in the testes, MAGs, ovaries, but not CA. Addition of MF to incubation medium also led to JH III, from which we inferred the epoxidation of MF to JH III. JH biosynthesis from farnesol in the testes, MAGs, and ovaries of A. germari proceeds via oxidation to FA, methylation to MF, and epoxidation to JH III. This is a well-known pathway to JH III, described here for the first time in reproductive tissues of longhorned beetles. C 2010 Wiley Periodicals, Inc. Keywords: juvenile hormone; biosynthetic pathway; biosynthetic ability; reproductive tissue; corpora allata INTRODUCTION Juvenile hormones (JHs) are pleiotropic hormones responsible for influencing insect development, metamorphosis, pheromone biosynthesis, behavior, caste determination and diapause (Goodman and Granger, 2005). JHs also act in many aspects of reproduction, including gonadal maturation (Raikhel et al., 2005). The chemistry of JHs is fairly complex with at least nine biologically active forms. Lepidopterans produce JH 0, JH I, JH II and 4-methyl JH I; JH III is associated with several insect orders; higher dipterans produce a diepoxide JH called JH III bisepoxy; at least some orthopterans biosynthesize hydroxylated JHs, 40-, 80- and 120-hydroxy JH III (Goodman and Granger, 2005). The JHs are biosynthesized via the classical mevalonate pathway, known for 25 years, with variations appropriate to specific hormones (Schooley and Barker, 1985; Goodman and Granger, 2005). JHs have been studied in a relatively small range of insect species and discovery of additional forms of JH is not unexpected. JH is generally produced and released from the corpora allata (CA; Tobe and Stay, 1985) or CA cells associated with the ring gland of dipterans, although the detailed picture is more complicated because the CA is not the sole source of JHs. In some lepidopterans and dipterans, the male CA lacks the enzyme JH acid methyltransferase (JHAMT), the enzyme responsible for transferring a methyl group from S-adenosyl-L- methionine to the carboxyl group of farnesoic acid (FA) and JH acids. In these species, including the moth Heliothis virescens (Park and Ramaswamy, 1998; Park et al., 1998) and the boll weevil Anthonomus grandis (Taub-Montemayor et al., 2005), JH acid from the CA is transported via hemolymph to other organs, including male accessory glands (MAGs) and ovaries, where it is converted into JH. In other species, for example the Archives of Insect Biochemistry and Physiology Juvenile Hormone Biology in Apriona germari 277 yellow fever mosquito, Aedes aegypti, JH is biosynthesized de novo in the MAGs (Borovsky et al., 1994a) and the ovaries express a JHAMT to produce JH III (Borovsky et al., 1994b). More recent work by Minakuchi et al. (2008) revealed the presence of three methyltransferase genes in the red flour beetle, Tribolium castaneum, TcMT1,-2, and -3. TcMT3 protein, but not the other two, methylated FA and JH III acid. Treating flour beetle larvae with RNAi designed to silence TcMT-3 led to precocious larval–pupal metamorphosis, which could be rescued by applying methoprene to the experimental larvae. Some, but certainly not all, methyltransferases act in JH biosynthesis. Overall, the emerging picture of JH biology is becoming more complicated as on-going research leads to continued discovery of variations on the classical view. We have been investigating JH biology in the mulberry longhorned beetle, Apriona germari, a serious and widely distributed forest pest in China (Shui et al., 2009). 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. In this study, we report on the outcomes of experiments designed to test our hypothesis. MATERIALS AND METHODS Insect Rearing Newly emerged A. germari adults were collected on each day of June on the campus of Nanjing Forestry University, China. They were maintained in laboratory at 25711C with fresh biennial branch of paper mulberry, Broussonetia papyrifera. Whether the male and female adults had been mated was determined by ‘‘mating blots.’’ 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). Chemicals JH III, Ficoll 400, bovine serum albumin, farnesol, and Grace’s medium were purchased from Sigma-Aldrich (St. Louis, Missouri); Medium 199 purchased from GibcoBRL (Grand Island, New york); FA and methyl farnesoate (MF) purchased from Echolon (Utah University, Salt Lake, Utah City); 3H-methionine purchased from Perkin Elmer (NET061, Waltham, Massachusetts); Methionine purchased from Sangon Biotech Co., Ltd (Shanghai, China); NaCl, CaCl2, KCl, MgCl2.6H2O, NaHCO3, glucose, hexane, isooctane, ethyl acetate and dimethylbenzene purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China); 2,5-Diphenyloxazole purchased from Shanghai Hufeng chemical industry Co., Ltd (Shanghai, China). Archives of Insect Biochemistry and Physiology 278 Archives of Insect Biochemistry and Physiology, December 2010 Identification of JH Synthetic Precursors in A. germari Tissues by Reverse Phase High- Performance Liquid Chromatography and Gas Chromatography–Mass Spectrometry We carried out the analytical biochemistry in the State Key Laboratory of Pharmaceutical Biotechnology in Nanjing University. We produced JH III acid by saponification of synthetic JH III (Teal, 2002), purified JH III acid and confirmed its identity on liquid chromatography–mass spectrometry (TSQ7000; Finnigon, California) using an ion trap mass spectrometer with electrospray ionization at 4.5 KV performed at 2501C. We isolated MAGs, ovaries, and CA from 10 adults, age 7 days in modified saline buffer (116.2 mM NaCl, 1.8 mM CaCl2, 2.7 mM KCl, 1.0 mM MgCl2.6H2O, 1.8 mM NaHCO3, 42.8 mM glucose, and pH 6.5). Without trying to separate the CA from corpora cardiaca (Teal and Proveaux, 2006), we homogenized the organs and extracted for potential intermediates in the JH biosynthetic pathway in hexane with ultrasonic dispersion on ice for 10 min (SB-5200 DTD; Ningbo Scientz Biotechnology Co., Ltd., China), and then centrifuged at 12,700g 41C for 10 min, collected upper organic layer, and repeated the extraction three times. The aqueous phase was further extracted for FA and JH acids with 1 ml chromatographic grade ethyl acetate for three times. We combined the total hexane extract and ethyl acetate extract and concentrated