Genomewide Screening and Transcriptional Profile Analysis Of

Insect Science (2012) 19, 55–63, DOI 10.1111/j.1744-7917.2011.01427.x

ORIGINAL ARTICLE Genome-wide screening and transcriptional proﬁle analysis of desaturase genes in the European corn borer moth

Bingye Xue1,†, Alejandro P. Rooney2 and Wendell L. Roelofs1 1Department of Entomology, NYSAES-Cornell University, Geneva, NY, 2Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL, USA

Abstract Acyl-coenzyme A (Acyl-CoA) desaturases play a key role in the biosynthesis of female moth sex pheromones. Desaturase genes are encoded by a large multigene family, and they have been divided into five subgroups on the basis of biochemical functionality and phylogenetic affinity. In this study both copy numbers and transcriptional levels of desaturase genes in the European corn borer (ECB), Ostrinia nubilalis, were investigated. The results from genome-wide screening of ECB bacterial artificial chromosome (BAC) library indicated there are many copies of some desaturase genes in the genome. An open reading frame (ORF) has been isolated for the novel desaturase gene ECB ezi-11β from ECB gland complementary DNA and its functionality has been analyzed by two yeast expression systems. No functional activities have been detected for it. The expression levels of the four desaturase genes both in the pheromone gland and fat body of ECB and Asian corn borer (ACB), O. furnacalis, were determined by real-time polymerase chain reaction. In the ECB gland, 11 is the most abundant, although the amount of 14 is also considerable. In the ACB gland, 14 is the most abundant and is 100 times more abundant than all the other three combined. The results from the analysis of evolution of desaturase gene transcription in the ECB, ACB and other moths indicate that the pattern of 11 gene transcription is significantly different from the transcriptional patterns of other desaturase genes and this difference is tied to the underlying nucleotide composition bias of the genome. Key words bacterial artificial chromosome (BAC) library, desaturase, European corn borer, G+C, sex pheromone

Introduction fatty acids that generate a blend of pheromones unique to each species. Desaturase genes are encoded by a large Acyl-coenzyme A (Acyl-CoA) desaturases play a key role multigene family. A considerable amount of research has in the biosynthesis of female moth sex pheromones. They been conducted on the structure, function and evolution are responsible for the production of distinct, desaturated of moth sex pheromone desaturase genes (e.g., Knipple et al., 1998, 2002; Liu et al., 1999, 2002a,b, 2004; Rosen- field et al., 2001; Hao et al., 2002; Roelofs et al., 2002; Correspondence: Wendell L. Roelofs, Department of Ento- Xue et al., 2007; Lassance & Lofstedt,¨ 2009), and they mology, New York State Agricultural Experiment Station, Cor- have been divided into five subgroups on the basis of bio- nell University, 630 West North Street, Geneva, New York, NY chemical functionality and phylogenetic affinity (Roelofs 14456, USA. Tel: +1 315 789 1426; fax: +1 315 787 2326; et al., 2002; Roelofs & Rooney, 2003; Liu et al., 2004; email: [email protected] Xue et al., 2007; Rooney, 2009). †Current address: Department of Plant Pathology, North Car- Previously, we characterized a novel class of desaturase- olina State University, Raleigh, NC 27607, USA retroposon fusion genes from the European corn borer

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(ECB), Ostrinia nubilalis, and the Asian corn borer cDNA cloning and functional assay for ECB ezi-11β (ACB), Ostrinia furnacalis (Xue et al., 2007). The novel and ACB ezi-11β desaturase genes indicate that the evolution of moth sex pheromone desaturases is much more complex than pre- Complementary DNAs (cDNAs) were made from viously recognized due to the gene duplication and retro- pheromone gland and fat body of both ECB and ACB poson invasion to the genome. Thus, questions arise on (Roelofs et al., 2002). Nested PCRs were used for whether these novel desaturase genes are transcribed and cDNA cloning of ECB ezi-11β and ACB ezi-11β functional, and how these genes are distributed in the ECB by two sets of primers designed from the genomic genome. DNA sequences (GenBank accession nos. EF113395, Another more interesting scenario in the study of the EF113393). The primers are ECB-E/Z11–2-F1 (5- structure, function and evolution of moth sex pheromone CTAATATAAACATGGCCGAAAT-3) and ECB-E/Z11– desaturases is the pattern of reciprocal gene activation– 2-R1 (5-CTTGTTCAATAAATTATAAT-3) and ECB- deactivation observed in ECB and ACB. The female E/Z11–2-F2 (5-ACATGGCCGAAATACCAAC-3) and ECB pheromone gland contains four different desaturase ECB-E/Z11–2-R2 (5-TAATTTAGACTTCATTCTAC- gene messenger RNA (mRNA) transcripts (11, 14, 3). One fragment (GenBank accession no. FJ906800) 9 [16>18] and 9 [18>16]), but only the 11 gene was isolated from the ECB gland cDNA and the open is expressed to produce an enzyme that functions in fe- reading frame (ORF) of 966 bp for the ECB ezi-11β male sex pheromone biosynthesis (Roelofs et al., 2002). gene was deduced from this fragment and ligated into Likewise, the 11, 14, 9 (16>18) and 9 (18>16) YEpOLEX (Knipple et al., 1998; Liu et al., 1999) and genes are also transcribed in the female ACB pheromone pYES2 (Toke & Martin, 1996; Liu et al., 2002a,b) gland, but in this case it is the 14 gene that is ex- expression vectors and transformed to yeast ole1 and pressed to produce an enzyme that functions in female Elo1 cells. sex pheromone biosynthesis. This odd pattern was diffi- Functionality was subsequently analyzed by mass spec- cult to explain until Lassance & Lofstedt¨ (2009) showed tral analysis of DMDS (Dimethyl disulfide) adducts. that the ECB 14 gene is expressed in the male hair- pencil tissue where it functions in the male sex pheromone biosynthetic pathway. Thus, one might speculate that the Quantitative real-time PCR 14 gene product would be found in the ACB male hair pencil tissue as well, but it is not (Lassance & Lofstedt,¨ To determine the expression level of desaturase genes, 2009). These paradoxical observations lead one to wonder mRNAs were isolated from 2–3-day-old ECB and ACB how this complex system of desaturase gene expression female glands and fat-body and reverse-transcribed into evolved. cDNA. A real-time PCR system, Cepheid’s Smart Cycler In the present paper, we report the results of ECB System (Cepheid, Inc., Sunnyvale, CA, US) was used for Bacterial Artificial Chromosome (BAC) library screen- the amplification of target cDNAs. Reactions were car- ing, novel gene functional analysis, and the analy- ried out in a total volume of 25 μL/tube. Each reaction sis of desaturase gene transcriptional patterns in the master mixture contained 3 mmol/L MgCl2,0.1μmol/L fat body and pheromone gland of the ECB and of each primer, 0.2 mmol/L deoxynucleotide triphos- ACB. phate (dNTP), 0.5 μL10× SYBR Green I, 1.25 U Hot Start Ex Taq (TaKaRa Bio USA, Madison, WI, USA), 2.5 μL10× buffer, 5 μL template DNA and water. In Materials and methods every run, one negative control (water), seven standard cDNA, and eight tissue samples including two replica- ECB BAC library screening tions of ECB gland and fat body cDNA, ACB gland and fat body cDNA, were included. The following protocol European corn borer BAC library was constructed was used: initial denaturation for 2 min at 95◦C followed by Clemson University Genomics Institute (CUGI). by 45 cycles of: (i) 15 s at 95◦C; (ii) 20 s at 60◦C; and (iii) Primers were designed based on the sequences (Table 1) 20 s at 72◦C. The reaction was heated above the melting deposited in the GenBank and polymerase chain reaction temperature of the dimers for each gene for an additional (PCR) products labeled with 32P were used as probes. 10s and fluorescence measurements were made at the end Hybridization followed the protocol provide by CUGI of the additional extension phase. The threshold line was (http://www.genome.clemson.edu/resources/protocols). set at 30 fluorescent units on the Y-axis and the primary The screening procedure is summarized in Fig. 1. curve was selected in the curve analysis.

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Table 1 Polymerase chain reaction (PCR) primers for making probes to screen European corn borer bacterial artificial chromosome (ECB BAC) library.

Name of genes PCR product size (bp) PCR primer sequence Accession no.

OnuZ/E11 309 bp 5-AATTATATTTTATTTTTTTGCTGAG-3 AF441221 5-CTATATAAATCAGCCACATCACAC-3 OnuZ/E14 211 bp 5-CAGACATAGACGCCACCAACCCT-3 AF441220 5-GACGCGATTTTTACATTTCCTCC-3 OnuZ9(16>18) (Z9–1) 208 bp 5-CTCCTAATATTAAGGACGGAGCT -3 AF243047 5-TGCCATTTCGCTGAAAACAGGAAT-3 OnuZ9(18>16) (Z9–2) 194 bp 5-TGCCGCCACAAGGTGCAGAGAG-3 AF430246 5-CATCACAGTTGTGAGCATCAGG-3 ECB-ezi-11α 266 bp 5-TCTATACTCGGAATTACTGCAGCTGCT-3 EF113398 5-GATTTGCCTTGTCGTTTCACCTCTTCG-3 ECB-ezi-11β 303 bp 5-TATGTATCACATAACTATAATAGGC-3 EF113395 5-TTGTATAAGTCGGACATGTCCATAGAC-3 ECB-ezi-11ψ 335 bp 5-AGTGCTGATGCATATATTGGGTACC-3 EF113404 5-CATTCTGGAGGCGTATGACAGGA-3

A specific primer pair (Table 2) was used to determine ECB-ezi-11β, ECB-11ψ (Fig. 1, Table S1). Further the transcription level for each of the four desaturase analysis by colony PCR and DNA sequencing confirmed genes in ECB and ACB. Melting curve analyses were that there were two distinct clones of ECB-11 (75M1, performed immediately following PCR to identify spe- 2I23), 8 clones of ECB-ezi-11α and ECB-ezi-11β cific PCR products and primer dimers as the temperature (32A7, 32H12, 51K12, 76E13, 76N10, 79K15, 32G3, slowly increased from 60 to 95◦C. The reactions were 88O9), two clones of ECB-14 (33F4, 54 C14) and six also checked with agarose gel electrophoresis to corre- clones of ECB-9 (16>18) (31B16, 48C4, 57P12, 83A15, late product length with melting peaks and to determine 89B2, 92 M13) (Fig. 1). No clones of ECB-9 (18>16) if primer dimers or nonspecific amplification products and ECB-11ψ were found. ECB-ezi-11α and ECB- were formed. Analysis of data was accomplished using ezi-11β were found in the same clone. The restriction the Smart Cycler Software (Cepheid). Relative copy num- patterns of the clones and DNA sequences indicate that bers of unknown samples were determined according to a each clone is different from the others, but two clones standard curve equation (Fig. 3). of ECB-11 could be from the same loci in the genome (Fig. 2).

Sequence analysis Functional assay of the ECB ezi-11β gene LasergeneR sequence analysis software (DNASTAR Inc., Madison, WI, USA) was used to edit resultant DNA One fragment (GenBank accession no. FJ906800) was sequences, which were subsequently aligned using the isolated from the ECB gland cDNA library, but no computer program CLUSTALW (Thompson et al., 1994). other fragments were isolated from ECB fat-body, ACB Analyses of G+C content were completed using the com- gland and ACB fat-body cDNAs. The ORF was success- puter program MEGA4 (Tamura et al., 2007). fully cloned into YEpOLEX (Knipple et al., 1998; Liu et al., 1999), designated YEpOLEX-ezi-11β and pYES2 (Toke & Martin, 1996; Liu et al., 2002b), des- Results ignated pYES2-ezi-11β. The functional assay was conducted under identical conditions to those used to assay Distribution of desaturase genes in the ECB genome the activity of other active 11 ORFs (Toke & Martin, 1996; Knipple et al., 1998; Liu et al., 1999, 2002a,b; Hao A total of 77 clones were isolated after twice screen- et al., 2002). One-half mmol saturated fatty acid MAME ing with a mixture of probes from ECB-9 (16>18), (myristic acid methyl ester) or 0.5 mmol/L monounsat- ECB-9 (18>16), ECB-11, ECB-14, ECB-ezi-11α, urated fatty acids, oleic acid and palmitoleic acid, were

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European corn borer (ECB) BAC Library

First screening: Second screening: - Mixture of five probes from D11, D11 V1, - Mixture of five probes from Z9-1, Z9-2, D11, D11 V2, D11V3, D14 was used ezi-D11 psi, D14 was used - 37 positive colonies were pulled out - 40 positive colonies were pulled out

Colony PCR

- One D11 (75M1); - 6 Z9-1 (31B16, 48C4, 57P12, 83A15, 89B2, - 10 ezi-D11 alpha (32A7, 32G3, 32H12, 92M13); 51K12, 76E13, 76N10, 79K15, 88O9, - No Z9-2 92H6, 95E24); - One D11 (2I23); - 10 ezi-D11 beta (same clone number as - 4 ezi-D11 psi (1D14, 3G20, 11P22, 59G21); ezi-D11 alpha) - 2 D14 (33F4, 54C14) - One ezi-D11 psi (54O10); Were detected - One D14 (64H19) Were detected

PCR product sequencing

One D11 (75M1); One D11 (2I23); 8 ezi-D11 alpha and beta (32A7, 32G3, 2 D14 (33F4, 54C14) 32H12, 51K12, 76E13, 76N10, 79K15, 6 Z9-1 (31B16, 48C4, 57P12, 83A15, 89B2, 92M13) 88O9) Were confirmed Were confirmed

2 D11 (2I23&75M1), 2 D14 (33F4&54C14), 3 Z9-1 (31B16, 48C4, 57P12) and 6 ezi-D11 alpha and beta (32A7, 32H12, 51K12, 76E13, 76N10, 79K15) were selected to isolate BAC DNA for digesting and further sequencing analysis.

Fig. 1 Diagram of genomic DNA bacterial artificial chromosome (BAC) library screening.

Table 2 Real-time polymerase chain reaction (PCR) primers and standard graph formulae.

Real-time PCR Real-time Formula of Gene product size (bp) PCR primers standard graph

9(16>18) 308 5-GGAACTGAAGAGAAAGGGCAAGGGACT-3 y =−0.219x + 7.931, r2 = 0.997 5-CCAACGCGAATAAGGAGACTGAGAGGT-3 9(18>16) 545 5-AGATCAAAGCCAAGGGACACACCATC-3 y =−0.272x + 8.785, r2 = 0.997 5-GACTTTCTTGTCCTCCTCGGGGATGTT-3 11 249 5-CACAAAACGTTCTTCTGAATCTTGCTGTG-3 y =−0.242x + 9.482, r2 = 0.999 5-GTTTATCGGCACTGTGACCCCATAAGTC-3 14 389 5-TCGCTGTTCTTCACCTTCGGCTTCCTCCT-3 y =−0.212x + 8.47, r2 = 0.994 5-ATTATCCTTTTGTTTAGCGTCTCAGGGTCG-3

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Fig. 2 Restriction enzyme digestion analysis of the bacterial artificial chromosome (BAC) clones. E: EcoRI, H: HindIII, S: SacI. 1: 32A7; 2: 32H12; 3: 51K12; 4: 76E13; 5: 76N10; 6: 79K15; 7: 31B16, 8: 48C4; 9: 57P12; 10, 12, 14: 2I23; 11,13, 15: 75M1; 16,18,20: 33F4; 17,19,21: 54C14. M: DNA ladder. added to YEpOLEX and pYES2 yeast expression sys- found for the ECB transcripts. In the ACB gland cDNA, tems. The products were analyzed by GC/MS, but no un- 14 was the most abundant transcript and found at levels saturated product from the inserted ezi-11β ORF were that exceed 100 times more than the next most abundant detected. However, we did not assay this ORF to see if it transcript, which was the 11. As in the ECB pheromone had hydrolase or other enzymatic activities. gland, the 9 (16>18) and 9 (18>16) gene transcripts were also detected, but their abundance was significantly lower than the 14 and 11 transcripts. In the ACB fat Expression level of desaturases in pheromone gland and body cDNA, the 14, 9 (16>18) and 9 (18>16) gene fat body transcripts were found at low levels, whereas the ACB 11 gene transcript was found at a higher level (Fig. 3). Quantitative real-time PCR was conducted to amplify all four desaturase genes (11, 14, 9 [16>18] and 9 Correlation between expression level and GC4 content [18>16]) from the pheromone-gland cDNA and fat-body cDNA. The results (Fig. 3) show that the key pheromone An interesting correlation was found when the tran- desaturase 11 is the most abundant transcript in the scription level against the percentage of guanine and cy- ECB pheromone gland and the amount of 14 is also tosine nucleotides (G+C) at fourfold degenerate codon considerable. The fact that the 14 is the second-most sites (GC4) of desaturase genes were plotted (Fig. 4). The abundant transcript is interesting considering that it does 11 desaturases formed one cluster whereas the other not function in female sex pheromone biosynthesis. The desaturases formed a separate cluster; this result was ob- 9 (16>18) and 9 (18>16) gene transcripts were also tained in the case of both the fat body and pheromone detectable in the pheromone gland, but their abundance gland transcripts (Fig. 4). It is readily apparent from the was significantly lower than the 11 and 14 transcripts. plot in Fig. 4 that the 11 genes all possess GC4 content In the fat body cDNA of the ECB, all four desaturase genes levels that are lower than non-11 desaturases (i.e., 9 were expressed at low levels (Fig. 3). [16>18], 9 [18>16], 9 [14–26], 14 and NF). The av- The results from the quantitative real-time PCR study erage percent GC4 value for 11 genes (41.6 ± 11.6) of ACB desaturase gene transcripts were similar to those was significantly different (P<0.001, t-test) than the

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unique cDNAs isolated exceeds the minimum number of transcripts required to encode enzymatically distinct desaturases capable of producing the known sex pheromone components (Roelofs et al., 2002; Knipple et al., 2002). Discovery of many ‘cryptic’ desaturase genes in both ECB and ACB genomes by the GenomeWalker technique revealed that the evolution of these desaturases is much more complex than once believed due to transposon invasion after gene duplication in the evolution of insects (Xue et al., 2007). However, it remained to be shown if these ‘novel’ genes were functional. To answer this question, we screened the cDNAs from the pheromone gland and fat body of ECB and ACB. We generated a 1 003 bp cDNA fragment for the ECB ezi-11β gene from the ECB gland cDNA, but failed to get any sequences for other ‘novel’ genes from this screening. Further functional analysis revealed that ECB ezi-11β has an ORF, but no functional product was detected. During evolution, the different desaturase genes were probably generated through gene duplication, but it is difficult to know how the duplicated copies diversified into different func- Fig. 3 Quantification of the desaturase genes in European corn tional genes after duplication (Roelofs & Rooney, 2003). borer (ECB) and Asian corn borer (ACB) by quantitative real- Initially the gene could duplicate and evolve into many time polymerase chain reaction. copies due to deletion of sequences and mutation or as a result of retrotransposon invasion (Xue et al., 2007). Eventually one copy could have evolved a new function, average percent GC value for non-11 genes 4 whereas others could remain in the genome with no func- (58.9 ± 6.6) (Table 3). tion or perhaps a different function. An important question is whether the ezi-11 gene duplicates are pseudogenes. Discussion If that were the case, one would have expected a frame- shift or nonsense mutation to have occurred in the reading This study was conducted to increase knowledge on the frames of these genes over the course of this time period. evolution of the diverse desaturases found in female moth Our results indicate that one of the ECB ezi-11β genes sex pheromone glands. In any given species, the number of has an ORF, but lost functionality. Since it has an ORF, it could potentially become functional and serve as raw material from which new pheromone blends could arise if the genes were co-opted into sex pheromone biosynthesis pathways. However, it is possible that these genes do not function in ECB sex pheromone biosynthesis at all, and they may have been co-opted to perform some other unrelated function. Another question that we sought to answer regards how many copies of these ‘novel’ genes exist within the genome. An ECB BAC library was used to screen with probes from all the desaturase genes discovered by our group (Xue et al., 2007). BAC clones that contained a desaturase sequence were sequenced. Not surprisingly, the results from ECB BAC library screening indicated that the Fig. 4 Relationship between underlying nucleotide composi- α β tion bias and transcription level of sex pheromone desaturase copies of ECB ezi- 11 and ezi- 11 are the most abun- genes. Transcription level reflects the ranked level (i.e., from dant and are found in the same clone. The average size of highest to lowest) for each gene in the specific tissue examined the BAC clone is 125 kb, which shows that the ECB ezi- (fat body or pheromone gland). 11α and ezi-11β sequences are located closely in the

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Table 3 G+C content at fourfold degenerate sites in representative moth species studied.

Non-11 11

Species/gene GC4 Species/gene GC4

Ostrinia furnacalis 14 66.9 Ostrinia furnacalis 11 46.8 Ostrinia furnacalis 9 (18>16) 58.9 Ostrinia nubilalis 11 46.8 Ostrinia furnacalis 9 (16>18) 54.0 Choristoneura parallela 11 41.6 Ostrinia nubilalis 14 66.4 Choristoneura rosaceana 11 20.5 Ostrinia nubilalis 9 (18>16) 58.2 Helicoverpa zea 11 44.6 Ostrinia nubilalis 9 (16>18) 53.2 Epiphyas postvittana 11 42.7 Choristoneura parallela 9 (14–26) 60.8 Argyrotaenia velutinana 11 40.4 Choristoneura parallela 9 (18>16) 52.2 Planotortrix octo 11 64.9 Choristoneura parallela 9 (16>18) 64.5 Trichoplusia ni 11 38.3 Choristoneura parallela NF 54.9 Bombyx mori 11 29.3 Choristoneura rosaceana 9 (16>18) 65.3 Choristoneura rosaceana NF 53.5 Helicoverpa zea 9 (18>16) 57.4 Helicoverpa zea 9 (16>18) 57.0 Epiphyas postvittana 9 (16>18) 70.9 Epiphyas postvittana 9 (16>18) 65.0 Argyrotaenia velutinana 9 (16>18) 65.9 Planotortrix octo 9 (16>18) 58.6 Trichoplusia ni 9 (16>18) 48.1 Bombyx mori 9 (16>18) 47.0 genome. Recently, Coates et al. reported that they iden- more transcripts could be detected in the pheromone gland tified two ezi-like LINEs (Long Interspersed Elements) than required for pheromone blend production, the func- that were shown to be multicopies within the BAC li- tional gene is expressed over 100 times more abundantly brary OnB1 (Coates et al., 2009). Since we identified the than the other desaturase genes in the pheromone gland transposon LINEs in the process of genomic cloning of (Fig. 3). This is similar to what Sakai et al. found for desaturase genes, we were expecting to get the BAC clones 11 and 14 genes in Ostrinia scapulalis and ACB. that contain ECB ezi-11α and ezi-11β, including de- Although it is not clear which element controls the desat- saturase domains and transposon domains. Unfortunately, urase gene’s transcription, Sakai et al. (2009) found that we could not go further to get the retroposon sequences a recessive gene (di14) in O. scapulalis suppresses the that linked to ECB ezi-11. It would definitely be worth- transcription from the 14-desaturase gene (OscZ/E14), while to sequence them further to provide a better under- and an allele (di11) in ACB suppresses the transcription standing of how the ezi-11 evolved and what (if any) from the 11-desaturase gene (OfurZ/E11). The relative impact this might have on sex pheromone diversification expression levels of 11 and 14 in ECB/ACB (Fig. 3) in moths. and O. scapulalis/ACB (Sakai et al., 2009) could indicate The results from both our research on ECB and ACB that transcription inhibition could be key in controlling (Roelofs et al., 2002) and Sakai et al.’s research on ACB desaturase activity. and adzuki bean borer (Ostrinia scapulalis) (Sakai et al., However, concerning the evolution of female sex 2009) indicated different species can have the same set of pheromone blends in Ostrinia spp. another question is different desaturase genes transcribed, but only one is ex- whether or not there was any evidence for selection acting pressed to produce an enzyme that functions in female sex on transcription and expression. To do this, we examined pheromone biosynthesis (Roelofs et al., 2002; Sakai et al., the correlation between transcription levels and the under- 2009). Transcriptional analysis of the desaturase gene ex- lying nucleotide composition bias of the genomic region pression in moths could provide important information on where desaturase genes are located (Fig. 4). As a proxy for how biosynthesis is controlled, which has not been accu- this region, we looked at fourfold degenerate codon sites. rately determined as yet. Our results showed that although These are third codon positions in which a change in any

C 2011 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 19, 55–63 62 B. Xue et al. nucleotide does not alter the encoded amino acid. The use References of such sites as proxies for the underlying genomic nucleotide content is well established (Nei, 1987; Li, 1997; Coates, B.S., Sumerford, D.V., Hellmich, R.L. and Lewis, L.C. Rooney, 2003). The result indicates that 11 GC4 lev- (2009) Repetitive genome elements in a European corn borer, els are significantly different from non-11 GC4 levels Ostrinia nubilalis, bacterial artificial chromosome library (Fig. 4, Table 3). What might have caused this disparity were indicated by bacterial artificial chromosome end se- in GC4 levels between 11 and non-11 desaturases? quencing and development of sequence tag site markers: im- One explanation is that it results from a phylogenetic plications for lepidopteran genomic research. Genome, 52, correlation. If closely related species are examined (e.g., 57–67. sibling species or species from the same genus), GC4 lev- Hao, G., Liu, W., O’Connor, M. and Roelofs, W.L. (2002) els for a particular gene could be similar simply due to Acyl-CoA Z9-and Z10-desaturase genes from a New Zealand their taxonomic relatedness. To guard against this pos- leafroller moth species, Planotortrix octo. Insect Biochem- sibility, we examined several different species (Table 3) istry and Molecular Biology, 32, 961–966. from a range of taxonomic genera and families (Harvey & Harvey, P.H. and Pagel, M. (1991) The Comparative Method Pagel, 1991). An alternative explanation for differences in Evolutionary Biology. Oxford University Press, Oxford. in GC4 levels is that the 11 gene has moved to a region 248 pp. of the genome that possesses a different underlying nu- Knipple, D.C., Rosenfield, C.L., Miller, S.J., Liu, W., Tang, J., cleotide composition than non-11 genes. Whereas the Ma, P.W.K. and Roelofs, W. L. (1998) Cloning and functional exact reason for why different regions of the genome have expression of a cDNA encoding a pheromone gland-specific different G+C contents is still open to debate, one poten- acyl-CoA Delta(11)-desaturase of the cabbage looper moth, tial possibility is that the differences help regulate gene Trichoplusia ni. Proceedings of the National Academy of Sci- expression. In fact, the correlation between transcription ences of the United States of America, 95, 15287–15292. level and GC4 content was highly significant (r = 0.73, Knipple, D.C., Rosenfield, C.L., Nielsen, R., You, K.M. and P < 0.000 5) for non-11 transcripts in both the fat body Jeong, S.E. (2002) Evolution of the integral membrane desat- and the pheromone gland (Fig. 4). This result should not urase gene family in moths and flies. Genetics, 162, 1737– happen unless transcription is somehow tied to the un- 1752. derlying nucleotide composition of the genomic region Lassance, J-M. and Lofstedt,¨ C. (2009) Concerted evolution of in which these genes lie. However, it should be pointed male and female display traits in the European corn borer, out that the correlation was not significant for the 11 Ostrinia nubilalis. BMC Biology, 7, 10. desaturase group (Table 3), most likely because the Li, W.H.(1997) Molecular Evolution. Sinauer, Sunderland, MA. number of observable data points was too small to 487 pp. make a meaningful comparison. Thus, future studies Liu, W., Ma, P.W.K., Marsella-Herrick, P., Rosenfield, C.L., should be undertaken to further investigate this possibility Knipple, D.C. and Roelofs, W.L. (1999) Cloning and func- further. tional expression of a cDNA encoding a metabolic acyl-CoA Delta 9-desaturase of the cabbage looper moth, Trichoplu- Data deposition sia ni. Insect Biochemistry and Molecular Biology, 29, 435– 443. The sequences reported in this paper have Liu, W., Jiao, H., O’Connor, M. and Roelofs, W.L. (2002a) Gene been deposited in the GenBank database (accession characterized for membrane desaturase that produces (E)-11 nos. FJ906800; GQ166859–GQ166860; GQ225747– isomers of mono- and diunsaturated fatty acids. Proceedings GQ225767). of the National Academy of Sciences of the United States of America, 99, 620–624. Acknowledgments Liu, W., Jiao, H., O’Connor, M. and Roelofs, W.L. (2002b) Moth desaturase characterized that produces both Z and E This project was supported by Award Number 2002– isomers of Delta 11-tetradecenoic acids. Insect Biochemistry 35302-12287 of the USDA-CSREES NRI Competitive and Molecular Biology, 32, 1489–1495. Grants Program. The authors thank Dr. Richard G. Har- Liu, W., Rooney, A.P., Xue, B. and Roelofs, W.L. (2004) Desat- rison and his laboratory for supplying the European urases from the spotted fireworm moth (Choristoneura par- corn borer bacterial artificial chromosome (BAC) library, allela) shed light on the evolutionary origins of novel moth Steven M. Bogdanowicz for technical assistance, and sex pheromone desaturases. Gene, 342, 303–311. Sepp Kohlwein and Charles Martin for supplying the elo1 Nei, M. (1987) Molecular Evolutionary Genetics. Columbia and ole1 mutant yeast strains, respectively. University Press, New York. 512 pp.

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Roelofs, W.L., Liu, W.,Hao, G., Jiao, H., Rooney, A.P.and Linn, Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) C.E. Jr. (2002) Evolution of moth sex pheromones via ances- CLUSTAL W: improving the sensitivity of progressive tral genes. Proceedings of the National Academy of Sciences multiple sequence alignment through sequence weighting, of the United States of America, 99, 13621–13626. position-specific gap penalities and weight matrix choice. Roelofs, W.L. and Rooney, A.P. (2003) Molecular genetics and Nucleic Acids Research, 22, 4673–4680. evolution of pheromone biosynthesis in Lepidoptera. Pro- Toke, D.A., and Martin, C.E. (1996) Isolation and characteri- ceedings of the National Academy of Sciences of the United zation of a gene affecting fatty acid elongation in Saccha- States of America, 100, 9179–9184. romyces cerevcisiae. Journal of Biological Chemistry, 271, Rooney, A.P. (2003) Selection for highly biased amino acid 18413–18420. frequency in the TolA cell envelope protein of proteobacteria. Xue, B., Rooney, A.P., Kajikawa, M., Okada, N. and Roelofs, Journal of Molecular Evolution, 57, 731–736. W.L.(2007) Novel sex pheromone desaturases in the genomes Rooney, A.P. (2009) Evolution of moth sex pheromone desat- of corn borers generated through gene duplication and urases. Annals of the New York Academy of Sciences, 1170, retroposon fusion. Proceedings of the National Academy 506–510. of Sciences of the United States of America, 104, 4467– Rosenfield, C.L., You, K.M., Marsellla-Herrick, P., Roelofs, 4472. W.L. and Knipple, D.C. (2001) Structural and functional Accepted March 6, 2011 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 Supporting information Molecular Biology, 31, 949–964. Sakai, R., Fukuzawa, M., Nakano, R., Tatsuki, S. and Ishikawa, Additional Supporting Information may be found in the Y. (2009) Alternative suppression of transcription from online version of this article: two desaturase genes is the key for species-specific sex Table S1 List of accession number of the genes. pheromone biosynthesis in two Ostrinia moths. Insect Bio- chemistry and Molecular Biology, 39, 62–67. Please note: Wiley-Blackwell are not responsible for Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: the content or functionality of any supporting materials Molecular Evolutionary Genetics Analysis (MEGA) software supplied by the authors. Any queries (other than missing version 4.0. Molecular Biology and Evolution, 24, 1596– material) should be directed to the corresponding author 1599. for the article.

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