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Identification of a diacylglycerol acyltransferase 2 gene involved in pheromone biosynthesis activating neuropeptide stimulated pheromone production in Bombyx mori
Journal of Insect Physiology 58 (2012) 699–703
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Identification of a diacylglycerol acyltransferase 2 gene involved in pheromone biosynthesis activating neuropeptide stimulated pheromone production in Bombyx mori Mengfang Du, Songdou Zhang, Bin Zhu, Xinming Yin, Shiheng An ⇑ College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, PR China
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Article history: Received 15 November 2011 Received in revised form 2 February 2012 Accepted 10 February 2012 Available online 3 March 2012 Keywords: Bombyx mori Pheromone gland Diacylglycerol acyltransferase (DGAT) Sex pheromone
a b s t r a c t Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triacylglycerol biosynthesis. In the present study, a DGAT2 gene from Bombyx mori was characterized. Temporal expression profiles indicated that BmDGAT2 steadily increased from 96 h before eclosion (96 h) to an expression peak in the pheromone glands (PGs) of new-emerged female (0 h), a key stage for sex pheromone production. Spatial expression analysis revealed that the BmDGAT2 transcript was most richly expressed in PGs. Decapitation and subsequent methoprene, a juvenile hormone (JH) analog, treatment experiments revealed that JH had no influence on the expression of BmDGAT2 transcript before emergence, but inhibited the expression of BmDGAT2 transcript when administered to newly emerged adults. Further RNAi analysis confirmed that the decrease in BmDGAT2 mRNA level caused a significant reduction in sex pheromone production. Thus, DGAT2 is a key enzyme regulating B. mori sex pheromone synthesis and release. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Sex pheromones are important in moth chemical communication and successful mating. These sex pheromones are produced and released by a specific organ, the female pheromone gland (PG), situated between the ultimate and penultimate terminal segments of the abdomen (Rafaeli and Gileadi, 1995; Rafaeli, 2009). Sex pheromone synthesis and release increase during a specific scotophase when the male is sexually receptive to sex pheromones. The synthesis and release of sex pheromones in moths are triggered by pheromone biosynthesis activating neuropeptide (PBAN), a neurohormone released from the suboesophageal ganglion into hemolymph during the scotophase (Rafaeli, 2005). PBAN binds to specific G-protein-coupled receptors located in PGs, followed by activation of second messengers. Stimulation of these receptors by PBAN triggers an influx of calcium and cAMP production in Helicoverpa zea PG cells (Rafaeli and Gileadi, 1995). In Bombyx mori, however, PBAN only opens calcium channels, triggering calcium influx, but is not engaged in cAMP-dependent signaling (Hull et al., 2007). Although there are differences in the signaling pathways engaged by PBAN in B. mori and H. zea, the origin of the sex pheromones is the similar. Sex pheromones
⇑ Corresponding author. E-mail address: [email protected] (S. An). 0022-1910/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2012.02.002
in both species are derived from acetyl-CoA through fatty acid synthesis, desaturation, and chain-shortening reactions, followed by reductive modification of the carbonyl carbon (Tillman et al., 1999). In Bombyx and Helicoverpa species, the precursor of sex pheromones is contained within lipid droplets (LDs) consisting of various triacylglycerols (TAGs). Before adult emergence, PG cells rapidly accumulate LDs. The accumulation of LDs parallels the production of sex pheromones following PBAN stimulation in Bombyx (Fónagy et al., 2001). In Helicoverpa, suppression of acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme for fat synthesis, significantly decreases the production of sex pheromone (Tsfadia et al., 2008). These results provide strong evidence that sex pheromones synthesis and release are linked to fatty acid synthesis pathways in the pheromone gland (Tsfadia et al., 2008). Triacylglycerols serve as a source for the de novo synthesis of bombykol. Diacylglycerol acyltransferase (DGAT) is an endoplasmic reticulum (ER) membrane protein that catalyzes the final step in TAG biosynthesis (Bell and Coleman, 1980). Overexpression of DGAT results in increased triglyceride synthesis (Liu et al., 2009). Our previous results revealed that a B. mori DGAT 2 (BmDGAT2) expressed at significant transcript levels in newly emerged females, indicating that this gene is likely involved in sex pheromone biosynthesis. In this study, the expression profile and function of BmDGAT2 in sex pheromone synthesis were studied.
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2. Materials and methods 2.1. Insect culture B. mori (Zhengzhu Chunlei) were kindly provided by the Sericultural Research Institute, Henan Academy of Agricultural Sciences and were reared on mulberry leaves at 26 °C under 14 h light/10 h dark cycles. Wandering stage larvae were collected for pupal age determination. Pupae were sexually separated, and adult males and females emerged in separate cages until tissue dissection. Pupal age was determined following the previous methods (Matsumoto et al., 2002).
2.2. Chemicals B. mori PBAN was synthesized by Songon Biotech (Shanghai) Co. Ltd. The main sex pheromone component, bombykol, was a gift from Shogo Matsumoto (RIKEN, Advanced Science Institute, Japan). Bombykol was used as the standard for pheromone quantification by gas chromatography–mass spectrometry (GC/MS).
2.3. Temporal and spatial expression profile of BmDGAT2 To analyze the temporal expression profile of BmDGAT2 transcripts at different developmental stages, female PGs were collected at 96 (96 h before eclosion), 72 (72 h before eclosion), 48 (48 h before eclosion), 24 (24 h before eclosion), 0 (newemergence), 24 (24 h after eclosion), 48 (48 h after eclosion), and 72 h (72 h after eclosion), and immediately frozen at 80 °C for later use. Different tissues, including pheromone glands, muscles, fat body, midgut, head, epidermis, and eggs were collected from the newly emerged females and immediately stored at 80 °C for later use. The PG and other tissue samples collected above were homogenized in Trizol reagent (Invitrogen, Gaithersburg, MD, USA) and used for total RNA extraction according to the manufacturer’s instruction. RNA concentration was determined by measuring the absorbance at 260 nm on a spectrophotometer. The first-strand cDNA was synthesized from 1 lg total RNA of each sample using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Kyoto, Japan). To measure the temporal expression profile of BmDGAT2, the synthesized cDNA was used as a template for analysis of BmDGAT2 transcripts by real-time PCR with gene specific primers (Forward 50 -CGCATCGCTATGAAGTCAC-30 ; Reverse 50 -AACAGTTGTAACGGG ACTC-30 ). The Bombyx ribosomal protein 49 (rp49) gene (Forward 50 -CAGGCGGTTCAAGGGTCAATAC-30 ; Reverse 50 -TGCTGGGCTCT TTCCACGA-30 ) was used as the internal control for normalization. Real-time PCR amplification and analysis was carried out on an Applied Biosystems 7500 Fast Real-Time PCR System (ABI, Carlsbad, CA, USA) using SYBR Green Supermix (TaKaRa). The thermal cycle conditions used in the real-time PCR were 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s and 60 °C for 1 min. The specificity of the SYBR green PCR signal was further confirmed by melting curve analysis and agarose gel electrophoresis. Comparative expression analysis was performed using the comparative CT method (Cross Threshold or CT is defined as the PCR cycle number where the signal crosses the signal threshold) (Livak and Schmittgen, 2001). The CT of the RP49 gene was subtracted from the CT of the target gene to obtain DCT. The normalized fold changes of the target gene mRNA were expressed as 2DDCT, where DDCT is equal to DCT treated sample – DCT control.
To examine the tissue distribution of BmDGAT2 transcripts, PCR was performed using synthesized cDNA and gene-specific primers. The thermal cycle conditions were 95 °C for 2 min, followed by 30 cycles of 95 °C for 15 s and 60 °C for 1 min. PCR products were further analyzed by agarose gel electrophoresis. The Rp49 gene also was used as an internal control. 2.4. Decapitation experiments The females were decapitated at different developmental stages from pupa to adult (96, 72, 48, 24, and 0 h) and maintained on a moist plate under identical photoperiod and temperature conditions. Pheromone glands were dissected at 24, 48, and 72 h after decapitation. The changes in BmDGAT2 transcript levels were analyzed using real-time PCR following the above methods. The pheromone glands from normal developmental females were used as control. 2.5. Treatment by methoprene in vitro Females at different developmental stages (72, 48, 24, and 0 h) were decapitated. Pheromone glands were dissected 24 h after decapitation and immediately placed in tissue plates containing 1 mL Grace’s insect culture medium (Invitrogen). After 1 h incubation, the medium was replaced with fresh Grace’s medium containing 2 lM methoprene. Pheromone glands were then harvested at designated time intervals (0, 2, 4, and 6 h) and stored at 80 °C for later real-time PCR analysis. Total RNA was extracted, the first strand was synthesized, and real-time PCR was performed according to the above methods. 2.6. Double-stranded RNA (dsRNA) synthesis Double stranded RNAs were synthesized using the MEGAscript RNAi kit (Ambion, Huntingdon, UK) according to the manufacturer’s instructions. PCR was performed using gene-specific primers containing T7 polymerase sites (Forward primer 50 -GATCACTAA TACGACTCACTATAGGGAGAATCACGGAGTGTGGGGATA-30 ; Reverse primer 50 -GATCACTAATACGACTCACTATAGGGAGACTACCATTATAG CCACACA-30 ). PCR was performed at 94 °C for 3 min, followed by 35 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min, and a final elongation at 72 °C for 10 min. PCR product was then treated with DNase and RNase to eliminate template DNA and single-stranded RNA. Double stranded RNA was purified using MEGAclearTM columns (Ambion, Huntingdon, UK) and eluted in diethyl pyrocarbonate (DEPC)-treated nuclease-free water. The dsRNA concentrations were measured using a biophotometer (Eppendorf, Hamburg, Germany). Our previous experiments confirmed that either dsGFP RNA or ddH2O are suitable negative controls. In the present study, DEPC-treated nuclease-free water was used as a negative control. The effects of RNAi on transcript expression were analyzed by RT-PCR. The primers used in this experiment were the same as those used for real-time PCR analysis. 2.7. Injection of dsRNA and in vivo bombykol analysis The 72 h female pupae were decapitated. After 24 h, 20 lg dsRNA of BmDAGT2 was injected into the decapitated females. Control females were injected with DEPC-treated nuclease-free water. The females were maintained for 48 h under normal conditions and then injected with either 5 pmol B. mori PBAN or again with DEPC-treated nuclease-free water. The PGs were dissected 90 min after injection and the pheromone component was extracted in hexane.
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3. Results 3.1. Temporal and spatial expression profile of BmDGAT2 To determine the temporal expression profile of BmDGAT2, realtime PCR was performed on PG tissue isolated at different developmental stages, from 96 h before eclosion to 72 h after eclosion. The newly emerged females were designated as 0 h females. The result reveals that BmDGAT2 transcript steadily increased with age (Fig. 1A), which indicating that expression pattern of BmDGAT2 transcript is age-dependent. To examine the tissue distribution of BmDGAT2 transcripts, PCR was performed on RNAs isolated from various adult tissues. The BmDGAT2 transcript was present in all tissues examined, but most richly expressed in PGs (Fig. 1B). Fig. 1. Temporal and spatial expression profile of BmDGAT2 in the PG of B. mori during pupal–adult development (the zero time point indicates the time of eclosion). (A) Temporal and spatial expression profile of BmDGAT2. PGs were collected at different developmental stages (96, 72, 48, 24, 0, 24, 48, and 72 h) and total RNA was extracted for real-time PCR analysis. The Rp49 gene was used as the housekeeping gene for normalization. The data are presented as mean ± SD of three biological replicates. (B) Tissue distributions of BmDGAT2. Different tissues, including PG, muscles (Ms), fat body (Fb), midgut (Mg), head (Hd), epidermis (Ep), egg (Eg) were collected from newly emerged females and total RNA was extracted for PCR analysis. The Rp49 gene was used as the housekeeping gene.
Bombykol accumulation was measured by GC/MS (Trace GC Ultra Trace DSQ; MS-Thermo Scientific DSQ II) equipped with a 30 m capillary column (RTX-5SILMS, Restek, 0.25 mm diameter). Each sample contained a pooled hexane extract from at least five B. mori pheromone glands. Each sample was then subjected to GC/MS analysis. 2.8. Statistical analysis The real-time PCR experiments were performed in triplicate, and expressed as mean ± SD. Real-time PCR results were compared using Student’s t-tests.
3.2. Decapitation experiments The expression levels of BmDGAT2 transcript in PGs from Bombyx females decapitated at various ages (96, 72, 48, 24, and 0 h) were measured by real-time PCR (Fig. 2). These BmDGAT2 transcripts still increased significantly with age despite head detachment and the expression temporal pattern in decapitated females was similar to that observed in normally developing females, although BmDGAT2 transcripts significant decrease compared to normally developing females.
3.3. Treatments of methoprene in vitro The expression pattern of BmDGAT2 transcript was further examined after treatment of decapitated females with methoprene, a JH analog. Females at different developmental stages were decapitated. After 24 h, PGs were collected from the decapitated females and treated with methoprene. The results show that BmDGAT2 transcript remained almost unchanged in 72, 48, and 24 h PGs despite methoprene treatment (Fig. 3), whereas methoprene significantly inhibited BmDGAT2 expression in 0 h PGs.
Fig. 2. The effect of head detachment after 24, 48, and 72 h on BmDGAT2 expression levels during pupal–adult development. Females at different developmental stages (A: 96 h; B: 72 h; C: 48 h; D: 24 h; E: 0 h) were decapitated. The PGs were collected at 24, 48, and 72 h after decapitation and total RNA was extracted for real-time PCR analysis. The Rp49 gene was used as the housekeeping gene for normalization. The blank bars present the normally developed female (control). The black bars represent the decapitated females. The data represent the mean ± SD of three biological replicates. ⁄⁄p < 0.01.
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Fig. 3. The effect of JH on BmDGAT2 expression levels during pupal–adult development. Females at different developmental stages (A: 72 h; B: 48 h; C: 24 h; D: 0 h) were decapitated. The PGs were collected 24 h after decapitation, treated with 2 lM methoprene, and harvested at different incubated time intervals (0, 2, 4, and 6 h) for real-time PCR analysis. The Rp49 gene was used as the housekeeping gene for normalization. The data represent the mean ± SD of three biological replicates. ⁄⁄⁄p < 0.01.
3.4. Injection of dsRNA and in vivo bombykol analysis To confirm the function of BmDGAT2 during the sex pheromone synthesis and release, RNAi-mediated knockdown was performed. First, 20 lg BmDGAT2 dsRNA was injected into 72 h decapitated females and PCR performed 48 h later. Expression of BmDGAT2 mRNA was significantly decreased compared to decapitated females injected with DEPC-treated nuclease-free water (Fig. 4A). To examine the effect of BmDGAT2 on bombykol production, PBAN-induced bombykol production was determined by GC/MS in control females and females treated with BmDGAT2-specific RNAi. Chromatographic analysis revealed a significant reduction
Fig. 4. Effects of RNAi treatment on bombykol production. (A) RNAi-induced reduction of BmDGAT2 transcript. RT-PCR was performed using cDNA generated from the total RNA extracted from PGs of females injected with vehicle or 20 lg dsRNAs for the BmDGAT2 gene. (B) Effects of RNAi for the BmDGAT2 gene on bombykol production. Females at 72 h before elcosion were decapitated and injected with double stranded RNAs. Decapitated females were injected with 5 pmol PBAN 48 h after dsRNA injection. Bombykol production was measured by GC/MS from PGs 90 min after injection of PBAN by GC/MS. Bars indicate mean values ± SD from independent experimental animals (n P 5). Statistically significant differences from the PBAN alone are denoted by ⁄⁄⁄(p < 0.01) as determined by the Student’s t-test.
in bombykol production after successful reduction of BmDGAT2 transcript by RNAi. Bombykol production was reduced to 25% of control in the knockdown females, suggesting that BmDGAT2 is required for bombykol production (Fig. 4B). 4. Discussion The successful propagation of Bombyx depends on highefficiency mating, which must be regulated by sex pheromones synthesized and released in adult stages. The key biosynthetic steps to generate specific sex pheromone in moths include de novo biosynthesis of fatty acid precursors, followed by various chemical modifications, including desaturation, reduction, acetylation, and oxidation (Tillman et al., 1999; Bjostad et al., 1987). In B. mori, lipid must be synthesized and accumulated in PGs in the form of TAGs before adult emergence to meet the substrate requirements for the large amounts of pheromones that must be synthesized by newly emerged females (Foster, 2000). Previous works indicated that TAGs are synthesized by two pathways, the monoacylglycerol (MAG) and glycerol phosphate pathways (Bell and Coleman, 1980; Coleman and Lee, 2004). The MAG pathway plays the predominant role in TAG synthesis in animal intestines. In contrast, the glycerol phosphate pathway is a de novo TAG synthesis pathway in most tissues. These two pathways share a common final step in which a fatty acyl-CoA and diacylglycerol (DG) molecule are covalently joined to form TAG. This final reaction is rate-limiting and is catalyzed by DGAT (Bell and Coleman, 1980; Coleman and Lee, 2004; Lehner and Kuksis, 1996). As the sex pheromone precursor material, a decrease in TAG production unavoidably causes a substantial decrease in sex pheromone production. Thus, DGAT should play a key role in sex pheromone production. At present, at least two DGAT family members, DGAT1 and DGAT2, have been identified in animals and plants (Hofmann, 2000). DGAT1 is a member of the membrane-bound O-acyltransferases with nine transmembrane domains. In contrast, DGAT2 belongs to a family that includes acyl-CoA:monoacylglycerol acyltransferase and acyl-CoA:wax alcohol acyltransferase, with only two transmembrane domains (Stone et al., 2004; Turkish et al., 2005). Although they share the same DGAT enzyme reaction, DGAT1 and DGAT2 bear little sequence resemblance. Amino acid sequence analysis indicated that DGAT1 is composed of 7 sequence blocks, whereas DGAT2s include 6 sequence blocks (Cao, 2011). In fact it has been proposed DGAT1 and DGAT2 have different physiological functions. Mice deficient DGAT1 was found to be viable and has modest decreases in TAG, compared to mice deficient DGAT2, which
M. Du et al. / Journal of Insect Physiology 58 (2012) 699–703
cause severe reduction of TAGs (Stone et al., 2004; Smith et al., 2000). In plants, two enzymes seem to be species and tissues dependent (Shockey et al., 2006). In addition, In Vernonia galamensis, both DGAT1 and DGAT2 were found to increase epoxy fatty acid accumulations, but DGAT2 had a greater effect (Li et al., 2010). Our previous digital gene expression (DGE) profile in PGs from different developmental stages identified a DGAT encoded by the BGIBMGA008049-TA transcript, which was richly expressed in newly emerged females, a key stage for sex pheromone synthesis and release. Further sequence analysis revealed that the amino acid sequence encoded by BGIBMGA008049-TA shared classic characteristics with other DGAT2 members and belonged to the DGAT2 family (data not shown here). Temporal expression profiling was consistent with DGE results and temporal expression patterns mirrored those of many other key sex pheromone synthesis genes, including acyl-CoA desaturase, fatty acyl reductase, PBAN receptor, fatty acid transport protein, and acyl-CoA binding protein (Rafaeli and Gileadi, 1995; Ohnishi et al., 2009; Matsumoto et al., 2001, 2010; Moto et al., 2003). Knockdown of BmDGAT2 by RNAi confirmed that a decrease in BmDGAT2 mRNA expression caused a significant reduction in sex pheromone production. Interestingly, our DGE result did not identify DGAT1 that richly expressed in newly emerged females. Whether DGAT1 also present in Bombyx PGs and if present, then what is the function of DGAT1 in sex pheromone synthesis and release require further investigation. Methoprene challenge experiment suggested Methoprene, a JH analog, significantly inhibited the expression of BmDGAT2 transcript in newly emerged females (mature adults), similar to results from Helicoverpa arimgera showing that fenoxycarb upregulated pheromone production and PBANR in pharate adults, but downregulated both in mature adults (Rafaeli et al., 2003; Bober et al., 2010). This developmentally regulated effect of JH may be due to the increase in JH concentration in the haemolymph of mature Bombyx females caused by constitutive production, synthesis induced by seminal peptides produced by the male accessory gland, or direct transfer of JH produced in the male accessory gland (Park et al., 1998). An overabundance of JH in mature adult females inhibits BmDGAT2 expression. Temporal expression profiles also confirmed that BmDGAT2 transcript levels started to decrease after 0 h, consistent with the methoprene treatment experiment. These results appear at odds with the 0 h decapitation experiment, however, in which BmDGAT2 transcript declined compared to normally developed females (Fig. 2). The loss of hemolymph after decapitation or direct harm induced by decapitation may activate other mechanisms that inhibit BmDGAT2 expression. Although many characteristics of BmDGAT2 regulation and expression were identified in the present study, these uncertainties deserve further study on the regulation of BmDGAT2 transcripts and the function of the encoded protein. Acknowledgements This study was supported by a Distinguished Professor Foundation of Henan Agricultural University (No: 30600022) and Natural Science Foundation of Henan Province (No: 2011B180021). References Bell, R.M., Coleman, R.A., 1980. Enzymes of glycerolipid synthesis in eukaryotes. Annual Review of Biochemistry 49, 459–487. Bjostad, L.B., Wolf, W.A., Roelofs, W.L., 1987. Pheromone biosynthesis in lepidopterans: desaturation and chain shortening. In: Prestwich, G.D., Blomquist, G.J. (Eds.), Pheromone Biochemistry. Academic Press, New York, pp. 77–120. Bober, R., Azrielli, A., Rafaeli, A., 2010. Developmental regulation of the pheromone biosynthesis activating neuropeptide-receptor (PBAN-R): re-evaluating the role of juvenile hormone. Insect Molecular Biology 19, 77–86. Cao, H., 2011. Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms. BMC Research Notes 4249.
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Identification of a diacylglycerol acyltransferase 2 gene involved in pheromone biosynthesis activating neuropeptide stimulated pheromone production in Bombyx mori Mengfang Du, Songdou Zhang, Bin Zhu, Xinming Yin, Shiheng An ⇑ College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, PR China
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Article history: Received 15 November 2011 Received in revised form 2 February 2012 Accepted 10 February 2012 Available online 3 March 2012 Keywords: Bombyx mori Pheromone gland Diacylglycerol acyltransferase (DGAT) Sex pheromone
a b s t r a c t Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triacylglycerol biosynthesis. In the present study, a DGAT2 gene from Bombyx mori was characterized. Temporal expression profiles indicated that BmDGAT2 steadily increased from 96 h before eclosion (96 h) to an expression peak in the pheromone glands (PGs) of new-emerged female (0 h), a key stage for sex pheromone production. Spatial expression analysis revealed that the BmDGAT2 transcript was most richly expressed in PGs. Decapitation and subsequent methoprene, a juvenile hormone (JH) analog, treatment experiments revealed that JH had no influence on the expression of BmDGAT2 transcript before emergence, but inhibited the expression of BmDGAT2 transcript when administered to newly emerged adults. Further RNAi analysis confirmed that the decrease in BmDGAT2 mRNA level caused a significant reduction in sex pheromone production. Thus, DGAT2 is a key enzyme regulating B. mori sex pheromone synthesis and release. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Sex pheromones are important in moth chemical communication and successful mating. These sex pheromones are produced and released by a specific organ, the female pheromone gland (PG), situated between the ultimate and penultimate terminal segments of the abdomen (Rafaeli and Gileadi, 1995; Rafaeli, 2009). Sex pheromone synthesis and release increase during a specific scotophase when the male is sexually receptive to sex pheromones. The synthesis and release of sex pheromones in moths are triggered by pheromone biosynthesis activating neuropeptide (PBAN), a neurohormone released from the suboesophageal ganglion into hemolymph during the scotophase (Rafaeli, 2005). PBAN binds to specific G-protein-coupled receptors located in PGs, followed by activation of second messengers. Stimulation of these receptors by PBAN triggers an influx of calcium and cAMP production in Helicoverpa zea PG cells (Rafaeli and Gileadi, 1995). In Bombyx mori, however, PBAN only opens calcium channels, triggering calcium influx, but is not engaged in cAMP-dependent signaling (Hull et al., 2007). Although there are differences in the signaling pathways engaged by PBAN in B. mori and H. zea, the origin of the sex pheromones is the similar. Sex pheromones
⇑ Corresponding author. E-mail address: [email protected] (S. An). 0022-1910/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2012.02.002
in both species are derived from acetyl-CoA through fatty acid synthesis, desaturation, and chain-shortening reactions, followed by reductive modification of the carbonyl carbon (Tillman et al., 1999). In Bombyx and Helicoverpa species, the precursor of sex pheromones is contained within lipid droplets (LDs) consisting of various triacylglycerols (TAGs). Before adult emergence, PG cells rapidly accumulate LDs. The accumulation of LDs parallels the production of sex pheromones following PBAN stimulation in Bombyx (Fónagy et al., 2001). In Helicoverpa, suppression of acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme for fat synthesis, significantly decreases the production of sex pheromone (Tsfadia et al., 2008). These results provide strong evidence that sex pheromones synthesis and release are linked to fatty acid synthesis pathways in the pheromone gland (Tsfadia et al., 2008). Triacylglycerols serve as a source for the de novo synthesis of bombykol. Diacylglycerol acyltransferase (DGAT) is an endoplasmic reticulum (ER) membrane protein that catalyzes the final step in TAG biosynthesis (Bell and Coleman, 1980). Overexpression of DGAT results in increased triglyceride synthesis (Liu et al., 2009). Our previous results revealed that a B. mori DGAT 2 (BmDGAT2) expressed at significant transcript levels in newly emerged females, indicating that this gene is likely involved in sex pheromone biosynthesis. In this study, the expression profile and function of BmDGAT2 in sex pheromone synthesis were studied.
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2. Materials and methods 2.1. Insect culture B. mori (Zhengzhu Chunlei) were kindly provided by the Sericultural Research Institute, Henan Academy of Agricultural Sciences and were reared on mulberry leaves at 26 °C under 14 h light/10 h dark cycles. Wandering stage larvae were collected for pupal age determination. Pupae were sexually separated, and adult males and females emerged in separate cages until tissue dissection. Pupal age was determined following the previous methods (Matsumoto et al., 2002).
2.2. Chemicals B. mori PBAN was synthesized by Songon Biotech (Shanghai) Co. Ltd. The main sex pheromone component, bombykol, was a gift from Shogo Matsumoto (RIKEN, Advanced Science Institute, Japan). Bombykol was used as the standard for pheromone quantification by gas chromatography–mass spectrometry (GC/MS).
2.3. Temporal and spatial expression profile of BmDGAT2 To analyze the temporal expression profile of BmDGAT2 transcripts at different developmental stages, female PGs were collected at 96 (96 h before eclosion), 72 (72 h before eclosion), 48 (48 h before eclosion), 24 (24 h before eclosion), 0 (newemergence), 24 (24 h after eclosion), 48 (48 h after eclosion), and 72 h (72 h after eclosion), and immediately frozen at 80 °C for later use. Different tissues, including pheromone glands, muscles, fat body, midgut, head, epidermis, and eggs were collected from the newly emerged females and immediately stored at 80 °C for later use. The PG and other tissue samples collected above were homogenized in Trizol reagent (Invitrogen, Gaithersburg, MD, USA) and used for total RNA extraction according to the manufacturer’s instruction. RNA concentration was determined by measuring the absorbance at 260 nm on a spectrophotometer. The first-strand cDNA was synthesized from 1 lg total RNA of each sample using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Kyoto, Japan). To measure the temporal expression profile of BmDGAT2, the synthesized cDNA was used as a template for analysis of BmDGAT2 transcripts by real-time PCR with gene specific primers (Forward 50 -CGCATCGCTATGAAGTCAC-30 ; Reverse 50 -AACAGTTGTAACGGG ACTC-30 ). The Bombyx ribosomal protein 49 (rp49) gene (Forward 50 -CAGGCGGTTCAAGGGTCAATAC-30 ; Reverse 50 -TGCTGGGCTCT TTCCACGA-30 ) was used as the internal control for normalization. Real-time PCR amplification and analysis was carried out on an Applied Biosystems 7500 Fast Real-Time PCR System (ABI, Carlsbad, CA, USA) using SYBR Green Supermix (TaKaRa). The thermal cycle conditions used in the real-time PCR were 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s and 60 °C for 1 min. The specificity of the SYBR green PCR signal was further confirmed by melting curve analysis and agarose gel electrophoresis. Comparative expression analysis was performed using the comparative CT method (Cross Threshold or CT is defined as the PCR cycle number where the signal crosses the signal threshold) (Livak and Schmittgen, 2001). The CT of the RP49 gene was subtracted from the CT of the target gene to obtain DCT. The normalized fold changes of the target gene mRNA were expressed as 2DDCT, where DDCT is equal to DCT treated sample – DCT control.
To examine the tissue distribution of BmDGAT2 transcripts, PCR was performed using synthesized cDNA and gene-specific primers. The thermal cycle conditions were 95 °C for 2 min, followed by 30 cycles of 95 °C for 15 s and 60 °C for 1 min. PCR products were further analyzed by agarose gel electrophoresis. The Rp49 gene also was used as an internal control. 2.4. Decapitation experiments The females were decapitated at different developmental stages from pupa to adult (96, 72, 48, 24, and 0 h) and maintained on a moist plate under identical photoperiod and temperature conditions. Pheromone glands were dissected at 24, 48, and 72 h after decapitation. The changes in BmDGAT2 transcript levels were analyzed using real-time PCR following the above methods. The pheromone glands from normal developmental females were used as control. 2.5. Treatment by methoprene in vitro Females at different developmental stages (72, 48, 24, and 0 h) were decapitated. Pheromone glands were dissected 24 h after decapitation and immediately placed in tissue plates containing 1 mL Grace’s insect culture medium (Invitrogen). After 1 h incubation, the medium was replaced with fresh Grace’s medium containing 2 lM methoprene. Pheromone glands were then harvested at designated time intervals (0, 2, 4, and 6 h) and stored at 80 °C for later real-time PCR analysis. Total RNA was extracted, the first strand was synthesized, and real-time PCR was performed according to the above methods. 2.6. Double-stranded RNA (dsRNA) synthesis Double stranded RNAs were synthesized using the MEGAscript RNAi kit (Ambion, Huntingdon, UK) according to the manufacturer’s instructions. PCR was performed using gene-specific primers containing T7 polymerase sites (Forward primer 50 -GATCACTAA TACGACTCACTATAGGGAGAATCACGGAGTGTGGGGATA-30 ; Reverse primer 50 -GATCACTAATACGACTCACTATAGGGAGACTACCATTATAG CCACACA-30 ). PCR was performed at 94 °C for 3 min, followed by 35 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min, and a final elongation at 72 °C for 10 min. PCR product was then treated with DNase and RNase to eliminate template DNA and single-stranded RNA. Double stranded RNA was purified using MEGAclearTM columns (Ambion, Huntingdon, UK) and eluted in diethyl pyrocarbonate (DEPC)-treated nuclease-free water. The dsRNA concentrations were measured using a biophotometer (Eppendorf, Hamburg, Germany). Our previous experiments confirmed that either dsGFP RNA or ddH2O are suitable negative controls. In the present study, DEPC-treated nuclease-free water was used as a negative control. The effects of RNAi on transcript expression were analyzed by RT-PCR. The primers used in this experiment were the same as those used for real-time PCR analysis. 2.7. Injection of dsRNA and in vivo bombykol analysis The 72 h female pupae were decapitated. After 24 h, 20 lg dsRNA of BmDAGT2 was injected into the decapitated females. Control females were injected with DEPC-treated nuclease-free water. The females were maintained for 48 h under normal conditions and then injected with either 5 pmol B. mori PBAN or again with DEPC-treated nuclease-free water. The PGs were dissected 90 min after injection and the pheromone component was extracted in hexane.
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3. Results 3.1. Temporal and spatial expression profile of BmDGAT2 To determine the temporal expression profile of BmDGAT2, realtime PCR was performed on PG tissue isolated at different developmental stages, from 96 h before eclosion to 72 h after eclosion. The newly emerged females were designated as 0 h females. The result reveals that BmDGAT2 transcript steadily increased with age (Fig. 1A), which indicating that expression pattern of BmDGAT2 transcript is age-dependent. To examine the tissue distribution of BmDGAT2 transcripts, PCR was performed on RNAs isolated from various adult tissues. The BmDGAT2 transcript was present in all tissues examined, but most richly expressed in PGs (Fig. 1B). Fig. 1. Temporal and spatial expression profile of BmDGAT2 in the PG of B. mori during pupal–adult development (the zero time point indicates the time of eclosion). (A) Temporal and spatial expression profile of BmDGAT2. PGs were collected at different developmental stages (96, 72, 48, 24, 0, 24, 48, and 72 h) and total RNA was extracted for real-time PCR analysis. The Rp49 gene was used as the housekeeping gene for normalization. The data are presented as mean ± SD of three biological replicates. (B) Tissue distributions of BmDGAT2. Different tissues, including PG, muscles (Ms), fat body (Fb), midgut (Mg), head (Hd), epidermis (Ep), egg (Eg) were collected from newly emerged females and total RNA was extracted for PCR analysis. The Rp49 gene was used as the housekeeping gene.
Bombykol accumulation was measured by GC/MS (Trace GC Ultra Trace DSQ; MS-Thermo Scientific DSQ II) equipped with a 30 m capillary column (RTX-5SILMS, Restek, 0.25 mm diameter). Each sample contained a pooled hexane extract from at least five B. mori pheromone glands. Each sample was then subjected to GC/MS analysis. 2.8. Statistical analysis The real-time PCR experiments were performed in triplicate, and expressed as mean ± SD. Real-time PCR results were compared using Student’s t-tests.
3.2. Decapitation experiments The expression levels of BmDGAT2 transcript in PGs from Bombyx females decapitated at various ages (96, 72, 48, 24, and 0 h) were measured by real-time PCR (Fig. 2). These BmDGAT2 transcripts still increased significantly with age despite head detachment and the expression temporal pattern in decapitated females was similar to that observed in normally developing females, although BmDGAT2 transcripts significant decrease compared to normally developing females.
3.3. Treatments of methoprene in vitro The expression pattern of BmDGAT2 transcript was further examined after treatment of decapitated females with methoprene, a JH analog. Females at different developmental stages were decapitated. After 24 h, PGs were collected from the decapitated females and treated with methoprene. The results show that BmDGAT2 transcript remained almost unchanged in 72, 48, and 24 h PGs despite methoprene treatment (Fig. 3), whereas methoprene significantly inhibited BmDGAT2 expression in 0 h PGs.
Fig. 2. The effect of head detachment after 24, 48, and 72 h on BmDGAT2 expression levels during pupal–adult development. Females at different developmental stages (A: 96 h; B: 72 h; C: 48 h; D: 24 h; E: 0 h) were decapitated. The PGs were collected at 24, 48, and 72 h after decapitation and total RNA was extracted for real-time PCR analysis. The Rp49 gene was used as the housekeeping gene for normalization. The blank bars present the normally developed female (control). The black bars represent the decapitated females. The data represent the mean ± SD of three biological replicates. ⁄⁄p < 0.01.
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Fig. 3. The effect of JH on BmDGAT2 expression levels during pupal–adult development. Females at different developmental stages (A: 72 h; B: 48 h; C: 24 h; D: 0 h) were decapitated. The PGs were collected 24 h after decapitation, treated with 2 lM methoprene, and harvested at different incubated time intervals (0, 2, 4, and 6 h) for real-time PCR analysis. The Rp49 gene was used as the housekeeping gene for normalization. The data represent the mean ± SD of three biological replicates. ⁄⁄⁄p < 0.01.
3.4. Injection of dsRNA and in vivo bombykol analysis To confirm the function of BmDGAT2 during the sex pheromone synthesis and release, RNAi-mediated knockdown was performed. First, 20 lg BmDGAT2 dsRNA was injected into 72 h decapitated females and PCR performed 48 h later. Expression of BmDGAT2 mRNA was significantly decreased compared to decapitated females injected with DEPC-treated nuclease-free water (Fig. 4A). To examine the effect of BmDGAT2 on bombykol production, PBAN-induced bombykol production was determined by GC/MS in control females and females treated with BmDGAT2-specific RNAi. Chromatographic analysis revealed a significant reduction
Fig. 4. Effects of RNAi treatment on bombykol production. (A) RNAi-induced reduction of BmDGAT2 transcript. RT-PCR was performed using cDNA generated from the total RNA extracted from PGs of females injected with vehicle or 20 lg dsRNAs for the BmDGAT2 gene. (B) Effects of RNAi for the BmDGAT2 gene on bombykol production. Females at 72 h before elcosion were decapitated and injected with double stranded RNAs. Decapitated females were injected with 5 pmol PBAN 48 h after dsRNA injection. Bombykol production was measured by GC/MS from PGs 90 min after injection of PBAN by GC/MS. Bars indicate mean values ± SD from independent experimental animals (n P 5). Statistically significant differences from the PBAN alone are denoted by ⁄⁄⁄(p < 0.01) as determined by the Student’s t-test.
in bombykol production after successful reduction of BmDGAT2 transcript by RNAi. Bombykol production was reduced to 25% of control in the knockdown females, suggesting that BmDGAT2 is required for bombykol production (Fig. 4B). 4. Discussion The successful propagation of Bombyx depends on highefficiency mating, which must be regulated by sex pheromones synthesized and released in adult stages. The key biosynthetic steps to generate specific sex pheromone in moths include de novo biosynthesis of fatty acid precursors, followed by various chemical modifications, including desaturation, reduction, acetylation, and oxidation (Tillman et al., 1999; Bjostad et al., 1987). In B. mori, lipid must be synthesized and accumulated in PGs in the form of TAGs before adult emergence to meet the substrate requirements for the large amounts of pheromones that must be synthesized by newly emerged females (Foster, 2000). Previous works indicated that TAGs are synthesized by two pathways, the monoacylglycerol (MAG) and glycerol phosphate pathways (Bell and Coleman, 1980; Coleman and Lee, 2004). The MAG pathway plays the predominant role in TAG synthesis in animal intestines. In contrast, the glycerol phosphate pathway is a de novo TAG synthesis pathway in most tissues. These two pathways share a common final step in which a fatty acyl-CoA and diacylglycerol (DG) molecule are covalently joined to form TAG. This final reaction is rate-limiting and is catalyzed by DGAT (Bell and Coleman, 1980; Coleman and Lee, 2004; Lehner and Kuksis, 1996). As the sex pheromone precursor material, a decrease in TAG production unavoidably causes a substantial decrease in sex pheromone production. Thus, DGAT should play a key role in sex pheromone production. At present, at least two DGAT family members, DGAT1 and DGAT2, have been identified in animals and plants (Hofmann, 2000). DGAT1 is a member of the membrane-bound O-acyltransferases with nine transmembrane domains. In contrast, DGAT2 belongs to a family that includes acyl-CoA:monoacylglycerol acyltransferase and acyl-CoA:wax alcohol acyltransferase, with only two transmembrane domains (Stone et al., 2004; Turkish et al., 2005). Although they share the same DGAT enzyme reaction, DGAT1 and DGAT2 bear little sequence resemblance. Amino acid sequence analysis indicated that DGAT1 is composed of 7 sequence blocks, whereas DGAT2s include 6 sequence blocks (Cao, 2011). In fact it has been proposed DGAT1 and DGAT2 have different physiological functions. Mice deficient DGAT1 was found to be viable and has modest decreases in TAG, compared to mice deficient DGAT2, which
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cause severe reduction of TAGs (Stone et al., 2004; Smith et al., 2000). In plants, two enzymes seem to be species and tissues dependent (Shockey et al., 2006). In addition, In Vernonia galamensis, both DGAT1 and DGAT2 were found to increase epoxy fatty acid accumulations, but DGAT2 had a greater effect (Li et al., 2010). Our previous digital gene expression (DGE) profile in PGs from different developmental stages identified a DGAT encoded by the BGIBMGA008049-TA transcript, which was richly expressed in newly emerged females, a key stage for sex pheromone synthesis and release. Further sequence analysis revealed that the amino acid sequence encoded by BGIBMGA008049-TA shared classic characteristics with other DGAT2 members and belonged to the DGAT2 family (data not shown here). Temporal expression profiling was consistent with DGE results and temporal expression patterns mirrored those of many other key sex pheromone synthesis genes, including acyl-CoA desaturase, fatty acyl reductase, PBAN receptor, fatty acid transport protein, and acyl-CoA binding protein (Rafaeli and Gileadi, 1995; Ohnishi et al., 2009; Matsumoto et al., 2001, 2010; Moto et al., 2003). Knockdown of BmDGAT2 by RNAi confirmed that a decrease in BmDGAT2 mRNA expression caused a significant reduction in sex pheromone production. Interestingly, our DGE result did not identify DGAT1 that richly expressed in newly emerged females. Whether DGAT1 also present in Bombyx PGs and if present, then what is the function of DGAT1 in sex pheromone synthesis and release require further investigation. Methoprene challenge experiment suggested Methoprene, a JH analog, significantly inhibited the expression of BmDGAT2 transcript in newly emerged females (mature adults), similar to results from Helicoverpa arimgera showing that fenoxycarb upregulated pheromone production and PBANR in pharate adults, but downregulated both in mature adults (Rafaeli et al., 2003; Bober et al., 2010). This developmentally regulated effect of JH may be due to the increase in JH concentration in the haemolymph of mature Bombyx females caused by constitutive production, synthesis induced by seminal peptides produced by the male accessory gland, or direct transfer of JH produced in the male accessory gland (Park et al., 1998). An overabundance of JH in mature adult females inhibits BmDGAT2 expression. Temporal expression profiles also confirmed that BmDGAT2 transcript levels started to decrease after 0 h, consistent with the methoprene treatment experiment. These results appear at odds with the 0 h decapitation experiment, however, in which BmDGAT2 transcript declined compared to normally developed females (Fig. 2). The loss of hemolymph after decapitation or direct harm induced by decapitation may activate other mechanisms that inhibit BmDGAT2 expression. Although many characteristics of BmDGAT2 regulation and expression were identified in the present study, these uncertainties deserve further study on the regulation of BmDGAT2 transcripts and the function of the encoded protein. Acknowledgements This study was supported by a Distinguished Professor Foundation of Henan Agricultural University (No: 30600022) and Natural Science Foundation of Henan Province (No: 2011B180021). References Bell, R.M., Coleman, R.A., 1980. Enzymes of glycerolipid synthesis in eukaryotes. Annual Review of Biochemistry 49, 459–487. Bjostad, L.B., Wolf, W.A., Roelofs, W.L., 1987. Pheromone biosynthesis in lepidopterans: desaturation and chain shortening. In: Prestwich, G.D., Blomquist, G.J. (Eds.), Pheromone Biochemistry. Academic Press, New York, pp. 77–120. Bober, R., Azrielli, A., Rafaeli, A., 2010. Developmental regulation of the pheromone biosynthesis activating neuropeptide-receptor (PBAN-R): re-evaluating the role of juvenile hormone. Insect Molecular Biology 19, 77–86. Cao, H., 2011. Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms. BMC Research Notes 4249.
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