Ada Rafaeli, Institute for Technology and Storage of Agricultural Products, Volcani Center, ARO, P.O. Box 6, Bet-Dagan 50250, Israel. Tel.: + 972 3 9683729; fax: + 972 3 9604428; e-mail: firstname.lastname@example.org
Sex pheromone production in Helicoverpa armigera is regulated by pheromone-biosynthesis-activating neuropeptide (PBAN), which binds to a G-protein coupled receptor at the pheromone gland. We demonstrate the temporal differential expression levels of the PBAN receptor (PBAN-R) gene, reaching peak levels at a critical period of 5 h post-eclosion. Previous studies implied a possible regulatory role for juvenile hormone (JH). We herein demonstrate that PBAN-R expression levels increase normally when females are decapitated or head-ligated, removing the source of JH, before peak transcript levels are reached. Similarly, sex pheromone production can be induced by PBAN in such decapitated females. These results indicate that up-regulation, at this critical time, is not dependent on JH originating from the head. Conversely, JH injected in vivo at this critical period significantly inhibits PBAN-R transcript levels.
The PBAN-receptor (PBAN-R) was first identified by us from the pheromone gland of female Helicoverpa zeamoths (HezPBAN-R) as a G-protein coupled receptor (GPCR) (Choi et al., 2003). Since then, a number of additional receptors from pheromone glands belonging to other moth species have been sequenced based on homology to HezPBAN-R or identified from genome sequencing projects (Hull et al., 2004; Lee & Boo, 2006 NCBI Accession Number AY974334; Rafaeli et al., 2007; Zheng et al., 2007; Kim et al., 2008; see reviews: Bober & Rafaeli, 2009; Rafaeli, 2009) and two types may be distinguished by the presence of a C-terminal extension, allegedly important for internalization of the receptor after activation (Hull et al., 2004; Kim et al., 2008). The presence of both the PBAN-R protein, based on a binding assay using a photoaffinity-biotin labelled PBAN analogue, and the transcript was also demonstrated in neural tissues (brain, thoracic ganglion and ventral ganglia) of adult females as well as in the aedeagi of the male, which is a tissue homologous to the female pheromone gland (Rafaeli et al., 2007). Quantitative analysis (quantitative real-time reverse transcription PCR, qPCR) showed that the PBAN-R gene is expressed at lower levels in neural tissues and aedeagi relative to the pheromone glands (Rafaeli et al., 2007). The physiological significance of PBAN-R in these other tissues remains to be elucidated.
Sex pheromone titres in many adult moth species follow an age-dependent pattern. Under natural conditions an increase in sex pheromone production occurs, reaching peak values, and thereafter sex pheromone titres decline with increasing age (Raina et al., 1986; Teal et al., 1990; Foster et al., 1995; Delisle & Simard, 2003). Studies in our laboratory confirmed an age-dependent pattern of sex pheromone production in female Helicoverpa armigera in vitro, as was reported for other moth species in vivo, where PBAN activates sex pheromone production only in newly emerged and adult females (Fan et al., 1999a; Rafaeli et al., 2003; Rafaeli & Bober, 2005). In H. armigera, an age dependence was also reflected by the presence of the PBAN-R protein, evident already in pharates (1 and 2 days pre-eclosion) as well as in adults, but not in female pupae 3 days pre-eclosion (–3 day-old) (Rafaeli et al., 2003). However, pheromone glands were not responsive to PBAN in pre-eclosion females despite the presence of the PBAN-R protein (Rafaeli et al., 2003).
Insects have adapted to various strategies for vitellogenesis and egg development, which may be regulated by hormones such as juvenile hormone (JH), ecdysteroids and neurosecretions (Nijhout, 1994). Based on the various reproductive strategies, the lepidopterans can be grouped (Ramaswamy et al., 1997) into those that are in concert with the metamorphic process relying on ecdysteroid levels and emerging as fully vitellogenic adult females (eg Bombyx mori,Ohnishi, 1987) and those that depend on rising JH levels post-eclosion (eg H. zea, Satyanarayana et al., 1992). In addition to its central role in oogenesis, JH can affect dispersal and flight activity, calling behaviour, post-copulatory changes in female behaviour and oviposition behaviour (Hartfelder, 2000). Hartfelder (2000) eloquently described JH's role in insect reproduction as a conserved ‘integrator’ providing a platform for flexibility in life history strategies.
The role of JH in mating and the regulation of sex pheromone production has been demonstrated in a few species of migratory moths that display a unique behaviour (Cusson & McNeil, 1989; Gadenne, 1993; Cusson et al., 1994; Picimbon et al., 1995), but the direct involvement of JH in sex pheromone production is controversial in other moth species. Treatment with JH II or analogues of –1 day pre-eclosion pharate females (but not of younger pharates or pupae) was shown to trigger pheromone glands to respond to PBAN; however, at a lower level than adult female pheromone glands (Fan et al., 1999a; Rafaeli et al., 2003). Although JH and analogues did initiate specific binding to the PBAN-R protein in –3 day female pupae, these pupae did not respond to PBAN and did not produce sex pheromone (Rafaeli et al., 2003). Taken together, these results do not demonstrate a clear-cut stimulatory role of JH on sex pheromone production, but do imply that JH might be required for the primary induction of the gland to PBAN stimulation. In contrast, treatment of adult females with the JH analogue, fenoxycarb (FX) caused a significant inhibition of both sex pheromone production and specific binding to PBAN-R, thereby suggesting an inhibitory role for JH (Rafaeli & Bober, 2005).
Clarifying the molecular regulatory mechanisms associated with sex pheromone production and reproductive behaviour will advance our knowledge concerning the regulation and evolution of this GPCR and could lead to the development of specific strategies for disruption of the reproductive process. In the present study, we aimed to elucidate the temporal differential pattern of PBAN-R gene expression levels and the role of JH in adults of H. armigera with comparison to the silkworm, B. mori, thereby comparing representative species that both employ different reproductive strategies and also express different PBAN-receptor types.
Temporal regulation of the PBAN-R transcript levels in females and males
In order to verify the age-dependent pattern of the PBAN-R transcript in H. armigera, pheromone glands were removed and analysed at various time intervals from 3 day pre-eclosion pupae (−72 h) up to 3 day post-eclosion adult females (72 h) (Fig. 1A). Our results indicate that the PBAN-R gene expression levels in H. armigera pheromone glands increase and reach maximum levels within the first 5 h post-eclosion, whereas pheromone glands of pupae, pharate and adults up to 3 h post-eclosion express low levels (Fig. 1A). At this latter time females do not respond to PBAN and sex pheromone production cannot be induced (see below; Table 2– Intact 3 h). For comparison, the differential expression pattern was also elucidated for B. mori pheromone glands using 5 day pre-eclosion (−120 h) up to 2 day post-eclosion females (48 h), time points that correspond to the earlier maturation in this species. Up-regulation of B. mori PBAN-R indeed occurs 1 day prior to eclosion, increasing 1–2 days post-eclosion (Fig. 1B), but the precise timing was not delineated in this species.
Table 2. The effect of decapitation at 3 h post-eclosion on pheromone biosynthesis activating neuropeptide-receptor (PBAN-R)-stimulated sex pheromone production by Helicoverpa armigera
Age post-eclosion at pheromone quantification (h)
Differences in Z-11 hexadecenal levels after stimulation for 2 h in vivo (5 µM PBAN-stimulated levels – control levels) (ng/female) Mean ± SEM (n)
Fold stimulation in response to injected PBAN (5 µM PBAN-stimulated levels – control levels/control levels) Mean ± SEM (n)
Previously, we have shown the presence of PBAN-R in the aedeagus of H. armigera males (Rafaeli et al., 2007). To examine whether the differential expression of the PBAN-R transcript observed in females is restricted only to the temporal regulation of female sex pheromone production, we were motivated to also investigate the age-dependent pattern of the PBAN-R transcript in the aedeagi of males of both moth species. The PBAN-R transcript was quantified from H. armigera aedeagus at different time intervals from 3 day pre-eclosion pharates (−72 h) up to 3 day post-eclosion adults (72 h). Although the precise timing of up-regulation was not delineated, this tissue also revealed a developmental pattern with up-regulation occurring between 1 and 2 days post-eclosion (Fig. 1A). This finding suggests that the male and female transcripts both undergo transcriptional regulation, and that it is not specific to sex pheromone production in the female pheromone gland. To verify that this regulation of the PBAN-R expression is not limited to H. armigera males, we conducted a similar study using B. mori. The B. mori PBAN-R transcript was successfully detected in the adult aedeagus although at an eightfold lower magnitude than the PBAN-R transcript found in the B. mori pheromone gland (data not shown). Aedeagi, taken from different male age groups, showed a temporal differential gene expression profile with up-regulation occurring 1 day post-eclosion (Fig. 1B). As the PBAN-R transcript is also present in adult neural tissues, but at lower levels compared to adult pheromone glands (Rafaeli et al., 2007), quantitative analysis comparing 1 day pre-eclosion and 1 day post-eclosion female H. armigera brains and thoracic ganglia was also performed. The results revealed that pupal neural tissues did not contain measurable PBAN-R transcript levels. As levels of PBAN-R transcript were detected in 1 day post-eclosion females, it can be concluded that age dependence also occurs in female brains and thoracic ganglia (data not shown).
Effect of decapitation at various times on the PBAN-R transcript levels of females
The PBAN-R transcript levels of pheromone glands from H. armigera females that were decapitated (adults) or head-ligated (pupa) at various ages were compared to levels before head detachment (Table 1). The results demonstrate that transcript levels significantly increase with age despite the head detachment. Levels comparable to intact 5 h post-eclosion females were attained by pre-eclosion pharates after ligation for 48 or 24 h in –2 day-old and –1 day-old pharates, respectively. Similarly, decapitation at 1 or 3 h post-eclosion, when transcript levels are still low, did not prevent females from reaching normal transcript levels 24 h later (Table 1). In addition, head detachment did not affect PBAN-R transcript levels in 5 h post-eclosion or older females.
Table 1. The effect of head detachment of female Helicoverpa armigera at various ages on pheromone biosynthesis activating neuropeptide-receptor (PBAN-R) expression levels after 24 or 48 h
Age of female at head detachment* (h pre/post-eclosion)
PBAN-R transcript levels before head detachment Mean ± SEM (n)
PBAN-R transcript levels after 24 h† Mean ± SEM (n)
PBAN-R transcript levels after 48 h† Mean ± SEM (n)
As a result of a high pupal mortality rate after decapitation, pre-eclosion pupae were head-ligated, whereas post-eclosion adults were decapitated.
No PBAN-R transcript levels, attained 24 or 48 h after head detachment, were significantly different from levels in intact females 24 or 48 h later; that is, not differing from the expected PBAN-R transcript levels for the respective age (P≥ 0.05, Student's t-tests).
Differences relate to levels before and after head detachment for each group (Student's t-tests).
0.46 ± 0.04 (14)
0.42 ± 0.05 (14)
1.04 ± 0.07 (4)
0.47 ± 0.07 (11)
0.99 ± 0.08 (4)
0.49 ± 0.03 (13)
1.60 ± 0.13 (11)
1.12 ± 0.06 (25)
1.34 ± 0.21 (4)
1.33 ± 0.10 (32)
1.45 ± 0.14 (7)
1.12 ± 0.10 (13)
0.96 ± 0.05 (8)
1.02 ± 0.29 (14)
Effect of decapitation at 3 h post-eclosion on sex pheromone production
When PBAN was injected into 1 h post-eclosion H. armigera females and incubated in vivo for 2 h, pheromone glands of these 3 h post-eclosion females were not significantly stimulated by PBAN to produce sex pheromone (Table 2: intact 3 h), as expected from the low PBAN-R transcript levels (Fig. 1A: females). These 3 h post-eclosion females, containing low PBAN-R transcript levels (Table 1), were decapitated and challenged with PBAN for 2 h in vivo thereby reaching the critical period of 5 h post-eclosion. At this time, even though the PBAN-R transcript levels reached their maximum (Fig. 1A: females), the levels of sex pheromone attained were very low and not significantly different from levels of 3 h post-eclosion PBAN-stimulated females [Table 2: decapitated 5 h, Student–Newman–Keuls (SNK) rank1]. However, the level of stimulation reached by these females was significant, thereby indicating a minimal PBAN response (SNK rank2). Similar results were also evident in 6 h post-eclosion decapitated females (Table 2: decapitated 6 h). Although these maximum levels of transcript at 5 and 6 h post-eclosion did not reflect the ability to attain maximum sex pheromone levels in response to PBAN, after 24 h the decapitated females could be stimulated to produce sex pheromone at levels that did not differ from those observed in intact 24 h post-eclosion females (Table 2).
Effect of JH injections and FX topical applications on PBAN-R transcript levels in females
We have previously shown that treatment of –3 day-old female pupae with FX can initiate specific binding to the PBAN-R protein although such pupae could not respond to PBAN and did not produce sex pheromone (Rafaeli et al., 2003). This experiment was repeated in order to investigate the effect of FX at the same concentration (3 µg/female) on PBAN-R transcript expression levels. Therefore, FX was topically applied to female pupae 5 days pre-eclosion and after 48 h the PBAN-R transcript was quantified from pheromone glands of these, now –3 day-old pupae. Topical application of acetone served as a control. No significant difference (P= 0.7447, Student's t-test) was found in the relative gene expression levels of FX-treated pupae (0.42 ± 0.12, n= 7) when compared to the acetone treatment (0.36 ± 0.12, n= 6). Thus, the JH analogue did not induce up-regulation of the PBAN-R transcript.
To determine the regulatory role of JH on PBAN-R gene expression levels at the critical period, JH I, JH II and JH III were separately injected into 3 h post-eclosion females, with oil injections serving as controls. After 2 h incubation, thereby reaching the 5 h critical period for induction, the PBAN-R transcript was quantified from pheromone glands (Fig. 2). The results clearly show that all forms of JH significantly inhibit the normal induction of the transcript levels (oil treatment). JH II, the predominant JH in H. armigera (Fan et al., 1999a) also inhibited the levels of expression of the PBAN-R gene in 1 day-old females (Fig. 2), corroborating previous observations regarding its inhibitory effect on the PBAN-R protein and sex-pheromone production in adult females (Rafaeli & Bober, 2005).
In the present study, an age-dependent pattern of PBAN-R transcript levels, comparable to that of sex pheromone production by intact females in response to PBAN, was evident. Three hours post-eclosion females revealed low PBAN-R transcript levels and females were unable to produce sex pheromone in response to PBAN at this time. The increase in expression levels of the PBAN-R transcript was delineated to occur at a critical period of 5 h post-eclosion. Thus, by demonstrating an age-dependent pattern of expression we allude to the fact that regulation indeed must occur; however, in view of the fact that the PBAN-R transcript levels of decapitated newly emerged females or head-ligated pharates continued to increase and adhere to an age-dependent pattern, it can be deduced that factors originating from the female head during this critical period had no influence on PBAN-R transcription regulation. Although maximum levels of transcript at 5 h post-eclosion did not reflect the ability to attain maximum pheromone levels in response to PBAN, a significant increase in stimulation was evident, indicating a process in progress with full maturity and ability of sex-pheromone production requiring more than 5 or 6 h post-eclosion but independent of JH from the head. Thus, the present study contests the hypothesis that PBAN-R gene transcription is under the control of JH as was previously implied (Fan et al., 1999a; Rafaeli et al., 2003).
Our previous results that showed JH initiation of specific binding to the PBAN-R protein in –3 day-old female pupae of H. armigera (Rafaeli et al., 2003) can possibly be explained as an action of JH on translation of previously transcribed mRNA (Ilan et al., 1970), because low levels of the transcript do appear in pupae 3 days pre-eclosion. However, it should be emphasized that treatment with JH analogue on such pupae did not influence either the levels of PBAN-R transcript (in the present study) or the responsiveness to PBAN (Rafaeli et al., 2003). During administration of the JH analogue (for 48 h) these pupae are 5 days pre-eclosion, a time in which a dramatic switch in gene expression is observed as a result of the absence of JH after the reprogramming period for the adult moth and during the differentiation phase, which could be drastically disrupted by applications of JH and stable analogues (Bouhin et al., 1992; Zhou & Riddiford, 2002).
Additional evidence against the hypothesis that JH plays a role in up-regulating the PBAN-R transcript is evident in our observations of the analogous temporal up-regulation profiles of the PBAN-R transcript in the male aedeagus of bothH. armigera and B. mori. Sexually dimorphic production of JH in adults has been demonstrated in a number of insect species. In Diploptera punctata, males have both smaller corpora allata (CA) and less synthetic ability (Szibbo & Tobe, 1982): JH, produced by female CA, closely tracks the gonotrophic cycle (Tobe & Stay, 1977; Tobe, 1980), whereas synthesis rates in males remain at a constant and relatively low level (Tobe et al., 1979). Corpora allata in males of many moth species including Hyalophora cecropia, H. zea and Manduca sexta have been reported to lack the enzyme JH acid methyltransferase and thus their CA produce and release only JH acid rather than JH (Bhaskaran et al., 1988). JH acid is converted to JH locally in the male accessory gland (Shirk et al., 1980). Similarly, CA of B. mori males cannot produce JH, only female CA produce JH in the adult stage (Kinjoh et al., 2007). Therefore, as it is not produced by male CA, JH is unlikely to be the basis for the temporal pattern evident in the PBAN-R of males. It can be assumed that the mechanisms responsible for up-regulating PBAN-R in males most likely also acts on females. The identification and characterization of these transcription factors remain unresolved.
Correlations of simultaneous increases in JH titres and up-regulation of the PBAN-R can be argued to preclude cause and effect of these two events and provide corroborative evidence against the involvement of JH in up-regulation. Corpora allata from pharates and newly emerged adults of H. armigera produce very low titres of JH and up-regulation, occurring between 3 and 6 h post-eclosion (Fan et al., 1999a), coincides with the time observed for up-regulation of PBAN-R between 3 and 5 h post-eclosion. PBAN-R transcript regulation may not necessarily be downstream to JH regulation as both increases occur at much the same time. In B. mori, Northern blot analyses of the stage-specific expression of the B. mori PBAN-R transcript within the pheromone gland indicated that the PBAN-R undergoes significant up-regulation 1 day prior to adult eclosion (Hull et al., 2004). Our qPCR analyses comparing B. mori with H. armigera corroborate these findings. The observation that up-regulation of the PBAN-R occurs between 2 and 3 days prior to eclosion in B. mori is not surprising because in this species egg development and reproductive competence also occur before eclosion (Ohnishi, 1987). Indeed, elevations of JH levels in female B. mori also occur between 2 and 3 days pre-eclosion (Kinjoh et al., 2007) coinciding with the timing of up-regulation of PBAN-R in this moth. Thus, the coincident temporal patterns of JH with the PBAN-R transcript, that reflect the different reproductive strategies of the two moth species, may preclude up-regulation of PBAN-R transcription by JH. Furthermore, a number of B. mori pheromone gland-specific genes have been reported to be up-regulated at this time including: acyl-CoA desaturase (Yoshiga et al., 2000); acyl-CoA-binding protein (Matsumoto et al., 2001); fatty-acyl reductase (Moto et al., 2003); and fatty-acyl desaturase (Moto et al., 2004). Moreover, Northern blot analyses obtained from pheromone glands of decapitated B. mori females also showed that desaturase 1 and desaturase 2 gene expression levels, genes that are involved in the sex pheromone biosynthetic pathway, were unaffected by decapitation (Yoshiga et al., 2000) and therefore cannot depend on JH.
We suggest that the PBAN-R transcript may be regulated early during reproductive maturation of the female by a developmental programme that primes the tissues for the later PBAN-R expression and, because injections of JH significantly inhibited the levels of PBAN-R transcript at the critical period when up-regulation occurs, we conclude that the absence of JH in pharate adults, as well as its absence immediately after eclosion, is critical for normal up-regulation of PBAN-R gene expression levels. Furthermore, JH administered to adult females also significantly down-regulates the PBAN-R transcript, corroborating our previous observations concerning its effect on the PBAN-R protein and sex-pheromone production (Rafaeli & Bober, 2005). JH has long been implicated in the switch from virgin to mated behaviour (Webster & Cardé, 1984). Significant increases in JH titres of adult female moths that occur after mating have been reported in several moth species (Edwards et al., 1995; Ramaswamy et al., 1997; Park et al., 1998; Shu et al., 1998; Cusson et al., 1999; Webb et al., 1999). This increase in JH of mated female haemolymph may be the result of an allatotropic effect on female CA from the transfer of male accessory gland-produced peptides, such as sex-peptide (Fan et al., 1999b, 2000; Nagalakshmi et al., 2004, 2007), or because of a direct transfer of JH produced in the male accessory glands (Park et al., 1998). Sex peptide has been shown to terminate PBAN-stimulated sex pheromone production in female H. armigera (Fan et al., 1999b, 2000). Post-copulatory suppression of sex pheromone production may thus be hypothesized as a direct, or a combination of both direct and indirect, influences because of the rising JH haemolymph titres in mated females and its action on the transcription of PBAN-R.
Helicoverpa armigera were raised on an artificial diet (Heliothis Premix, Stonefly Industries, Inc, Bryan, TX, USA) at a constant temperature of 26 ± 1 °C and 14:10 h (light : dark) photoperiod. Larvae, at their wandering stage, were isolated in transparent culture cells (J-2 cavities, Nu-Trend Container, Jacksonville, FL, USA) and the date of pupation was noted for each larva to enable precise age definition of the pharate adult. As reported previously, the normal duration of the pupal period in H. armigera is 12–13 days (Rafaeli et al., 2003). Larvae of B. mori (third to fourth instars) were obtained as a gift from a private culture (Ms Shlomit Weisler, Raanana, Israel) and were reared on a daily supply of fresh mulberry leaves at Israeli spring temperatures (April–May) under a natural photoperiod cycle. Pupae of both species were sexed and adult males and females were allowed to emerge in separate cages until tissue dissection. Observations of each individual female/male at onset of eclosion during the scotophase enabled the definition of the exact hours post-eclosion.
Helicoverpa armigera decapitation and head-ligation experiments
Three hours post-eclosion H. armigera females were decapitated and kept separately on moist paper towels under the same photoperiod regime for 2 or 24 h before in vivo experiments were initiated. In addition, pheromone glands of females, decapitated at various time intervals post-eclosion, were collected 24 h after decapitation for differential PBAN-R transcript level quantification. For the experiments requiring decapitation before female eclosion, preliminary tests showed a high mortality rate after decapitation of pharates, 1–2 days pre-eclosion. Thus, decapitation at these times was replaced by ligation between the head and thorax using a fine thread. Pheromone glands were collected 24 or 48 h after head ligation for differential PBAN-R transcript level quantification in pharate pheromone glands.
Pheromone glands of H. armigera and B. mori from females of different and strictly defined developmental ages and from the various treatments were frozen in liquid nitrogen. Likewise, aedeagi from males of both species were dissected and frozen. Total RNA isolation, reverse-transcription, DNase I digestion and qPCR assays were performed using the same methodologies as described previously (Rafaeli et al., 2007) and analysis was performed using the ΔΔCt (Livak & Schmittgen, 2001) and Pfaffel quantification methods (Pfaffel et al., 2002). We also used the same H. armigera gene-specific primers as designed and published previously (Rafaeli et al., 2007). Three day-old adults served as calibrators in these qPCR analyses. For the B. mori studies, gene-specific primers were designed to correspond to the following amino acids in B. mori PBAN-R trans-membrane domain II: sense: HTATNF; antisense: LYRLWNP. All of the B. mori PBAN-R transcripts were normalized to the rp49 expression level, which served as a reference gene and 2 day-old adults served as calibrators in these qPCR analyses. Amplified PCR products were analysed on a 1% agarose gel, extracted using Qiagen MiniElute Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA) and the sequences were verified (Hylab, Rehovot, Israel).
Sex pheromone production in vivo
Helicoverpa armigera post-eclosion females were injected with 10 µl Pipes buffered incubation medium (21 mM KCl, 12 mM NaCl, 3 mM CaCl2, 18 mM glucose, 43 mM trehalose, 5 mM Pipes buffer brought to pH 6.6 using 0.1 N KOH) in the presence or the absence of 5 µM HezPBAN (custom synthesized by Peptide 2.0, Herndon, VA, USA) for 2 h. Pheromone glands were removed, cleared from internal tissues by gentle squeezing and sex pheromone levels were then analysed by extracting them in hexane containing 25 ng tridecanyl acetate (Sigma Chemical Co., St. Louis, MO, USA) for 20 min at room temperature. The main sex pheromone component, Z-11 hexadecenal, of H. armigera was determined using gas chromatography and the internal standard quantification method as described previously (Rafaeli & Soroker, 1989).
Treatments of JH and FX in vivo
JH I and JH II were purchased from SciTech (Prague, Czech Republic) and JH III from Sigma. The three JHs (I, II and III) were each dissolved in olive oil (1 µg/µl) and individually injected to intact, newly emerged females (1 µl/female) at 3 h post-eclosion. For control treatments 1 µl olive oil was injected into each female. At 5 h post-eclosion (2 h later) injected females were killed and pheromone glands were removed for RNA extraction and differential PBAN-R transcript level quantification. To test a possible JH effect on transcript levels of pupae, 1 µl of a stock solution (3 µg/µl acetone) of the stable JH analogue, FX (obtained as a gift from Dr Victoria Soroker, Plant Protection Institute, Volcani Center, ARO, Bet-Dagan, Israel) was topically applied to female pupae 5 days pre-eclosion (Rafaeli et al., 2003). For control treatments 1 µl acetone was applied. After 48 h (when females were –3 day-old) the pheromone glands were removed from the acetone- and FX-treated females for RNA extraction and differential PBAN-R transcript level quantification.
Statistical analysis was performed using SAS v. 8.2 (SAS Institute Inc., Cary, NC, USA) using a general linear modelling (GLM) procedure. For sex pheromone production, because variances were nonparametric, the data were transformed to [y= log (1 +x)] and subjected to the SNK test for multiple comparisons. For the qPCR assays (differential PBAN-R transcript level quantification) the data were analysed using one-way anova, Tukey–Kramer ranking or the Student's t-test.
The research was supported by a research grant award IS-4163-08C from the United States – Israel Binational Agricultural Research and Development Fund. This is contribution no. 556/09 from the Agricultural Research Organization, Volcani Center, Bet Dagan, Israel. This research represents a part of the PhD dissertation of R. B.