Sex desaturase functioning in a primitive is cryptically conserved in congeners’ genomes

Takeshi Fujiia, Katsuhiko Itob, Mitsuko Tatematsua, Toru Shimadab, Susumu Katsumab, and Yukio Ishikawaa,1

aLaboratory of Applied Entomology, and bLaboratory of Genetics and Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657,

Edited* by Wendell L. Roelofs, Cornell University, Geneva, NY, and approved February 25, 2011 (received for review December 28, 2010) (E)-11- and (Z)-11-tetradecenyl acetate are the most common (Z)-11-tetradecenoate (E11- and Z11-14:Acyl) from the sub- female sex pheromone components in Ostrinia . The Δ11- strate tetradecanoate (14:Acyl) (20). desaturase expressed in the pheromone gland (PG) of female moths We are interested in the sex pheromone of a primitive species is a key enzyme that introduces a double bond into pheromone of Ostrinia, the Far-Eastern knotweed borer , molecules. A single Δ11-desaturase of Ostrinia nubilalis, OnubZ/ because the single sex pheromone component of this species, (E)- E11, has been shown to produce an ∼7:3 mixture of (E)-11- and 11-tetradecenol (E11-14:OH), is unique in that it is not acetylated (Z)-11-tetradecenoate from the substrate tetradecanoate. In con- and has no Z isomer (9). In the present study, through the cloning trast, the sex pheromone of Ostrinia latipennis, a primitive species and functional analysis of a PG-specific Δ11-desaturase in O. of Ostrinia, is (E)-11-tetradecenol. This pheromone is unique in that latipennis, we show that the absence of the Z isomer in the it is not acetylated, and includes no Z isomer. In the present study, pheromone is attributable to the strict product specificity of the through the cloning and functional analysis of a PG-specific Δ11- Δ11-desaturase in this species, LATPG1. We also show that desaturase in O. latipennis, we showed that the absence of the LATPG1 is closely related to retroposon-linked cryptic Δ11- Z isomer in the pheromone is attributable to the strict product spec- desaturases (ezi-Δ11) found in the genomes of O. nubilalis and fi Δ i city of the 11-desaturase in this species, LATPG1. Phylogenetic (21). The evolution of Δ11-desaturases in analysis revealed that LATPG1 was not closely related to OnubZ/ Ostrinia is discussed in light of these findings. E11. Rather, it was closely related to retroposon-linked cryptic Δ11- desaturases (ezi-Δ11) found in the genomes of O. nubilalis and Results Ostrinia furnacalis . Taken together, the results showed that an un- Pheromone Precursors in the PG of O. latipennis. To elucidate the Δ O. latipennis usual 11-desaturase is functionally expressed in , al- crucial biosynthetic step that determines the absence of the Z though the genes encoding this enzyme appear to be cryptic isomer in the pheromone of O. latipennis, we analyzed the Z/E in congeners. ratio of pheromone precursor acids in the PG of O. latipennis. GC-MS analysis revealed that only the E isomer of the phero- baculovirus expression system | speciation mone precursor was present in the PG (Fig. 1), suggesting that the Δ11-desaturase in this species produces only E11-14:Acyl. ost moths use a mate-finding system that involves the long- Mdistance attraction of males with female sex Cloning of a Desaturase Gene Expressed in the PG of O. latipennis. To (1). The great majority of moth pheromones are a blend of un- identify the O. latipennis Δ11-desaturase that produces only the E – saturated C10 C18 primary acetates, alcohols, or aldehydes, and isomer, we performed PCR using degenerate primers designed the combination of components and the blend ratios confer high based on the histidine-cluster sequences conserved in nonheme species specificity to the signal (1). Given the primary role of sex desaturases (16, 17). We cloned and sequenced ∼570-bp cDNA pheromones in reproductive behavior, the divergence of phero- fragments, and found that 44 of 47 clones showed the same se- mone signals should have profoundly affected premating re- quence, which was different from those of Δ11-desaturase genes productive isolation and speciation in moths (for a review, see ref. identified from the congeners. We named this unique gene latpg1 2). Moths of the Ostrinia (: ), which (latipennis pheromone gland-specific desaturase 1). The remain- show distinct differences in sex pheromones (3), are useful for ing three clones were fragments of Δ9-desaturase or ubiquitous studying the evolution of sex pheromone communication systems. genes, such as 16S rRNA. The Ostrinia nubilalis has long been a model The full-length sequence of latpg1 was obtained by 3′- and 5′- – for studies on this subject (4 7). Given the wealth of information RACE experiments. The latpg1 gene comprised a 957-bp ORF available on the genetic, biochemical, and molecular aspects of encoding a protein of 319 amino acid residues with a predicted sex pheromone production and reception in this species, com- molecular mass of 37.8 kDa. Sequence analysis revealed that parative studies using congeners should shed new light on the LATPG1 has features conserved in insect nonheme desaturases: subject. Among the 21 species of Ostrinia recorded worldwide (4, four transmembrane domains, three histidine boxes, and a sig- 8), the sex pheromones of nine species, including O. nubilalis, have been characterized to date (8, 9). (E)-11- and (Z)-11-

tetradecenyl acetate (E11- and Z11-14:OAc) are the most com- Author contributions: T.F., T.S., S.K., and Y.I. designed research; T.F., K.I., M.T., and S.K. mon female sex pheromone components in Ostrinia (10). The sex performed research; T.F., K.I., M.T., T.S., S.K., and Y.I. analyzed data; and T.F., K.I., T.S., pheromone components in Ostrinia are produced de novo in the S.K., and Y.I. wrote the paper. pheromone gland (PG) of female moths from a common fatty The authors declare no conflict of interest. acid, palmitic acid, through several enzymatic reactions (i.e., *This Direct Submission article had a prearranged editor. β limited oxidation, desaturation, reduction, and acetylation) Data deposition: The sequence reported in this paper has been deposited in the GenBank (11–14). A desaturase specifically expressed in the PG is the key database (accession no. AB465511). enzyme that introduces a double bond into the pheromone mol- 1To whom correspondence should be addressed. E-mail: [email protected]. – Δ ecules (15 19). A single 11-desaturase of O. nubilalis, OnubZ/ This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. E11, has been shown to produce an ∼7:3 mixture of (E)-11- and 1073/pnas.1019519108/-/DCSupplemental.

7102–7106 | PNAS | April 26, 2011 | vol. 108 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1019519108 Downloaded by guest on September 29, 2021 Fig. 3. Expression profiles of latpg1 analyzed by RT-PCR. Tissue distribution of latpg1 mRNA. RT-PCR analysis was performed using cDNA prepared from different tissues of adult virgin females (1- to 3-d-old). The actin gene was used as a control. FM, flight muscle; Lg, legs; Ep, epidermis; FB, fat body; Eg, eggs; PG, pheromone gland. Fig. 1. Fatty acid analysis of PG extracts from O. latipennis by GC-MS. Total ion chromatograms of methyl ester derivatives of fatty acids in the PG (solid line), and of authentic standards (broken line), (E)-11-tetradecenoic acid introduces a double bond with E geometry at position 11 of tetra- methyl ester (E11-14:COOMe; 9.32 min), and (Z)-11-tetradecenoic acid methyl ester (Z11-14:COOMe; 9.98 min). decanoic acid. Given that only the E isomer of the pheromone pre- cursor was found in the PG, we conclude that O. latipennis produces its pheromone using an unusual Δ11-desaturase, LATPG1. nature motif (Fig. 2). The amino acid sequence of LATPG1 showed 55.7 to 56.7% identity to Δ11-desaturases of congeners, Comparison of LATPG1 with Other Δ11-Desaturases. LATPG1 has ∼ Δ , O. furnacalis, and O. nubilalis (Fig. 2). only limited homology ( 55% amino acid identity) to the 11- Next, we examined the expression of latpg1 in several tissues desaturases of congeners (referred to as Z/E11-desaturases) of O. latipennis by RT-PCR. The latpg1 mRNA was specifically (Fig. 2). To gain insight into the evolutionary relationship be- expressed in the PG (Fig. 3), suggesting that LATPG1 is the major tween LATPG1 and other insect desaturases, a phylogenetic tree desaturase involved in pheromone synthesis in O. latipennis. of lepidopteran Δ9-, Δ11-, and Δ14-desaturases was recon- structed. As shown in Fig. 5A, LATPG1 was included in the Δ11 In Vitro Functional Assay of LATPG1. To verify the enzymatic activity clade, consistent with the enzyme’s function. Interestingly, how- of LATPG1, we generated recombinant baculoviruses expressing ever, LATPG1 was located far from Z/E11-desaturases, also LATPG1 (LATPG1-AcNPV) or His-tagged LATPG1 (LATPG1- consistent with the difference in product specificity. It should be His-AcNPV). Western blot analysis of proteins extracted from noted that LATPG1 is not closely related to CpaE11, a Δ11- Sf9 cells infected with LATPG1-His-AcNPV demonstrated that desaturase known to produce only the E isomer (16). a His-tagged LATPG1 with a molecular mass of ∼37 kDa was A BLAST search of public databases revealed that LATPG1 is expressed (Fig. 4A). In the presence of tetradecanoic acid, only closely related to retroposon-linked cryptic Δ11-desaturases (ezi- (E)-11-tetradecenoic acid was produced in Sf9 cells infected with Δ11) found in the genomes of O. nubilalis and O. furnacalis (Fig. LATPG1-His-AcNPV or LATPG1-AcNPV (Fig.4B). Sf9 cells 5B). As shown in Fig. 2, the N-terminal region of LATPG1 infected with the control virus (Bac1-AcNPV) did not show Δ11- exhibited little homology to Z/E11-desaturases, consistent with

desaturase activity. These results clearly demonstrated that LATPG1 the inference that latpg1 and Z/E11 are not orthologs. EVOLUTION

Fig. 2. Comparison of deduced amino acid sequences of LATPG1 and Δ11 desaturases from three other Ostrinia species (Ostrinia Z/E11). Amino acid sequences of three Ostrinia Z/E11-desaturases were aligned with LATPG1 by the Clustal W program. Histidine-rich motifs (box1–box3), putative trans- membrane domains (TM1–TM4), and a signature motif are indicated. Amino acids identical to the top sequence are represented by hyphens (-), and the gaps added to optimize the alignment are shown by dots (•).

Fujii et al. PNAS | April 26, 2011 | vol. 108 | no. 17 | 7103 Downloaded by guest on September 29, 2021 A A

B

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Fig. 4. Enzymatic activity of LATPG1 protein expressed by recombinant baculoviruses. (A) Expression of recombinant LATPG1 protein. Recombinant LATPG1 protein with or without a His-tag at the C terminus was expressed using a baculovirus expression system. Protein samples from AcNPV-infected Sf9 cells were analyzed by SDS/PAGE and stained with CBB (Right). The same Fig. 5. Phylogenetic analysis of LATPG1 and other desaturases. (A)Aneighbor- samples were also analyzed by Western blotting with the anti-His antibody joining tree of LATPG1, and Δ11- and Δ14-desaturases. The Δ9-desaturase of (Left). Lane 1, LATPG1-AcNPV; lane 2, LATPG1-His-AcNPV; lane 3, Bac1- Spodoptera littoralis was used as an out-group. Bootstrap values after 1,000 AcNPV (control). (B) GC-MS analysis. Total ion chromatogram (TIC) for the replications are shown near the branches. GenBank accession numbers of fatty acid methyl esters prepared from Sf9 cells infected with Bac1-AcNPV, sequences are shown in parentheses. (B) A neighbor-joining tree of LATPG1 LATPG1-AcNPV, and LATPG1-His-AcNPV. Mass chromatograms of the base and ezi-linked cryptic desaturases in the genomes of O. nubilalis and O. fur- Δ – ion (m/z = 67) and a molecular ion (m/z = 240) of Z- and E-isomers of 11 14: nacalis. Bootstrap values after 1,000 replications are shown near the branches. COOMe are also shown. GenBank accession numbers of sequences are shown in parentheses.

We next examined the expression of latpg1 homolog in the PGs genes appeared to maintain an intact ORF over long evolutionary of other Ostrinia species by RT-PCR. The expression of latpg1 time periods (10–30 million years) (21). homolog was not observed in other congeners examined (Fig. In the present study, a Δ11-desaturase which shows homology to 6A). On the other hand, the expression pattern of Z/E11 homolog cryptic desaturases was found to be expressed in an extant Ostrinia was consistent with the presence/absence of Z11-isomer in the species, O. latipennis. Interestingly, LATPG1 exhibited stricter pheromone; that is, Z/E11 homolog was observed in species with product specificity than the Z/E11-desaturases in congeners. At Z11-isomer (O. scapulalis, , and ), present, no information is available on the nature of the common whereas not observed in species without Z11-isomer (O. furna- ancestor of latpg1 and Z/E11. It is possible that the stricter product calis and O. latipennis). specificity of LATPG1 is a derivative trait. This mutation might have acted to diminish the diversity of sex pheromone compo- Discussion nents, and so did not prevail in Ostrinia. Alternatively, the simul- Relationship Between latpg1 and ezi-Δ11. The genome of O. nubi- taneous production of E and Z isomers may be a derivative trait. lalis contains five retroposon-linked cryptic Δ11-desaturase genes This mutation might have contributed to the diversification of sex (ECB ezi-Δ11α1–3, ECB ezi-Δ11β1–2) and a pseudogene (ECB pheromone components in Ostrinia together with the advent of ψΔ11) in addition to the genes encoding “normal” Z/E11- fatty acid reductases varying in substrate specificity (7, 14, 21, 22). desaturases (21). The evolution of Δ11-desaturases in O. nubilalis At present, we do not know which scenario is correct, but the was inferred by Xue et al. (21). After the duplication of an ancestral expression of pheromone receptors specific for E11-14:OH in the Δ11-desaturase gene, a LINE retroposon named the ezi element male antennae of all Ostrinia species examined to date (23) seems was inserted into one of the copies. The copy without the retro- to support the latter scenario. Whether the latpg1 gene contains an poson evolved into the Z/E11-desaturase gene. Meanwhile, the ezi element or not remains to be determined. other duplicate gene underwent unequal crossovers with another ezi-linked gene, resulting in several ezi-Δ11 gene variants (21). The Acetyltransferase. The sex pheromone of O. latipennis, E11-14: most intriguing finding on ezi-Δ11 genes was that these cryptic OH, is distinct from others also in that it is not acetylated

7104 | www.pnas.org/cgi/doi/10.1073/pnas.1019519108 Fujii et al. Downloaded by guest on September 29, 2021 68 °C for 40 s, 30 cycles of 94 °C for 30 s, 42 °C for 30 s, and 68 °C for 40 s, and A finally 72 °C for 7 min. The full-length cDNA of latpg1 was obtained using a GeneRacer Kit (Invitrogen) with gene-specific primer sets (Table S1). The nucleotide sequence of latpg1 was submitted to the DDBJ/EMBL/GenBank Data Libraries with the accession no. AB465511.

Profiling of latpg1 Expression by RT-PCR. Complementary DNA was prepared from total RNA (1 μg) extracted from the PGs of virgin females of five Ostrinia species using a PrimeScript II first strand cDNA Synthesis Kit (Takara Bio). PCR was performed with the gene-specific primer sets shown in Table S1.

Phylogenetic Analyses. For phylogenetic analysis, we used an online service B provided by the DNA Data Bank of Japan (http://clustalw.ddbj.nig.ac.jp/top-e. html). Amino acid sequences were aligned using Clustal W, and the phylo- genetic tree was constructed by the neighbor-joining method with the DIS- TANCE option set to “Kimura.” We evaluated the reliability of the inferred tree by bootstrap analysis with 1,000 resamplings. The following sequences, which showed high scores in a Blast P search of the National Center for Bio- technology Information database with LATPG1 as a query, were used for this analysis: Trichoplusia ni, AF035375; Spodoptera littoralis, AY362879; Heli- coverpa zea, AF272342; Bombyx mori, AF182405 and AB166851; Epiphyas postvittana, AY049741; Argyrotaenia velutinana, AF416738; Choristoneura parallela, AF518014; Choristoneura rosaceana, AF545481 and AF518018; O. furnacalis AF441861 and AY062023; O. scapulalis AB232855 and AB264085; and O. nubilalis AF441220 and AF441221. A Δ9-desaturase of Scinax littoralis (AY362880) was used as an outgroup.

Construction of Recombinant Baculoviruses. Recombinant Autographa cal- ifornica nucleopolyhedroviruses (AcNPVs) were constructed using a Bac-to- Bac system (Invitrogen), as described previously (25). The coding region of latpg1 with or without a His-tag at the C terminus was PCR-amplified with Fig. 6. Comparative studies and biosynthetic pathways of Ostrinia sex a set of gene-specific primers (Table S1) containing restriction enzyme rec- pheromones. (A) Expression of latpg1 and Ostrinia Z/E11 genes in the PG of ognition sites (Bam-latpg1F, latpg1R-His-Sph, and latpg1R-Sph). The latpg1 five Ostrinia species. (B) Proposed sex pheromone biosynthetic pathway in O. fragments were then cloned into a vector, pFastBac1 (Invitrogen), and the latipennis, which produces E11-14:OH as a single sex pheromone component. bacmids containing the latpg1s were isolated. Recombinant AcNPVs, desig- nated as Latpg1-His-AcNPV and Latpg1-AcNPV, were generated by trans- fection with bacmid DNAs, and used in desaturase assays. The parental virus, Bac1-AcNPV (25), was used as the control.

(Fig. 6B). It appears that an enzyme responsible for acetylation EVOLUTION (i.e., fatty alcohol acetyltransferase) is not functioning in the PG of O. latipennis. At present, no information is available on the SDS/PAGE and Western Blotting. SDS/PAGE and Western blotting were per- molecular nature of lepidopteran acetyltransferases involved in formed as described previously (24). The samples were separated on 12% pheromone biosynthesis. Studies on the control of acetyltransferases gels by SDS/PAGE, and transferred to polyvinylidene fluoride membranes would help to understand the evolution of sex pheromone pro- (Immobilon-P; Millipore). Expression of the recombinant LATPG1 in Sf9 cells was detected with an anti-His antibody (Qiagen; 1:3,000). duction systems in Ostrinia because similar mechanisms may un- Δ derlie the selective expression of 11-desaturases (latpg1 or Z/ Desaturase Assay. Sf9 cells seeded in a 60-mm cell-culture dish were infected E11) and acetyltransferases. with LATPG1-His-AcNPV, LATPG1-AcNPV, or Bac1-AcNPV. At 48 h postinfection, 40 mg of free tetradecanoic acid was added to the dish as a substrate (15). At 72 Materials and Methods h postinfection, cells were collected by centrifugation at 800 × g for 5 min. Insect and Cell Lines. Adult female moths of O. latipennis were collected in Fatty acid in the cell pellet was extracted with 100 μL of chloroform/methanol Semboku, Akita, Japan (39.4°N, 140.4°E), and the offspring were reared on (2:1), saponified in 200 μL of 0.5 N KOH/methanol at room temperature for 1 h, an artificial diet (Nosan Corp.) at 25 °C under a photoperiod of 16-h light 8-h and subsequently acidified with 1 N HCl. To perform the GC-MS analysis of free dark (9). The Sf9 cells were maintained in TC-100 medium with 10% FBS, as fatty acids, the samples were methyl-esterified with diazomethane. described previously (24). Chemicals and GC-MS Analysis. Authentic (E)-11-tetradecanoic acid (E11–14: fl RNA Extraction. PG, ight muscle, legs, epidermis, fat bodies, and eggs were COOH) and (Z)-11-tetradecanoic acid (Z 11–14:COOH) were synthesized from dissected from 1- to 3-d-old virgin female moths in PBS [2.5 mM KCl, 141 mM corresponding alcohols purchased from Pherobank. The fatty acid phero- NaCl, 8.1 mM Na2HPO4, and 2.5 mM KH2PO4 (pH 7.0)]. Total RNA was iso- mone analogs in the extract from the female sex pheromone gland of O. lated from each tissue using RNAiso (Takara Bio), treated with DNase I latipennis were transesterified by alkaline methanolysis according to the – (Qiagen), and stored at 80 °C. method of Foster (26). A QP5050 GC-mass analyzer system (Shimadzu) equipped with a capillary column (DB-Wax, 30 m × 0.25 mm i.d., J & W Cloning of latpg1 cDNA. One microgram of total RNA was reverse-transcribed Scientific) was used for the analysis of fatty acid methyl esters. All analytical with an oligo-dT adaptor primer using an RNA PCR Kit, Ver.3.0 (Takara Bio). conditions were as described previously (27). The resultant cDNA was used as a template for cloning of latpg1 cDNA. De- generate primers (Table S1) were designed based on sequence information ACKNOWLEDGMENTS. We thank Dr. Takuma Takanashi for his valuable on the histidine-cluster (His-box), a typical motif in nonheme desaturases (Fig. comments during the course of this study. This work was supported by 3). PCR was performed with ExTaq polymerase (Takara Bio) under the fol- Grants 19208005 (to Y.I.), 19688004 (to S.K.), and 22128004 (to T.S.) from lowing conditions: 94 °C for 2 min, 5 cycles of 94 °C for 30 s, 37 °C for 30 s, and the Ministry of Education, Culture, Sports, Science, and Technology.

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