Comparative Biochemistry and Physiology, Part D 15 (2015) 28–38

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Comparative Biochemistry and Physiology, Part D journal homepage: www.elsevier.com/locate/cbpd

Identification and expression pattern of candidate olfactory genes in Chrysoperla sinica by antennal transcriptome analysis Zhao-Qun Li a,b, Shuai Zhang a, Jun-Yu Luo a, Si-Bao Wang a,b, Chun-Yi Wang a, Li-Min Lv a, Shuang-Lin Dong b,⁎, Jin-Jie Cui a,⁎⁎ a b

State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang 455000, China Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

a r t i c l e

i n f o

Article history: Received 21 January 2015 Received in revised form 15 April 2015 Accepted 27 May 2015 Available online 3 June 2015 Keywords: Transcriptomic analysis Odorant-binding proteins Chemosensory proteins Odorant receptors Chrysoperla sinica

a b s t r a c t Chrysoperla sinica is one of the most prominent natural enemies of many agricultural pests. Host seeking in insects is strongly mediated by olfaction. Understanding the sophisticated olfactory system of insect antennae is crucial for studying the physiological bases of olfaction and could also help enhance the effectiveness of C. sinica in biological control. Obtaining olfactory genes is a research priority for investigating the olfactory system in this species. However, no olfaction sequence information is available for C. sinica. Consequently, we sequenced female- and male-antennae transcriptome of C. sinica. Many candidate chemosensory genes were identified, including 12 odorant-binding proteins (OBPs), 19 chemosensory proteins (CSPs), 37 odorant receptors (ORs), and 64 ionotropic receptors from C. sinica. The expression patterns of 12 OBPs, 19 CSPs and 37 ORs were determined by RT-PCR, and demonstrated antennae-dominantly expression of most OBP and OR genes. Our finding provided large scale genes for further investigation on the olfactory system of C. sinica at the molecular level. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Chrysoperla sinica is one of the most important natural predators of various pests, such as aphids, coccids, thrips, mites, planthoppers, and the eggs and young larvae of lepidopteran insects (Winterton and de Freitas, 2006), in many different cropping systems, including maize, cotton, and rice (Brooks, 1994; Bai et al., 2005). Thus, they are very useful auxiliaries in biological control. Understanding the olfactory systems of predatory insects can improve biological control in sustainable agriculture. Host seeking in insects is strongly mediated by olfaction (Zhou, 2010). In insects, olfactory genes are expressed in and around olfactory receptor neurons (ORN). The antennae, which are covered with several different types of sensilla, are specialized insect organs for olfaction. According to current thinking the major olfactory-related gene families in insects are odorant-binding proteins (OBP), chemosensory proteins (CSP), odorant receptors (OR), and ionotropic receptors (IR) (Leal, 2012). OBPs, abundant small proteins found in the sensilla lymph, are the liaison between the external environment and ORs (Vogt and

⁎ Correspondence to: S.-L. Dong, Entomology Department, Nanjing Agricultural University, No. 1, Weigang, Nanjing 210095 P.R. China. Tel./fax: +86 25 84399062. ⁎⁎ Correspondence to: J.-J. Cui, Plant Protection Department, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, No. 38, Huanghe Road Anyang 455000, Henan, P.R. China. Tel./fax: +86 372 2562296. E-mail addresses: [email protected] (S.-L. Dong), [email protected] (J.-J. Cui).

http://dx.doi.org/10.1016/j.cbd.2015.05.002 1744-117X/© 2015 Elsevier Inc. All rights reserved.

Riddiford, 1981; Pelosi et al., 2006). External hydrophobic odorant molecules reaching the pore tubules enter an antennal sensillum and are captured and solubilized by OBPs (Zhou, 2010; Leal, 2012; Li et al., 2013a) then delivered to the surface of ORN dendrites through the sensillum lymph (Pelosi and Maida, 1995; Pelosi et al., 2006). CSPs are also small soluble proteins found in abundance within the sensilla lymph (Gong et al., 2007). CSPs are broadly expressed in many organs, including antennae (Jacquin-Joly et al., 2001; González et al., 2009), proboscises (Nagnan-Le Meillour et al., 2000), legs (Kitabayashi et al., 1998), wings (Ban et al., 2003), and pheromone glands (Jacquin-Joly et al., 2001), as well as other tissues. Although CSPs are thought to affect insect chemoreception by enhancing the solubility of semiochemicals and delivering them to the chemosensory receptors (Jacquin-Joly et al., 2001; Zhang et al., 2014), little is known about how they work. Insect ORs are seven-transmembrane-domain proteins located in the dendrite membrane of ORNs and have an intracellular N-terminus and an extracellular C-terminus (Rutzler and Zwiebel, 2005; Hallem et al., 2006). Since the first insect ORs were discovered in the fruit fly (Clyne et al., 1999; Vosshall et al., 1999), multiple homologs have been identified (Robertson and Wanner, 2006; Bohbot et al., 2007; Engsontia et al., 2008; Li et al., 2013b; Zhang et al., 2013; Cao et al., 2014b). ORs play a key role in detecting odorants and triggering the transduction of chemical signals to electric signals (Liu et al., 2013). This signal transduction process involves the odorant receptor coreceptor (ORco), which is highly conserved among insect species (Nakagawa et al., 2012). It forms stand-alone heteromeric complexes with other ORs as a ligand-gated ion channel and enhances odorant

Z.-Q. Li et al. / Comparative Biochemistry and Physiology, Part D 15 (2015) 28–38

2. Materials and methods

Table 1 Summary of Chrysoperla sinica transcriptomes.

2.1. Insect samples

C. sinica

Total number of raw reads Total number of clean reads Total number of transcripts Total number of combined transcripts

29

Female

Male

68,882,138 63,977,252 75,937 68,646

70,468,008 65,061,400 89,541

responsiveness without altering ligand specificity (Neuhaus et al., 2005; Benton et al., 2006). IRs are a large and highly divergent family of ionotropic glutamate receptor (iGluR)-related genes whose products concentrate in the sensory cilia where chemical detection takes place. IRs are thought to play important roles in insect chemoreception (Benton et al., 2009; Rytz et al., 2013). Previous studies revealed that IRs also coexpress in a single sensory neuron, thus, these receptors also form multimeric protein assemblies with subunit-dependent characteristics (Benton et al., 2009). Based on their widespread homologs in different species, IRs represent a more ancient lineage of chemosensory receptors than ORs (Benton et al., 2009). Deep sequencing data can provide extensive information about genes and gene expression. A large number of chemosensory genes have been identified from many insects by high-throughput sequencing (Li et al., 2013b; Younus et al., 2014; Zhou et al., 2014). To identify more chemosensory genes of C. sinica, the female- and male-antennal transcriptomes were sequenced in this study. In total, 19 CSP, 12 OBP, 36 OR, and 64 IR genes from C. sinica were obtained. Furthermore, the gene expression profiles of the OBP, CSP, and OR genes were compared among tissues, and the antennal abundances of CSP, OBP, OR, and IR transcripts were examined.

The C. sinica used in this experiment were collected from a cotton field at the Institute of Cotton Research, CAAS. Experimental insects were the offspring of a single female and reared in the laboratory on Acyrthosiphon pisum. Rearing conditions were 25 ± 1 °C, 14:10 L:D cycle, and 65 ± 5% relative humidity (RH). To keep the insects in virgin status, pupae were kept in separate cages for eclosion. The pupae of C. sinica were checked daily for emergence and supplied with 10% honey solution. Antennae from 600 3-day-old virgin females and males of C. sinica were collected respectively, immediately frozen in liquid nitrogen, and stored at −80 °C for transcriptome sequencing. One hundred 3-day-old virgin females and males of each species were used to collect antennae, heads, wings, legs, thoraxes, and abdomens. Tissue samples were kept at − 80 °C until RNA isolation, with three replicates. 2.2. cDNA library construction and Illumina sequencing Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and the quality of the RNA was checked with Agilent 2100. cDNA library construction and Illumina sequencing of the samples were performed at the Beijing Genomics Institute, Shenzhen, China (Zhang et al., 2010). Briefly, Poly-adenylated RNAs were isolated from 20 μg of pooled total RNA using oligo (dT) magnetic beads and fragmented into short fragments in the presence of divalent cations in fragmentation buffer at 94 °C for 5 min. Using these cleaved, short fragments as templates, random hexamer primers were used to synthesize first-strand cDNA. Second-strand cDNA was generated

Fig. 1. Distribution of Chrysoperla sinica genes annotated by GO. The x axis shows three categories and their subcategories. The y axis shows the percentage of sequences. The analysis level was 3.

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Table 2 BLASTX results for putative OBPs, CSPs and ORs in the C. sinica. Gene Name

ID

Length (bp)

FPKM

Best Blastx Match

Female

Male

Name

Species

E-value

Identity

Acc.number

CsinOBPs OBP1 OBP2 OBP3 OBP4 OBP5 OBP6 OBP7 OBP8 OBP9 OBP10 OBP11 OBP12

CL2433-1 CL3032-2 CL3668-2 CL3786-2 CL3942-2 CL4062-1 CL4336-1 CL6620-3 CL6814-1 CL7150-1 U14525 U17780

678 637 625 783 585 909 1265 933 862 836 1098 767

2478.63 4469.20 1283.83 1497.97 261.17 847.88 23.20 2628.27 2204.96 1209.83 3.13 423.40

2218.82 3986.45 1210.41 1408.75 316.89 754.18 29.29 2843.80 1830.03 1354.96 5.61 371.56

odorant binding protein 15 odorant-binding protein pheromone-binding protein 6 odorant-binding protein 8 odorant binding protein pheromone binding protein 1 AgamOBP56 odorant-binding protein pheromone binding protein 1 odorant binding protein 6 odorant binding protein odorant-binding protein 18a

Tribolium castaneum Dendroctonus ponderosae Tribolium castaneum Sogatella furcifera Spodoptera exigua Loxostege sticticalis Anopheles gambiae Delia antiqua Loxostege sticticalis Tribolium castaneum Dendrolimus houi Lygus lineolaris

1.00E-16 1.00E-35 3.00E-29 3.00E-12 3.00E-13 9.00E-19 1.00E-06 5.00E-14 2.00E-09 5.00E-46 6.00E-56 4.00E-11

33% 47% 44% 34% 36% 37% 33% 28% 30% 59% 52% 33%

EFA12066 AFI45061 XP_008197708 AHB59654.1 ADY17884 ACF48467 AAQ16288 BAN59724 ACF48467 EFA04594 AII00978.1 AHF71046.1

CsinCSPs CSP1 CSP2 CSP3 CSP4 CSP5 CSP6 CSP7 CSP8 CSP9 CSP10 CSP11 CSP12 CSP13 CSP14 CSP15 CSP16 CSP17 CSP18 CSP19

CL425-1 CL632-2 CL3308-2 CL4535-2 CL6215-1 U6038 U9462 U9723 U9941 U10090 U16878 U19710 U20431 U22439 U24278 U28705 U33225 U42589 U44935

614 376 521 801 370 338 294 367 459 417 366 237 579 631 517 704 550 299 330

1749.69 800.43 1.79 994.37 3841.88 2.21 2.42 6.42 67.21 433.01 3.17 57.11 95.00 33.36 9.26 434.84 5.85 1.75 1.81

1066.83 674.79 5.59 1096.80 3293.09 4.42 2.66 7.73 139.39 808.76 3.87 30.38 77.64 181.69 6.78 445.40 18.11 4.12 3.50

chemosensory protein 11 CSP1 chemosensory protein 11 CSP1 chemosensory protein 6 CSP2 chemosensory protein 6 chemosensory protein 14 chemosensory protein 3 chemosensory protein 3 chemosensory protein 2 chemosensory protein 3 chemosensory protein 11 chemosensory protein 2 chemosensory protein 7 CSP chemosensory protein 8 chemosensory protein 11 chemosensory protein 2

Helicoverpa armigera Spodoptera exigua Tribolium castaneum Plutella xylostella Dendroctonus ponderosae Helicoverpa armigera Tribolium castaneum Tribolium castaneum Glossina morsitans Glossina morsitans Apis mellifera Glossina morsitans Helicoverpa armigera Dendroctonus ponderosae Tribolium castaneum Stomoxys calcitrans Dendroctonus ponderosae Helicoverpa armigera Glossina morsitans

2.00E-23 3.00E-18 4.00E-34 1.00E-40 4.00E-19 4.00E-67 1.00E-12 5.00E-19 2.00E-34 5.00E-24 1.00E-35 6.00E-14 5.00E-35 5.00E-40 9.00E-27 2.00E-39 4.00E-42 7.00E-56 1.00E-18

43% 53% 50% 53% 55% 100% 42% 55% 51% 41% 86% 52% 53% 53% 58% 56% 56% 93% 45%

AFR92095 ABM67688 NP_001039279 ABM67686 AGI05162 AEX07265 NP_001039288 NP_001039282 CBA11329 CBA11329 NP_001071278 CBA11329 AFR92095 AGI05172 NP_001039289 ADG96053 AGI05164 AFR92095 CBA11328

CL1552-4 CL1761-2 CL2040-1 CL278-1 CL368-3 CL469-8 CL5285-1 CL7802-1 CL840-1 U10075 U10247 U11112 U11697 U14839 U17093 U20337 U20586 U20820 U20990 U21956 U22246 U22590 U27481 U28219 U28638 U28949 U29213 U29908 U3089 U38428 U4259 U44584 U47556 U4930 U7706 U9287 U7957

1930 551 2084 561 1170 1361 331 613 819 570 444 940 729 1372 651 489 650 431 290 1298 634 589 589 359 595 1537 1154 963 435 231 1700 334 413 1027 583 326 987

12.48 1.15 13.53 0.20 4.89 1.22 4.21 7.56 5.07 3.28 2.69 4.65 3.85 4.99 4.77 3.14 4.49 3.21 3.22 4.03 3.66 6.03 7.32 6.67 15.27 2.65 7.03 6.37 2.92 3.88 4.97 1.46 1.82 4.88 3.78 3.33 50.27

15.48 2.98 11.42 0.13 5.74 1.25 4.08 7.85 4.60 2.81 3.70 4.09 3.17 4.92 3.21 3.59 4.53 4.24 2.83 4.17 2.18 5.32 5.80 9.14 19.56 3.16 5.14 5.58 2.83 4.52 4.92 3.24 1.99 4.00 3.33 3.32 40.63

odorant receptor Or1 odorant receptor 59 odorant receptor OR28 odorant receptor 12 odorant receptor 44 odorant receptor Or2 odorant receptor 24 odorant receptor 64 odorant receptor 64 olfactory receptor 16 odorant receptor 66 odorant receptor 37 olfactory receptor, odorant receptor 13a odorant receptor OR28 odorant receptor 58 odorant receptor 64 odorant receptor 73 odorant receptor 43 Or83a odorant receptor 10 odorant receptor 63a olfactory receptor 4 candidate olfactory odorant receptor 59 odorant receptor 64 odorant receptor 37 odorant receptor 37 odorant receptor 73 candidate olfactory receptor odorant receptor 102 odorant receptor 93 odorant receptor 70 odorant receptor 16 odorant receptor 102 odorant receptor 73 olfactory receptor 16

Megachile rotundata Tribolium castaneum Cydia pomonella Tribolium castaneum Tribolium castaneum Bombus impatiens Tribolium castaneum Tribolium castaneum Tribolium castaneum Danaus plexippus Tribolium castaneum Tribolium castaneum Aedes aegypti Harpegnathos saltator Cydia pomonella Tribolium castaneum Tribolium castaneum Tribolium castaneum Tribolium castaneum Drosophila pseudoobscur Tribolium castaneum Megachile rotundata Helicoverpa armigera Bombyx mori Tribolium castaneum Tribolium castaneum Tribolium castaneum Tribolium castaneum Tribolium castaneum Bombyx mori Tribolium castaneum Tribolium castaneum Tribolium castaneum Tribolium castaneum Tribolium castaneum Tribolium castaneum Tribolium castaneum

2.00E-19 1.00E-29 1.00E-38 7.00E-14 1.00E-36 4.00E-38 8.00E-06 7.00E-18 5.00E-10 2.00E-13 2.00E-05 2.00E-36 6.00E-27 6.00E-29 3.00E-10 6.00E-20 2.00E-11 6.00E-24 1.00E-08 1.00E-15 9.00E-14 4.00E-12 5.00E-09 1.00E-04 2.00E-11 5.00E-49 2.00E-54 2.00E-22 7.00E-23 1.00E-10 2.00E-14 7.00E-10 0.006 1.00E-06 2.00E-15 0.067 5.00E-141

23% 44% 25% 36% 29% 30% 31% 35% 20% 39% 24% 34% 30% 34% 30% 32% 31% 50% 38% 21% 29% 27% 29% 47% 47% 32% 35% 39% 51% 54% 34% 31% 32% 30% 39% 36% 72%

XP_003707057 EEZ99171 AFC91736.1 EFA09172 EEZ99412 XP_003488417 EFA10799 EFA10800 EFA10800 EHJ70341 EEZ97727 EEZ99229 XP_001651429 EFN78840 AFC91736 EEZ99414 EFA10800 EFA05710 EEZ99411 XP_001359366 EFA09294 XP_003706296 ACF32962 NP_001091788 EEZ99171 EFA10800 EEZ99229 EEZ99229 EFA05710 NP_001091788 EEZ97750 EFA02941 EEZ99312 EFA09170 EEZ97750 EFA05710 CAM84014

CsinORs OR1 OR2 OR3 OR4 OR5 OR6 OR7 OR8 OR9 OR10 OR11 OR12 OR13 OR14 OR15 OR16 OR17 OR18 OR19 OR20 OR21 OR22 OR23 OR24 OR25 OR26 OR27 OR28 OR29 OR30 OR31 OR32 OR33 OR34 OR35 OR36 ORco CsinIRs

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Table 2 (continued) Gene Name

ID

Length (bp)

FPKM Female

IR1 IR2 IR3 IR4 IR5 IR6 IR7 IR8 IR9 IR10 IR11 IR12 IR13 IR14 IR15 IR16 IR17 IR18 IR19 IR20 IR21 IR22 IR23 IR24 IR25 IR26 IR27 IR28 IR29 IR30 IR31 IR32 IR33 IR34 IR35 IR36 IR37 IR38 IR39 IR40 IR41 IR42 IR43 IR44 IR45 IR46 IR47 IR48 IR49 IR50 IR51 IR52 IR53 IR54 IR55 IR56 IR57 IR58 IR59 IR60 IR61 IR62 IR63 IR64

CL1079-1 CL1079-2 CL1212-1 CL1212-2 CL1360-1 CL1360-2 CL1858-1 CL3277-1 CL3589-2 CL362-1 CL392-1 CL4309-2 CL5116-1 CL5275-2 CL5550-1 CL5880-1 CL5880-3 CL6515-3 CL7099-1 CL7099-2 CL7778-1 CL7944-4 CL8150-1 U11173 U12102 U13547 U15037 U16248 U18087 U18195 U18250 U18427 U18489 U19043 U21141 U21611 U21612 U22829 U25174 U25328 U25336 U26307 U27967 U27974 U28841 U29115 U29138 U29810 U29889 U30540 U30969 U31103 U3160 U32197 U32592 U3354 U35912 U37707 U38780 U39025 U3922 U6999 U7493 U9577

1996 1142 513 1022 401 1480 1090 2008 960 2701 3906 1351 522 1243 2477 216 1412 492 449 223 1894 1619 422 661 1170 527 600 1712 1715 709 1393 1233 3264 1715 318 709 1549 763 1812 1465 1210 1819 486 527 1712 667 959 1153 1969 354 257 376 281 870 1109 263 401 263 206 333 984 446 931 375

31.42 12.21 6.12 1.87 2.98 2.70 3.02 9.79 8.65 55.32 71.64 6.66 2.44 3.40 4.39 7.10 8.10 0.68 8.66 5.20 49.24 51.64 1.51 3.34 5.08 4.61 6.42 5.05 9.81 2.16 15.60 5.40 118.81 8.72 22.11 11.44 15.71 5.10 16.43 7.17 11.40 9.97 6.23 3.69 5.29 8.41 5.73 3.15 8.83 2.64 2.18 9.55 3.06 7.01 12.41 2.42 3.17 3.13 2.54 5.39 6.12 3.77 6.43 5.09

Best Blastx Match Male 25.72 10.62 6.40 1.93 1.95 3.13 2.46 10.20 8.08 36.53 52.47 5.32 2.43 2.91 3.64 2.94 5.97 0.46 8.97 7.03 29.59 36.43 2.65 2.20 6.54 3.89 4.41 4.23 10.14 3.05 19.90 7.23 110.48 8.33 18.65 9.52 15.61 3.72 15.22 6.57 14.06 8.47 5.14 3.89 5.16 7.10 5.52 3.07 7.48 1.37 2.18 3.18 2.66 6.48 16.05 1.99 2.23 0.99 1.99 2.69 7.01 3.43 4.65 4.78

Name

Species

E-value

Identity

Acc.number

ionotropic glutamate receptor ionotropic glutamate receptor ionotropic receptor ionotropic receptor ionotropic receptors ionotropic receptor 75d ionotropic receptor ionotropic receptor ionotropic receptor 60a ionotropic receptor ionotropic receptor IR75q2 IR41a IR41a ionotropic glutamate receptor ionotropic receptors IR75q2 ionotropic receptors IR75q2 ionotropic receptors ionotropic receptors ionotropic receptor 8a Ir6 ionotropic receptors IR41a IR21a ionotropic receptor IR68a ionotropic receptors IR41a IR75q.2 ionotropic receptors ionotropic receptors Ir87a ionotropic glutamate receptor ionotropic receptor IR41a IR41a ionotropic receptors ionotropic receptors ionotropic receptors IR41a ionotropic receptors ionotropic receptors IR41a ionotropic glutamate receptor ionotropic receptor IR41a ionotropic glutamate receptor ionotropic receptors ionotropic receptor 75d ionotropic receptor ionotropic receptors glutamate receptor ionotropic glutamate receptor ionotropic glutamate receptor ionotropic glutamate receptor ionotropic IR41a IR87a IR41a ionotropic receptors ionotropic receptor IR25a IR41a ionotropic receptor IR87a ionotropic receptors

Danaus plexippus Danaus plexippus Calliphora stygia Calliphora stygia Dendrolimus houi Drosophila melanogaster Calliphora stygia Aedes aegypti Drosophila melanogaster Tribolium castaneum Tribolium castaneum Cydia pomonella Spodoptera littoralis Spodoptera littoralis Danaus plexippus Dendrolimus houi Cydia pomonella Dendrolimus houi Cydia pomonella Dendrolimus houi Acromyrmex echinatior Schistocerca gregaria Pleurobrachia bachei Tribolium castaneum Spodoptera littoralis Spodoptera littoralis Helicoverpa armigera Spodoptera littoralis Bombyx mori Spodoptera littoralis Spodoptera littoralis Tribolium castaneum Tribolium castaneum Cydia pomonella Pediculus humanus corporis Cydia pomonella Spodoptera littoralis Dendrolimus houi Bombyx mori Dendrolimus kikuchii Spodoptera littoralis Dendrolimus kikuchii Homarus americanus Spodoptera littoralis Aedes aegypti Cydia pomonella Aedes aegypti Dendrolimus kikuchii Drosophila melanogaster Calliphora stygia Tribolium castaneum Nasonia vitripennis Tribolium castaneum Nasonia vitripennis Bombyx mori Spodoptera littoralis Cydia pomonella Spodoptera littoralis Dendrolimus houi Cydia pomonella Spodoptera littoralis Calliphora stygia Cydia pomonella Tribolium castaneum

7.00E-32 3.00E-37 8E-12 4.00E-17 5.00E-29 6.00E-111 1.00E-28 2E-72 1.00E-05 5.00E-168 0.00E + 00 4.00E-47 3E-27 2.00E-46 2.00E-85 3.00E-11 3.00E-55 2E-18 7.00E-15 4.00E-05 3.00E-159 2.00E-123 0.7 1.00E-53 3.00E-25 1.00E-05 5.00E-09 0 1.00E-49 1.00E-18 9.00E-63 2.00E-06 0 1.00E-30 2.00E-05 5.00E-38 1E-65 2.00E-22 2.00E-81 1.00E-114 7.00E-50 3E-105 7.00E-17 2.00E-23 4.00E-70 3.00E-50 9E-38 9.00E-33 3.00E-110 6.00E-20 5.00E-19 0.00005 7.00E-35 3.00E-36 3.00E-38 5.00E-09 3.00E-08 2.00E-05 8.00E-17 2.00E-07 5E-33 1.00E-19 4.00E-09 3.00E-32

32% 30% 27% 24% 43% 43% 51% 32% 29% 50% 76% 34% 36% 29% 31% 55% 32% 35% 41% 43% 44% 69% 25% 50% 33% 28% 29% 55% 31% 34% 33% 30% 53% 26% 33% 41% 30% 33% 31% 47% 47% 44% 69% 34% 34% 45% 40% 30% 36% 47% 58% 36% 93% 31% 34% 33% 28% 37% 74% 34% 27% 38% 26% 57%

EHJ76038 EHJ76038 AID61276 AID61276 AII01117 NP_649074.2 AID61290 XP_001651553 NP_611901 XP_975120 XP_008200796 AFC91752 ADR64681 ADR64681 EHJ76038 AII01119 AFC91752 AII01116 AFC91752 AII01119 EGI68048 AHA80144 AEX15544 XP_008200796 ADR64681 ADR64678 AIG51919 ADR64682 XP_004929596 ADR64681 ADR64685 XP_008195465 XP_008201658 AFC91760 XP_002424150 AFC91758 ADR64681 AII01116 XP_004933515 AII01129 ADR64681 AII01129 AAM47017 ADR64681 XP_001661131 AFC91758 XP_001661131 AII01127 NP_649074.2 AID61284 XP_008198539 XP_008203135 XP_008200796 XP_008208508 XP_004929596 ADR64681 AFC91760 ADR64681 AII01113 AFC91757 ADR64681 AID61277 AFC91760 XP_008200796

using buffer, dNTPs, RNAseH, and DNA polymerase I. Following end repair and adaptor ligation, short sequences were amplified by PCR and purified with a QIAquick® PCR extraction kit (Qiagen, Venlo, The Netherlands), and sequenced on a HiSeq™ 2000 platform (San Diego, CA, USA).

2.3. Assembly and function annotation Transcriptome de novo assembly was carried out with the short read assembly program Trinity (Grabherr et al., 2011), which generated two classes of transcripts: clusters (prefix CL) and singletons (prefix U).

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Transcripts larger than 150 bp were aligned by BLASTX to the protein database, including Nr, Swiss-Prot, KEGG (Kanehisa et al., 2008), and COG (E-value b10− 5) databases, retrieving proteins with the highest sequence similarity with the given transcripts along with their protein functional annotations. We then used the Blast2GO program (Conesa et al., 2005) for GO annotation of the transcripts and WEGO software (Ye et al., 2006) to plot the GO annotation results. 2.4. RNA isolation and cDNA synthesis for RT-PCR Total RNA was isolated using SV Total Isolation System (Promega, Madison, WI, USA) according to the manufacturer’s instructions, and a spectrophotometer (NanoDrop™ 2000c, Thermo Fisher Scientific, Waltham, MA, USA) was used to check mRNA quality. Single-stranded cDNA templates were synthesized using 1 μg of RNA from various samples with the Reverse Transcription System (Promega, Madison, WI, USA) following the instruction manual.

calculating expression abundance (Mortazavi et al., 2008). The formula is: FPKM ðAÞ ¼

C  106 NL 103

FPKM (A) is the expression of gene A; C is the number of reads that uniquely align to gene A; N is the total number of fragments that uniquely align to all transcripts; and L is the number of bases in gene A. 2.7. Expression analysis by semi-quantitative RT-PCR analysis

The amino acid sequence alignment of the candidate OBPs and CSPs were performed using CLUSTALX 2.0 (Larkin et al., 2007) and then arranged by Jalview 2.4.0 b2 (Waterhouse et al., 2009). The candidate OBPs, CSPs and ORs of C. sinica were chosen for phylogenetic analysis along with CSPs from other insect species. All the phylogenetic trees were constructed using the neighbour-joining method implemented in MEGA6 (Tamura et al., 2011) with default settings and 1000 bootstrap replicates.

Gene-specific primers of putative olfactory genes were designed using Beacon Designer 7.7 (PREMIER Biosoft, Palo Alto, CA, USA) (listed in Table S1). PCR experiments, including negative controls (no cDNA template), were carried out in a MyCyclerTM (Bio-Rad, Hercules, CA, USA) as follows: 94 °C for 2 min; followed by 28 cycles of 94 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 30 sec. The reactions were performed in 20-μL RT-PCR reaction mixes containing 2.0 μL of 10 × ExTaq PCR buffer, 1.6 μL of deoxyribonucleotide triphosphate (10 mM), 0.8 μL of forward primer (10 μM), 0.8 μL of reverse primer (10 μM), 15 ng of single-stranded cDNA, and 0.2-μL Ex-Taq (5 U/μL). The PCR products were analysed by electrophoresis on 2.0% w/v agarose gels in TAE buffer, and the resulting bands were visualised with GluRed. The marker of DL2000 (TaKaRa, Dalian, Liaoning, China) were used and the PCR products reviewed single bands of the expected size. The GTPbinding protein gene was selected as the endogenous control to check the integrity of the cDNA templates (Li et al., 2013b). Each reaction was run in three independent biological replicates.

2.6. Analysis of transcript expression in the two lacewing species antennae

3. Results

The transcript expression abundances were calculated by the FPKM (Fragments Per Kilobase per Million mapped Reads) method (Mortazavi et al., 2008), which can eliminate the influence of different gene lengths and sequencing discrepancies in

3.1. Overview of transcriptomes

2.5. Phylogenetic analysis

The female- and male-antennal transcriptomes of C. sinica were sequenced by Illumina sequencing. A total of 65.7 and 67.2 million raw

Fig. 2. Alignment of amino acid sequences of candidate OBPs and CSPs from Chrysoperla sinica. (A): Amino acid alignment of the candidate OBPs. (B): Amino acid alignment of the candidate CSPs. Predicted signal peptides are boxed, and the conserved cysteines are labelled with red stars.

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reads were obtained from the C. sinica female- and male-antennae libraries (Table 1). After removing low-quality, adaptor, and contaminating sequences, 61.0 and 62.0 million clean reads were generated and assembled into 75,937 and 89,541 distinct transcripts, respectively. The clean reads are available from the NCBI/SRA database (accession SRR1653342). To annotate the transcripts, we combined the female- and maleantennal transcriptomes of the same insect and searched against the Nr, Swiss-prot, KEGG, COG, and GO databases by BLASTX with a cutoff e-value of 10−5. Totals of 28,132 (40.98% of all 68,646 distinct sequences), 23,131 (45.41%), 20,587 (40.29%), 12,632 (18.4%), and 14,445 (21.04%) transcripts were annotated by those respective databases in C. sinica. GO annotation was used to classify the functions of these transcripts from the transcriptome of C. sinica. Biological process, cellular process and metabolic process were the most abundant GO terms. Cell and cell part were the most prevalent within cellular component. Most transcripts that corresponded to molecular function were related to binding and

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catalytic activity (Fig. 1). The species distributions of the transcripts in the NR protein database were noted. The C. sinica sequences exhibited substantial matches with Tribolium castaneum.

3.2. Identification of putative OBP genes Sequence annotation led to the identification of 12 different candidate OBPs in C. sinica (Table 2). Sequence analysis revealed that 11 OBPs had full-length open reading frames (ORF) with a predicted signal peptide sequence. Sequences alignment showed that OBP5 belonged to the minus-C OBP family (with four conserved cysteines); OBP7, OBP8, and OBP12 were the members of the atypical OBP family (10 conserved cysteines); OBP11 have 5 conserved cysteines; and the other 7 OBPs were classic OBPs (with the typical signature of six conserved cysteines) (Fig. 2A). We use the 12 CsinOBPs along with OBPs from other species to construct a phylogenetic tree based on amino acid sequences (Fig. 3). The tree revealed that all the candidate OBPs belong to orthologous

Fig. 3. Phylogenetic analysis of putative odorant binding proteins of Chrysoperla sinica. The tree was constructed in MEGA6.0 using the neighbour-joining method. Values at the nodes are bootstrap percentages (1000 replicates) greater than 50%. Genes from C. sinica are labelled with red. Tcas: Tribolium castaneum, Dmel: Drosophila melanogaster.

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sequence groups in the other species. Nine of them show showed higher homology to OBPs from T. castaneum. 3.3. Identification of putative CSP genes By homology analysis, we identified 19 transcripts that belong to CSP gene family in C. sinica (Table 2). Sequence analysis displayed that 12 of the 19 CSP genes were full-length CSP genes had intact open reading frames (ORF) with four conserved cysteine residues, a predicted signal peptide sequence, and a general length of 400 bp, which are characteristic of insect CSPs. These 19 candidate CsinCSPs were phylogenetically analyzed with CSPs from five other species based on amino acid sequences (Fig. 4). The tree showed that most of CSPs from the same specie formed monophyletic groups. Eight CsinCSPs grouped into one orthologous family which was expanded in C. sinica.

3.4. Identification of candidate chemosensory receptors In total, we identified 37 ORs, and 64 IRs from C. sinica antennal transcriptomes (Table 2). Sequence analysis revealed that 8 of the 37sequences were full-length OR genes with characteristics typical of insect OR genes, full length ORFs about 1200 bp, and seven transmembrane domains. The CsinORs were used for phylogenetic clustering with ORs from T. castaneum (Fig. 5). Most CsinORs were distributed among the orthologous groups in the phylogenetic tree. ORs from C. sinica and T. castaneum were clustered into four expansion families, with one lineage-specific expansion. CsinORco were tightly associated with TcasOR1 with high bootstrap support. These two members of the ORco family serve as chaperones with the other ORs. As a novel family of candidate chemosensory ionotropic receptors, 64 IRs were identified from C. sinica antennal transcriptome (Table 2).

Fig. 4. Phylogenetic analysis of putative chemosensory proteins of and Chrysoperla sinica. The tree was constructed in MEGA6.0 using the neighbour-joining method. Values at the nodes are bootstrap percentages (1000 replicates) greater than 50%. Genes from and C. sinica are labelled with red. Tcas: Tribolium castaneum, Dmel: Drosophila melanogaster, Bmor: Bombyx mori, Apis: Acyrthosiphon pisum, Amel: Apis mellifera.

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Fig. 5. Phylogenetic analysis of putative odorant receptors of Chrysoperla sinica. The tree was constructed in MEGA6.0 using the neighbour-joining method. Values at the nodes are bootstrap percentages (1000 replicates) greater than 50%. Genes from C. sinica are labelled with red. Tcas: Tribolium castaneum.

Additionally, 67 putative chemoreception genes, including 19 CSPs, 12 OBPs, and 36 ORs, were confirmed by sequencing their PCR products. The sequencing results showed that the 67 putative chemoreception genes had more than 99% identities at the nucleic acid level with corresponding sequences from the transcriptome. 3.5. Tissue expression profiles of putative olfactory genes The expression patterns of the 12 OBP candidates in different tissues were investigated using semi-quantitative RT-PCR (Fig. 6). The results showed that most OBPs were abundant mainly in female and male antennae, except OBP5 and OBP8. Six of the ten antennae abundant OBPs, OBP3, OBP4, OBP6, OBP7, OBP9, and OBP12, had antennae-specific expressions. OBP5 were predominantly expressed in antennae and wings of female and male. OBP8 was mainly expressed in antennae, head, and legs. The expression patterns of the 19 CSP candidate genes in different tissues were also investigated using semi-quantitative RT-PCR (Fig. 6). Results showed that the CSP4, CSP6, CSP14, and CSP16 were only

expressed in antennae. Four CSP genes, CSP5, CSP6, CSP13 and CSP15, were expressed in antennae and wings. Among them, CSP6 and CSP15 were expressed particularly highly in antennae and CSP13 in wings. The CSP10 was mainly expressed in legs, while CSP8 was only expressed in legs. The other CSP genes were ubiquitous in most tissues tested at relatively high levels. We further characterized the expression levels and tissue distributions of OR genes (Fig. 6). The results showed that the OR genes were uniquely or more strongly expressed in antennae compared with other tissues. Most ORs were especially expressed in antennae, except for OR24 and OR25, which were expressed in other tissues. 3.6. Abundance analysis of putative olfactory genes The gene expression abundances in female and male antennae were calculated as fragments per kilobase per million mapped reads (FKPM). Among the 12 OBPs, OBP2 showed the highest expression, following by OBP8, OBP1, OBP9, OBP4, OBP10, OBP3 and etc. (Table 2). Of the 37 ORs, ORco showed the most abundant in antennae of C. sinica.

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Fig. 6. Tissue expression profiles of candidate OBPs, CSPs and ORs of Chrysoperla sinica. (A) Tissue expression profiles of candidate OBPs. (B) Tissue expression profiles of candidate CSPs. (C) Tissue expression profiles of candidate ORs. A: antennae, T: thorax, Ab: abdomen, H: heads without antennae, L: legs, W: wings. GTP binding protein was used as the positive control.

4. Discussion Chrysoperla sinica are important natural enemies of pests in many different cropping systems (Brooks, 1994; Bai et al., 2005; Winterton and de Freitas, 2006). Comparing with a large amount of studies on pests’ olfaction (Vogt et al., 2009; Gu et al., 2013; He and He, 2014; Sparks et al., 2014; Zhou et al., 2014; Liu et al., 2015), studies on natural enemy insects were scarce (Zhang et al., 2009; Li et al., 2013b; Donnell, 2014). Host seeking in insects is strongly mediated by olfaction, which is dependent on chemosensory genes (Zhou, 2010; Rinker et al., 2013). Understanding the olfactory systems of predatory insects can improve the effectiveness of C. sinica in biological control. Obtaining olfactory genes is a research priority for investigating the olfactory system in this species. However, no olfactory sequence information is available for C. sinica. Consequently, the female- and male-antennal transcriptomes of C. sinica were sequenced in this study. A total of 14 Gb of C. sinica antennae transcriptome data was obtained, which is larger than that from many other insects (Grosse-Wilde et al., 2011; Glaser et al., 2013; Zhang et al., 2013; Cao et al., 2014a; Cao et al., 2014b). After extensive sequencing and assembly, 19 CSPs, 12 OBPs, 36 ORs, and 64 IRs, were identified. Antennae-restricted expression is a useful criterion to identify genes involving in specific olfactory functions. Tissue distribution profiles of all

CSP, OBP, and OR genes were investigated in our study. The expression patterns of candidate olfactory genes in C. sinica may help characterize the function of these proteins in future researches. Our study showed that most of OBPs had relatively high expressed in antennae compared with other tissues, consistent with the antennae being the main olfactory organ in C. sinica and suggesting an olfactory role for these genes. OBP5 is rather broadly expression with relatively high levels of expression in antennae and wings of both female and male; therefore it may also be involved in gustatory function, as wings play somewhat gustatory roles in insects (Zhou et al., 2008). OBP8 was expressed in antennae, head, and legs at relatively high levels. This broad expression of OBPs may suggest different functions in chemoreception. Insect CSPs serve varied functions, including chemosensation (González et al., 2009) and development (Maleszka et al., 2007), as well as other processes (Kulmuni and Havukainen, 2013). The broad and diverse expression patterns suggest that different CSPs play different roles during the adult stage. Those genes highly expressed in antennae, CSP4, CSP6, CSP14, and CSP16, may be involved in insect chemoreception. All of the 37 ORs displayed relatively high expression levels in antennae, suggesting that they may play crucial roles in olfaction of C. sinica. Among the ORs, ORco gene was the most abundant in antennae of

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C. sinica, which is similar to AgosORco in A. gossypii (Cao et al., 2014a). Since ORco is a co-receptor in insects, which generally functions as a chaperoning receptor for the other ORs (Sato et al., 2008; Wicher et al., 2008), more ORco transcripts were detected in the antennae. In the meanwhile, besides ORco, three odorant receptors of OR1, OR3 and OR25 were also more abundant in the antennae compared to other ORs. This suggests that these three OR genes might play more important roles in the detection of C. sinica to host volatiles or sex pheromones, and further functional studies are needed in future. In conclusion, the female- and male-antennal transcriptomes of C. sinica were sequenced using next-generation sequencing technology. We identified 19 CSP, 12 OBP, 36 OR, and 64 IR genes from C. sinica. This large number of insect chemosensory genes will provide the basis for functional studies. As the crucial and first step towards understanding their functions, we conducted a comprehensive and comparative examination for the expression patterns of all the OBP, CSP and OR genes, and demonstrating predominantly chemosensory organ expression of most OBP and OR genes. Additionally, we provided large-scale sequence data for C. sinica. This large number of insect chemosensory and nonolfactory genes will provide the basis for further functional studies. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbd.2015.05.002. Acknowledgements This study was funded by research grants from the Ministry of Agriculture of China (2014ZX08011-002). References Bai, Y.Y., Jiang, M.X., A, C.J., 2005. Effects of transgenic cry1Ab rice pollen on the voviposition and adult longevity of Chrysoperla sinica Tjeder. Acta Phys. Sin. 32 (3), 225–230 (In Chinese with English abstract). Ban, L., Scaloni, A., Brandazza, A., Angeli, S., Zhang, L., Yan, Y., Pelosi, P., 2003. Chemosensory proteins of Locusta migratoria. Insect Mol. Biol. 12, 125–134. Benton, R., Sachse, S., Michnick, S.W., Vosshall, L.B., 2006. Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol. 4, e20. Benton, R., Vannice, K.S., Gomez-Diaz, C., Vosshall, L.B., 2009. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136, 149–162. Bohbot, J., Pitts, R.J., Kwon, H.W., Rutzler, M., Robertson, H.M., Zwiebel, L.J., 2007. Molecular characterization of the Aedes aegypti odorant receptor gene family. Insect Mol. Biol. 16, 525–537. Brooks, S.J., 1994. A taxonomic review of the common green lacewing genus Chrysoperla (Neuroptera: Chrysopidae). Bull. Brit. Mus. (Nat. Hist.)Entamol. Sin. 63, 137–210. Cao, D., Liu, Y., Walker, W.B., Li, J., Wang, G., 2014a. Molecular characterization of the Aphis gossypii olfactory receptor gene families. PLoS One 9, e101187. Cao, D., Liu, Y., Wei, J., Liao, X., Walker, W.B., Li, J., Wang, G., 2014b. Identification of candidate olfactory genes in Chilo suppressalis by antennal transcriptome analysis. Int. J. Biol. Sci. 10, 846–860. Clyne, P.J., Warr, C.G., Freeman, M.R., Lessing, D., Kim, J., Carlson, J.R., 1999. A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22, 327–338. Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M., Robles, M., 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3676. Donnell, D.M., 2014. Analysis of odorant-binding protein gene family members in the polyembryonic wasp. Copidosoma floridanum: Evidence for caste bias and host interaction. J. Insect Physiol. 60, 127–135. Engsontia, P., Sanderson, A.P., Cobb, M., Walden, K.K., Robertson, H.M., Brown, S., 2008. The red flour beetle's large nose: an expanded odorant receptor gene family in Tribolium castaneum. Insect Biochem. Mol. Biol. 38, 387–397. Glaser, N., Gallot, A., Legeai, F., Montagne, N., Poivet, E., Harry, M., Calatayud, P.A., JacquinJoly, E., 2013. Candidate chemosensory genes in the Stemborer Sesamia nonagrioides. Int. J. Biol. Sci. 9, 481–495. Gong, D.P., Zhang, H.J., Zhao, P., Lin, Y., Xia, Q.Y., Xiang, Z.H., 2007. Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 37, 266–277. González, D., Zhao, Q., McMahan, C., Velasquez, D., Haskins, W.E., Sponsel, V., Cassill, A., Renthal, R., 2009. The major antennal chemosensory protein of red imported fire ant workers. Insect Mol. Biol. 18, 395–404. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q.D., Chen, Z.H., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B.W., Nusbaum, C., Lindblad-Toh, K., Friedman, N.,

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Identification and expression pattern of candidate olfactory genes in Chrysoperla sinica by antennal transcriptome analysis.

Chrysoperla sinica is one of the most prominent natural enemies of many agricultural pests. Host seeking in insects is strongly mediated by olfaction...
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