The regulation of Δ11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis

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The regulation of D11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis Gabriella Köblös a,1, Tamás Dankó b,1, Kitti Sipos b, Ágnes Geiger c, Tamás Szlanka d, József Fodor a, Adrien Fónagy b,⇑ a

Department of Pathophysiology, Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Herman Ottó út 15, H-1022 Budapest, Hungary Ecotoxicology and Environmental Analysis Group, Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Herman Ottó út 15, H-1022 Budapest, Hungary c Department of Entomology, Faculty of Horticultural Science, Corvinus University of Budapest, H-1118 Ménesi út, 44, H-1118 Budapest, Hungary d Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary b

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Article history: Available online xxxx Keywords: Mamestra brassicae Pheromone gland (PG) Pheromone biosynthesis activating neuropeptide (PBAN) Pheromonotropin (PT) Pheromone biosynthesis D11-desaturase gene expression Z11-Hexadecenyl acetate

a b s t r a c t Cabbage moth (Mamestra brassicae) females produce sex pheromones to attract conspecific males. In our M. brassicae colony, the pheromone blend is composed of Z11-hexadecenyl acetate (Z11-16Ac) and hexadecyl acetate in a 93:7 ratio. A fatty acyl D11-desaturase is involved in the production of the main pheromone component. The release of Pheromone Biosynthesis Activating Neuropeptide (PBAN) regulates the pheromone production in the pheromone gland (PG). We cloned a cDNA encoding the MambrD11-desaturase and analyzed its expression profile over time in M. brassicae tissues. Transcript levels of the D11-desaturase in larvae, pupal PGs, fat body, brain and muscle tissues were <0.1% of that in female PGs, whereas expression in male genitalia was 2%. In the PGs of virgin females the expression level increased continuously from eclosion to the end of the 1st day when it reached a plateau without further significant fluctuation up to the 8th day. In contrast, we recorded a characteristic daily rhythmicity in pheromone production with a maximum around 200 ng Z11-16Ac/ PG. In some experiments, females were decapitated to prevent PBAN release and thereby inhibit pheromone production, which remarkably increased after treatment with Mambr-Pheromonotropin. Further experiments revealed that mating resulted in a significant suppression of pheromone production. However, expression of the D11-desaturase was not affected by any of these interventions, suggesting that it’s not regulated by PBAN. Fluorescent microscopy was used to study the potential role of lipid droplets during pheromone production, however, no lipid droplets were identified indicating that pheromonogenesis is regulated via de novo fatty acid synthesis. Ó 2015 Elsevier Inc. All rights reserved.

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1. Introduction

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Female moths produce species-specific volatile sex pheromones to attract conspecific males (Tamaki, 1985). The pheromone components (blends) are synthesized in the pheromone gland (PG), which is generally located between the 8th and 9th abdominal segments and consists of a single layer of modified epidermal cells in the intersegmental membrane. The major class of sex pheromones (Type I) produced by female moths is composed of straight-chain

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⇑ Corresponding author. 1

E-mail address: [email protected] (A. Fónagy). Equal contribution.

C10–C18 unsaturated and saturated aliphatic compounds with limited oxygenated functional groups (Ando et al., 2004). They are synthesized de novo from acetyl-CoA via modified fatty acid (FA) biosynthetic pathways through 16 (or 18):acyl, and undergo chain shortening and/or desaturation, followed by a final reduction to alcohol or acetylation to produce acetate esters or oxidation to produce aldehydes (Rafaeli, 2002; Tillman et al., 1999). Recent investigations have provided a better understanding of pheromone biosynthetic pathways (Matsumoto et al., 2007; Rafaeli, 2011; Vogel et al., 2010). The biosynthesis and emission from the surface of the extruded gland during the event of calling are synchronized to the photoperiodic cycle governed by the pheromone biosynthesis activating neuropeptide (PBAN). PBAN is a 33–

http://dx.doi.org/10.1016/j.ygcen.2015.03.004 0016-6480/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Köblös, G., et al. The regulation of D11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.03.004

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34-amino acid (aa) peptide, which was first isolated from Heliothis (Helicoverpa) zea (Raina et al., 1989). It is present in numerous species (Rafaeli, 2009) including Mamestra brassicae (Jacquin-Joly et al., 1998) and characterized by a C-terminal amidated pentapeptide FXPRL motif that is essential for pheromonotropic activity, i.e. induction of pheromone biosynthesis (Teal et al., 1996). PBAN is produced in the suboesophageal ganglion (SOG) and released from the corpus cardiacum into the hemolymph according to a daily rhythm (Bloch et al., 2013). In Bombyx mori, the PBAN gene encodes a common polyprotein precursor (Kitamura et al., 1989) which when post-translationally processed yields multiple peptides including PBAN, diapause hormone, and three additional shorter pheromonotropins (PTs) termed a- b- and c-SGNPs (SOG Neuro Peptides) all bearing the Cterminal amidated FXPRL motif (Sato et al., 1993). In M. brassicae, production of the pheromone blend is also under PBAN control (Bestmann et al., 1987, 1989; Jacquin et al., 1994). A PT identified in M. brassicae (Mambr-PT) (Fónagy et al., 2008, 1998) is a 18-aa peptide that shows close sequence similarity with a b-SGNP purified from Pseudaletia separata (Matsumoto et al., 1992) as well as predicted b-PTs, deduced from DNA sequences of various lepidopteran species (Rafaeli, 2009). The effect of PBAN on the different steps in the pheromone biosynthetic pathway has been investigated in numerous lepidopteran species. For pheromonogenesis, PBAN action on the PG cells is indispensable as the process is initiated by specific binding of PBAN to its receptor (PBANR). PBANR was first cloned and characterized as a G protein-coupled receptor in H. zea (Choi et al., 2003) and subsequently in B. mori (Hull et al., 2004) and a number of other species since then (Lee et al., 2012; Nusawardani et al., 2013). It was postulated that for some species such as Argyrotaenia velutinana (Tang et al., 1989; Jurenka et al., 1991a), H. zea (Jurenka et al., 1991b) and M. brassicae (Jacquin et al., 1994; Jacquin-Joly et al., 1998) PBAN regulates pheromonogenesis by controlling a step at or prior to fatty acid synthesis (reviewed by Jurenka and Rafaeli, 2011; Rafaeli, 2009; Tillman et al., 1999). Two-step regulation of pheromone biosynthesis by PBAN was demonstrated in Heliothis virescens, where the stimulation of acyl-CoA-carboxylase and the final reduction step are evidenced, providing a delicate regulation in pheromone production (push and pull theory) (Eltahlawy et al., 2007b). Regulation of the fatty acyl-CoA reductase by PBAN has been extensively investigated in B. mori (Hull et al., 2007; Matsumoto, 2010; Matsumoto et al., 1995), and it has been demonstrated that the depletion of B. mori pheromone precursors from lipid reservoirs, followed by their final reduction are under direct PBAN control (Fónagy et al., 2001). In Ostrinia nubilalis (Eltahlawy et al., 2007a; Ma and Roelofs, 1995), Thaumetopoea pityocampa (Fabrias et al., 1995), Spodoptera littoralis (Fabrias et al., 1994) and in Manduca sexta (Fang et al., 1996) PBAN likewise acts on the reduction of fatty acyl precursors. In yet other species, PBAN acts on other steps in the pheromone biosynthetic pathway such as the D11-desaturation step in Chrysodeixis chalcites (Altstein et al., 1989) and the final acetylation step in Sesamia nonagrioides (Mas et al., 2000). Acyl-CoA-desaturases are particularly important in generating structurally diverse components of lepidopteran sex pheromone, thus they have been studied from various aspects including gene identification, expression profiling and evolutionary analyses (Bjostad et al., 1987; Knipple et al., 2002; Liénard et al., 2008; Matousková et al., 2007; Rafaeli and Jurenka, 2003; Serra et al., 2007, 2006). Insect desaturases are endoplasmic reticular proteins with four transmembrane domains, sharing homology with D9acyl-CoA-desaturases of plants, fungi and animals (Shanklin and Cahoon, 1998; Stukey et al., 1990). They are encoded by a dynamically evolving gene family, whose members are thought to have originated from an ancestral desaturase prior to the divergence

of the dipteran and lepidopteran lineages (Knipple et al., 2002). The extant moth D9-desaturases may function in essential metabolic functions as well as pheromone production, while D11-desaturases function exclusively in pheromonogenesis as summarized by Liénard et al. (2010). It was also demonstrated that in contrast to D9-desaturases, distinct members of the D11-desaturase subfamily possess broad substrate-, stereo- and regio-specificity including Z10, Z11, Z/E11, E11, D11/10,12 and D11/D11,13 desaturase activities (Matousková et al., 2007; Moto et al., 2004; Serra et al., 2007, 2006). As reviewed by Blomquist et al. (2005) conjugated dienes are generally formed by the immediate action of a bifunctional desaturase with isomerizing around the first double bond or by two consecutive desaturation steps. In M. brassicae, it was demonstrated that a palmitate precursor undergoes desaturation during pheromonogenesis (Bestmann et al., 1989, 1987). It was also concluded that conversion of the precursor palmitic acid to the final Z11-16Ac, including desaturation, is strongly neurohormone dependent (Bestmann et al., 1989), while other studies suggest that PBAN does not affect desaturases (Gosalbo et al., 1992; Jacquin et al., 1994; Jurenka and Rafaeli, 2011; Tillman et al., 1999). Recently, four desaturases were cloned from M. brassicae PGs (Park et al., 2008). The most abundant desaturase-encoding transcript in M. brassicae pheromone glands was MbraLPAQ having D11 regioselectivity. Two pheromone glandspecific KPSE (D916>18) desaturases (MbraKPSE-a and MbraKPSEb) and a MbraNPVE (D918>16) desaturase with D9 regioselectivity were found not to be involved in pheromone biosynthesis in M. brassicae (Park et al., 2008). However, until now, no detailed time-dependence of desaturase activity, intermediate screening, or possible role of PBAN regulation have been elucidated. Previous reports indicate that calling by M. brassicae occurs mainly in the second half of scotophase (Attygalle et al., 1987; Bestmann et al., 1988; Noldus and Potting, 1990). In M. brassicae, the extracted blend was described as a mixture of Z11-16Ac, 16Ac and Z9-16Ac in a ratio of 90:10:1 (Attygalle et al., 1987). However, other blends may exist since examples of variation in pheromone composition between populations in different geographical regions have been reported (Pefia et al., 1988). Similar deviations of blend composition were observed for the oriental armyworm, P. separata, which inhabits distant regions (Fónagy et al., 2011). Mating in most insect species leads to a temporary or permanent loss in sexual receptivity of females and is often accompanied by a decline in sex pheromone production and the absence of calling. This phenomenon may be transient or permanent depending on whether the species is monandrous or polyandrous (Delisle et al., 2000). Studies on the regulation of the post-mating inhibition of pheromone production (pheromonostasis) showed that this process may be mediated by either hormonal or neural mechanisms as reviewed by Rafaeli (2011). However, in most species investigated to date, the PGs of mated females respond to PBAN treatment suggesting that pheromonostasis may be associated with cessation of PBAN release from the corpus cardiacum rather than its reduced synthesis (Ando et al., 1996; Delisle et al., 2000; Foster, 1993; Jurenka and Fabriás, 1993; Raina et al., 1994). The cellular dynamism of PG cells may show a close relation with pheromone production rate: i.e. storage reservoirs in the form of lipid droplets (LD) may be key elements in the process. The presence of reservoirs was observed in Trichoplusia ni (Jefferson and Rubin, 1973; Percy, 1979) M. sexta (Fang et al., 1996) and Epiphyas postvittana (Foster, 2001), whereas a significant accumulation and depletion of neutral lipids was described in B. mori (Fónagy et al., 2001; Yokoyama et al., 2003). Although LDs were not identified in P. separata, the changes in PG cell morphology were recognized to be in accordance with cell activity (i.e. pheromonogenesis), based on the daily analysis of the blend at

Please cite this article in press as: Köblös, G., et al. The regulation of D11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.03.004

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scotphase or following PBAN treatments in vivo and in vitro (Fónagy et al., 2011). M. brassicae, a key pest in Hungary (and Eurasia) can be a good comparative model for examining the mechanisms of pheromone production and their regulation in relation to those described in B. mori (Matsumoto, 2010). Our main objective is to provide a detailed description of pheromone blend production in our laboratory strain and focus on the daily rhythms in parallel with D11-desaturase gene expression to complement studies of Park et al. (2008). The better understanding of potential neuroendocrine regulation of production, and the effect of mating on these processes are also targeted. Morphological studies are aimed to clarify whether lipid reservoirs (droplets formed from triglycerides) are utilized or not in pheromone precursor storage or in pheromonogenesis.

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2. Materials and methods

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2.1. Insects

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A M. brassicae (Lepidoptera; Noctuidae) colony was established from individuals collected in three different regions of Hungary in August 2012. The stock colony was maintained in a rearing room at 25 °C, 60% relative humidity under a 16L (light):8D (dark) regime with larvae kept on a semi-artificial diet (Nagy, 1970). Reverse photoperiod conditions were used to harmonize with working hours. Pupae were sexed and females and males were kept separately. The large majority of adults emerged in the last hours of photophase. After eclosion, adults were kept separately in glass jars (12  10 cm) covered with fine mesh, fed with 10% honey solution applied on cotton wool and furnished with shredded paper. The newly emerged individuals were designated as day 0 (D0) and pooled for 24 h, except where indicated otherwise (see at Sections 2.2.4 and 2.2.5). For all observations and treatments, adults were transferred to an experimental room equipped with a dim red light suitable for scotophase monitoring, and maintained under the same conditions as above.

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2.2. Studies on pheromone blend production under different experimental conditions 2.2.1. Sampling and preparation of pheromone gland extracts For pheromone blend analysis 3 PGs were excised with the help of tweezers and a blade and pooled for extraction in 300 ll n-hexane and concentrated to 15 ll under N2 stream. For each time point or treatment the extracts were collected in five replicates and stored at 80 °C until measurements. 2.2.2. Gas chromatography mass spectrometry measurements An aliquot of 1 ll from each sample was measured (automatic injection method, splitless mode) under the following conditions using a RESTEC (Rxi-5SI) column (0.25 mm internal diameter  30 m and 0.25 lm film thickness) with helium as a carrier gas at a flow rate of 1 ml/min on a Gas Chromatograph-Mass Selective Detector (Hewlett Packard GC 6890, HP MSD 5973). The following running conditions were applied: 50 °C for the first min, then increased to 180 °C until 7.5 min (20 °C/min) and 190 °C until 8.17 min (15 °C/min), followed by an increase of 5 °C/min up to 220 °C at 14.17 min ending by 30 °C/min to 300 °C at 17.83 min. This allows a rapid, but sensitive fractionation of the two main components: Z11-16Ac (222 m/z; Ret. time 13.64 min) and 16Ac (224 m/z; Ret. time 13.77 min). Quantification was performed using the internal standard method [internal standard: Pentadecanol (Pherobank BV)] and MSD Chemstation ver. D.01.02.16 software.

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2.2.3. Time course studies for pheromone production in adult females Samplings were done daily beginning on day D0 until day 8 (D8) after emergence. On the first 5 days pheromone samples were collected 7 times per day with 2 h intervals. The first sample was taken at the 1st h of scotophase, whereas the last at the 5th h of photophase (covering a total period of 12 h/day). From D6 to D8 only 4 samplings were performed (at the 5th and 7th h of scotophase, and at the 3rd and 5th h of photophase). Timing of the samplings was based on behavioral observations, since virgin females of our colony showed intensive calling behavior in the early photophase as well (see Fig. 1a).

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2.2.4. In vivo effect of Mambr-PT on pheromone production To study the effect of a synthetic 18-aa pheromonotropin (Mambr-PT) on pheromone production, two sets of experiments were designed. First, females were decapitated on the second day (3rd h of scotophase), placed into Petri dishes that contained moist filter paper and maintained in the experimental room. They were injected with a fine Hamilton syringe on the third day (6th h of scotophase) with 5.6 pmol/2 ll Mambr-PT (synthetized at the Department of Medical Chemistry, University of Szeged, Hungary) or distilled water (DW) alone. In a set of separate experiments, adult females were decapitated within 4 h after emergence. These decapitated females were maintained as described above and then injected with 5.6 pmol Mambr-PT on D0, D1 or D2 at the 6th h of scotophase. Pheromone glands were collected for GC–MS analysis at 60 or 90 min after injections and processed as described in Sections 2.2.1. and 2.2.2.

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2.2.5. The effect of mating on pheromone production For mating experiments, individually tagged, newly emerged females (n = 5–6) and males (n = 7–8) were placed together into glass jars and maintained in the experimental room (see Section 2.1). Mating was confirmed by direct observation. Pheromone glands were extracted and analyzed from D2 to D5 females during the last hour of scotophase as described above (see Sections 2.2.1 and 2.2.2). Non-mated D5 virgin females were sampled as a control at the last hour of scotophase, whereas another group of D5 mated females (also in five replicates) was injected with Mambr-PT (see Section 2.2.4).

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2.3. cDNA cloning of D11-desaturase

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In order to clone the open reading frames (ORFs) of D11-desaturase and b-actin genes, total RNA from 15 PGs of 2–3-day-old calling females were purified with EXTRACTME Total RNA Kit (BLIRT, DNA Gdansk). The cDNAs were reverse transcribed with a HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems) and ORFs were amplified by PCR using Phusion high-fidelity DNA polymerase (Thermo Scientific) with primers: 50 -ATGGATCA AAGCGTACGGA30 (50 primer) and 50 -TTATTCGTCTTTTCCTTCGTT T30 (30 primer) for M. brassicae D11-desaturase (MbraLPAQ, GenBank accession No. EU285580) as well as 50 -ATGTGTGAC GAGGATGTTGC-30 (50 primer) and 50 -TTAGAAGCACTTGCGGTGG A-30 (30 primer) for M. brassicae b-actin (accession No. EU035314). The resulting PCR products (1014-bp and 1131-bp for D11-desaturase and b-actin, respectively) were purified with a High Pure PCR product purification kit (Roche) and directly ligated into a pJET1.2/blunt cloning vector (CloneJET PCR Cloning Kit, Thermo Scientific). Ligates were transformed into Escherichia coli Top10 cells following the manufacturer’s instructions. Recombinant clones were sequenced in both directions using pJET1.2 forward and reverse primers. Plasmid DNA containing the insert was purified using a PureLink Quick plasmid Miniprep Kit (Invitrogen) and quantified on a NanoDrop ND-1000 spectrophotometer.

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Fig. 1. Time course study of pheromone production and D11-desaturase gene expression in M. brassicae. (a) Pheromone content in pheromone gland (PG) extracts from pupal stage (prior to emergence; designated as 1), at emergence (arrow) and on days 0–8 of adult stage. Samples were collected 7 times per day in 2 h intervals from the 1st h of scotophase. Data are expressed as mean pheromone content in nanograms per PG of pooled samples of 3 PGs using 5 replicates for each time point. Scotophases are marked by light gray columns. nd = not detectable. (b) D11-desaturase transcript expression levels in PGs. Samples were collected 1 h prior to scotophase, the 3rd and 7th h of scotophase as well as the 3rd h of photophase. Gene expression levels were measured by RT-qPCR and normalized to the expression levels of b-actin. No D11-desaturase or bactin amplification products were observed when non-reverse transcribed RNA samples were used as templates for PCR reactions (data not shown). Data are based on 3 independent experiments (n = 5) and run in triplicate. Upper vertical bars show standard deviations. Mean ± SD of expression levels for pupae is shown numerically in the graph.

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2.4. Quantitative real-time PCR

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Real-time PCR primers were designed using Primer-BLAST online software (National Center for Biotechnology Information) and used at a final concentration of 0.4 lM. The primers were 50 ACAACAATCCTGTGCTGAGGT-30 (50 primer) and 50 -CACGGGAACAC GTGGTGAT-30 (30 primer) to amplify a 298 bp long region of the M. brassicae D11-desaturase cDNA, and 50 -CCCTCTTCCAGCCCTC ATTC-30 (50 primer) and 50 -CGATACCGGGGTACATGGTG-30 (30 primer) for a 150 bp long region of the M. brassicae b-actin cDNA. Quantitative real-time PCR was carried out in a 20 ll reaction volume using SensiFAST SYBR No-ROX Kit (Bioline) on a C1000Touch Thermal Cycler and CFX96 Real-Time PCR Detection System (Bio-Rad) according to the manufacturer’s specifications. Each PCR reaction was performed in triplicate. Both non-template and non-reverse transcribed RNA samples were used as negative controls. PCRs were carried out under the following conditions: initial denaturation for 2 min at 95 °C followed by 40 cycles of denaturation for 5 s at 95 °C, annealing for 10 s at 60 °C and elongation for 20 s at 72 °C. The specificity of amplifications was verified by analysis of melting curves (generated between 65 °C and 95 °C, reading every 0.5 °C). Cq (quantitation cycle) values were calculated with Bio-Rad CFX Manager 3.1 in Single Threshold mode. In some cases, the specificity of the PCR reactions were also tested by agarose gel electrophoresis (1.5%) and sequencing of the PCR products. Expression levels of b-actin transcript were used as an internal reference. Calibration curves (log of DNA concentration versus cycle threshold) for quantification were determined with a sixpoint (10-fold) dilution series of known amount of recombinant plasmid DNA containing ORFs for D11-desaturase and b-actin genes. Standard curve analysis showed a PCR efficiency of 96.1% and 97.2% and R2 values of 0.997 and 0.998 for b-actin and D11-desaturase primer pairs, respectively.

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2.5. Gene expression studies of MambrD11-desaturase in pheromone glands

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For D11-desaturase gene expression studies, PGs were excised from females kept under various experimental conditions and pooled in groups of 5 in 1.5 ml tubes, frozen in liquid nitrogen and stored at 80 °C before RNA extraction. Samples were collected from at least three independent experiments. Control tissue samples were obtained from 2nd and 5th instar larvae, PGs of female pupae (pre-emergence) and genitalia of D2–D3 adult males. In addition, fat bodies and brain tissues were also dissected, and legs were cut from D2 to D3 females. For the time course study of MambrD11-desaturase gene expression, PGs were excised from newly emerged and D0–D8 females 1 h prior to scotophase, in the 3rd and 7th h of scotophase, and the 3rd h of photophase. In parallel with sampling for pheromone titer measurements, PG samples were also obtained from females for MambrD11-desaturase gene expression studies in order to investigate the effects of in vivo Mambr-PT treatments and mating. Treatments and sampling times were identical to those described in Sections 2.2.3– 2.2.5.

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2.6. Morphological studies of lipid droplets and/or triglyceride reservoirs

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Fluorescence microscopy was performed as described previously (Fónagy et al., 2011). In brief, excised PGs were trimmed from surrounding tissues in PBS, cut longitudinally and spread open. Tissues were fixed in 4% formaldehyde/PBS for 10 min, rinsed three times in PBS and then transferred to freshly prepared staining solution. For staining of neutral lipids and nuclei, Nile Red (Molecular Probes Inc. Eugene, OR, USA) and Hoechst 33342 (Molecular Probes Inc. Eugene, OR, USA) were used, respectively.

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Please cite this article in press as: Köblös, G., et al. The regulation of D11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.03.004

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Six microliter of saturated Nile Red dissolved in acetone was added to 94 ll PBS and mixed with 0.5 ll Hoechst 33342 stock solution (1 mg/ml dissolved in distilled water). Samples on the slide were incubated in the staining solution at room temperature in dark for 10 min, then rinsed three times in PBS. Samples were prepared at emergence, D1–D4 at the 5th–6th h of scotophase (when daily pheromone production peaks) and an hour prior to the onset of scotophase (no pheromone production is expected). In addition, PGs were also isolated from D2 decapitated females treated with and without synthetic Mambr-PT. For details of treatment, see Section 2.2.4. Imaging was performed on an OLYMPUS BX-51 system equipped with a digital exposure unit. Nile Red (bright yellow– red) and Hoechst (blue) emissions were observed simultaneously using the following spectral settings: a 360–370 nm band pass excitation filter, a 400 nm dichroic mirror and a 420 nm pass barrier filter (OLYMPUS cube WU). Images were processed using Olympus StreamÒ.

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2.7. Statistical analyses

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Five independent replicates were performed for pheromone quantification measurements and at least three independent experiments were carried out in the gene expression studies. Statistical significance was determined with Student’s t-test and ANOVA followed by a Tukey post hoc test (Statistica 6.1, Statsoft, Tulsa, OK, U.S.A.). Differences were considered to be significant at P < 0.05.

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3. Results

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3.1. Time course studies of pheromone production in adult virgin females

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Pheromone was hardly detectible on the day of emergence (D0) (Fig. 1a). In D1 virgin females, the pheromone blend gradually increased from the 3rd h of scotophase and peaked at the beginning of photophase. Daily maximal pheromone levels were detected around the onset of each photophase throughout the 9day study. The highest level of total daily pheromone production was recorded on D2, including two time points above 200 ng/PG, then it gradually decreased (Fig. 1a). Analysis of the extracted blend composition showed that in our strain the ratio was 93:7 (Z11-16Ac:16Ac), but Z9-16Ac was not detected. Only 10% of females began calling on the 1st day (D1), during the last 2 h in the scotophase), which behavior continued at least until the 5th h of photophase. Calling increased daily by roughly 10%. The most intense activity (around 70–80% of the individuals investigated) was exhibited by older females (D6-7), while D8 females displayed consistently lower calling activity (unpublished observations). The above results formulated the basis for all further experiments.

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3.2. Cloning M. brassicae D11-desaturase

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In order to analyze the gene expression levels of MambrD11-desaturase in PGs, we first cloned and sequenced cDNA coding the

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D11-desaturase enzyme. The nucleotide and deduced amino acid sequences of the cDNA clone (GeneBank accession No.: KM28 3200) were aligned with those of the previously published D11-desaturase cDNA clone (MbraLPAQ, accession No. EU285580, Park et al., 2008). While the nucleotide sequence was 99.9% identical to that reported by Park et al. (2008), the deduced amino acid sequence was the same. The M. brassicae D11-desaturase shares greater than 82% amino acid sequence identity with D11-desaturases from other noctuid species. Since we used b-actin as a reference gene (internal control) for quantification of real-time reverse transcription PCR (RT-qPCR) data, RT-PCR primers were designed according to the published sequence of b-actin of M. brassicae (accession No.: EU035314) and the resulting cDNA sequences were cloned into plasmid vector and sequenced. Sequence comparison of our cloned transcripts with the one published in GenBank revealed 100% identity at the nucleotide level. Thereafter, specific primers were designed for RT-qPCR experiments based on sequence analysis of cloned cDNA fragments.

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3.3. Transcript expression profiling of D11-desaturase in the pheromone gland

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The mRNA levels of D11-desaturase were determined by RTqPCR assay using gene-specific primers. To evaluate the tissue specificity of MambrD11-desaturase expression, we analyzed the relative levels of the transcript derived from the PGs of pupae and adult virgin females, whole larvae, reproductive male tissues, as well as brain, leg muscle and fat body tissues of adult females. PGs from adult females had the highest expression levels, which were about three orders of magnitude higher than that in larvae and PGs removed from pupae (Table 1). In brain and muscle tissues, MambrD11-desaturase levels were about four orders of magnitude lower than in adult female PGs. Furthermore, no amplification was observed in the fat body of adult females. In contrast, MambrD11-desaturase was expressed at a relatively high level (only 50 times lower than that of adult female PG) in the hairpencil–aedeagus complexes of adult males (Table 1). These data show that MambrD11-desaturase is expressed in a tissue-specific manner, and is most abundant in PGs of adult female moths. Since sex pheromone production and release show a diurnal rhythm with peak titers during the late scotophase, or at very early photophase (Fig. 1a), relative gene expression levels were monitored over a time course of 9 days to evaluate the periodicity of D11-desaturase gene expression. After adult emergence, MambrD11-desaturase transcript levels increased until the second day (or second scotophase) of adult emergence (D1), reaching levels 10 times higher than that observed in newly emerged (1 h old, indicated by an arrow in Fig. 1b) females, and remained consistently high throughout the remainder of the 9-day period with relatively small fluctuations (Fig. 1b). The mean expression value of MambrD11-desaturase at D0 was less than one-third than at D2, a difference that was statistically significant (P < 0.05). During the period between D2 and D8, MambrD11-desaturase expression plateaued and no evidence for diurnal periodicity was detected (Fig. 1b).

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Table 1 Expression levels of MambrD11-desaturase transcripts in different tissues. PG = pheromone gland. Data are expressed as means ± SD of 3 independent experiments run in triplicates. Gene expression levels were measured by RT-qPCR and normalized to the expression levels of b-actin as an internal control. No D11-desaturase or b-actin amplification products were observed when non-reverse transcribed RNA samples were used as templates for PCR reactions (data not shown). Adult PG

Larvae

Pupal PG

Brain

Leg muscle

Fat body

Male genitalia

22.04 ± 3.08

0.017 ± 0.015

0.017 ± 0.019

0.008 ± 0.003

0.001 ± 0.001

Undetectable

0.415 ± 0.126

Please cite this article in press as: Köblös, G., et al. The regulation of D11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.03.004

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Fig. 2. Effect of in vivo administration of M. brassicae pheromonotropin (Mambr-PT) on D11-desaturase transcript expression and pheromone production. Females were decapitated 2 days after emergence and treated with Mambr-PT or distilled water (DW) on the third day. (a) D11-desaturase expression was detected in pheromone glands (PGs) 60 (PT60) or 90 (PT90) min after treatment. Transcript expression levels were measured by RT-qPCR and normalized to the expression levels of bactin. No D11-desaturase or b-actin amplification products were observed when non-reverse transcribed RNA samples were used as templates for qPCR reactions (data not shown). Data are expressed as means and error bars represent standard deviations of 3 independent experiments (n = 5) run in triplicate. (b) Pheromone content in PG extracts 60 and 90 min after injection of Mambr-PT or DW into decapitated females. Data are expressed as mean weights of Z11-hexadecenyl acetate (Z11-16Ac) and hexadecyl acetate (16Ac) in nanograms per PG. Upper and lower vertical bars show standard deviations for 16Ac and Z11-16Ac, respectively. Data are based on pooled samples of 3 PGs using 5 replicates for each time point. Asterisk (*) and double asterisk (**) denote significant differences at P < 0.05 and P < 0.01, respectively, in the levels of Z11-16Ac between Mambr-PT and DW-treated females (Student’s t-test). The 16Ac was not detectable in DW-treated females.

3.4. The effects of various in vivo Mambr-PT treatments on pheromone production and MambrD11-desaturase expression

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We also examined the effects of various treatments with Mambr-PT on MambrD11-desaturase expression. In order to eliminate the effect of endogenous PBAN, virgin females were decapitated in these experiments. Decapitation of D2 females resulted in a drastic reduction in pheromone production to nearly undetectable levels 27 h post-decapitation. Neither decapitation nor Mambr-PT treatments affected the expression levels of MambrD11-desaturase significantly in PGs of the above females (Fig. 2a). However, we observed that 60 or 90 min after MambrPT treatment of D3 females at the 6th h of scotophase (decapitated 27 h earlier) pheromone titers increased considerably (Fig. 2b). This increase in pheromone production following Mambr-PT treatment was statistically significant (P < 0.05). Moreover, the ratio of extracted pheromone blend components was similar to that of corresponding non-treated virgins. In another set of experiments, newly emerged females were decapitated within 4 h of emergence. The decapitated females were kept alive and injected with Mambr-PT on D0, D1 or D2 at the 6th h of scotophase. MambrD11-desaturase expression exhibited a similar pattern (significantly lower at D0 compared to the ensuing days) to the one measured during the time course studies, irrespective of Mambr-PT treatment or decapitation (compare Figs. 1b and 3a). On the contrary, a statistically significant (P < 0.01) increase in pheromone blend production was apparent when these females were exposed to Mambr-PT on the day of, or 1 or 2 days after decapitation. This finding indicates that the PG has the capacity to synthesize the pheromone blend even on D0 (Fig. 3b), which is remarkable in the light of the fact that pheromone levels are very low in PGs of intact D0 virgin females (Fig. 1a).

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Fig. 3. Effect of in vivo administration of M. brassicae pheromonotropin (Mambr-PT) on D11-desaturase transcript expression and pheromone production in pheromone glands (PGs) of females decapitated immediately after emergence. Females were injected with Mambr-PT (PT) or distilled water (DW) on day 0 (D0), day 1 (D1) or day 2 (D2). (a) D11-desaturase expression was detected by RT-qPCR in PGs 90 min after treatments. Expression levels of b-actin was used for reference. No D11-desaturase or b-actin amplification products were observed when non-reverse transcribed RNA samples were used as templates for PCR reactions (data not shown). Data are expressed as means and error bars represent standard deviations of 3 independent experiments (n = 5) run in triplicate. Asterisks (*) denote a significantly lower (P < 0.05) gene expression level on D0. (b) Pheromone content in PGs extracted 90 min after injection of Mambr-PT or DW into decapitated females. Data are expressed as mean weights of Z11-hexadecenylacetate (Z11-16Ac) and hexadecyl-acetate (16Ac) in nanograms per PG. Upper and lower vertical bars show standard deviations for 16Ac and Z11-16Ac, respectively. Data are based on pooled samples of 3 PGs using 5 replicates for each time point. Double asterisks (**) denote significant difference (P < 0.01) in the levels of Z11-16Ac between Mambr-PT and DW-treated females (Student’s t-test). The 16Ac was not detectable in DW-treated females.

Please cite this article in press as: Köblös, G., et al. The regulation of D11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.03.004

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3.5. The effect of mating on pheromone production and MambrD11desaturase expression Freshly emerged females and males were paired in glass jars and expression levels of MambrD11-desaturase in PGs as well as pheromone production was monitored daily on D2–D5 at the last hour of scotophase, when pheromone production is generally at its peak (Fig. 1a). Mating did not notably affect the expression level of MambrD11-desaturase (Fig. 4a). In contrast, the pheromone level was significantly lower (P < 0.01) in mated females on D4 and on D5 than that in D2–D3 (Fig. 4b). When D5 mated females were stimulated with injected Mambr-PT (D5/PT), pheromone production was comparable to that detected in control D5 virgin females (Fig. 4b). 3.6. Morphological studies of lipid droplets and/or triglyceride reservoirs We performed fluorescence microscopy using trimmed PGs double stained for LDs (Nile Red) and nuclei (Hoechst 33342) at different times of the adult stage as well as following decapitation and/or Mambr-PT treatments. Double staining was very effective for identifying cells and their orientation. For a positive control of lipid staining, we used fat body tissue remnants (results are not shown, but some droplets in Fig. 5a are visible as a reference). The Nile red fluorescent dye provided a characteristic pinkish background that clearly outlined cell borders. Fig. 5a shows the cellular arrangement of PG cells prepared from a newly emerged female. The single layer of modified epidermal cells is clearly visible. A PG from a D2 female dissected at late scotophase (when the highest pheromone content was recorded; see Fig. 1a) is shown in Fig. 5b. A PG with clearly distinguishable cells from a D3 decapitated female is shown at a higher magnification in Fig. 5c. On Fig. 5d a PG trimmed 90 min following the injection of Mambr-PT is presented. Since we were unable to find any neutral lipid

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material at any of the chosen sampling time points, we conclude that M. brassicae does not utilize LDs as storage reservoirs for pheromone blend precursors.

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We investigated in detail the time course of M. brassicae pheromone production in relation to the expression of MambrD11-desaturase, which encodes a key enzyme in the formation of the pheromone blend (Bjostad et al., 1987). As a first step, we sought to support or rule out the hypothesis that gene expression of D11-desaturase is under direct neuroendocrine control via the PBANR cascade. Since contradictory results have been published in the literature (Bestmann et al., 1989; Iglesias et al., 1999, 1998; Jacquin et al., 1994) regarding the role of PBAN in desaturase activation, we thus aimed to clarify this issue by directly measuring D11-desaturase transcript levels. In fatty-acyl chains, FA-desaturases catalyze the formation of double bonds (the positions of which are determined by enzyme specificity) and are responsible for most of the structural variations present in pheromone precursors (Rafaeli, 2009; Tillman et al., 1999). The involvement of desaturases in pheromonogenesis is reported to be a secondary derived function of these enzymes which to date have only been found in insects (Roelofs and Rooney, 2003). We used different experimental approaches in order to understand the neuroendocrine regulation of MambrD11-desaturase gene expression by PBAN, the main regulator of pheromonogenesis (Jurenka and Rafaeli, 2011; Matsumoto, 2010; Raina, 1993; Tillman et al., 1999).

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4.1. Time course study of pheromone production in M. brassicae

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We recorded a characteristic daily rhythmicity in M. brassicae pheromone production (Fig. 1a) in accordance with reports from other species (Blomquist et al., 2005; Fónagy et al., 2011; Raina,

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Fig. 4. Effect of mating on D11-desaturase transcript expression and pheromone production in pheromone glands (PGs) of M. brassicae females. Newly emerged females (n = 5–6) and males (n = 7–8) were placed together into glass jars. Gene expression levels as well as pheromone production were analyzed from days 2–5 (D2–D5). D5 mated females were injected with Mambr-PT and sampled 90 min following the treatment (D5/PT). D5 virgin females were used as a reference. (a) D11-desaturase transcript expression in PGs. Transcript expression levels were measured by RT-qPCR and normalized to the expression levels of b-actin. No D11-desaturase or b-actin amplification products were observed when non-reverse transcribed RNA samples were used as templates for PCR reactions (data not shown). Data are expressed as means and error bars represent standard deviations of 3 independent experiments (n = 5) run in triplicate. Asterisks (*) denote a significant (P < 0.05) but not remarkable difference between expressions in mated females sampled at D2 and D5 virgins (Student’s t-test). (b) Pheromone content in PG extracts. Data are expressed as mean weights of Z11-hexadecenyl acetate (Z11-16Ac) and hexadecyl acetate (16Ac) in nanograms per PG. Upper and lower vertical bars show standard deviations for 16Ac and Z11-16Ac, respectively. Data are based on pooled samples of 3 PGs using 5 replicates for each time point. Double asterisks (**) denote significant differences (P < 0.01) in levels of Z11-16Ac (as well as in that of 16Ac for D4 mated females) between mated females and D5 virgins (Student’s t-test). The 16Ac component was not detectable in D5 mated females.

Please cite this article in press as: Köblös, G., et al. The regulation of D11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.03.004

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Fig. 5. Fluorescent microscopy of M. brassicae pheromone glands (PGs) dissected and spread out onto a slide. Double staining (Nile red for potential lipid reservoirs and Hoechst 33342 for nuclei) images were obtained at different times of the adult stage as well as following decapitation and/or Mambr-PT treatments. (a) The cellular arrangement of PG cells prepared from a newly emerged female is shown by blue nuclei staining. The single layer of modified epidermal cells is clearly visible. (b) The structure of PG from a D2 female dissected at late scotophase is demonstrated at higher magnification. (c) Representative area of PG cells of a D3 decapitated female is shown at higher magnification. Cell boundaries are clearly distinguishable. (d) PG trimmed 90 min following the injection of Mambr-PT into a virgin female decapitated 27 h earlier. The yellow arrow indicates the retractor muscles of the PG. Lipid droplets originating from fat body are shown (yellow arrowhead). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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1993). Pheromone production reached a maximum towards the end of scotophase and at the beginning of photophase under long day conditions. This is a common phenomenon in Noctuids (Raina, 1993), while some other moth species, like B. mori, produce pheromones throughout the photophase (Arima et al., 1991; Fónagy et al., 1992). In accordance with the above results, calling activity in M. brassicae also shows diurnal fluctuations (Noldus and Potting, 1990). Based on these findings, we performed experiments to simultaneously investigate the production of the three previously reported pheromone blend components (Attygalle et al., 1987) over an extended period. Surprisingly, we found that instead of the previously described Z11-16Ac:16Ac:Z916Ac = 90:10:1 composition (Attygalle et al., 1987), the ratio of Z11-16Ac and 16Ac was consistently found to be 93:7 in extracts from PGs of our strain and that Z9-16Ac was not detectable. It should be noted that, similarly to other moth species (Fónagy et al., 2011), the highest pheromone content could be detected close to the end of the third scotophase (D2). 4.2. Cloning and time course expression of M. brassicae D11desaturase The sequence of a PG-specific D11-desaturase cDNA was first isolated from T. ni (Knipple et al., 1998). Since that time, a number of D11-desaturases (in addition to other novel desaturases) have been identified from multiple species and shown to function in pheromone biosynthesis (Dallerac et al., 2000; Hao et al., 2002; Liu et al., 2004; Rodríguez et al., 2004; Rosenfield et al., 2001; Yoshiga et al., 2000). Early studies on the stereochemistry of D11-desaturases indicated that most of these enzymes have extreme selectivity (Boland et al., 1993), while some of the characterized D11-desaturases have been shown to be bi- or multifunctional (Matousková et al., 2007; Moto et al., 2004; Serra et al., 2007, 2006). It was also reported that some of the desaturase genes have become functionless (Roelofs and Rooney, 2003). In

M. brassicae, four acyl-CoA desaturases were cloned from PGs (Park et al., 2008). The two most abundant, differentially expressed transcripts encoding acyl-CoA desaturases were studied in more detail. Functional expression experiments demonstrated that the most abundant transcript encodes a D11-desaturase. The other desaturase was a non-functional D9-desaturase that had presumably lost functionality because of two mutations (Park et al., 2008). In our study, we determined that MambrD11-desaturase is expressed in large quantities exclusively in female PGs (Table 1). The tissue with the next highest expression level, albeit significantly lower than that found in the PG, was the male hairpencil– aedeagus. This structure is involved in male pheromone production (Jacquin et al., 1991), however, it is very unlikely that the D11-desaturase plays role in biosynthesis of the male pheromones, since they are different in chemical structure from the female-produced pheromones. The D11-desaturase ORF sequence identified in our study differed from that published by Park et al. (2008) in only 1 out of 1014 nucleotides. This difference does not alter the amino acid sequence. It is unlikely to be the result of a PCR or cloning artifact, since we sequenced a dozen clones containing amplification products obtained from the initial RT-PCR reaction. This strong conservation over the coding region is somewhat unexpected given the geographical distance between Central Europe and East Asia. There are two hypotheses regarding D11-desaturase regulation. One claims that D11-desaturase is under PBAN control in M. brassicae (Bestmann et al., 1989) similarly to that in C. chalcites (Altstein et al., 1989). However, the majority of studies suggest that PBAN does not affect desaturases (Gosalbo et al., 1992; Jacquin et al., 1994; Jurenka and Rafaeli, 2011; Tillman et al., 1999). In the Z strain of O. nubilalis, it was postulated that although more myristic acid underwent D11-desaturation in the gland in the presence of PBAN, this was counterbalanced by less D11-desaturation of palmitic acid, indicating that desaturase activity did not change overall (Eltahlawy et al., 2007a). This is in agreement

Please cite this article in press as: Köblös, G., et al. The regulation of D11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.03.004

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with our finding that in PGs of the Z strain of O. nubilalis the expression level of D11-desaturase measured during the period of daily maximum pheromone production does not differ significantly from that measured during the period of minimum activity (unpublished results). The gene expression level of D11-desaturase in PGs dissected from pre-emergence pupae is about 0.1% of the maximum value in 2–8-day-old females. In the PGs of virgin females the expression level increased significantly from eclosion to the end of the D1 when it reached the plateau phase. It is in agreement with earlier studies focusing on the onset of calling behavior, which was first observed close to the end of scotophase on D1 (Noldus and Potting, 1990). MambrD11-desaturase transcript expression was largely induced in PGs after adult eclosion. The relative transcriptional level was significantly lower (10%) in newly emerged females compared to that in calling ones from D1 at late scotophase. Accordingly, pheromone compounds were almost undetectable in D0 females. In some experiments, females were decapitated to prevent the effect of endogenous PBAN. We found that neither decapitation of females nor the subsequent injection with Mambr-PT influenced significantly MambrD11-desaturase expression levels in PGs (Figs. 2a and 3a). These results indicate that D11-desaturase expression is not induced or even influenced by PBAN. Nevertheless, it does not exclude the possibility that the enzyme activity varies under PBAN control, even if the gene expression is constant. 4.3. Evaluation of the effect of Mambr-PT treatments and mating on pheromone production Our studies confirmed that pheromone production in M. brassicae, like in the vast majority of Lepidopteran species (Blomquist et al., 2005; Jurenka and Rafaeli, 2011; Matsumoto, 2010; Rafaeli, 2002; Raina, 1993; Tillman et al., 1999), is under humoral control. This conclusion is based on the effect of Mambr-PT injections under various experimental conditions. Temporal changes of PBAN-like immunoreactivity in hemolymph in M. brassicae females were described previously in relation to sex pheromone production and calling behavior (Iglesias et al., 1999). However, that study was limited to analysis of the main component only (Z11-16Ac) and restricted to the third scotophase. In experiments where females were decapitated to prevent the effect of endogenous PBAN, decapitation resulted in a drop in the pheromone blend to almost an undetectable level, which was dramatically increased by Mambr-PT treatment (Fónagy et al., 2008, 1998). Strikingly, D0 females could be stimulated to produce significant amounts of pheromone in the appropriate ratio, in spite of the fact that untreated females at this time period do not synthesize the pheromone blend nor show calling behavior (Noldus and Potting, 1990; Fig. 1a and our unpublished observations). Last but not least, Mambr-PT treatment was able to trigger pheromone blend production more than 48 h after decapitation. This indicates that the enzyme machinery, including the D11-desaturase, is still intact in these females and fully capable of de novo synthesizing the pheromone blend (Fig. 3b). In a number of moth species, the uptake of carbohydrates is a prerequisite for sex pheromone production, which is indispensable for mating and fertile egg deposition (Foster, 2009; Foster and Johnson, 2010; O’Brien et al., 2004; Wheeler, 1996). In M. brassicae, we detected high pheromone titer in females decapitated immediately after emergence and maintained for 1or 2 days before Mambr-Pt treatment. This suggests that feeding is not absolutely necessary for the pheromone production. It is well known in species like B. mori, H. zea, Lymantria dispar, and Helicoverpa armigera that mating results in a significant

9

suppression in pheromone production accompanied by a halt in calling behavior and male receptivity (Ando et al., 1996; Kingan et al., 1995; Nagalakshmi et al., 2007; Raina et al., 1994). This phenomenon has been reviewed in detail by Rafaeli (2011) who focused on a H. zea pheromone suppressive peptide characterized from the accessory gland and duplex of male H. zea (Kingan et al., 1995). In E. postvittana (Tortricidea), mating apparently triggers the cessation of PBAN release resulting in a permanent decline in pheromone production. It was suggested that this decline in pheromone titer is due to reduced competency of the gland in older females to produce pheromone rather than to a decrease in PBAN titer (Foster and Roelofs, 1994). The senescent decline is mainly attributed to a decrease in FA synthesis and FA reduction, whereas b-oxidation, desaturation and acetylation steps remain unchanged (Greenwood and Foster, 1997). This seems to be the case in M. brassicae as well, since D11-desaturase gene expression reached its plateau within 2 days of adult eclosion and persisted throughout the 9-day study. Furthermore, Mambr-PT treatment of mated D5 females restored pheromone production to levels observed in D5 virgin females (Fig. 4b).

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Structural organization of PG cells in relation to pheromone production showed that, unlike in B. mori (Fónagy et al., 2001; Yokoyama et al., 2003) or T. ni (Percy, 1979), no Nile red positive LDs were observed in the PG cells. A close correlation between pheromone production and dynamic changes in lipid reservoirs were demonstrated in B. mori, while similar detailed studies were not conducted in T. ni (Percy and Weatherston, 1974). Recently, a comprehensive study was performed in P. separata (Fónagy et al., 2011), in which LDs were not detected either. During photophase or one day after decapitation, PG cells of P. separata were compact with gaps between cells, but during intensive natural pheromone production or following synthetic PBAN treatment, the pheromone producing cells became swollen and tightly packed. Such dynamism in PG cell morphology was not observed in M. brassicae. The absence of LD reservoirs indicates that pheromonogenesis is regulated via de novo FA synthesis (Jurenka and Rafaeli, 2011; Rafaeli, 2009; Tillman et al., 1999).

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Acknowledgments

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The research was supported by Hungarian Research Funds OTKA K104011 and OTKA K100421, in parallel with TÉT_12_FR2-2014-0009 international collaborative agreement. The help of László Peregovits, Csaba Szabóky and Dr. Antal Nagy is acknowledged for the establishment of the M. brassicae stock colony. Thanks are due to Gyöngyi Vajdics for excellent technical assistance. Dr. Kitti Sipos acknowledges that this research was also supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-1-2012-0001 ‘National Excellence Program’. The authors would like to express their special thanks to the three anonymous reviewers for comments that significantly contributed to the improvement of the manuscript.

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References

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Altstein, M., Harel, M., Dunkelblum, E., 1989. Effect of a neuroendocrine factor on sex pheromone biosynthesis in the tomato looper, Chrysodeixis chalcites (Lepidoptera: Noctuidae). Insect Biochem. 19, 645–649. Ando, T., Inomata, S.-I., Yamamoto, M., 2004. Lepidopteran sex pheromones. Top. Curr. Chem. 239, 51–96.

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Ando, T., Kasuga, K., Yajima, Y., Kataoka, H., Suzuki, A., 1996. Termination of sex pheromone production in mated females of the silkworm moth. Arch. Insect Biochem. Physiol. 31, 207–218. Arima, R., Takahara, K., Kadoshima, T., Numazaki, F., Ando, T., Uchiyama, M., Nagasawa, H., Kitamura, A., Suzuki, A., 1991. Hormonal regulation of pheromone biosynthesis in the silkworm moth, Bombyx mori (Lepidoptera: Bombycidae). Appl. Entomol. Zool. 26, 137–147. Attygalle, A.B., Herrig, M., Vostrowsky, O., Bestmann, H.J., 1987. Technique for injecting intact glands for analysis of sex pheromones of Lepidoptera by capillary gas chromatography: reinvestigation of pheromone complex of Mamestra brassicae. J. Chem. Ecol. 13, 1299–1311. Bestmann, H., Herrig, M., Attygalle, A., Hupe, M., 1989. Regulatory steps in sex pheromone biosynthesis in Mamestra brassicae L. (Lepidoptera: Noctuidae). Experientia 45, 778–781. Bestmann, H., Herrig, M., Attygalle, A.B., 1987. Terminal acetylation in pheromone biosynthesis by Mamestra brassicae L. (Lepidoptera: Noctuidae). Experientia 43, 1033–1034. Bestmann, H.J., Erler, J., Vostrowsky, O., 1988. Determination of diel periodicity of sex pheromone release in three species of Lepidoptera by ‘‘closed-loopstripping’’. Experientia 44, 797–799. Bjostad, L., Wolf, W., Roelofs, W., 1987. Pheromone biosynthesis in lepidopterans: desaturation and chain shortening. In: Prestwich, G.D., Blomquist, G.J. (Eds.), Pheromone Biochemistry. Academic Press, Orlando, pp. 77–120. Bloch, G., Hazan, E., Rafaeli, A., 2013. Circadian rhythms and endocrine functions in adult insects. J. Insect Physiol. 59, 56–69. Blomquist, G.J., Jurenka, R.A., Schal, C., Tittiger, C., 2005. Biochemistry and molecular biology of pheromone production. In: Gilbert, L.I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science, vol. 3. Elsevier, Oxford, pp. 705–751. Boland, W., Frössl, C., Schöttler, M., Tóth, M., 1993. Sex pheromone biosynthesis in Mamestra brassicae l. (Lepidoptera: Noctuidae); stereochemistry of the D11 desaturation. J. Chem. Soc. Chem. Commun., 1155–1157 Choi, M.-Y., Fuerst, E.-J., Rafaeli, A., Jurenka, R.A., 2003. Identification of a G proteincoupled receptor for pheromone biosynthesis activating neuropeptide from pheromone glands of the moth Helicoverpa zea. Proc. Natl. Acad. Sci. U.S.A. 100, 9721–9726. Dallerac, R., Labeur, C., Jallon, J.M., Knipple, D.C., Roelofs, W.L., Wicker-Thomas, C., 2000. A D9 desaturase gene with a different substrate specificity is responsible for the cuticular diene hydrocarbon polymorphism in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 97, 9449–9454. Delisle, J., Picimbon, J.-F., Simard, J., 2000. Regulation of pheromone inhibition in mated females of Choristoneura fumiferana and C. rosaceana. J. Insect Physiol. 46, 913–921. Eltahlawy, H., Buckner, J., Foster, S., 2007a. Regulation of pheromone biosynthesis in the ‘‘Z Strain’’ of the European corn borer, Ostrinia nubilalis. Arch. Insect Biochem. Physiol. 65, 29–38. Eltahlawy, H., Buckner, J., Foster, S., 2007b. Evidence for two-step regulation of pheromone biosynthesis by the pheromone biosynthesis-activating neuropeptide in the moth Heliothis virescens. Arch. Insect Biochem. Physiol. 64, 120–130. Fabrias, G., Barrot, M., Camps, F., 1995. Control of the sex pheromone biosynthetic pathway in Thaumetopoea pityocampa by the pheromone biosynthesis activating neuropeptide. Insect Biochem. Mol. Biol. 25, 655–660. Fabrias, G., Marco, M.P., Camps, F., 1994. Effect of the pheromone biosynthesis activating neuropeptide on sex pheromone biosynthesis in Spodoptera littoralis isolated glands. Arch. Insect Biochem. Physiol. 27, 77–87. Fang, N., Teal, P.E.A., Tumlinson, J., Fang, N.B., 1996. Effects of decapitation and PBAN injection on amounts of triacylglycerols in the sex pheromone gland of Manduca sexta (L.). Arch. Insect Biochem. Physiol. 32, 249–260. Fónagy, A., Marco, H.G., König, S., Gäde, G., 2008. Biological activity and identification of neuropeptides in the neurosecretory complexes of the cabbage pest insect, Mamestra brassicae (Noctuidae; Lepidoptera). Acta Biol. Hung. 59, 385–402. Fónagy, A., Matsumoto, S., Uchiumi, K., Orikasa, C., Mitsui, T., 1992. Action of pheromone biosynthesis activating neuropeptide on pheromone glands of Bombyx mori and Spodoptera litura. J. Pestic. Sci. 17, 47–54. Fónagy, A., Moto, K., Ohnishi, A., Kurihara, M., Kis, J., Matsumoto, S., 2011. Studies of sex pheromone production under neuroendocrine control by analytical and morphological means in the oriental armyworm, Pseudaletia separata, Walker (Lepidoptera: Noctuidae). Gen. Comp. Endocrinol. 172, 62–76. Fónagy, A., Teal, P., Meredith, J., Körmendy, C., Tumlinson, J., 1998. Partial identification of a new pheromonotropic peptide from Mamestra brassicae. Ann. New York Acad. Sci. 839, 488–490. Fónagy, A., Yokoyama, N., Matsumoto, S., 2001. Physiological status and change of cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori (Lepidoptera, Bombycidae). Arthropod Struct. Dev. 30, 113–123. Foster, S., 2009. Sugar feeding via trehalose haemolymph concentration affects sex pheromone production in mated Heliothis virescens moths. J. Exp. Biol. 212, 2789–2794. Foster, S.P., 1993. Neural inactivation of sex pheromone production in mated lightbrown apple moths, Epiphyas postvittana (Walker). J. Insect Physiol. 39, 267–273. Foster, S.P., 2001. Fatty acyl pheromone analogue-containing lipids and their roles in sex pheromone biosynthesis in the lightbrown apple moth, Epipyhas postvittana (Walker). J. Insect Physiol. 47, 433–443.

Foster, S.P., Johnson, C.P., 2010. Feeding and hemolymph trehalose concentration influence sex pheromone production in virgin Heliothis virescens moths. J. Insect Physiol. 56, 1617–1623. Foster, S.P., Roelofs, W.L., 1994. Regulation of pheromone production in virgin and mated females of two tortricid moths. Arch. Insect Biochem. Physiol. 25, 271– 285. Gosalbo, L., Fabrias, G., Arsequell, G., Camps, F., 1992. Inhibition of sex pheromone biosynthesis in Spodoptera littoralis by cyclopropene fatty acids. Insect Biochem. Mol. Biol. 22, 687–690. Greenwood, D.R., Foster, S.P., 1997. Change in reductase activity is responsible for senescent decline in sex pheromone titre in the lightbrown apple moth, Epiphyas postvittana (Walker). J. Insect Physiol. 43, 1093–1100. Hao, G., Liu, W., O’Connor, M., Roelofs, W.L., 2002. Acyl-CoA Z9- and Z10-desaturase genes from a New Zealand leafroller moth species, Planotortrix octo. Insect Biochem. Mol. Biol. 32, 961–966. Hull, J.J., Kajigaya, R., Imai, K., Matsumoto, S., 2007. Sex pheromone production in the silkworm, Bombyx mori, is mediated by store-operated Ca2+ channels. Biosci. Biotechnol. Biochem. 71, 1993–2001. Hull, J.J., Ohnishi, A., Moto, K., Kawasaki, Y., Kurata, R., Suzuki, M.G., Matsumoto, S., 2004. Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor from the silkmoth, Bombyx mori. Significance of the carboxyl terminus in receptor internalization. J. Biol. Chem. 279, 51500–51507. Iglesias, F., Jacquin-Joly, E., Marco, M.-P., Camps, F., Fabrias, G., 1999. Temporal distribution of PBAN-like immunoreactivity in the hemolymph of Mamestra brassicae females in relation to sex pheromone production and calling behavior. Arch. Insect Biochem. Physiol. 40, 80–87. Iglesias, F., Marco, M.-P., Jacquin-Joly, E., Camps, F., Fabrias, G., 1998. Regulation of sex pheromone biosynthesis in two noctuid species, S. littoralis and M. brassicae, may involve both PBAN and the ventral nerve cord. Arch. Insect Biochem. Physiol. 37, 295–304. Jacquin, E., Jurenka, R.A., Ljungberg, H., Nagnan, P., Löfstedt, C., Descoins, C., Roelofs, W.L., 1994. Control of sex pheromone biosynthesis in the moth Mamestra brassicae by the pheromone biosynthesis activating neuropeptide. Insect Biochem. Mol. Biol. 24, 203–211. Jacquin, E., Nagnan, P., Frerot, B., 1991. Identification of hairpencil secretion from male Mamestra brassicae (L.) (Lepidoptera: Noctuidae) and electroantennogram studies. J. Chem. Ecol. 17, 239–246. Jacquin-Joly, E., Burnet, M., François, M.C., Ammar, D., Meillour, P.N., Descoins, C., 1998. CDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of Mamestra brassicae: a new member of the PBAN family. Insect Biochem. Mol. Biol. 28, 251–258. Jefferson, R., Rubin, R., 1973. Sex pheromones of Lepidoptera. XXXV. Pupal and postemergence development of the pheromone gland of Trichoplusia ni (Lepidoptera: Noctuidae). Ann. Entomol. Soc. Am. 66, 277–279. Jurenka, R.A., Fabrias, E., Roelofs, W.L., 1991a. Hormonal control of female sex pheromone biosynthesis in the redbanded leafroller moth Argyrotaenia velutinana. Insect Biochem. 21, 81–89. Jurenka, R.A., Jacquin, E., Roelofs, W.L., 1991b. Stimulation of pheromone biosynthetic pathway in Helicoverpa zea: action of a brain hormone on pheromone glands involves Ca++ and cAMP as second messengers. Proc. Natl. Acad. Sci. U.S.A. 88, 8621–8625. Jurenka, R., Fabriás, G., 1993. Control of pheromone biosynthesis in mated redbanded leafroller moths. Arch. Insect Biochem. Physiol. 137, 129–137. Jurenka, R.A., Rafaeli, A., 2011. Regulatory Role of PBAN in sex pheromone biosynthesis of Heliothine moths. Front. Endocrinol. (Lausanne) 2, 1–8. Kingan, T.G., Bodnar, W.M., Raina, A.K., Shabanowitz, J., Hunt, D.F., 1995. The loss of female sex pheromone after mating in the corn earworm moth Helicoverpa zea: identification of a male pheromonostatic peptide. Proc. Natl. Acad. Sci. U.S.A. 92, 5082–5086. Kitamura, A., Nagasawa, H., Kataoka, H., Inoue, T., Matsumoto, S., Ando, T., Suzuki, A., 1989. Amino acid sequence of pheromone biosynthesis activating neuropeptide (PBAN) of the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 163, 520–526. Knipple, D.C., Rosenfield, C.L., Miller, S.J., Liu, W., Tang, J., Ma, P.W., Roelofs, W.L., 1998. Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA D11-desaturase of the cabbage looper moth, Trichoplusia ni. Proc. Natl. Acad. Sci. U.S.A. 95, 15287–15292. Knipple, D.C., Rosenfield, C.-L., Nielsen, R., You, K.M., Jeong, S.E., 2002. Evolution of the integral membrane desaturase gene family in moths and flies. Genetics 162, 1737–1752. Lee, J.M., Hull, J.J., Kawai, T., Goto, C., Kurihara, M., Tanokura, M., Nagata, K., Nagasawa, H., Matsumoto, S., 2012. Re-evaluation of the PBAN receptor molecule: characterization of PBANR variants expressed in the pheromone glands of moths. Front. Endocrinol. (Lausanne) 3, 1–12. Liénard, M.A., Lassance, J.-M., Wang, H.-L., Zhao, C.-H., Piskur, J., Johansson, T., Löfstedt, C., 2010. Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus (Lepidoptera: Lasiocampidae) reveals D11- and D9-desaturases with unusual catalytic properties. Insect Biochem. Mol. Biol. 40, 440–452. Liénard, M.A., Strandh, M., Hedenström, E., Johansson, T., Löfstedt, C., 2008. Key biosynthetic gene subfamily recruited for pheromone production prior to the extensive radiation of Lepidoptera. BMC Evol. Biol. 8, 1–5. Liu, W., Rooney, A.P., Xue, B., Roelofs, W.L., 2004. Desaturases from the spotted fireworm moth (Choristoneura parallela) shed light on the evolutionary origins of novel moth sex pheromone desaturases. Gene 342, 303–311.

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Ma, P.W.K., Roelofs, W.L., 1995. Calcium involvement in the stimulation of sex pheromone production by PBAN in the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae). Insect Biochem. Mol. Biol. 25, 467–473. Mas, E., Llòria, J., Quero, C., Camps, F., Fabriàs, G., 2000. Control of the biosynthetic pathway of Sesamia nonagrioides sex pheromone by the pheromone biosynthesis activating neuropeptide. Insect Biochem. Mol. Biol. 30, 455–459. Matousková, P., Pichová, I., Svatos, A., 2007. Functional characterization of a desaturase from the tobacco hornworm moth (Manduca sexta) with bifunctional Z11- and 10,12-desaturase activity. Insect Biochem. Mol. Biol. 37, 601–610. Matsumoto, S., 2010. Molecular mechanisms underlying sex pheromone production in moths. Biosci. Biotechnol. Biochem. 74, 223–231. Matsumoto, S., Fónagy, A., Kurihara, M., Uchiumi, K., Nagamine, T., Chijimatsu, M., Mitsui, T., 1992. Isolation and primary structure of a novel pheromonotropic neuropeptide structurally related to leucopyrokinin from the armyworm larvae, Pseudaletia separata. Biochem. Biophys. Res. Commun. 182, 534–539. Matsumoto, S., Hull, J.J., Ohnishi, A., Moto, K., Fónagy, A., 2007. Molecular mechanisms underlying sex pheromone production in the silkmoth, Bombyx mori: characterization of the molecular components involved in bombykol biosynthesis. J. Insect Physiol. 53, 752–759. Matsumoto, S., Ozawa, R., Nagamine, T., Kim, G.H., Uchiumi, K., Shono, T., Mitsui, T., 1995. Intracellular transduction in the regulation of pheromone biosynthesis of the silkworm, Bombyx mori: suggested involvement of calmodulin and phosphoprotein phosphatase. Biosci. Biotechnol. Biochem. 59, 560–562. Moto, K., Suzuki, M.G., Hull, J.J., Kurata, R., Takahashi, S., Yamamoto, M., Okano, K., Imai, K., Ando, T., Matsumoto, S., 2004. Involvement of a bifunctional fatty-acyl desaturase in the biosynthesis of the silkmoth, Bombyx mori, sex pheromone. Proc. Natl. Acad. Sci. U.S.A. 101, 8631–8636. Nagalakshmi, V.K., Applebaum, S.W., Azrielli, A., Rafaeli, A., 2007. Female sex pheromone suppression and the fate of sex-peptide-like peptides in mated moths of Helicoverpa armigera. Arch. Insect Biochem. Physiol. 64, 142–155. Nagy, B., 1970. Rearing of the European corn borer (Ostrinia nubilalis Hbn.) on a simplified artificial diet. Acta Phytopathol. Acad. Sci. Hung. 5, 73–79. Noldus, L.P.J.J., Potting, R.P.J., 1990. Calling behaviour of Mamestra brassicae: effect of age and photoperiod. Entomol. Exp. Appl. 56, 23–30. Nusawardani, T., Kroemer, J.A., Choi, M.-Y., Jurenka, R.A., 2013. Identification and characterization of the pyrokinin/pheromone biosynthesis activating neuropeptide family of G protein-coupled receptors from Ostrinia nubilalis. Insect Mol. Biol. 22, 331–340. O’Brien, D.M., Boggs, C.L., Fogel, M.L., 2004. Making eggs from nectar: the role of life history and dietary carbon turnover in butterfly reproductive resource allocation. Oikos 105, 279–291. Park, H.Y., Kim, M.S., Paek, A., Jeong, S.E., Knipple, D.C., 2008. An abundant acyl-CoA (D9) desaturase transcript in pheromone glands of the cabbage moth, Mamestra brassicae, encodes a catalytically inactive protein. Insect Biochem. Mol. Biol. 38, 581–595. Pefia, A., Arn, H., Buser, H., Rauscher, S., Bigler, F., Brunetti, R., Main, S., Tóth, M., 1988. Sex pheromone of European corn borer, Ostrinia nubilalis: polymorphism in various laboratory and field strains. J. Chem. Ecol. 14, 1359–1366. Percy, J.E., 1979. Development and ultrastructure of sex-pheromone gland cells in females of the cabbage looper moth, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae). Can. J. Zool. 57, 220–236. Percy, J.E., Weatherston, J., 1974. Gland structure and pheromone production in insects, in: Frontiers of Biology, vol. 32, Pheromones. pp. 11–34. Rafaeli, A., 2002. Neuroendocrine control of pheromone biosynthesis in moths. Int. Rev. Cytol. 213, 49–91. Rafaeli, A., 2009. Pheromone biosynthesis activating neuropeptide (PBAN): regulatory role and mode of action. Gen. Comp. Endocrinol. 162, 69–78. Rafaeli, A., 2011. Moth sex-pheromone production: biosynthetic pathways, regulatory physiology, inhibitory processes and disruption. In: Cauterruccio, L. (Ed.), Moths: Types, Ecological Significance and Control. Nova Science Publisher Inc., pp. 1–29.

11

Rafaeli, A., Jurenka, R.A., 2003. PBAN regulation of pheromone biosynthesis in female moths. In: Blomquist, G., Vogt, R. (Eds.), Insect Pheromone Biochemistry and Molecular Biology. Academic Press, New York, pp. 107–136. Raina, A.K., 1993. Neuroendocrine control of sex pheromone biosynthesis in Lepidoptera. Annu. Rev. Entomol. 38, 329–349. Raina, A.K., Jaffe, H., Kempe, T.G., Keim, P., Blacher, R.W., Fales, H.M., Riley, C.T., Klun, J.A., Ridgway, R.L., Hayes, D.K., 1989. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Science 244, 796– 798. Raina, A.K., Kingan, T.G., Giebultowicz, J.M., 1994. Mating-induced loss of sexpheromone and sexual receptivity in insects with emphasis on Helicoverpa zea and Lymantria dispar. Arch. Insect Biochem. Physiol. 25, 317–327. Rodríguez, S., Camps, F., Fabriàs, G., 2004. Inhibition of the acyl-CoA desaturases involved in the biosynthesis of Spodoptera littoralis sex pheromone by analogs of 10,11-methylene-10-tetradecenoic acid. Insect Biochem. Mol. Biol. 34, 283– 289. Roelofs, W.L., Rooney, A.P., 2003. Molecular genetics and evolution of pheromone biosynthesis in Lepidoptera. Proc. Natl. Acad. Sci. U.S.A. 100, 9179–9184. Rosenfield, C.L., You, K.M., Marsella-Herrick, P., Roelofs, W.L., Knipple, D.C., 2001. Structural and functional conservation and divergence among acyl-CoA desaturases of two noctuid species, the corn earworm, Helicoverpa zea, and the cabbage looper, Trichoplusia ni. Insect Biochem. Mol. Biol. 31, 949–964. Sato, Y., Oguchi, M., Menjo, N., Imai, K., Saito, H., Ikeda, M., Isobe, M., Yamashita, O., 1993. Precursor polyprotein for multiple neuropeptides secreted from the suboesophageal ganglion of the silkworm Bombyx mori: characterization of the cDNA encoding the diapause hormone precursor and identification of additional peptides. Proc. Natl. Acad. Sci. U.S.A. 90, 3251–3255. Serra, M., Piña, B., Abad, J.L., Camps, F., Fabriàs, G., 2007. A multifunctional desaturase involved in the biosynthesis of the processionary moth sex pheromone. Proc. Natl. Acad. Sci. U.S.A. 104, 16444–16449. Serra, M., Piña, B., Bujons, J., Camps, F., Fabriàs, G., 2006. Biosynthesis of 10,12dienoic fatty acids by a bifunctional D11 desaturase in Spodoptera littoralis. Insect Biochem. Mol. Biol. 36, 634–641. Shanklin, J., Cahoon, E., 1998. Desaturation and related modifications of fatty acids. Annu. Rev. Plant Physiol. Mol. Biol. 49, 611–641. Stukey, J., Mc Donough, V., Martin, C., 1990. The OLE 1 gene of Saccharomyces cerevisiae encodes the D9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene. J. Biol. Chem. 265, 20144–20149. Tamaki, Y., 1985. Sex pheromones. In: Kerkut, G., Gilbert, L. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, 145–191, vol. 9. Oxford, pp. 145–191. Tang, J.D., Charlton, R.E., Jurenka, R.A., Wolf, W.A., Phelan, P.L., Roelofs, W.L., 1989. Regulation of pheromone biosynthesis by a brain hormone in two moth species. Proc. Natl. Acad. Sci. U.S.A. 86, 1806–1810. Teal, P.E.A., Abernathy, R.L., Nachman, R.J., Fang, N., Meredith, J.A., Tumlinson, J.H., 1996. Pheromone biosynthesis activating neuropeptides: functions and chemistry. Peptides 17, 337–344. Tillman, J.A., Seybold, S.J., Jurenka, R.A., Blomquist, G.J., 1999. Insect pheromones – an overview of biosynthesis and endocrine regulation. Insect Biochem. Mol. Biol. 29, 481–514. Vogel, H., Heidel, A.J., Heckel, D.G., Groot, A.T., 2010. Transcriptome analysis of the sex pheromone gland of the noctuid moth Heliothis virescens. BMC Genomics 11, 29–49. Wheeler, D., 1996. The role of nourishment in oogenesis. Annu. Rev. Entomol. 41, 407–431. Yokoyama, N., Fónagy, A., Tatsuki, S., Arie, T., Yamashita, S., Matsumoto, S., 2003. Ultrastructural studies on the pheromone-producing cells in the silkmoth, Bombyx mori: formation of cytoplasmic lipid droplets before adult eclosion. Acta Biol. Hung. 54, 299–311. Yoshiga, T., Okano, K., Mita, K., Shimada, T., Matsumoto, S., 2000. CDNA cloning of acyl-CoA desaturase homologs in the silkworm, Bombyx mori. Gene 246, 334– 339.

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