Possible Roles of Juvenile Hormone and Juvenile

Physiological Entomology (2013) 38, 183–191 DOI: 10.1111/phen.12010

Possible roles of Juvenile Hormone and Juvenile Hormone binding protein on changes in the integument during termination of larval diapause in the bamboo borer Omphisa fuscidentalis

EAKARTIT RITDACHYENG1, MANAPORN MANABOON1, STEPHEN S. TOBE2 andTIPPAWAN SINGTRIPOP1 1Department of Biology, Chiang Mai University, Chiang Mai, Thailand and 2Department of Cell and System Biology, The University of Toronto, Toronto, Ontario, Canada

Abstract. To understand the role of Juvenile Hormone (JH) on changes in the integument of Omphisa fuscidentalis Hampson (Lepidoptera: Pyralidae) during larval–pupal development, the expression patterns of transcripts of the O. fuscidentalis JH binding protein gene (OfJHBP) are determined in the integument of diapausing larvae. Quantitative real-time polymerase chain reaction (qRT-PCR) studies show that the relative levels OfJHBP mRNA in the integument do not differ signiﬁcantly in different segments of the body. Application of the JH analogue (JHA) methoprene to diapausing larvae results in a relatively low expression of OfJHBP mRNA from days 0 to 8 after JHA application and reaches maximal levels in the pupal stage. Changes in OfJHBP expression after 20-hydroxyecdysone (20E) injection also show low levels of OfJHBP mRNA from days 0 to 8 and show a peak of expression in the pupal stage. Incubation of the integument in vitro in the presence of JHA (3.22 μm) induces high levels of OfJHBP expression within 120 min, whereas incubation with 20E (2.08 μm) induces gene expression at 150 min. To study the cooperative effect on OfJHBP expression of these two hormones, dose–response experiments are performed. Larval integument is maintained in the presence of 0.32 μm JHA or 2.08 μm 20E or a combination of both hormones in vitro for 120 min. Induction of OfJHBP expression by JHA and 20E in combination is signiﬁcantly higher than that of either hormone alone. The results impact not only on our understanding of how methoprene (and hence JH) terminates larval diapause through an increase in ecdysteroid titre in the haemolymph, but also on how JH acts directly on the integument to stimulate the expression of a JH binding protein gene. Key words. 20-hydroxyecdysone, bamboo borer, integument, Juvenile Hormone analogue, Juvenile Hormone binding protein, methoprene.

Introduction the expression of genes related to reproductive maturation (Gilbert et al., 2000). Juvenile Hormone (JH) is essential for insect development Diapause is a form of developmental arrest in insects that and reproduction (Riddiford, 1985). In the larval stages, JH is similar to hibernation in higher animals. It enables insects suppresses the genes responsible for larval metamorphosis, and related arthropods to circumvent adverse seasons. Winter is whereas, in the adults of many insect species, it stimulates most commonly avoided in temperate zones, although diapause is also used to avoid hot, dry summers and periods of food Correspondence: Tippawan Singtripop, Department of Biology, shortage in the tropics (Denlinger, 1981, 1985). Diapause in Chiang Mai University, Chiang Mai 50200, Thailand. Tel.: +66 53 larval stages, as in the south-western corn borer Diatraea 943346; e-mail: [email protected] grandiosella, is induced by a high haemolymph titre of JH

© 2013 The Royal Entomological Society 183 184 E. Ritdachyeng et al. that is maintained throughout diapause (Yin & Chippendale, mainly in the fat body, which is the primary site of JHBP 1973). The high JH titre prevents the brain from releasing synthesis. Interestingly, a modest level of OfJHBP expression prothoracicotropic hormone, resulting in the failure of the is detected in the integument. From these observations, it is prothoracic gland to synthesize ecdysteroids. hypothesized that the integument is one of the target tissues In Northern Thailand, Laos and Myanmar, larvae of the for JH in O. fuscidentalis. Knowledge of the expression pat- bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: tern of OfJHBP in the integument may not only provide an Pyralidae) experience a severe annual dry season. In Chiang understanding of how JH regulates metamorphosis in O. fus- Mai Province, Northern Thailand, adults appear in August, cidentalis, but also aid in unravelling the molecular action of and lay egg clusters on new bamboo shoots. After completing JH in the termination of larval diapause. larval growth in September, the larvae enter diapause and In the present study, the expression pattern of OfJHBP remain inside the internode of bamboo culm for 9 months until transcripts in integument from different segments of the body, they pupate during the subsequent June (Singtripop et al., as well as after hormonal induction, is examined both in vivo 1999). Application of the JH analogue (JHA) methoprene and in vitro. to diapausing larvae induces pupation, which results in an increase in the secretory activity of prothoracic gland in vitro, followed by an increase in ecdysteroid titre starting Materials and methods on day 12 after JHA application (Singtripop et al., 2000). These results show that JHA and hence JH might be involved Animals in the termination of larval diapause in O. fuscidentalis,in contrast to previous ﬁndings showing that JH inhibits the Larvae of O. fuscidentalis were collected from bamboo brain–prothoracic gland axis and thus maintains the larval forests in Amphur Maewang, Chiang Mai Province, Thailand, diapause (Denlinger, 1985). and kept in containers (12 × 14 × 8 cm) lined with sheets of ◦ After topical treatment of diapausing larvae with 1 μgof paper towel. The containers were maintained at 25 C under JHA, the larvae turn brown and develop a hard and pigmented continuous darkness (Singtripop et al., 1999). cuticle. Treatment of diapausing larvae with a lower amount of JHA (0.1 μg) yields larvae that become inactive before formation of the brown cuticle. The body colour then changes Hormones and analogues from creamy to light yellow and these larvae are designated as G1. The next day, the dorsal epidermis becomes light The JHA (S -methoprene; SDS Biotech, Japan) was dis- brown (designated G2) as a result of the deposition of solved in acetone at a concentration of 5 mg mL−1 and stored ◦ a pigmented pupal cuticle beneath the old larval cuticle. at −35 C as a stock solution. The stock solution was diluted to Approximately 1 day later, the entire body becomes brown 1.0 μgper5μL with acetone, and a 5-μL aliquot was applied (G3). The body colour turns darker and the integument topically to the dorsal surface of individual larvae. Control hardens a further 2 days after G2, and this stage is designated larvae received acetone alone. After JHA treatment, the lar- G4. Approximately 3 days after deposition of the pupal cuticle vae (designated as G0) became motionless and their integu- (G2), pupae enter stage G5. None of the G5 larvae shed the ment turned brown and formed a hard, pigmented cuticle. old cuticle. If the old cuticle is removed with forceps, the The physiological age of these larvae was divided into six treated insects are found to possess evaginated appendages, stages, as described above, from G0 to G5, based on body such as antennae, compound eyes and mandibles. They also colour and deposition of the pigmented pupal cuticle (Fig. 1) have forewings covered with a tanned cuticle, hind wings with (Singtripop et al., 2000). 20-hydroxyecdysone (20E; Sigma, St almost no tanned cuticle, and legs with a tanned cuticle. The Louis, Missouri) was dissolved in ethanol at a concentration ◦ larval prolegs with crochets disappear. These morphological of 1 mg mL−1 and stored at −35 C until use. The stock solu- characteristics indicate that the animals with tanned cuticles tion was diluted with distilled water, and diapausing larvae are complete pupae (Singtripop et al., 2000). were injected with a 5-μL aliquot through the second pro- Hidayat & Goodman (1994) report that, in a lepidopteran, leg (Singtripop et al., 2002). Control larvae received distilled over 99.9% of JH in the haemolymph is bound to JH binding water alone. The amounts of JHA and 20E applied were based protein (JHBP). They suggest further that interaction of the on Singtripop et al. (2000). JH–JHBP complex with target cell membranes may be the route by which JH reaches the cytoplasm. Moreover, JHBP is found in cytosol of larval and pupal epidermis of Galleria RNA isolation and cDNA synthesis mellonella (Wisniewski´ et al., 1988) and a JHBP with receptor properties has been detected in the epidermis of the Dipteran Total RNA was isolated from the integument of the second Drosophila hydei (Klages et al., 1980, 1981). dorsal abdominal segments using RiboZol RNA Extraction Ritdachyeng et al. (2012) report the characterization and reagent (Amresco, Solon, Ohio) in accordance with the cloning of the gene encoding JHBP from O. fuscidentalis manufacturers instructions. Before cDNA synthesis, the RNA (OfJHBP, GenBank accession number JQ658990). The expres- was treated with RNase-free DNaseI (Fermentas, Lithuania) sion patterns of OfJHBP in various tissues reveal the target to eliminate contaminating genomic DNA. An aliquot of organs of JH. The results show that the OfJHBP is expressed 200 ng of total RNA was used to generate ﬁrst-strand cDNA

GAC AGA GAG GCG-3) and reverse primer (5-CTC ACA CGT TAT AGT CTC AGG-3) and, for endogenous control, OfRpL3 (O. fuscidentalis ribosomal protein L3, accession number EF453378), forward primer (5-TCT ACC CCA AGA AGA GGT CTC G-3) and reverse primer (5-ACG ACA GTC CTC AGA CAT GTG C-3). These primers were based on the respective sequences of the cloned full- length OfJHBP and OfRpL3 (Ritdachyeng et al., 2012). The THUNDERBIRD SYBR qPCR Mix (Toyobo, Japan) was used to perform qRT-PCR in an iCycler iQ5 Real-time PCR Detection System (Bio-Rad, Hercules, California). Each of the PCR amplifications was run in triplicate from three independent samples, in a 20-μL reaction volume, which contained 1 × THUNDERBIRD SYBR® qPCR Mix, 0.2 μm Fig. 1. Induction of pupal development in diapausing larvae of each of forward and reverse primers and 1.0 μL of cDNA. ◦ Omphisa fuscidentalis by treatment with the Juvenile Hormone The thermal parameters were 94 C for 5 min, followed by μ ◦ ◦ ◦ analogue (JHA) S -methoprene. Larvae were treated once with 1 g 40 cycles at 94 C for 20 s, 60 C for 30 s and 72 Cfor JHA by topical application. Progression of pupal metamorphosis 40 s. To verify that the primers for qRT-PCR could amplify graded from 1 to 5 (G1–G5) (Singtripop et al., 2000). the corresponding genes, automated melting point analysis and agarose gel electrophoresis of the PCR product were using an oligo-dT primer and M-MuLV-Reverse Transcriptase carried out. Amplification efficiencies for both reference and (Fermentas). target genes were determined. After qRT-PCR, the relative expression of OfJHBP from samples with different treatments was estimated by a comparative C T method (C T)for Effects of JHA and 20E on OfJHBP gene expression in vitro relative quantitation of gene expression. The C T (cycle threshold) is defined as the number of cycles required for A diluted stock solution of JHA (0.02 μg μL−1) was the fluorescent signal to cross the background level. The prepared in ethanol. The solution was evaporated in air and dynamic range of both target (OfJHBP) and normalizer resuspended in Grace’s insect cell culture medium (Gibco (OfRpL3 ) was determined. After normalization with OfRpL3 Invitrogen Corporation, Grand Island, New York). All equip- [i.e. C T = C T(OfJHBP) – C T(OfRpL3 )], the C T value of ment used in the JHA experiment was coated with polyethylene the treatment group was compared with that of the control glycol using a 1% solution. For the 20E experiments, stock group known as calibrator (Livak & Schmittgen, 2001) (i.e. −1 solutions (0.02 μg μL ) were prepared in distilled water. CT). All results of qRT-PCR were analyzed using Pfaffl’s In brief, the diapausing larvae were anaesthetized on ice mathematical model (Pfaffl, 2001). Expression of OfRpL3 for 30 min. The integument of the second dorsal abdominal mRNA in the fat body and other tissues in O. fuscidentalis was segment was dissected. Then, the fat body was removed com- constant throughout development and during the period after pletely from the integument under a dissecting microscope, and hormone treatments. Expression of OfRpL3 as a reference gene the segment was rinsed five times in ice-cold Ringer’s solution has also been used to study mRNA expression levels in many (130 mm NaCl, 4.7 mm KCl and 1.9 mm CaCl2) to eliminate genes; for example, Ofhsp70 , Ofhsc70 , Ofhsp90 , OfSP1&2 any additional potential contamination by the fat body. The and OfTreh-1&2 (Tatun et al., 2008; Tungjitwitayakul et al., tissue was rinsed again in culture medium five times. The 2008a, b). The relative expression of OfJHBP was calculated stock solutions of JHA and 20E were diluted serially with from three samples of independent experiments. Data were culture medium to the appropriate working concentrations subjected to one-way analysis of variance followed by Tukey’s (JHA; 16.1 nm to 32.2 μm, 20E; 10.4 nm to 20.8 μm). Individ- test for multiple comparisons. P < 0.01 was considered ual pieces of integument were incubated in 100 μL of medium significantly different. Data are presented as the mean ± SEM. containing hormones in a 96-well culture plate (Sero-Wel, ◦ Bibby Sterilin, U.K.) at 25 C with gentle shaking. For the controls, the integument was incubated in equal volumes of Results the medium without JHA or 20E. Total RNAs from the integument in both hormone treatments and control experiments Expression pattern of OfJHBP transcripts in the integument were isolated at the same time points after incubation. The expression pattern of OfJHBP in the integument from various body segments of diapausing larvae was examined by qRT-PCR qRT-PCR (Fig. 2). The integument was dissected from six different body segments: second dorsal thoracic segment, second The gene-specific primers used for amplifying cDNA ventral thoracic segment, second dorsal abdominal segment, fragments corresponding to OfJHBP (GenBank accession second ventral abdominal segment, seventh dorsal abdominal number JQ658990) were: forward primer (5-TTC AAA ATG segment and seventh ventral abdominal segment. The relative

To examine the effect of JHA on the change in OfJHBP expression as a function of time, the integument was incubated in medium containing 3.22 μm of JHA for varying periods (Fig. 4A). During the incubation period, the relative expression of OfJHBP was low between 0 and 30 min, and then increased, reaching a maximum at 120 min. Expression then decreased to a low level at 150–180 min after the addition of methoprene to the bathing medium. On the basis of results of the time course experiment, the dose–response of OfJHBP mRNA expression to JHA was determined by adding various concentrations of methoprene (i.e. 16.1 nm to 32.2 μm) to the medium and analyzing 120 min after treatment. The relative level of mRNA expression was low at JHA concentrations from 16.1 nm to 0.161 μm, μ Fig. 2. Expression of OfJHBP mRNA in the integument from increasing to a maximal value at 0.322 m JHA (Fig. 4B). μ various body segments of Omphisa fuscidentalis as determined by Expression then decreased on exposure to 1.61 m JHA, and the quantitative real-time polymerase chain reaction. 2DT, second remained at steady low levels when incubated with higher dorsal thoracic segment; 2VT, second ventral thoracic segment; 2DAb, concentrations (i.e. 3.22–32.2 μm). second dorsal abdominal segment; 2VAb, second ventral abdominal The effect of 20E on the expression of OfJHBP in segment; 7DAb, seventh dorsal abdominal segment and 7VAb, seventh the integument in vitro was also examined. For the time ventral abdominal segment. The level of gene expression in various course experiment (Fig. 5A), the explants of integument were body parts were normalized relative to the level in the second maintained in vitro in the presence of 2.08 μm 20E. The dorsal abdominal segment. Data are presented as the mean of three levels of OfJHBP mRNA increased slightly from 0 to 120 min independent samples. A one-way analysis of variance with Tukey’s test after exposure to 20E, and peaked at 150 min, followed by a for multiple comparisons was used to assess differences in OfJHBP transcripts levels among different body segments. decrease at 180 min. Based on the previous results of the time course experiment, the expression of OfJHBP was determined over a range of expression of OfJHBP mRNA was not signiﬁcantly different 20E concentrations (10.4 nm to 20.8 μm) at 150 min (Fig. 5B). between integument from various body segments (P = 0.075). Total RNA was collected and isolated from the integument. The relative expression of OfJHBP was low at concentrations of 20E ranging from 10.4 nm to 1.04 μm. The level of OfJHBP Effects of JHA and 20E on OfJHBP gene expression in the mRNA increased to a maximum level at 2.08 μm; however, integument in vivo higher concentrations of 20E (10.4 and 20.8 μm)resultedina decrease in the level of OfJHBP expression. The effect of JHA on OfJHBP mRNA expression was examined using integument from the second dorsal abdominal segment. Pupation was induced by treating diapausing larvae Additive effects of JHA and 20E on the expression of OfJHBP with methoprene. After the application of 0.1 μgofJHAper in the integument in vitro animal, the relative levels of OfJHBP mRNA were low from days 0 to 8, and then increased rapidly to a maximum in the The potential for a cooperative effect of JHA and 20E on G1 stage (Fig. 3A). Thereafter, expression declined abruptly the expression of OfJHBP was examined under the conditions to low levels until the G3 stage. established in the previous experiments. The integument was The effects of 20E on the expression of OfJHBP were maintained in medium in the presence of 0.322 μm JHA or also examined. In larvae injected with 0.1 μgof20E,levels 2.08 μm 20E alone or with 0.322 μm JHA and 2.08 μm 20E of OfJHBP mRNA were low from days 0 to 2, and then together for 120 min. The relative level of OfJHBP mRNA decreased on days 4 and 6 (Fig. 3B). The relative expression expression in integument incubated in the presence of JHA then remained at low levels from day 8 to G1, reaching a peak and 20E in combination was signiﬁcantly higher (P = 0.0002) in the G2 stage and decreasing during the G3 stage. than that in medium with either JHA or 20E alone (Fig. 6). To determine whether the stimulatory effect by both JHA and 20E together was a synergistic or additive effect, the calculated Effects of JHA and 20E on the expression of OfJHBP mRNA sum of the increase in relative OfJHBP mRNA level was in the integument in vitro compared with the observed increase, namely the increased level of OfJHBP expression after treatment with JHA and 20E To understand how hormones induce JHBP gene expression together in vitro. The observed increase in OfJHBP expression directly in the integument, integument from the second dorsal was equal (relative expression = 23.8) to the calculated sum abdominal segment was isolated from diapausing larvae and of the increase (relative expression: 17.69 + 5.72 = 23.4). This incubated in vitro in the presence of hormones under various supports the notion that JHA and 20E stimulate OfJHBP conditions. expression in an additive manner.

Fig. 3. Induction of OfJHBP mRNA expression in the integument of diapausing larvae after topical application of 0.1 μg of Juvenile Hormone analogue (JHA) (A) or injection of 0.1 μg of 20-hydroxyecdysone (20E) (B) as a function of stage. The levels of OfJHBP mRNA were determined from the total pool of RNA after treatment; D0−D8 = 0–8 days after treatment, G0–G3 = stages in pupal development. The larvae became motionless before the formation of a brown cuticle (G0). The body colour then changes from creamy to light yellow (G1). The next day, the dorsal epidermis turns light brown (G2). Approximately 1 day later, the entire body becomes brown (G3) (Singtripop et al., 2000). For the controls, larvae were treated with acetone or distilled water, in the JHA and 20E experiments, respectively. Total RNA was isolated from the integument of staged Omphisa fuscidentalis larvae (as above) and mRNA expression levels were analyzed using the quantitative real-time polymerase chain reaction. The results are expressed as the relative expression after normalization against the expression of endogenous OfRpL3 . Values are relative to the level of expression in the different controls collected at the same time points as the treated samples. Each value is the mean ± SEM of three independent experiments. Means with different letters are signiﬁcantly different (analysis of variance, n = 3, F 8,18 = 6.885, P < 0.01).

Discussion for the integument comprising a JH-target organ. Knowledge of the expression pattern of OfJHBP in the integument after JHA In the bamboo borer O. fuscidentalis, topical application of the treatment may thus provide insight into how JH is involved in JHA methoprene to diapausing larvae induces pupation. The the termination of larval diapause of O. fuscidentalis. prothoracic glands of these larvae become active, followed by On the basis of the relatively constant levels of OfJHBP an increase in haemolymph ecdysteroid titre. Subsequently, mRNA in the integument collected from different body the larvae turn brown with a hard and pigmented cuticle, segments of diapausing larvae, integument from the second which indicates pupation of these larvae (Singtripop et al., dorsal abdominal segment was selected for further study of 2000). These results indicate that JH may be involved in the the effects of a hormone and analogue on the expression of termination of larval diapause in O. fuscidentalis. OfJHBP under various conditions. It is possible that JHBP mRNA levels correlate with JH titre The effects of methoprene and 20E treatment in vivo show during the development of O. fuscidentalis (Ritdachyeng et al., a similar stimulatory action on the relative levels of OfJHBP 2012). Moreover, the changes in the cuticle and expression of expression in the integument. After hormone (20E or JHA) OfJHBP in the integument after JHA application are evidence application, levels of OfJHBP mRNA are relatively low in

Fig. 4. Induction of OfJHBP gene expression by Juvenile Hormone analogue (JHA) in the integument of diapausing larvae in vitro. (A) Time course of OfJHBP mRNA expression after treatment with 3.22 μm JHA determined at 30-min intervals. (B) Effect of JHA doses on OfJHBP expression at 120 min after treatment with various concentrations of JHA (16.1 nm to 32.2 μm). Total RNA was extracted and mRNA expression levels were determined using the quantitative real-time polymerase chain reaction. The results are expressed as the relative expression after normalization against endogenous OfRpL3 . Expression is relative to gene expression in the different controls (integument incubated in medium without addition of JHA was collected at the same time points as the treated samples). Each value represents the mean ± SEM of three independent experiments. Means with different letters are significantly different (analysis of variance, n = 3, F 6,14 = 18.660, P < 0.01). the larval stage and reach a maximum after ecdysis to the period corresponds to the decrease in JH binding activity in the pupal stage, followed by a subsequent decrease in relative epidermal cytosol as recorded in G. mellonella (Wisniewski´ expression. These results in O. fuscidentalis correspond to et al., 1988). The JH titre increases again during the prepupal the reported increase in JH binding activity in the cytosol of period in many insects (Schooley et al., 1976; Hsiao & Hsiao, the epidermis of the greater wax moth G. mellonella after 1977). These results support the assumption that JH must be each ecdysis (Wisniewski´ et al., 1988). Moreover, work by present during the pupal stage (G1 and G2) of O. fuscidentalis, Nakato et al. (1994) demonstrates that JHA (methoprene) which corresponds to a maximal level of relative OfJHBP activates transcription of the LCP30 gene, a major larval expression. There is evidence suggesting that an increase in cuticle protein occurring in the integument throughout the JH titre is necessary during this period (i.e. the pupal stage) larval stages. Each of the above findings supports a role for to prevent premature adult development (Williams, 1961; JH in the maintenance of larval characteristics by regulating Kiguchi & Riddiford, 1978). Another possible explanation for the expression of larval-specific genes such as larval cuticle OfJHBP expression being at its highest level after ecdysis to proteins in the integument (Riddiford, 1985). The observed the pupal stage is that JHBP facilitates the clearance of JH decrease in relative expression of OfJHBP to the lowest level from tissues by a specific JH esterase (Touhara et al., 1995) during the late last larval stadium of O. fuscidentalis after because a decline in JH titre is necessary to permit subsequent treatment with JHA, as shown in the present study, may be metamorphosis (Nijhout & Williams, 1974). explained by a decrease in JH sensitivity or an increase in To understand how hormones induce JHBP gene expression JH esterase activity in the final larval instars, which results in directly in the integument of diapausing larvae of O. fusciden- JH degradation, as reported in G. mellonella. In many tissues, talis, the expression of OfJHBP is examined by maintenance including the epidermis of G. mellonella, a rapid increase in of integument with JHA and 20E in vitro. Gene expression rate of JH degradation occurs, starting on the third day of the is up-regulated after JHA treatment, with a maximum relative last larval stadium (Wisniewski´ et al., 1986a, 1987). This time expression within the first 120 min. Expression of OfJHBP is

Fig. 5. Induction of OfJHBP gene expression by 20-hydroxyecdysone (20E) in the integument of diapausing larvae in vitro. (A) Time course of OfJHBP expression after treatment with 2.08 μm JHA determined at 30-min intervals. (B) Dose–response of the induction of OfJHBP expression at 150 min after treatment with several concentrations of 20E (10.4 nm to 20.8 μm). Total RNA was extracted and mRNA expression levels were determined using the quantitative real-time polymerase chain reaction. The results are expressed as the relative expression after normalization against endogenous OfRpL3 . Expression is relative to gene expression in the different controls (integument incubated in medium without addition of 20E was collected at the same time points as the treated samples). Each value represents the mean ± SEM of three independent experiments. Means with different letters are significantly different (analysis of variance, n = 3, F 6,14 = 13.044 P < 0.01). also stimulated by 20E within 150 min. These results provide vivo of both hormones on OfJHBP mRNA expression need to evidence that JH itself plays a crucial role in regulating the be examined further to confirm these results. Niewiadowska- expression of its carrier protein or receptor (Orth et al., 1999). Cimicka et al. (2011) used a luciferase reporter assay to The discovery of ecdysteroid response elements located in investigate the JHBP core promoter responses to JH and 20E the 5 promoter region of the Manduca sexta JHBP (Orth treatment in G. mellonella. No synergistic effect is observed et al., 2003) and Chorion factor 1/ultraspiracle (CF1/USP), after treatment of cells with both JH and 20E, suggesting that which are putative regulatory elements from the region of the the integument of G. mellonella and O. fuscidentalis may G. mellonella JHBP promoter (Sok et al., 2008), suggests respond to these two hormones similarly. The current gene that ecdysteroids may also be involved in the regulation expression data obtained in vivo and in vitro therefore should of OfJHBP expression. In M. sexta and G. mellonella,the affect not only our understanding of the previous finding that ecdysteroid response elements of MsJHBP and GmJHBP may JH might be involved in the termination of larval diapause be involved in down-regulation of JHBP expression (Orth in O. fuscidentalis through an increase in ecdysteroid titre in et al., 2003; Sok et al., 2008). Based on the dose–response the haemolymph (Singtripop et al., 2000), but also suggest data for methoprene and 20E on the relative levels of OfJHBP that one of the functions of JH in the termination of larval mRNA expression, both compounds are used to test a potential diapause is to stimulate OfJHBP expression directly in the cooperative effect. It is found that simultaneous treatment with integument. This could provide a new mechanism for the JHA and 20E increases OfJHBP mRNA expression in an addi- regulation of larval diapause in O. fuscidentalis. tive manner relative to the application of either JHA or 20E The mechanisms of regulation OfJHBP gene expression alone. This suggests that the two hormones regulate OfJHBP by steroid and terpenoid hormones are still unknown. In expression in the same way, or by a similar mechanism. It M. sexta and G. mellonella,the5 upstream promoter region appears that 20E may be involved in the up-regulation of of the gene for JHBP contains several transcription binding OfJHBP expression in the integument, although it may act sites, including an ecdysteroid response element and USP, through different pathways. However, the additive effects in as described above. Ultraspiracle has been suggested to be a

References

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Klages, G., Emmerich, H. & Peter, M.G. (1980) High-affinity binding Drosophila fuscidentalis. In the presence of both hormones, JHA may sites for juvenile hormone I in the larval integument of hydei. Nature, 286, 282–285. bind to USP, and 20E may bind to EcR (Jones et al., 2001). A Klages, G., Emmerich, H., & Peter, M.G. (1981) Uptake and high heterodimer of USP and EcR could be formed. In addition, affinity binding of juvenile hormone I to the integument cytosol a stable EcR/USP complex is formed upon the binding of of Drosophila hydei larvae. Regulation of Insect Development and ligands, resulting in the conformational changes. The binding Behavior (ed. by F. Sehnal, A. Zabza, J. J. Menn and B. Cym- of this stable heterodimer complex to the ecdysteroid response borowski), pp. 673–690. Wydawnictwo Politechniki Wroclawskiej, elements located in the 5 region of ecdysteroid responsive Poland. genes (i.e. the gene for JHBP in this case) could induce gene Livak, K.J. & Schmittgen, T.D. (2001) Analysis of relative gene transcription (Yao et al., 1993). expression data using real-time quantitative PCR and the 2(−Delta The present studies on the relative expression of OfJHBP Delta C(T)) method. Methods, 25, 402–408. in the integument indicate important functions for JH with Nakato, H., Shofuda, K., Izumi, S. et al. (1994) Structure and respect to regulating changes in the integument during the developmental expression of a larval cuticle protein gene of the development of O. fuscidentalis. In the larval stages, JH silkworm, Bombyx mori. Insect Biochemistry, 1218,65–74. et al may be involved in the maintenance of larval characteristics Niewiadowska-Cimicka, A., Schmidt, M., Ozyhar,˙ A. . (2011) Juvenile hormone binding protein core promoter is TATA-driven by regulating the synthesis and secretion of larval cuticle with a suppressory element. Biochimica et Biophysica Acta, 1809, proteins. 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Insect Biochemistry Chiang Mai University fellowship to E.R. and Molecular Biology, 33, 93–102.

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