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cDNA cloning and in situ hybridization of Δ11-desaturase, a key enzyme of pheromone biosynthesis in Ostrinia scapulalis (Lepidoptera: Crambidae)
ARTICLE IN PRESS
Journal of Insect Physiology 52 (2006) 430–435 www.elsevier.com/locate/jinsphys
cDNA cloning and in situ hybridization of D11-desaturase, a key enzyme of pheromone biosynthesis in Ostrinia scapulalis (Lepidoptera: Crambidae) Mai Fukuzawa, Xiaoyan Fu, Sadahiro Tatsuki, Yukio Ishikawa a
Laboratory of Applied Entomology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan Received 26 August 2005; received in revised form 8 November 2005; accepted 10 November 2005
Abstract Female sex pheromones are considered to be produced in a ‘‘pheromone gland’’ located in the terminal abdominal segments (8th–10th, TAS) of a moth; however, in many moth species, the cells that produce pheromones have not actually been specified. We investigated cells in the TAS that synthesize pheromones in the adzuki bean borer Ostrinia scapulalis, by locating pheromones and their precursors, and mRNA for D11-desaturase, a key enzyme in pheromone biosynthesis. We demonstrated that the pheromone components, (E)-11and (Z)-11-tetradecenyl acetates, and their fatty acyl precursors were specifically contained in the dorsal part of the TAS. A cDNA (OscaZ/E11) that encodes a D11-desaturase was cloned from the TAS. RT-PCR and in situ hybridization unequivocally showed that OscaZ/E11 is specifically expressed in the modified epidermal cells located at the dorsal end of the 8th–9th intersegmental membrane. r 2005 Elsevier Ltd. All rights reserved. Keywords: In situ hybridization; D11-desaturase; Pheromone biosynthesis; Ostrinia; Fatty acid pheromone analog
1. Introduction Many moth species use sex pheromones derived from fatty acids to attract mates. The sex pheromones usually have a straight chain frame with 10–18 carbons and one to three double bonds, and an oxygenated functional group such as an alcohol, acetate ester or aldehyde (Tamaki, 1985). Sex pheromones are considered to be produced in a specialized tissue, the ‘‘pheromone gland (PG)’’. Morphological investigations suggest that the PG in many lepidopteran species is a modified intersegmental membrane between the 8th and 9th segments (Percy-Cunningham and MacDonald, 1978). Due to difficulty in isolating PG, however, pheromones have been conventionally analyzed using isolated terminal abdominal segments (TAS, 8th to 10th segments). The presumed PG cells are generally columnar in shape and contain many lipid spheres within the cytoplasm. The abundant smooth endoplasmic reticula present in these Corresponding author. Tel./fax: +81 3 5841 5061.
E-mail address: [email protected] (Y. Ishikawa). 0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.11.004
cells indicate that PG cells synthesize lipid (Percy-Cunningham and MacDonald, 1978). Recently, ultrastructural features of the PG in several species were studied in detail. Females of Helicoverpa zea have an almost ring-shaped PG, which occupies most of the 8th–9th intersegmental membrane (Raina et al., 2000). The PG cells in this species have distinct features such as microvilli, pockets of granular material, and intercellular canals with abundant desmosomes (Raina et al., 2000). In contrast, the European corn borer Ostrinia nubilalis has PG cells only in the dorsal part of the 8th–9th intersegmental membrane (Ma and Roelofs, 2002). Consistent with the distribution of PG cells, the sex pheromone components, (E)- and (Z)-11tetradecenyl acetate (E11-14:OAc and Z11-14:OAc), and their fatty acid precursors, (E)- and (Z)-11-tetradecenoate, were found only in the dorsal portion (Ma and Roelofs, 2002). Along with the morphology of the PG, biosynthetic pathways of the sex pheromones derived from saturated fatty acids have been studied for more than two decades. These studies have shown the importance of acyl-CoA desaturases, D11-desaturase in particular, in lepidopteran
ARTICLE IN PRESS M. Fukuzawa et al. / Journal of Insect Physiology 52 (2006) 430–435
species (see Tillman et al., 1999 for a review). To date, cloning of D11-desaturase cDNA and a functional assay have been conducted in Trichoplusia ni (Knipple et al., 1998), H. zea (Rosenfield et al., 2001), Ostrinia nubilalis and O. furnacalis (Roelofs et al., 2002), Epiphyas postvittana (Liu et al., 2002a), Argyrotaenia velutinana (Liu et al., 2002b), Choristoneura rosaceana (Hao et al., 2002), C. parallela (Liu et al., 2004), Spodoptera littoralis (Rodrı´ guez et al., 2004) and Bombyx mori (Moto et al., 2004; B. mori Desat1 shows both D11- and D10,12desaturation activities). As described above, identification of the PG has been mostly based on morphology. Here, we used in situ hybridization to locate the site of desaturase gene expression, and visualize cells that synthesize pheromones in the adzuki bean borer O. scapulalis, a species phylogenetically very close to the European corn borer O. nubilalis (Kim et al., 1999). We have long been studying the evolution of sex pheromone communication systems in eight Ostrinia species in Japan with particular emphasis on Ostrinia scapulalis (Ishikawa et al., 1999), and hence have gathered much information on the genetics and biosynthesis of the sex pheromone in O. scapulalis (a blend of E11-14:OAc and Z11-14:OAc)(Huang et al., 1997, Huang et al., 2002; Takanashi et al., 2005). We cloned a cDNA of the D11desaturase gene from the TAS of O. scapulalis, and conducted in situ hybridization experiments with a probe prepared using this cDNA. 2. Materials and Methods 2.1. Insects Cultures of O. scapulalis that are monomorphic in terms of the production of E-type pheromone (499% E1114:OAc) were established from females collected at Matsudo, as described in Takanashi et al. (2005). Pupae were sexed by the morphology of the TAS, and separately maintained in 420 ml plastic cups. Newly emerged adults were collected every day and housed in new cups. 2.2. Chemical analysis The TAS of a 3-day-old female at mid-photophase were extruded by applying gentle pressure to the abdomen, and cut with microscissors. The TAS were then cut at the level of the lateral apophyses into dorsal and ventral parts under a binocular microscope. For pheromone extraction, these parts were separately immersed in 10 ml of hexane containing tridecyl acetate (an internal standard) for 30 min. For fatty acyl analysis, the respective parts were immersed overnight in 50 ml of a 2:1 mixture (v/v) of dichloromethane:methanol containing 175 ng of tritridecanoin (Sigma) as an internal standard. Prior to GC analysis, the lipid extract was subjected to base methanolysis according to the method of Foster (2001).
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A gas chromatograph (GC-17A, Shimadzu, Kyoto) fitted with a flame ionization detector and a fused silica capillary column (DB-Wax, 30 m 0.25 mm ID, J & W Scientific, USA) was used for analysis. The oven temperature was programmed to increase at 5 1C/min to 150 1C, then 3 1C/min to 200 1C and finally 5 1C/min to 240 1C. The authentic chemicals, tetradecanol, (Z)-11-tetradecenol, (E)11-tetradecenol, (Z)-11-tetradecenyl acetate and (E)-11tetradecenyl acetate, were obtained from Pherobank (Wageningen, The Netherlands). The alcohols were converted to corresponding acid methyl esters in our laboratory. 2.3. cDNA cloning and rapid amplification of cDNA ends (RACE) Total RNA was extracted from the TAS of 3-day-old O. scapulalis females using an RNeasy mini kit and RNasefree DNase (Qiagen). For 30 -RACE, the first-strand cDNA was synthesized using an RNA PCR kit (AMV) ver.2.1 with an oligo-dT adaptor primer that includes the M13 primer M4 sequence (Takara-bio Inc., Otsu). A forward primer desL1 (50 -ATCTGCGCTTTATGGGCTGT-30 ) specific for D11-desaturase was designed based on the cDNA sequences of D11-, D14- and D9-desaturase gene transcripts in the PGs of O. nubilalis (OnuZ/E11, GenBank accession no. AF441221; OnuZ/E14, AF441220;OnuD9, AF430246) and O. furnacalis (OfuZ/E11, AF441861; OfuZ/E14, AY062023; OfuD9, AY057863). PCR was performed using desL1 and the M13-M4 primer (Takarabio) and hot start Ex Taq polymerase (Takara-bio) under the following conditions: 95 1C for 15 min, followed by 35 cycles of 94 1C for 1 min, 55 1C for 1 min and 72 1C for 1 min, and a final extension at 72 1C for 10 min. Another first-strand cDNA was synthesized for 50 RACE using desR1 (50 -CAGAGTCGTGGGGTTCATTT-30 ) as the reverse primer specific for D11-desaturase gene. A poly A tail was attached to the 50 -end of the synthesized cDNA using a terminal deoxynucleotidyl transferase (Promega) and dATP (Amersham Bioscience). The first PCR was performed with the primer desR1 and an oligo dT-anchor primer (50 -GACCACGCGTATCGATGTCGAC(T)16V-30 ). Subsequently, the second PCR was performed using the resultant PCR product with the PCR anchor primer (50 -GACCACGCGTATCGATGTCGAC-30 ) and desR2 (50 -ATGGTGCACACGATGGTCT C-30 ). After separation of the PCR products on a 2% gel, cDNAs of 1 kbp were recovered from the gel and cloned using the pGEMs-T Easy Vector System (Promega). The resultant clones were sequenced. 2.4. Reverse transcription-polymerase chain reaction (RTPCR) Total RNA was extracted from the head, thorax, abdomen exclusive of the TAS, and TAS of an O. scapulalis female using an RNeasy mini kit and RNase-free DNase
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(Qiagen). First-strand cDNA was synthesized with the oligo-dT adaptor primer that includes the M13-M4 sequence (Takara-bio). PCR was performed using Ex Taq polymerase (Takara-bio) and the primers desL1 and desR2. Another pair of primers (30 -AACTGGGATGACATGGAGAAGATCTGGCA-50 and 30 -GAGATCCACATCTGCTGGAAGGTGGACAG-50 ) was used to amplify b-actin (control). The conditions for the PCR were 95 1C for 15 min, followed by 30 cycles of 94 1C for 1 min, 55 1C for 1 min and 72 1C for 1 min, and a final extension at 72 1C for 10 min. Amplified products were checked on a 2% agarose gel. 2.5. In situ hybridization The TAS of a female were extruded with gentle pressure to the abdomen, cut with microscissors, and immediately immersed into a fixative containing formaldehyde (STF-01, Genostaff Inc., Tokyo, Japan). Tissues were embedded in paraffin by the conventional method, and 6 mm sections were prepared with a microtome. Part of the OscZ/E11 sequence was amplified by PCR with desL1 and M13-M4 primers, and cloned using the pGEMs-T Easy Vector System (Promega). Digoxigeninlabeled sense and antisense RNA probes were prepared in vitro using the SP6 and T7 RNA polymerases supplied with a DIG RNA Labeling Kit (Roche Applied Science) according to the manufacturer’s instructions. The probe contained part of ORF (849 bases) and the 30 -untranslated region (181 bases). Sections were treated as described by Hoshino et al. (1999). Anti-digoxigenin antibody labeled with alkaline phosphatase and NBT/BCIP were used to visualize the signal. Counterstaining was performed using a Kernechtrot stain solution (Muto Chemical, Japan). 3. Results Pheromones (E11- and Z11-14:OAc) and fatty acid pheromone precursors (E11- and Z11-tetradecenoate) were found only in the dorsal portion of the TAS (Fig. 1a, b). On the other hand, tetradecanoate, a substrate of D11desaturase, was found in both dorsal and ventral portions of the TAS (14:Me in Fig. 1a, b). These results suggest that D11-desaturase is present only in the dorsal portion of the TAS. A 1217-base cDNA (OscZ/E11, AB232855) that included a complete open reading frame encoding 329 amino acid residues was cloned from the TAS of O. scapulalis females (Fig. 2). The deduced amino acid sequence (Fig. 2) has three histidine boxes which are highly conserved within acyl-CoA desaturases in animals and yeast (Los and Murata, 1998), and a CPTQ signature motif typical of D11-desaturase (Knipple et al., 2002). OscZ/E11 showed extremely high homology (amino acid identity499%) to the D11-desaturase genes of O. nubilalis (OnuZ/E11) and O. furnacalis (OfuZ/E11), and relatively low homology to insects from different genera, such as S. littoralis (67%,
Fig. 1. Gas chromatograms of pheromone components (a, b) and methyl esters of fatty acid pheromone precursors (c, d) extracted from the dorsal and ventral parts of the isolated terminal abdominal segments (TAS). The TAS were cut into dorsal and ventral parts at the level of lateral apophyses. IS: internal standards, tridecyl acetate (a, b) and tridecanoic acid methyl ester (c, d).
AY362879), A. velutinana (63%, AF416738), H. zea (65%, AF272342) and T. ni (64%, AF035375). RT-PCR was performed to confirm that the OscZ/E11 gene is not expressed in body parts other than the TAS. Analysis of the head, thorax, abdomen exclusive of the TAS, and TAS of an adult female showed that mRNA of OscZ/E11 is expressed only in the TAS (Fig. 3). In situ hybridization on thin sections of TAS was performed to visualize the cells in which the OscZ/E11 gene is expressed. The signal from an antisense probe was found only in the large, columnar cells present in the dorsal
ARTICLE IN PRESS M. Fukuzawa et al. / Journal of Insect Physiology 52 (2006) 430–435
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Fig. 2. Translated amino acid sequence of OscZ/E11 (O. scapulalis) aligned with those of OnuZ/E11 (O. furnacalis) and OfuZ/E11 (O. nubilalis). The regions underlined indicate histidine boxes, a unique structure found in animal acyl-CoA desaturases (Los and Murata, 1998). The four amino acids enclosed by a square is the ‘signature motif’, XXXQ, which is typically found in lepidopteran D11 acyl-CoA desaturases (Knipple et al., 2002).
Fig. 3. Expression of OscZ/E11 in a female adult of O. scapulalis. The body of a female was divided into the head (H), thorax (T), terminal abdominal segments (8–10th, TAS), and abdomen exclusive of TAS (A–TAS). Expression of b-actin is indicated as a positive control.
region (Fig. 4a, b). No specific signal was found when a control sense probe was used (Fig. 4c). 4. Discussion When the deduced amino acid sequences of OscZ/E11 (O. scapulalis), OnuZ/E11 (O. nubilalis) and OfuZ/E11 (O. furnacalis) are compared, OnuZ/E11 and OfuZ/E11 are identical, and OscZ/E11 differs from the others only at three positions (Fig. 2). Considering that OnuZ/E11 ( ¼ OfuZ/E11) is shown to exhibit D11-desaturase activity in a functional assay (Roelofs et al., 2002), and that the three variable positions are not in the regions where any functional or structural importance is attributed, it is rational to conclude that the OscZ/E11 gene encodes a functional D11-desaturase. Although transcripts of both D11- and D14-desaturase genes have been found in the PGs of O. nubilalis and O. furnacalis, only one or the other of these is functionally
expressed in a single species (Roelofs et al., 2002). For instance, D11-desaturase is not functionally expressed in O. furnacalis although the gene is transcribed into mRNA. It is curious that OfuZ/E11 and OnuZ/E11 are identical, considering that OfuZ/E11 is not functionally expressed, and that O. furnacalis is more distantly related to O. nubilalis than O. scapulalis (Kim et al. 1999). Further studies are needed to clarify the evolution of D11- and D14desaturases in the genus Ostrinia. The present findings indicate that in O. scapulalis, desaturation of pheromone precursors occurs specifically in the modified cells located at the dorsal end of the 8th–9th intersegmental membrane. In morphology and location, the cells with positive signals matched the PG cells in O. nubilalis, which were determined based on morphological and biochemical lines of evidence (Ma and Roelofs, 2002). In B. mori, Fo´nagy et al. (2000) have proven that a layer of cells isolated by papain treatment of the 8th–9th intersegmental membrane are the pheromone-producing cells, which, in tissue culture, retain the ability to produce bombykol in response to the pheromonotropic peptide TKYFSPRLamide, ionomycin and calcium ionophore A23187. In no other species are the functions of pheromone-producing cells demonstrated as elegantly as in this study. Our present study on Ostrinia, although involving a different approach, has also succeeded in identifying the pheromone-producing cells. The control of pheromone biosynthesis in Ostrinia is not well understood. Although pheromone-biosynthesisactivating neuropeptide (PBAN) released from the
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Fig. 4. Expression of OscZ/E11 in sections of the 8th–9th intersegmental membrane. (a) In situ hybridization with the antisense RNA probe. (b) Magnification of the area indicated by the square in (a). Pheromone-producing cells are found as typical column-shaped epidermal cells located in the dorsal part. (c) Hybridization with the sense probe (negative control). Hg, hind gut; Ov, oviduct; PG, pheromone-producing cells. Parts of the 8th segment (indicated by arrows) appear because the 8th–9th intersegmental membrane was not fully extruded from the 8th segment.
suboesophageal ganglion is believed to be involved in this control, our preliminary studies showed that PBAN does not affect the mRNA level of D11-desaturase (Fukuzawa, unpublished). Rather, mRNA for D11-desaturase is always expressed regardless of pheromone production. The present findings on the sequence of D11-desaturase and its localization would facilitate studies on the control of pheromone biosynthesis in O. scapulalis and related species. Acknowledgments We thank Dr. Chen Bin and Dr. Nami Miura for useful discussions during the course of the present study. This work was supported in part by Grant-in-Aid for Scientific Research No.16208005 from the Japan Society for the Promotion of Science (JSPS). References Fo´nagy, A., Yokoyama, N., Okano, K., Tatsuki, S., Maeda, S., Matsumoto, S., 2000. Pheromone-producing cells in the silkworm moth, Bombyx mori: identification and their morphological changes in response to pheromonotropic stimuli. Journal of Insect Physiology 46, 735–744. Foster, S.P., 2001. Fatty acyl pheromone analog-containing lipids and their roles in sex pheromone biosynthesis in the lightbrown apple moth, Epiphyas postvittana (Walker). Journal of Insect Physiology 47, 433–443.
Hao, G., O’Connor, M., Liu, W., Roelofs, W.L., 2002. Characterization of Z/E11- and Z9-desaturases from the obliquebanded leafroller moth, Choristoneura rosaceana. Journal of Insect Science 2, 26. Hoshino, M., Sone, M., Fukata, M., Kurodad, S., Kaibuchi, K., Nabeshima, Y., Hama, C., 1999. Identification of the stef gene that encodes a novel guanine nucleotide exchange factor specific for Rac1. Journal of Biological Chemistry 274, 17837–17844. Huang, Y., Tatsuki, S., Kim, C., Hoshizaki, S., Yoshiyasu, Y., Honda, H., Ishikawa, Y., 1997. Identification of sex pheromone of adzuki bean borer, Ostrinia scapulalis. Journal of Chemical Ecology 23, 2791–2802. Huang, Y., Takanashi, T., Hoshizaki, S., Tatsuki, S., Ishikawa, Y., 2002. Female sex pheromone polymorphism in adzuki bean borer, Ostrinia scapulalis, is similar to that in European corn borer, O. nubilalis. Journal of Chemical Ecology 28, 533–539. Ishikawa, Y., Takahashi, T., Kim, C., Hoshizaki, S., Tatsuki, S., Huang, H., 1999. Ostrinia spp. in Japan: their host plants and sex pheromones. Entomologia Experimentalis et Applicata 91, 237–244. Kim, C., Hoshizaki, S., Huang, Y., Tatsuki, S., Ishikawa, Y., 1999. Usefulness of mitochondrial COII gene sequences in examining phylogenetic relationships in the Asian corn borer, Ostrinia furnacalis, and allied species (Lepidoptera: Pyralidae). Applied Entomology and Zoology 34, 405–412. Knipple, D.C., Rosenfield, C., Miller, S., Liu, W., Tang, J., Ma, P.W.K., 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. Proceedings of the National Academy of Sciences of the USA 95, 15287–15292. Knipple, D.C., Rosenfield, C., Nielsen, R., You, K.M., Jeong, S.E., 2002. Evolution of the internal membrane desaturae gene family in moths and flies. Genetics 162, 1737–1752.
ARTICLE IN PRESS M. Fukuzawa et al. / Journal of Insect Physiology 52 (2006) 430–435 Liu, W., Jiao, H., Murray, C., O’connor, M., Roelofs, W.L., 2002a. Gene characterized for membrane desaturase that produces (E)-11 isomers of mono- and diunsaturated fatty acids. Proceedings of the National Academy of Sciences of the USA 99, 620–624. Liu, W., Jiao, H., O’Connor, M., Roelofs, W.L., 2002b. Moth desaturase characterized that produces both Z and E isomers of D11-tetradecenoic acids. Insect Biochemistry and Molecular Biology 32, 1489–1495. Liu, W., Roony, A.P., Xue, B.Y., Roelofs, W.L., 2004. Desaturases from the spotted fireworm moth (Choristneura parallela) shed light on the evolutionary origins of novel moth sex pheromone desaturases. Gene 342, 303–311. Los, D.A., Murata, N., 1998. Structure and expression of fatty acid desaturases. Biochemica et Biophysica Acta-Lipids and Lipid Metabolism 1394, 3–15. Ma, P.W.K., Roelofs, W.L., 2002. Sex pheromone gland of the female European corn borer moth, Ostrinia nubilalis (Lepidoptera: Pyralidae): ultrastrutural and biochemical evidences. Zoological Science 19, 501–511. 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. Proceedings of the National Academy of Sciences of the USA 101, 8631–8636. Percy-Cunningham, J.E., MacDonald, J.A., 1978. Biology and ultrastructure of sex pheromone-producing glands. In: Blomquist, G.J., Prestwich, G.D. (Eds.), Pheromone Biochemistry. Academic Press, Orland, Florida, pp. 27–75.
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Raina, A.K., Wergin, W.P., Murphy, C.A., Erbe, E.F., 2000. Structural organization of the sex pheromone gland in Helicoverpa zea in relation to pheromone production and release. Arthropod Structure and Development 29, 343–353. Rodrı´ guez, S., Hao, G.X., Liu, W., Pinˇa, B., Roony, A.P., Camps, F., Roelofs, W.L., Fabria`s, G., 2004. Expression and evolution of D9 and D11 desaturase genes in the moth Spodoptera littoralis. Insect Biochemistry and Molecular Biology 34, 1315–1328. Roelofs, W.L., Liu, W., Hao, G., Jiao, H., Rooney, A.P., Linn Jr., C.E., 2002. Evolution of moth sex pheromones via ancestral genes. Proceedings of the National Academy of Sciences of the USA 99, 13621–13626. 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 Biochemistry and Molecular Biology 31, 949–964. Takanashi, T., Huang, Y., Takahasi, R., Hoshizaki, S., Tatsuki, S., Ishikawa, Y., 2005. Genetic analysis and population survey of sex pheromone variation in the adzuki bean borer moth, Ostrinia scapulalis. Biological Journal of Linnean Society 84, 143–160. Tamaki, Y., 1985. Sex pheromones. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 9. Pergamon Press, Oxford, pp. 145–191. Tillman, J.A., Seybold, S.J., Jurenka, R.A., Blomquist, G.J., 1999. Insect pheromones—an overview of biosynthesis and endocrine regulation. Insect Biochemstry and Molecular Biology 29, 481–514.
Journal of Insect Physiology 52 (2006) 430–435 www.elsevier.com/locate/jinsphys
cDNA cloning and in situ hybridization of D11-desaturase, a key enzyme of pheromone biosynthesis in Ostrinia scapulalis (Lepidoptera: Crambidae) Mai Fukuzawa, Xiaoyan Fu, Sadahiro Tatsuki, Yukio Ishikawa a
Laboratory of Applied Entomology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan Received 26 August 2005; received in revised form 8 November 2005; accepted 10 November 2005
Abstract Female sex pheromones are considered to be produced in a ‘‘pheromone gland’’ located in the terminal abdominal segments (8th–10th, TAS) of a moth; however, in many moth species, the cells that produce pheromones have not actually been specified. We investigated cells in the TAS that synthesize pheromones in the adzuki bean borer Ostrinia scapulalis, by locating pheromones and their precursors, and mRNA for D11-desaturase, a key enzyme in pheromone biosynthesis. We demonstrated that the pheromone components, (E)-11and (Z)-11-tetradecenyl acetates, and their fatty acyl precursors were specifically contained in the dorsal part of the TAS. A cDNA (OscaZ/E11) that encodes a D11-desaturase was cloned from the TAS. RT-PCR and in situ hybridization unequivocally showed that OscaZ/E11 is specifically expressed in the modified epidermal cells located at the dorsal end of the 8th–9th intersegmental membrane. r 2005 Elsevier Ltd. All rights reserved. Keywords: In situ hybridization; D11-desaturase; Pheromone biosynthesis; Ostrinia; Fatty acid pheromone analog
1. Introduction Many moth species use sex pheromones derived from fatty acids to attract mates. The sex pheromones usually have a straight chain frame with 10–18 carbons and one to three double bonds, and an oxygenated functional group such as an alcohol, acetate ester or aldehyde (Tamaki, 1985). Sex pheromones are considered to be produced in a specialized tissue, the ‘‘pheromone gland (PG)’’. Morphological investigations suggest that the PG in many lepidopteran species is a modified intersegmental membrane between the 8th and 9th segments (Percy-Cunningham and MacDonald, 1978). Due to difficulty in isolating PG, however, pheromones have been conventionally analyzed using isolated terminal abdominal segments (TAS, 8th to 10th segments). The presumed PG cells are generally columnar in shape and contain many lipid spheres within the cytoplasm. The abundant smooth endoplasmic reticula present in these Corresponding author. Tel./fax: +81 3 5841 5061.
E-mail address: [email protected] (Y. Ishikawa). 0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.11.004
cells indicate that PG cells synthesize lipid (Percy-Cunningham and MacDonald, 1978). Recently, ultrastructural features of the PG in several species were studied in detail. Females of Helicoverpa zea have an almost ring-shaped PG, which occupies most of the 8th–9th intersegmental membrane (Raina et al., 2000). The PG cells in this species have distinct features such as microvilli, pockets of granular material, and intercellular canals with abundant desmosomes (Raina et al., 2000). In contrast, the European corn borer Ostrinia nubilalis has PG cells only in the dorsal part of the 8th–9th intersegmental membrane (Ma and Roelofs, 2002). Consistent with the distribution of PG cells, the sex pheromone components, (E)- and (Z)-11tetradecenyl acetate (E11-14:OAc and Z11-14:OAc), and their fatty acid precursors, (E)- and (Z)-11-tetradecenoate, were found only in the dorsal portion (Ma and Roelofs, 2002). Along with the morphology of the PG, biosynthetic pathways of the sex pheromones derived from saturated fatty acids have been studied for more than two decades. These studies have shown the importance of acyl-CoA desaturases, D11-desaturase in particular, in lepidopteran
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species (see Tillman et al., 1999 for a review). To date, cloning of D11-desaturase cDNA and a functional assay have been conducted in Trichoplusia ni (Knipple et al., 1998), H. zea (Rosenfield et al., 2001), Ostrinia nubilalis and O. furnacalis (Roelofs et al., 2002), Epiphyas postvittana (Liu et al., 2002a), Argyrotaenia velutinana (Liu et al., 2002b), Choristoneura rosaceana (Hao et al., 2002), C. parallela (Liu et al., 2004), Spodoptera littoralis (Rodrı´ guez et al., 2004) and Bombyx mori (Moto et al., 2004; B. mori Desat1 shows both D11- and D10,12desaturation activities). As described above, identification of the PG has been mostly based on morphology. Here, we used in situ hybridization to locate the site of desaturase gene expression, and visualize cells that synthesize pheromones in the adzuki bean borer O. scapulalis, a species phylogenetically very close to the European corn borer O. nubilalis (Kim et al., 1999). We have long been studying the evolution of sex pheromone communication systems in eight Ostrinia species in Japan with particular emphasis on Ostrinia scapulalis (Ishikawa et al., 1999), and hence have gathered much information on the genetics and biosynthesis of the sex pheromone in O. scapulalis (a blend of E11-14:OAc and Z11-14:OAc)(Huang et al., 1997, Huang et al., 2002; Takanashi et al., 2005). We cloned a cDNA of the D11desaturase gene from the TAS of O. scapulalis, and conducted in situ hybridization experiments with a probe prepared using this cDNA. 2. Materials and Methods 2.1. Insects Cultures of O. scapulalis that are monomorphic in terms of the production of E-type pheromone (499% E1114:OAc) were established from females collected at Matsudo, as described in Takanashi et al. (2005). Pupae were sexed by the morphology of the TAS, and separately maintained in 420 ml plastic cups. Newly emerged adults were collected every day and housed in new cups. 2.2. Chemical analysis The TAS of a 3-day-old female at mid-photophase were extruded by applying gentle pressure to the abdomen, and cut with microscissors. The TAS were then cut at the level of the lateral apophyses into dorsal and ventral parts under a binocular microscope. For pheromone extraction, these parts were separately immersed in 10 ml of hexane containing tridecyl acetate (an internal standard) for 30 min. For fatty acyl analysis, the respective parts were immersed overnight in 50 ml of a 2:1 mixture (v/v) of dichloromethane:methanol containing 175 ng of tritridecanoin (Sigma) as an internal standard. Prior to GC analysis, the lipid extract was subjected to base methanolysis according to the method of Foster (2001).
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A gas chromatograph (GC-17A, Shimadzu, Kyoto) fitted with a flame ionization detector and a fused silica capillary column (DB-Wax, 30 m 0.25 mm ID, J & W Scientific, USA) was used for analysis. The oven temperature was programmed to increase at 5 1C/min to 150 1C, then 3 1C/min to 200 1C and finally 5 1C/min to 240 1C. The authentic chemicals, tetradecanol, (Z)-11-tetradecenol, (E)11-tetradecenol, (Z)-11-tetradecenyl acetate and (E)-11tetradecenyl acetate, were obtained from Pherobank (Wageningen, The Netherlands). The alcohols were converted to corresponding acid methyl esters in our laboratory. 2.3. cDNA cloning and rapid amplification of cDNA ends (RACE) Total RNA was extracted from the TAS of 3-day-old O. scapulalis females using an RNeasy mini kit and RNasefree DNase (Qiagen). For 30 -RACE, the first-strand cDNA was synthesized using an RNA PCR kit (AMV) ver.2.1 with an oligo-dT adaptor primer that includes the M13 primer M4 sequence (Takara-bio Inc., Otsu). A forward primer desL1 (50 -ATCTGCGCTTTATGGGCTGT-30 ) specific for D11-desaturase was designed based on the cDNA sequences of D11-, D14- and D9-desaturase gene transcripts in the PGs of O. nubilalis (OnuZ/E11, GenBank accession no. AF441221; OnuZ/E14, AF441220;OnuD9, AF430246) and O. furnacalis (OfuZ/E11, AF441861; OfuZ/E14, AY062023; OfuD9, AY057863). PCR was performed using desL1 and the M13-M4 primer (Takarabio) and hot start Ex Taq polymerase (Takara-bio) under the following conditions: 95 1C for 15 min, followed by 35 cycles of 94 1C for 1 min, 55 1C for 1 min and 72 1C for 1 min, and a final extension at 72 1C for 10 min. Another first-strand cDNA was synthesized for 50 RACE using desR1 (50 -CAGAGTCGTGGGGTTCATTT-30 ) as the reverse primer specific for D11-desaturase gene. A poly A tail was attached to the 50 -end of the synthesized cDNA using a terminal deoxynucleotidyl transferase (Promega) and dATP (Amersham Bioscience). The first PCR was performed with the primer desR1 and an oligo dT-anchor primer (50 -GACCACGCGTATCGATGTCGAC(T)16V-30 ). Subsequently, the second PCR was performed using the resultant PCR product with the PCR anchor primer (50 -GACCACGCGTATCGATGTCGAC-30 ) and desR2 (50 -ATGGTGCACACGATGGTCT C-30 ). After separation of the PCR products on a 2% gel, cDNAs of 1 kbp were recovered from the gel and cloned using the pGEMs-T Easy Vector System (Promega). The resultant clones were sequenced. 2.4. Reverse transcription-polymerase chain reaction (RTPCR) Total RNA was extracted from the head, thorax, abdomen exclusive of the TAS, and TAS of an O. scapulalis female using an RNeasy mini kit and RNase-free DNase
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(Qiagen). First-strand cDNA was synthesized with the oligo-dT adaptor primer that includes the M13-M4 sequence (Takara-bio). PCR was performed using Ex Taq polymerase (Takara-bio) and the primers desL1 and desR2. Another pair of primers (30 -AACTGGGATGACATGGAGAAGATCTGGCA-50 and 30 -GAGATCCACATCTGCTGGAAGGTGGACAG-50 ) was used to amplify b-actin (control). The conditions for the PCR were 95 1C for 15 min, followed by 30 cycles of 94 1C for 1 min, 55 1C for 1 min and 72 1C for 1 min, and a final extension at 72 1C for 10 min. Amplified products were checked on a 2% agarose gel. 2.5. In situ hybridization The TAS of a female were extruded with gentle pressure to the abdomen, cut with microscissors, and immediately immersed into a fixative containing formaldehyde (STF-01, Genostaff Inc., Tokyo, Japan). Tissues were embedded in paraffin by the conventional method, and 6 mm sections were prepared with a microtome. Part of the OscZ/E11 sequence was amplified by PCR with desL1 and M13-M4 primers, and cloned using the pGEMs-T Easy Vector System (Promega). Digoxigeninlabeled sense and antisense RNA probes were prepared in vitro using the SP6 and T7 RNA polymerases supplied with a DIG RNA Labeling Kit (Roche Applied Science) according to the manufacturer’s instructions. The probe contained part of ORF (849 bases) and the 30 -untranslated region (181 bases). Sections were treated as described by Hoshino et al. (1999). Anti-digoxigenin antibody labeled with alkaline phosphatase and NBT/BCIP were used to visualize the signal. Counterstaining was performed using a Kernechtrot stain solution (Muto Chemical, Japan). 3. Results Pheromones (E11- and Z11-14:OAc) and fatty acid pheromone precursors (E11- and Z11-tetradecenoate) were found only in the dorsal portion of the TAS (Fig. 1a, b). On the other hand, tetradecanoate, a substrate of D11desaturase, was found in both dorsal and ventral portions of the TAS (14:Me in Fig. 1a, b). These results suggest that D11-desaturase is present only in the dorsal portion of the TAS. A 1217-base cDNA (OscZ/E11, AB232855) that included a complete open reading frame encoding 329 amino acid residues was cloned from the TAS of O. scapulalis females (Fig. 2). The deduced amino acid sequence (Fig. 2) has three histidine boxes which are highly conserved within acyl-CoA desaturases in animals and yeast (Los and Murata, 1998), and a CPTQ signature motif typical of D11-desaturase (Knipple et al., 2002). OscZ/E11 showed extremely high homology (amino acid identity499%) to the D11-desaturase genes of O. nubilalis (OnuZ/E11) and O. furnacalis (OfuZ/E11), and relatively low homology to insects from different genera, such as S. littoralis (67%,
Fig. 1. Gas chromatograms of pheromone components (a, b) and methyl esters of fatty acid pheromone precursors (c, d) extracted from the dorsal and ventral parts of the isolated terminal abdominal segments (TAS). The TAS were cut into dorsal and ventral parts at the level of lateral apophyses. IS: internal standards, tridecyl acetate (a, b) and tridecanoic acid methyl ester (c, d).
AY362879), A. velutinana (63%, AF416738), H. zea (65%, AF272342) and T. ni (64%, AF035375). RT-PCR was performed to confirm that the OscZ/E11 gene is not expressed in body parts other than the TAS. Analysis of the head, thorax, abdomen exclusive of the TAS, and TAS of an adult female showed that mRNA of OscZ/E11 is expressed only in the TAS (Fig. 3). In situ hybridization on thin sections of TAS was performed to visualize the cells in which the OscZ/E11 gene is expressed. The signal from an antisense probe was found only in the large, columnar cells present in the dorsal
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Fig. 2. Translated amino acid sequence of OscZ/E11 (O. scapulalis) aligned with those of OnuZ/E11 (O. furnacalis) and OfuZ/E11 (O. nubilalis). The regions underlined indicate histidine boxes, a unique structure found in animal acyl-CoA desaturases (Los and Murata, 1998). The four amino acids enclosed by a square is the ‘signature motif’, XXXQ, which is typically found in lepidopteran D11 acyl-CoA desaturases (Knipple et al., 2002).
Fig. 3. Expression of OscZ/E11 in a female adult of O. scapulalis. The body of a female was divided into the head (H), thorax (T), terminal abdominal segments (8–10th, TAS), and abdomen exclusive of TAS (A–TAS). Expression of b-actin is indicated as a positive control.
region (Fig. 4a, b). No specific signal was found when a control sense probe was used (Fig. 4c). 4. Discussion When the deduced amino acid sequences of OscZ/E11 (O. scapulalis), OnuZ/E11 (O. nubilalis) and OfuZ/E11 (O. furnacalis) are compared, OnuZ/E11 and OfuZ/E11 are identical, and OscZ/E11 differs from the others only at three positions (Fig. 2). Considering that OnuZ/E11 ( ¼ OfuZ/E11) is shown to exhibit D11-desaturase activity in a functional assay (Roelofs et al., 2002), and that the three variable positions are not in the regions where any functional or structural importance is attributed, it is rational to conclude that the OscZ/E11 gene encodes a functional D11-desaturase. Although transcripts of both D11- and D14-desaturase genes have been found in the PGs of O. nubilalis and O. furnacalis, only one or the other of these is functionally
expressed in a single species (Roelofs et al., 2002). For instance, D11-desaturase is not functionally expressed in O. furnacalis although the gene is transcribed into mRNA. It is curious that OfuZ/E11 and OnuZ/E11 are identical, considering that OfuZ/E11 is not functionally expressed, and that O. furnacalis is more distantly related to O. nubilalis than O. scapulalis (Kim et al. 1999). Further studies are needed to clarify the evolution of D11- and D14desaturases in the genus Ostrinia. The present findings indicate that in O. scapulalis, desaturation of pheromone precursors occurs specifically in the modified cells located at the dorsal end of the 8th–9th intersegmental membrane. In morphology and location, the cells with positive signals matched the PG cells in O. nubilalis, which were determined based on morphological and biochemical lines of evidence (Ma and Roelofs, 2002). In B. mori, Fo´nagy et al. (2000) have proven that a layer of cells isolated by papain treatment of the 8th–9th intersegmental membrane are the pheromone-producing cells, which, in tissue culture, retain the ability to produce bombykol in response to the pheromonotropic peptide TKYFSPRLamide, ionomycin and calcium ionophore A23187. In no other species are the functions of pheromone-producing cells demonstrated as elegantly as in this study. Our present study on Ostrinia, although involving a different approach, has also succeeded in identifying the pheromone-producing cells. The control of pheromone biosynthesis in Ostrinia is not well understood. Although pheromone-biosynthesisactivating neuropeptide (PBAN) released from the
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Fig. 4. Expression of OscZ/E11 in sections of the 8th–9th intersegmental membrane. (a) In situ hybridization with the antisense RNA probe. (b) Magnification of the area indicated by the square in (a). Pheromone-producing cells are found as typical column-shaped epidermal cells located in the dorsal part. (c) Hybridization with the sense probe (negative control). Hg, hind gut; Ov, oviduct; PG, pheromone-producing cells. Parts of the 8th segment (indicated by arrows) appear because the 8th–9th intersegmental membrane was not fully extruded from the 8th segment.
suboesophageal ganglion is believed to be involved in this control, our preliminary studies showed that PBAN does not affect the mRNA level of D11-desaturase (Fukuzawa, unpublished). Rather, mRNA for D11-desaturase is always expressed regardless of pheromone production. The present findings on the sequence of D11-desaturase and its localization would facilitate studies on the control of pheromone biosynthesis in O. scapulalis and related species. Acknowledgments We thank Dr. Chen Bin and Dr. Nami Miura for useful discussions during the course of the present study. This work was supported in part by Grant-in-Aid for Scientific Research No.16208005 from the Japan Society for the Promotion of Science (JSPS). References Fo´nagy, A., Yokoyama, N., Okano, K., Tatsuki, S., Maeda, S., Matsumoto, S., 2000. Pheromone-producing cells in the silkworm moth, Bombyx mori: identification and their morphological changes in response to pheromonotropic stimuli. Journal of Insect Physiology 46, 735–744. Foster, S.P., 2001. Fatty acyl pheromone analog-containing lipids and their roles in sex pheromone biosynthesis in the lightbrown apple moth, Epiphyas postvittana (Walker). Journal of Insect Physiology 47, 433–443.
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