JH BIOSYNTHESIS by REPRODUCTIVE TISSUES and CORPORA ALLATA in ADULT LONGHORNED BEETLES, Apriona Germari

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).

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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 it under a nitrogen stream, then analyzed the extract on reverse phase high-performance liquid chromatography (RP-HPLC; Angilent, Palo Alto, California) equipped with a Hypezsil BDS C18 column (150 4.6 mm) and gas chromatography– mass spectrometry (GC-MS). We identified the compounds by comparing retention times to synthetic FA, MF, JH III acid, and JH III standards. The RP-HPLC conditions were: 40–80% acetonitrile: H2O(H2O containing 0.1% trifluoroacetic acid, V: V; 1 ml/min) for 20 min, 100% acetonitrile for 10 min, monitoring absorbance at 218 nm. We confirmed the chemical identifications on GC-MS (CP-3800/Saturn 2200, Varian, Palo Alto, California) equipped with a VF-5 MS column (Varian, Palo Alto, California) in the Analysis Center of Nanjing Normal University. The chromatography conditions were initial injector temperature 5 801C for 1 min; Injector temperature increased at 501C/min to 2701C, initial column temperature 5 601C for 1 min; column temperature increased at 101C/min to 2601C; He carrier gas linear flow velocity 5 1ml/min. The MS was in the electron impact ion source under the following conditions: multiplier voltage 5 70 eV; mass acquisition range 5 60–350 amu; scan rate 5 1 sec. Identification of FA, MF, and JH III was based on retention time and the comparison of fragmentation patterns and retention indexes of compounds eluted during analysis of samples with synthetic standard FA, MF, and JH III mixture. JH Biosynthesis Assay for CA, Testes, and Ovaries via Methionine Incorporation In Vitro

We recorded the JH biosynthesis in CA, testes, and ovaries isolated as described just above. The radiochemical assay (RCA) followed (Tobe and Pratt, 1974; Cusson et al., 1999). Before incubation, we washed the organs 2 with saline buffer, blotted them dry, and determined their wet weight on a microbalance (BS110S, Sartorius, Go¨ttingen, Germany). We separated paired organs and incubated one half-gland (either left or right) in 1 ml modiﬁed medium 199 containing 0.714 nM 3H-methionine (ﬁnal methionine 250 mM, CaCl2 10 mM, 1% Ficoll 400, 0.1% BSA, pH 7.0; GibcoBRL) for 4 h at 281C in the dark. The other half-gland served as the control; we mixed the control half with 199 medium without incubation to remove the radioactivity carried by organ. After the incubations, the reactions were stopped by

Archives of Insect Biochemistry and Physiology Juvenile Hormone Biology in Apriona germari 279 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. We confirmed the results of the RCA after the methods just described. CA, MAGs, testes, and ovaries were isolated from 30 adults in modified saline. After removing external water by blotting, we cultured half-tissues, separately, in Medium 199 (final methionine 250 mM, CaCl2 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.

JH Biosynthesis in CA, MAGs, Testes, and Ovaries From Added Farnesol We isolated CA, MAGs, testes, and ovaries from 30 males and 30 females and prepared the tissues as just described. We incubated one half-organs in 1 ml Grace’s medium (ﬁnal CaCl2 10 mM, 1% Ficoll 400, 0.1% BSA, pH 7.0) supplemented with farnesol (1 mg) at 281C in the dark for 4 h. The complementary lobes of paired organs were quick-frozen as controls. We extracted FA and JH with hexane and the content was quantiﬁed by RP-HPLC using external standards and performed three independent biological replicates.

JH Biosynthesis in CA, MAGs, Testes, and Ovaries From Added FA and From Added MF We isolated CA, MAGs, testes, and ovaries from one set of ten females and ten males. We incubated half-organs in Grace’s medium (without methionine, CaCl2 10 mM, 1% Ficoll 400, 0.1% BSA, pH 7.0) supplemented with 4 mg FA for 4 h at 281C in the dark, the other side organs were quick-frozen as controls. After the incubation, we extracted and determined JH content by RP-HPLC, performing three independent biological replicates. We conﬁrmed JH biosynthesis in testes, MAGs, and ovaries from FA and, separately, MF. We isolated CA, MAGs, testes, and ovaries from 15 males and 15 females. Thirty half-organs were randomly divided into three groups, incubating one group in medium 199, one group in medium 199 supplemented with 4 mg FA, and the other group in medium 199 supplemented with 4 mg MF. After incubation for 4 h at 281C in the dark, we extracted and analyzed JH as just described and performed three independent biological replicates.

Statistical Analyses

Sample means were compared with two-tailed Student’s t-test with signiﬁcance set at Po0.05.

RESULTS

We rigorously identified FA, MF, and JH III in the testes, MAGs, ovaries, and CA from males and females by retention times on chromatography and by the presence of specific diagnostic ions on mass spectrometry. 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.,

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2002). Identification of MF was based on ion intensities of six diagnostic ions m/z 5 219, 191, 169, 137, 121, and 109. Identification of JH III was based on ion intensities of six diagnostic ions (JH III 5 m/e 235, 217, 189, 147, 121, and 111) (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. 1A). Incubations in the presence of unlabeled methionine yielded similar results (Fig. 1B). JH III increased by about 96% in the testes and about 49% in the ovaries. Again, JH III did not increase in the CA of males nor females. As can be seen in Figure 2A, the testes, MAGs, and ovaries converted exogenous farnesol into substantial quantities of FA. The CA from males and females also produced FA, however, the CA from males produced considerable more FA, up from controls by about 20-fold compared with about 2-fold in the CA from females. The outcomes were otherwise with respect to conversion of farnesol into JH (Fig. 2B). After incubations in the presence of farnesol, JH III contents significantly increased in the testes and ovaries, but not in MAGs, nor in the CA from both genders. The data presented in Figure 3A show that after incubations in the presence of exogenous FA and in the absence of exogenous methionine, JH III titers in the testes, MAGs, and ovaries increased significantly. JH III titers did not increase in the CA from both genders after incubations in the presence of FA. However, after incubation for 4 h in the presence of both exogenous FA and methionine, the JH III titer in ovaries was not increased compared with the ovaries incubated in the presence of only methionine (Fig. 3B). Incubations in the presence of MF led to significant increases in JH III titers in the testes, MAGs, and ovaries, but not in the CA of males nor females (Fig. 3B).

DISCUSSION

The outcomes of our experiments support our hypothesis that the testes, ovaries, and MAGs can biosynthesize JH III from long-chain intermediates in the JH biosynthetic pathway, beginning with farnesol. Several points are germane. First, we identified FA, MF, and JH III in the testes, MAGs, ovaries, and the CA from males and females by chemical analysis. Second, incubations in the presence of exogenous methionine led to increased JH III titers in the testes, ovaries, and MAGs, but not in CA from both genders. Third, incubations in the presence of exogenous farnesol let to increased titers of FA in all tested organs and led to increased JH III titers in the testes, MAGs, and ovaries, but not in CA. Fourth, incubations in the presence of exogenous FA yielded increased titers of JH III in three organs, but not in CA. Finally, incubations in the presence of exogenous MF were followed by significant increases in JH III in the testes, MAGs and ovaries, but, again, not in CA. Aside from bolstering our hypothesis, these data allow us to infer the biochemical pathway of JH III synthesis in A. germari reproductive tissues. There are two likely routes to JH III. In one, farnesol is oxidized to FA, followed by epoxidation to JH III acid and methylation to JH III. Although we recorded substantial amounts of FA, MF, and JH III, JH III acid did not appear in any of our chemical analyses, from which we favor an alternate pathway of methylating FA into MF and epoxidation directly to JH III, as outlined by Goodman and Granger (2005). We conclude that specific reproductive tissues in male and female longhorned beetles biosynthesize JH. This opens a door to future research on the biological role(s) of JH in post-mated adults.

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A 150 60 * *

100 40 weight) weight) 50 20 JH (nmol/ g fresh JH (nmol/ g fresh

0 0 Control testes Control ovaries 1.5 1.5

1 1

0.5 0.5 JH (nmol/ pair CA) JH (nmol/ pair CA) 0 0 Control male CA Control female CA

B 150 150 * * 100 100 weight) weight) 50 50 JH III (nmol/ g fresh JH III (nmol/ g fresh 0 0 Control testes Control ovaries 15 10

10 5 5 JH III (nmol/ pair CA) JH III (nmol/ pair CA) 0 0 Control male CA Control female CA

Figure 1. Incorporation of the methyl group of methionine into JH by isolated testes, ovaries, and CA of Apriona germari.(A) The JH biosynthesized by the RCA method in the testes, ovaries, male CA, and female CA. Histogram bars represent mean JH content (nmol/g fresh weight or nmol/pair CA)71SDin indicated tissues; black bar: control; gray bar: experimental. Three biological replicates were conducted. Ã Ã indicates signiﬁcant differences. Po0.05. (B) Incorporation of unlabeled methyl group from methionine into JH III by the testes, ovaries, male CA, and female CA. The histogram bars represent mean FA content (nmol/g fresh weight or nmol/pair CA)71 SD in indicated tissues; black bar: control; gray bar: experimental. Ten animals were used for each group and three biological replicates were conducted. Ã indicates signiﬁcant differences. JH, juvenile hormone; CA, corpora allata; FA, farnesoic acid; RCA, radiochemical assay.

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A 12000 * 30000 20000 * 15000 * 8000 20000 10000 weight) weight) weight) 4000 10000 5000 FA (nmol/g fresh FA FA (nmol/g fresh FA FA (nmol/g fresh FA 0 0 0 Control Testes Control MAGs Control Ovaries 150 40 * * 30 100 20 50 10 FA (nmol/pair CA) FA FA (nmol/pair CA) FA 0 0 Control Male CA Control Female CA B 150 * 100 100 * 100 50 50 weight) 50 weight) weight) JH III (nmol/g fresh 0 JH III (nmol/g fresh JH III (nmol/g fresh 0 0 Control Testes Control MAGs Control Ovaries 20 15 15 10 10 5 5

0 0 JH III (nmol/pair CA) JH III (nmol/pair CA) Control Male Control Female CA CA

Figure 2. Incorporation of exogenous farnesol into farnesoic acid and JH III by isolated reproductive tissues and CA. (A) FA content in the testes, MAGs, ovaries, male CA, and female CA after incubation in the presence of farnesol. The histogram bars represent mean FA content (nmol/g fresh weight or nmol/pair CA)71 SD in indicated tissues; black bar: control; gray bar: experimental. Three biological replicates were Ã conducted. indicates signiﬁcant differences. (B) JH III content the in testes, MAGs, ovaries, male CA and female CA after incubation in the presence of farnesol. The histogram bars represent mean JH III content (nmol/g fresh weight or nmol/pair CA)71 SD in indicated tissues; black bar: control; gray bar: experimental. Ã Three biological replicates were conducted. indicates signiﬁcant differences. JH, juvenile hormone; MAG, male accessory glands; CA, corpora allata; FA, farnesoic acid.

JH biosynthesis is age-related in some adults. The CA of older T. molitor biosynthesized JH III in vitro (Judy et al., 1975) but did not produce JH during the ﬁrst week after adult emergence. Female T. molitor did not lay eggs until 1 week after adult emergence (Trautmann et al., 1974a,b; Weaver et al., 1980) and reproductive tissues did not biosynthesize JH in this species. JH III was detected in the young A. germari adult reproductive tissues by GC-MS analysis and the adults produce eggs within 1 or 2 days after eclosion, regardless of whether or not they were mated (Tian et al., 2010). We note that the biology of JH biosynthesis in T. molitor and A. germari is quite different. The situation is different also in larvae of the cockroach, Diploptera punctata.InD. punctata, the CA ceases JH III synthesis during the last half of the fourth instar, but it continues to produce and release FA throughout this period (Cusson et al., 1991). This is superﬁcially similar to A. germari adults. The reproductive tissues but not

Archives of Insect Biochemistry and Physiology Juvenile Hormone Biology in Apriona germari 283

A 150 120 * * 80 *

100 80 60 40 weight) weight)

50 40 weight) 20 JH III (nmol/g fresh JH III (nmol/g fresh 0 0 JH III (nmol/g fresh 0 Control Testes Control MAGs Control Ovaries 10 15

10 * 5 5

0 0 JH III (nmol/ pair CA) Control Male CA JH III (nmol/ pair CA) Control Female CA B 200 a 200 a 100 a b 150 b 150 b 80 b c 60 100 100 c 40 weight) weight) weight) 50 50 20 JH III (nmol/g fresh JH III (nmol/g fresh 0 0 JH III (nmol/g fresh 0 Control Testes Control MAGs Control Ovaries 8 10 6 8 6 4 4 2 2 0 0 JH III (nmol/pair CA) JH III (nmol/pair CA) Control Male CA Control Female CA

Figure 3. Incorporation of exogenous precursors into JH after incubation in the presence of farnesoic acid and methyl farnesoate. (A) JH III content in testes, MAGs, ovaries, male CA and female CA after incubation in the presence of exogenous farnesoic acid compared with controls. The histogram bars represent mean JH III content (nmol/g fresh weight or nmol/pair CA)71 SD in indicated tissues; black bar:control;graybar:experimental. Ã Three biological replicates were conducted. indicates signiﬁcant differences. (B) JH III content in MAGs, ovaries, male CA, and female CA after incubation in the presence of exogenous methyl farnesoate compared with controls. The histogram bars represent mean JH III content (nmol/g fresh weight or nmol/pair CA)71 SD in indicated tissues; black bar: control; gray bar: exogenous FA; white bar: exogenous MF. Three biological replicates were conducted. Histogram bars annotated with the same letter are not signiﬁcantly different. JH, juvenile hormone; MAG, male accessory glands; CA, corpora allata; FA, farnesoic acid, MF, methyl farnesoate.

CA produced JH from long-chain intermediates while the CA produced only FA. We inferred that the reproductive tissues produce enough JH to promote reproduction regardless the JH biosynthetic capacity of CA. In A. aegypti, the CA, male MAGs, and female ovaries produce JH (Borovsky et al., 1994a,b). Whether such syntheses occur more generally and are physiologically relevant is not yet understood (Lafont, 2000). In two lepidopterans, the spruce budworm, Choristoneura fumiferana, and the related oblique banded leaf roller, C. rosaceana, JH biosynthesis increased over time from 1 to 5 days after copulation (Cusson et al., 1999). The authors considered the possibility that the males transfer JH to females during copulation, however, their radioimmunoassay data

Archives of Insect Biochemistry and Physiology 284 Archives of Insect Biochemistry and Physiology, December 2010 show that the MAGs of these two species do not produce JH in sufficient quantities for sexual transfer. The authors suggested an allatotropic factor that evokes JH synthesis in the female or a factor transferred by the male that stimulates female tissue other than CA to release an allatotropic substance may operate in this mating system (Cusson et al., 1999). The A. germari reproductive system differs from these two lepidopterans as JH biosynthesized in longhorned beetle MAGs is transferred to females during copulation (Tian et al., 2010). Aside from contributing JH, per se, one or more chemical mating factors (possibly JH from males) may stimulate JH biosynthesis in females after copulation (Tian et al., 2010). Sexual transfer of JH is not limited to beetles, however, as males of the moth H. virescens also transfer JH to females during copulation (Park et al., 1998). These examples demonstrate a tremendous variation in mating-related increases in JH titers in females, also noted by Cusson et al. (1999). JHs are highly pleiotropic hormones, acting in several aspects of insect reproduction. Among its important roles, JH can influence mating itself, in some insect species, through regulation of sex pheromone biosynthesis (Lemmen and Evenden, 2009). However, Bober et al. (2010) recently reported that JH does not influence the expression of the gene encoding the pheromone biosynthesis activating neuropeptide receptor (PBAN-R) in the moth Helicoverpa armigera. To the contrary, JH apparently inhibits PBAN-R transcript levels. Possibly one of the more far-reaching roles of JH lies the area of mediating trade-offs among physiological functions. Rolff and Siva-Jothy (2002) showed that the biological economy of mating necessitates a reduction in immunity in the beetle T. molitor. In this species, the trade-off between reproduction and immunity is mediated by JH. Zera et al. (1998) describe a different physiological trade-off, this one between reproduction and migration in the cricket Gryllus assimilis. Again, this trade-off is mediated by JH. These cases illustrate the wide range of JH actions in insect biology; we expect research into the biology and molecular biology of JH will continue to expand this wide range.

ACKNOWLEDGMENTS

The authors thank Zhijing He, State Key Laboratory of Pharmaceutical Biotechnology in Nanjing University. We are very grateful to Dr. William G. Bendena (Queen’s University, Canada) for the improvement and suggestions of this manuscript.

LITERATURE CITED

Bober R, Azrielli A, Rafaeli A. 2010. Developmental regulation of the pheromone biosynthesis activating neuropeptide-receptor (PBAN-R): re-evaluating the role of juvenile hormone. Insect Mol Biol 19:77–86. Borovsky D, Carlson DA, Hancock RG, Rembold H, Van HE. 1994a. De novo biosynthesis of juvenile hormone III and I by the accessory glands of the male mosquito. Insect Biochem Mol Biol 24:437–444. Borovsky DD, Carlson DA, Ujva´ry I, Prestwich GD. 1994b. Biosynthesis of (10R)- juvenile hormone III from farnesoic acid by Aedes aegypti ovary. Arch Insect Biochem Physiol 27:11–25. Burns SN, Teal PEA, Meer RKV, Nation JL, Vogt JT. 2002. Identiﬁcation and action of juvenile hormone III from sexually mature alate females of the red imported ﬁre ant, Solenopsis invicta. J Insect Physiol 48:357–365.

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Cusson M, Yagi KJ, Ding Q, Duve H, Thorpe A, McNeil JN, Tobe SS. 1991. Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: the search for a unifying arthropod endocrinology. Insect Biochem 21:1–6. Cusson M, Delisle J, Miller D. 1999. Juvenile hormone titers in virgin and mated Choristoneura fumiferana and C. rosaceana females: assessment of the capacity of males to produce and transfer JH to the female during copulation. J Insect Physiol 45:637–646. Goodman W, Granger N. 2005. The juvenile hormones. In: Gilbert LI, Iatrou K, Gill SS, editors. Comprehensive molecular insect science, vol. 3. Oxford: Elsevier Pergamon. p 319–408. Ji B-Z, Qian F-J, Wang Y-C. 1998. Effects of diflubenzuron on haemolymph proteins of adult Batocera horsfieldi Hope. J Nanjing Forest Univ 22:1–5. Judy KJ, Schooley DA, Troetschler RG, Jennings RC, Bergot BJ, Hall MS. 1975. Juvenile hormone production by corpora allata of Tenebrio molitor in vitro. Life Sci 16:1059–1066. Lafont R. 2000. Understanding insect endocrine systems: molecular approaches. Entomol Exp Appl 97:123–136. Lemmen J, Evenden M. 2009. Peripheral and behavioral plasticity of pheromone response and its hormonal control in a long-lived moth. J Exp Biol 212:2000–2006. Minakuchi C, Namiki T, Yoshiyama M, Shinoda T. 2008. RNAi-mediated knockdown of juvenile hormone acid O-methyltransferase gene causes precocious metamorphosis in the red flour beetle Tribolium castaneum. FEBS J 275:2919–2931. Park YI, Ramaswamy SB. 1998. Role of brain, ventral nerve cord, and corpora cardiaca- corpora allata complex in the reproductive behavior of female tobacco budworm (Lepidoptera: Noctuidae). Ann Entomol Soc Amer 91:329–334. Park YI, Shy S, Ramaswamy SB, Srinivasan A. 1998. Mating in Heliothis virescens: transfer of juvenile hormone during copulation by male to female and stimulation of biosynthesis of endogenous juvenile hormone. Arch Insect Biochem Physiol 38:100–107. Raikhel AS, Brown MR, Belles X. 2005. Hormonal control of reproductive processes. In: Gilbert LI, Iatrou K, Gill SS, editors. Comprehensive molecular insect science, vol. 3. Oxford: Elsevier Pergamon. p 433–491. Rolff J, Siva-Jothy MT. 2002. Copulation corrupts immunity: a mechanism for a cost of mating in insects. Proc Natl Acad Sci USA 99:9916–9918. Schooley DA, Barker FC. 1985. Juvenile hormone biosynthesis. In: Gilbert LI, editors. Comprehensive insect physiology, biochemistry and pharmacology, vol. 7. Oxford: Pergramon Press. p 363–389. Shui S-Y, Wen J-B, Chen M, Hu X-L, Kiu F, Li J. 2009. Chemical control of Apriona germari (Hope) larvae with zinc phosphide sticks. Forest Stud China 11:9–13. Taub-Montemayor ET, Min KJ, Chen Z, Bartlett T, Rankin MA. 2005. JH III production, titers and degradation in relation to reproduction in male and female Anthonomus grandis. J Insect Physiol 51:427–434. Teal PEA. 2002. Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta. Peptides 23:663–669. Teal PEA, Proveaux AT. 2006. Identification of methyl farnesoate from in vitro incubation of the retrocerebral complex of adult females of the moth, Heliothis virescens (Lepidoptera: Noctuidae) and its conversion to juvenile hormone III. Arch Insect Biochem Physiol 61:98–105. Tian L, Ji B-Z, Liu S-W, Jin F, Gao J, Li S. 2010. Juvenile hormone III produced in male accessory glands of the longhorned beetle, Apriona germari, is transferred to female ovaries during copulation. Arch Insect Biochem Physiol 75:57–67. Trautmann KH, Masner P, Schuler A, Suchy’ M, Wipf HK. 1974a. Evidence of the Juvenile hormone methyl (2E, 6E)-10,11-epoxy-3,7,11-trimethyl-2,6- dodecadienoate (JH-3) in insects of four orders. Z Naturforsch PT C 29:757–759.

Archives of Insect Biochemistry and Physiology 286 Archives of Insect Biochemistry and Physiology, December 2010

Trautmann KH, Schuler A, Suchy M, Wipf HK. 1974b. Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen von Insekten. Nachweis von 10,11- Epoxy-3,7,11-trimethyl-2-trans-6-trans dodecadiensaurementylester in Mellontha melolontha. Z Naturforsch PT C 29:161–168. Tobe SS, Pratt GE. 1974. The inﬂuence of substrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust in vitro. Biochem J 144:107–113. Tobe SS, Stay B. 1985. Structure and regulation of the corpus allatum. Adv Insect Physiol 18:305–432. Weaver RJ, Pratt GE, Hamnett AF, Jennings RC. 1980. The inﬂuence of incubation conditions on the rates of juvenile hormone biosynthesis by corpora allata isolated from adult females of the beetle Tenebrio molitor. Insect Biochem 10:245–254. Zera AJ, Potts J, Kobus K. 1998. The physiology of life-history trade-offs: experimental analysis of a hormonally induce life-history trade-off in Gryllus assimilis. Am Nat 152:7–23.

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