Pesticide Biochemistry and Physiology 107 (2013) 327–333

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Molecular cloning and mRNA expression of a ryanodine receptor gene in the cotton bollworm, Helicoverpa armigera Jian Wang, Yaping Liu, Jingkun Gao, Zhijuan Xie, Li Huang, Wenlong Wang, Jianjun Wang ⇑ College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, PR China

a r t i c l e

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Article history: Received 31 July 2013 Accepted 17 September 2013 Available online 29 September 2013 Keywords: Helicoverpa armigera Cotton bollworm Ryanodine receptor Calcium release channel Diamide insecticides RT-qPCR

a b s t r a c t Ryanodine receptors (RyRs) are the targets of novel diamide insecticides. The cotton bollworm, Helicoverpa armigera, is one of the most important cotton pests in the world. In this study, we report the full-length RyR cDNA sequence (named as HaRyR) of H. armigera. The 16,083-bp contiguous sequence encoded 5, 142 amino acid residues, which shares 80% and 78% overall identities with its homologues in Nilaparvata lugens (NlRyR) and Drosophila melanogaster (DmRyR), respectively. All hallmarks of RyR proteins are conserved in the HaRyR, including the GXRXGGGXGD motif conserved in the Ca2+ release channels and four copies of RyR domain unique to RyR channels. The previously identified seven lepidopteran-specific RyR residues were also found in HaRyR (N4977, N4979, N4990, L5005, L5036, N5068 and T5119). An amino acid sequence alignment showed that the N-terminal region of HaRyR (residues 188–295) shared high sequence identity with NlRyR (94%) and DmRyR (92%), and moderate sequence identity (47–50%) with three rabbit RyR isoforms, while the short segment of the C-terminal transmembrane region of HaRyR (residues 4632–4676) exhibited moderate sequence identity with NlRyR (69%) and DmRyR (67%), and low sequence identity (19–28%) with three rabbit RyR isoforms. In addition, expression analysis of HaRyR revealed that the mRNA expression level in eggs was significantly lower than in third instar larvae, pupae and adults, and anatomical regulation of HaRyR expression was also observed with the highest expression level in head compared with thorax and abdomen. Our results lay a foundation for comprehensive structural and functional characterization of HaRyR and for understanding of the molecular mechanisms of toxicity selectivity of diamide insecticides among different species. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Calcium (Ca2+) is a key second messenger that controls diverse cellular and developmental events, including muscle contraction, neurotransmitter release, hormone secretion, activation of enzymes, gene transcription, apoptosis and fertilization [1]. Ryanodine receptors (RyRs) are ligand-gated intracellular calcium channels located in the sarcoplasmic reticulum (SR) of muscle cells and endoplasmic reticulum (ER) of neurons and other non-muscle cells, which mediate the release of calcium ions from intracellular stores. RyRs derive their name from the natural insecticidal alkaloid ryanodine, a plant metabolite from Ryania speciosa, which has been found to affect calcium release by locking RyR channels in a subconductance state [2]. While high mammalian toxicity and limited insecticidal properties of ryanodine have precluded its continued use, recently two types of diamide insecticides targeting RyRs have been commercially developed, including the phthalic acid diamide derivative, flubendiamide, and the anthranilic diamides, chlorantraniliprole (Rynaxypyr™) and Cyantraniliprole (Cyazypyr™) ⇑ Corresponding author. Fax: +86 514 87347537. E-mail address: [email protected] (J. Wang). 0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.09.006

[2–7]. These diamides have received considerable interest because of their novel action mode, potent insecticidal activity, and low acute mammalian toxicity [7,8]. RyRs are the largest ion channels currently known, and exist as large homotetrameric protein of approximately 2300 kDa. To date, three types of ryanodine receptor (RyR1, RyR2 and RyR3) encoded by separate genes have been identified in mammals [9]. RyR1 is predominately found in skeletal muscles and is essential for excitation–contraction (EC) coupling [10]. RyR2 is expressed at the greatest abundance in the SR of mammalian heart, and play a critical role in cardiomyocyte EC-coupling [11,12]. RyR3 is the least understood of the RyR isoforms and seems to plays its most important role during development by involved in calcium signaling. It is ubiquitously distributed at low levels throughout different tissues, and is relatively abundant in brain and in certain skeletal tissues [13]. These three RyR isoforms are approximately 65% amino acid sequence identical, and are similar in structure, having a large N-terminal cytoplasmic domain modulating the gating of the channel pore located in the C–terminus. Due to alternative splicing, a variety of RyR variants have been isolated, and some of them showed differences in terms of the channel function properties [14–18].

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In contrast to mammals, only one RyR gene (DmRyR) was identified in Drosophila melanogaster, and showed only 45–47% amino acid identity with the three mammalian RyRs [19]. Functional expression of Drosophila RyR has subsequently been reported in CHO cells [20] and a Spodoptera frugiperda cell line (Sf9) [5,21], respectively. Recently, cDNAs encoding three novel lepidopterous RyRs (sRyR, PxRyR and CmRyR) and one homopterous RyR (NlRyR) were cloned and characterized from the silkworm, Bombyx mori, the diamondback moth, Plutella xylostella, the rice leaffolder, Cnaphalocrocis medinalis, and the brown planthopper, Nilaparvata lugens, respectively [22–26]. These insect RyRs share common structural features with mammalian RyR isoforms, and a single mutation (G4946E) in PxRyR was found to be associated with high levels of diamide resistance in two P. xylostella strains [27]. The cotton bollworm, Helicoverpa armigera, is one of the most important pests of cotton in the world. In China, the introduction of Bt cotton since 1997 has drastically reduced insecticide use for H. armigera control. However, the risk of H. armigera resistance development to Bt toxins necessitates the combination of transgenic crops with rational insecticide use. Recent study showed that chlorantraniliprole was highly active against H. armigera and could be integrated in Bt cotton management schemes to delay H. armigera resistance development [28]. In the present study, we isolated a full-length RyR cDNA (named HaRyR) from H. armigera. We report the structural features of HaRyR. We also document the expression patterns of HaRyR transcripts. Our results provide the basis for functional characterization of HaRyR and a good understanding of the mechanism of insecticide resistance targeting RyR in H. armigera.

Dalian, China) and gene specific primers (Table 1). The procedures for RT-qPCR were the same as described by Zhu et al. [29]. The elongation factor-1a (EF-1a) was used as an internal control [30]. The PCR reaction volume was 20 lL containing 2 lL of diluted cDNA, 0.4 lM of each primer, 10 lL SYBR Premix EX Taq™ II (2) and 0.4 lL ROX Reference Dye II (50). Two kinds of negative controls were set up including non-template reactions and a reverse transcription negative control. Thermocycling conditions were set as an initial incubation of 95 °C for 30 s; 40 cycles of 95 °C for 10 s and 60 °C for 15 s. Afterwards, a dissociation protocol with a gradient from 57 °C to 95 °C was used for each primer pair to verify the specificity of the qRT-PCR reaction and the absence of primer dimer. mRNA levels were normalized to EF-1a with DDCT method using Bio-Rad CFX Manager 2.1 software. Means and standard errors for each time point were obtained from the average of three independent sample sets. 2.5. Cloning and sequence analysis

2. Materials and methods

RT-PCR and RACE products were subcloned into the pMD18-T vector (TaKaRa, Dalian, China) and sequenced. Nucleotide sequences from individual clones were assembled into a full-length contig using the ContigExpress program, which is part of the Vector NTI Advance 9.1.0 (Carlsbad, CA, Invitrogen) suite of programs. The sequence alignment was performed using CLUSTALW [31] with the default settings. The aligned sequences were used to construct the phylogenetic tree in MEGA5 [32]. Transmembrane region predictions were made using the TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Conserved domains were predicted using the Conserved Domains Database (NCBI) or by alignment to other published RyRs.

2.1. Insects

2.6. Database entries

A laboratory strain of H. armigera, generously provided by Dr. Yongan Tang (Institute of Plant Protection, Jiangsu Academy of Agricultural Science, China), was maintained in an insectary kept at 28 °C with a photoperiod of 16 h light:8 h dark on artificial diet.

The entire coding sequence of HaRyR has been deposited in the GenBank and the accession number is KF641862.

2.2. Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNAs were extracted using an SV total RNA isolation system (Promega, Madison, WI), according to the manufacturer’s instructions. First-strand cDNA was synthesized from 5 lg of total RNA using the Primescript™ First-Strand cDNA Synthesis kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Degenerate primer pairs were designed against the amino acid residues that were conserved between insects, other invertebrates, and vertebrate RyR isoforms [25], and specific primers were designed to amplify across the gaps (Table 1). PCR was performed as described by Wang et al. [25]. A total of 13 RT-PCR products corresponding to the HaRyR cDNA sequence were finally obtained. 2.3. Rapid amplification of cDNA ends (RACE) To complete the cDNA sequence of HaRyR, 50 -RACE and 30 -RACE reactions were performed using 50 -full RACE core set and 30 -full RACE core set (Takara, Dalian, China), respectively, following the manufacturer’s instructions. Gene specific primers (GSP) used for the 50 - and 30 -RACE are listed in Table 1. 2.4. Reverse transcription quantitative PCR (RT-qPCR) RT-qPCR reactions were performed on the Bio-Rad CFX 96 Realtime PCR system using SYBRÒ PrimeScript™ RT-PCR Kit II (Takara,

3. Results 3.1. cDNA cloning and characterization of HaRyR To obtain full length cDNA of the RyR gene from H. armigera, a total of 15 overlapping cDNA fragments were amplified using RTPCR and RACE (Table 1). Compilation of the cDNA clones resulted in a 16,083-bp contiguous sequence containing 308 bp of 50 untranslated region and 346 bp of 30 -untranslated region. A typical polyadenylation signal ‘AATAAA’ was located 119-bp upstream of the 12-bp polyA tail. The 15,429 bp ORF of HaRyR encoded 5, 142 amino acid residues, and predicted a protein with molecular mass of 581.09 kDa and an isoelctric point (pI) of 5.47. An amino acid sequence alignment shows that HaRyR is 94%, 92%, 80% and 78% identical in pairwise comparisons with the CmRyR (AFI80904), PxRyR (AET09964), NlRyR (KF306296), and DmRyR (BAA41471) amino acid sequences, respectively, while identities of HaRyR with OcRyR1, OcRyR2 and OcRyR3 from the rabbit, Oryctolagus cuniculus, were 44%, 46% and 44%, respectively. Phylogenetic analysis also revealed that HaRyR was grouped with other lepdopteran RyR homologues, with high bootstrapping support in 1000 replications (Fig. 1). Additionally, the alignment of multiple cDNA clone sequences revealed two putative alternative splice sites in HaRyR (Fig. 2). The first site is located between amino acid residues 1393–1404 and forms the alternative splice region F encoding VTIQTYSNLQPG. The second site is located between amino acid residues 2954–2958 and forms the alternative splice region C encoding PPDIV. This splice site was also found in CmRyR [25].

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J. Wang et al. / Pesticide Biochemistry and Physiology 107 (2013) 327–333 Table 1 Oligonucleotide primers used for RT-PCR, RACE and RT-qPCR.

a

Primer name

Sequence (50 –30 )a

Description (cDNA position)

258.RYRF13 127.RYRR1 567.V1.10F1 597.U7.5R2 139.RYRF7 141.RYRR7 726.CBWU7.5F2 727.CBWU4.2R2 290.RYRF14 257.RYRR13 550.CBWU4.2F1 553.CBWU6.7R1 146.RYRF10 147.RYRR10 552.CBWU6.7F1 555.CBWU5.8R1 148.RYRF11 149.RYRR11 554.CBWU5.8F1 557.CBWWA1.1R 132.RYRF4 133.RYRR4 598.WA1.1F2 560.CBWWA2.1R1 134.RYRF5 136.RYRR5 568.V1.10R1 569.V1.10R2 558.CBWWA2.1F1 559.CBWWA2.1F2 846.CBWRyRF1 847.CBWRyRR1 343.CBWEF-F 344.CBWEF-R

TGGTGGACNGTNCAYCCNGC (WWTVHPA) TCCATYTTNCCYTCYTCRTG (HEEGKMD) AAGAAGGGCGTGGGTAAAGT TGGCATCGTCTACCTTCGGG GARAAYACNCAYAAYYTNTGG (ENTHNLW) TCCCARTTNGCYTTYTCCAT (MEKANWE) TGCCTCAAGATTTCCCACAA CCAGTTCAGTATTCTCCATC GCNATGTTYGAYCAYTTYGA (AMFDHFD) TCNCCRTTNACCCANACRCA (CVWVNGE) GAGCTGGTGGAGAAGGGGTA AGACCCAGCCGTTCTCAATC GARCAYTAYCAYGAYGCNTGG (EHYHDAW) GCNACCATYTCYTTYTCYTT (KEKEMVA) CTACTTCATAGCCGTTGCCA AGCGAGCGGCAGGAACGAAT CCNTGGATGACNMGNATHGC (PWMTRIA) TGYTGNGGRTGRTCDATCAT (MIDHPQQ) CTGTTGAAAGGATTGTGGCT ACTTGCGAAGCGACGCCTAT ATGGAYTTYTAYTGGCAYTA (MDFYWHY) GGYTCRTTNGGCATRTGYTC (EHMPNEP) GGTTTCTGCGACCGTTTCCA GAAGTCGTAAGTTTCAGAGT GTNAAYTAYTGGGAYAARTT (VNYWDKF) CATRTTCCANACRTANGTYTC (ETYVWNM) CCAGTTGATGAAGCCTCCTGCCCA TCCGACTCGCACCTTCTCACCCTC TGCTCGCTATTCTCCAGGGTCTGA GTTCCGCATGGGTTCGACACTCAC GTAACGTTCGTCTGCCCAAT CCAGAGGTTGTGGGTGTTCT GACAAACGTACCATCGAGAAG GATACCAGCCTCGAACTCAC

RT-PCR product P1 (750–1483) RT-PCR product P2 (1422–3423) RT-PCR product P3 (3300–4204) RT-PCR product P4 (4152–7458) RT-PCR product P5 (7335–7630) RT-PCR product P6 (7533–8955) RT-PCR product P7 (8904–9781) RT-PCR product P8 (9710–10754) RT-PCR product P9 (10665–11569) RT-PCR product P10 (11503–12625) RT-PCR product P11 (12534–13216) RT-PCR product P12 (13122–14840) RT-PCR product P13 (14775–15659) 50 -RACE product R5 (1–809) 30 -RACE product R3 (15531–16083) RT-qPCR (3181–3320) RT-qPCR

Corresponding amino acid sequences for degenerate primers were shown in parentheses.

97 99 100 71 100

HaRyR

Alternative splice region F

sRyR CmRyR PxRyR

Alternative splice region C

NlRyR DmRyR HsRyR2 HsRyR1

84

HsRyR3

0.05 Fig. 1. Phylogenetic tree of the RyR family. The HaRyR amino acid sequence was aligned to eight representative RyR isoforms from six species and used for phylogenetic analysis. The Neighbor-joining tree was generated in MEGA5 with 1000 bootstrap replicates. RyR sequences are obtained from the following GenBank entries: DJ085056 for Bombyx mori (sRyR); AFI80904 for Cnaphalocrocis medinalis (CmRyR); AET09964 for Plutella xylostella (PxRyR); KF306296 for Nilaparvata lugens (NlRyR), BAA41471 for Drosophila melanogaster (DmRyR); P21817 for human RyR1 (HsRyR1); Q92736 for human RyR2 (HsRyR2); Q15413 for human RyR3 (HsRyR3).

3.2. Multi-domain organization of HaRyR Analysis of the HaRyR amino acid sequence reveals a multi-domain protein organization, which can be grouped into two categories: those shared by RyRs and inositol-1,4,5-trisphosphate receptors (IP3Rs), which together form a superfamily of homotetrameric ligand-gated intracellular Ca2+ channels, and those shared with other proteins, but not with IP3Rs. The first group included the Ins145_P3_rec domain (pfam 08709) at positions 12–206, the

Fig. 2. Nucleotide and inferred amino acid sequences of alternative splice regions in the HaRyR gene.

MIR (Mannosyltransferase, IP3R and RyR [pfam02815]) domain at positions 217–398, two RIH (RyR and IP3R Homology [pfam01365]) domains at positions 445–654 and 2235–2462, and the RIH-associated domain (pfam08454) at positions 4016–4141. The MIR domain has been suggested to have a ligand transferase function and the RIH domain may form the IP3 binding site in combination with the MIR domain in IP3Rs [33]. However, the significance of both MIR and RIH domain in RyR regulation is not well understood to date. Additionally, six transmembrane helices (TM1–TM6) were predicted at positions 4477–4499, 4668–4690, 4752–4774, 4894– 4916, 4942–4964, and 5022–5041, respectively, and the sequence motif, GXRXGGGXGD, which constitutes part of the pore-forming segments of the Ca2+ release channels [34], was also highly conserved in HaRyR (residues 4994–5003). The second group included three copies of a SPRY (SPla and RyR) domain (residues 653–805, 1085–1217, and 1544–1695) and four copies of a RyR domain (residues 859–953, 972–1066, 2840–2933 and 2966–3054). The SPRY domains were originally identified in the Dictyostelium discoideum tyrosine kinase spore lysis A (SplA)

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Table 2 Percentage of amino acid sequence identity between the RyR domains, N-terminal region, a short segment of the C-terminal region of HaRyR and corresponding domains/regions of RyR isoforms from Cnaphalocrocis medinalis (CmRyR), Plutella xylostella (PxRyR), Bombyx mori (sRyR), Nilaparvata lugens (NlRyR), Drosophila melanogaster (DmRyR) and Oryctolagus cuniculus (OcRyR1, OcRyR2, OcRyR3).

HaRyR HaRyR HaRyR HaRyR HaRyR HaRyR

RyR domain 1 RyR domain 2 RyR domain 3 RyR domain 4 N-terminal region C-terminal region

CmRyR

PxRyR

sRyR

NlRyR

DmRyR

OcRyR1

OcRyR2

OcRyR3

96% 98% 97% 98% 99% 91%

94% 98% 96% 96% 94% 93%

93% 96% 97% 99% 99% 96%

78% 91% 84% 91% 94% 69%

74% 79% 70% 88% 92% 67%

54% 57% 40% 58% 50% 19%

56% 58% 45% 58% 49% 22%

55% 56% 47% 56% 47% 28%

and the mammalian RyRs [35], and are generally known to mediate protein–protein interactions [36]. Recently, the SPRY2 domain in RyR1 has been identified as an in vitro binding partner for the II– III loop of the a1S subunit of the skeletal muscle dihydropryidine receptor (DHPR) [37]. The RyR domains are unique to RyR channels, as these domains are not detectable in IP3Rs, nor in any other eukaryotic protein family. However, RyR domains are present in a plethora of bacterial, archaeal and bacteriophage proteins [38]. An amino acid sequence alignment shows that the RyR domains of HaRyR are highly conserved in other insect RyRs, especially the fourth RyR domain, which shares 88–99% identity (Table 2 and

Fig. 3), suggesting that they might play specialized roles for either cellular viability or protein–protein interaction in RyR-like channels. Recently, it was reported that the RyR domain contains a putative binding site for protein kinase [39,40]. 3.3. Specific regions putatively related to species selective toxicity of diamide insecticides Recent studies have revealed that two regions corresponding to the short segment of the C-terminal transmembrane region of DmRyR (residues 4610–4655) and the N- terminal region of sRyR

RyR domain 1 HaRyR CmRyR PxRyR sRyR NlRyR DmRyR OcRyR1 OcRyR2 OcRyR3

859 854 859 854 856 852 850 861 848

AFVPTPVDTLQITLPSYVEQIRDKLAENIHEMWAMNKIEAGWMYGDQREDLHKIHPCLVPFERLPPAEKRYDIQLAVQTLKTILALGYYISLDKP AFVPTPVDTLQVQLPGYVEQIRDKLAENIHEMWAMNKIEAGWMYGDQREDLHKIHPCLVPFERLPAAEKRYDIQLAVQTLKTILALGYYISLDKP AFVPMPVDTLTITLPSYIEQIRDKLAENIHEMWAMNKIEAGWMYGEHREDLHKIHPCLVPFERLPAAEKRYDIQLAVQTLKTILALGYYISLDKP AFVPTPVDTMTISLPTYVEQIRDKLAENIHEMWAMNKIEAGWMYGDQRDDIHKIHPCLVPFERLPPAEKRYDIQLAVQTLKTILALGYYITLDKP AFVPTPVDTTKVTLPNYIESIRDKLAENIHEMWAMNKVEAGWMFGEKRDDIRKVHPCLIQFDQLPPAEKRYDSQLAVQTLKTILALGYYITMDKP AFVPKPVDTTGVTLPSSVDQIKEKLAENIHEMWALNKIEAGWSWGEHRDDYHRIHPCLTHFEKLPAAEKRYDNQLAVQTLKTIISLGYYITMDKP DFVPCPVDTVQIVLPPHLERIREKLAENIHELWALTRIEQGWTYGPVRDDNKRLHPCLVNFHSLPEPERNYNLQMSGETLKTLLALGCHVGMADE AFTPIPVDTSQIVLPPHLERIREKLAENIHELWVMNKIELGWQYGPVRDDNKRQHPCLVEFSKLPEQERNYNLQMSLETLKTLLALGCHVGISDE SFIPCPIDTSQVVLPPHLEKIRDRLAENIHELWGMNKIELGWTFGKMRDDNKRQHPCLVEFSKLPETEKNYNLQMSTETLKTLLALGCHIAHVNP

RyR domain 2 HaRyR CmRyR PxRyR sRyR NlRyR DmRyR OcRyR1 OcRyR2 OcRyR3

972 967 972 967 969 965 964 975 962

GYKPAPLDLSAVTLTPKMDELVDQLAENTHNLWARERIQQGWTYGLNEDSDMHRSPHLVPYPKVDDAIKKANRDTASETVRTLLVYGYMLDPPTG GYKPAPLDLSAVTLTPKMDELVDQLAENTHNLWARERIQQGWTYGLNEDPDMHRSPHLVPYPKVDDAIKKANRDTASETVRTLLVYGYNLDPPTG GYKPAPLDLSAVTLTPKMDELVEQLAENTHNLWARERIQQGWTYGLNEDSDMHRSPHLVPYPKVDEAIKKANRDTASETVRTLLVYGYMLDPPTG GYKPAPLDLGAVTLTPKMDELVDQLAENTHNLWARERIQQGWTYGLNEDPEMHRSPHLVPYPKVDDAIKKANRDTASETVRTLLVYGYNLDPPTG GYKPAPLDLTAISLTPKMEELVDQLAENTHNLWAKERIQQGWTYGLNEDSDMLRSPHLVPYCKVDEAIKKANRDTASETVRTLLVYGYNLDPPTG GYKPAPLDLSAVTLTPKLEELVDQLAENTHNLWARERIQQGWTYGLNEDSENHRSPHLVPYAKVDEAIKKANRDTASETSANAPGLRICLGSSDG GYKPAPLDLSHVRLTPAQTTLVDRLAENGHNVWARDRVAQGWSYSAVQDIPARRNPRLVPYRLLDEATKRSNRDSLCQAVRTLLGYGYNIEPPDQ GYKPAPMDLSFIKLTPSQEAMVDKLAENAHNVWARDRIRQGWTYGIQQDVKNRRNPRLVPYTLLDDRTKKSNKDSLREAVRTLLGYGYNLEAPDQ GYKPAPLDLSDVKLLPPQEILVDKLAENAHNVWAKDRIKQGWTYGIQQDLKNKRNPRLVPYALLDERTKKSNRDSLREAVRTFVGYGYNIEPSDQ

RyR domain 3 HaRyR CmRyR PxRyR sRyR NlRyR DmRyR OcRyR1 OcRyR2 OcRyR3

2840 2802 2852 2807 2833 2815 2734 2700 2600

QYDPQPINTSSVALNNDLNTIVQKFSEHYHDAWASRKIENGWVYGEGWSDSQKTHPRLKPYNMLNDYEK-----ERYKEPVRESLKALLAIGWSVEHSE QYDPQPINTASVALNNDLNTIVQKFSEHYHDAWASRKIENGWVYGESWSDSQKSHPRLKPYNMLNDYEK-----ERYKEPVRESLKALLAIGWSVEHSE QYNPQPINTSSVALNNDLNTIVQKFSEHYHDAWASRKIENSWVYGENWSDSQKAHPRLKPYNMLNDYEK-----ERYKEPVRESLKALLAIGWSVEHSE QYDPQPINTSSVALNNDLNTIVQKFSEHYHDAWASRKIENGWVYGESWSDSQKTHPRLKPYNMLNDYEK-----ERYKEPVRESLKALLALGWSVEHSD PYNPQPINTQAVVLNNDLNTIVQKFSEHYHDAWASRKLENGWTYGEQWSDSQKTHPRLKPYNMLNDYVEPSIERERYKEPVRESIKALLAIGWTVEHSE QYMPNPIDTNNVHLDNDLNSLVQKFSEHYHDAWASRRLEGGWTYGDIRSDNDRKHPRLKPYNMLSEYER-----ERYRDPVRECLKGLLAIGWTVEHSE NFDPRPVETLNVIIPEKLDSFINKFAEYTHEKWAFDKIQNNWSYGENVDEELKTHPMLRPYKTFSEKDK-----EIYRWPIKESLKAMIAWEWTIEKAR NFNPQPVDTSNIIIPEKLEYFINKYAEHSHDKWSMDKLANGWIYGEIYSDSSKIQPLMKPYKLLSEKEK-----EIYRWPIKESLKTMLAWGWRIERTR NFDPKPINTINFSLPEKLEYIVTKYAEHSHDKWACEKSQSGWKYGISLDENVKTHPLIRPFKTLTEKEK-----EIYRWPARESLKTMLAVGWTVERTK

RyR domain 4 HaRyR CmRyR PxRyR sRyR NlRyR DmRyR OcRyR1 OcRyR2 OcRyR3

2966 2928 2991 2927 2959 2938 2854 2820 2718

NYNPHPVDMTNLTLSRETQNMAERLADNAHDIWAKKKKEELVTNGGGIHPQLVPYDLLTDKEKKKDRERSQEFLKYLQYQGYKLHRPSK NYNPHPVDMTNLTLSREMQNMAERLAENAHDIWAKKKKEELVTNGGGIHPQLVPYDLLTDKEKKKDRERSQEFLKYLQYQGYKLHRPSK NYNPHPVDMTNLTLSREMQNMAERLAENAHDIWAKKKKEELTVNGGGIHPQLVPYDLLTDKEKKKDRERSQEFLKYLQYQGYKLHRPSK NYNPHPVDMTNLTLSREMQNMAERLADNAHDIWAKKKKEELVTNGGGIHPQLVPYDLLTDKEKKKDRERSQEFLKYLQYQGYKLHRPSK NYHPNPIDMTNLTLSREMQNMAERLAENAHDIWAKKKKEELITCGGGIHPQLVPYDLLTDKEKKKDRERSQEFLKYLQYQGYKLHRPSR NYNPHPVDMSNLTLSREMQNMAERLAENSHDIWAKKKNEELNGCGGVIHPQLVPYDLLTDKEKKKDRERSQEFLKYMQYQGYKLHKPSK GYNPQPPDLSGVTLSRELQAMAEQLAENYHNTWGRKKKQELEAKGGGTHPLLVPYDTLTAKEKARDREKAQELLKFLQMNGYAVTRGLK GYSPRAIDMSNVTLSRDLHAMAEMMAENYHNIWAKKKKLELESKGGGNHPLLVPYDTLTAKEKAKDREKAQDILKFLQINGYAVSRGFK SYNPAPLDLSNVVLSRELQGMVEVVAENYHNIWAKKKKLELESKGGGSHPLLVPYDTLTAKEKFRDREKAQDLFKFLQVNGVIVSRGMK

Fig. 3. Amino acid sequence alignments of four copies of RyR domain predicted in HaRyR and other representative insect and mammal RyRs. Identical amino acids are shown in black boxes and similar amino acids are highlighted in gray boxes. Gaps have been introduced to permit alignment. Abbreviations and GenBank entries for sRyR, CmRyR, PxRyR, NlRyR, and DmRyR isoforms are described in Fig. 1. The other RyR sequences are obtained from the following GenBank entries: CAA33279 for Oryctolagus cuniculus RyR1 (OcRyR1); NP_001076226 for Oryctolagus cuniculus RyR2 (OcRyR2); NP_001076231 for Oryctolagus cuniculus RyR3 (OcRyR3).

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(residues 183–290) were critical to diamide insecticide sensitivity [22,41]. Therefore, these two critical diamide-sensitive regions in HaRyR were compared with other reported insect and mammalian RyRs, including CmRyR, PxRyR, sRyR, NlRyR, DmRyR, and three rabbit RyR isoforms (Fig. 4). The alignment showed that the N-terminal region in HaRyR (residues 188–295) exhibited high sequence identity (92–99%) to lepidopteran and non- lepidopteran insect RyRs and moderate sequence identity (47–50%) to three rabbit RyR isoforms, while the short segment of the C-terminal transmembrane region in HaRyR (residues 4632–4676) exhibited high sequence identity (91–96%) to lepidopteran insect RyRs, moderate sequence identity to NlRyR (69%) and DmRyR (67%), and low sequence identity (19–28%) to three rabbit RyR isoforms (Table 2, Fig. 4). Additionally, the previously reported seven lepdopteranspecific RyR residues [25] were also found in HaRyR (N4977, N4979, N4990, L5005, L5036, N5068 and T5119). 3.4. mRNA expression pattern of HaRyR The mRNA levels of HaRyR were analyzed using RT-qPCR at various developmental stages and different body parts of H. armigera. The developmental expression pattern revealed that there is no significant difference among third instar larvae, pupae and adults, but the mRNA expression level of egg was significantly lower than other developmental stages (Fig. 5). Anatomical regulation of HaRyR expression was also observed with the highest expression level in head compared with thorax and abdomen of third instar larvae (Fig. 6).

4. Discussion The phthalic acid diamides and anthranilic diamides are two novel classes of insecticides with a novel mode of action targeting the RyRs in insect muscle cells [7]. Treatment with both phthalic

A

HaRyR CmRyR PxRyR sRyR NlRyR DmRyR OcRyR1 OcRyR2 OcRyR3 HaRyR CmRyR PxRyR sRyR NlRyR DmRyR OcRyR1 OcRyR2 OcRyR3

B

HaRyR CmRyR PxRyR SRyR NlRyR DmRyR OcRyR1 OcRyR2 OcRyR3

188 183 188 183 187 182 187 199 189 267 262 267 262 266 261 262 277 266

Fig. 5. Relative mRNA expression levels of HaRyR in different developmental stages of Helicoverpa armigera. The relative expression level was expressed as mean ± SE (N = 3), with the third instar larvae as the calibrator. Different lowercase letters above the columns indicate the significant differences at the P < 0.05 level.

and anthranilic diamides were reported to elicit intracellular Ca2+ release in isolated insect neurons [3,5,42]. Flubendiamide and chlorantraniliprole, the first commercially available phthalic acid diamide and anthranilic diamide, respectively, have a favorable ecotoxicological profile and exhibit exceptional insecticidal activity against lepidopteran insect pests, and are now considered to be promising alternatives to conventional insecticides, especially for the control of lepidopterous pests of agricultural importance. Radioligand binding studies with house fly muscle membranes have provided the evidence that flubendiamide and chlorantraniliprole bind at different allosterically coupled sites of RyR [43]. While diamide insecticides exhibit exceptional insecticidal activity against lepidopteran insect pests and show a high selectivity for

EVSIVNASFHVTHWSVQPYGTGISRMKYVGYVFGGDVLRFFHG-GDECLTIPSTWTKDGGQNIVVYEGGSVMSQARSLWR EVSIVNASFHVTHWSVQPYGTGISRMKYVGYVFGGDVLRFFHG-GDECLTIPSTWTKDGGLNIVVYEGGSVMSQARSLWR EVSIVNASFHVTHWSVQPYGTGISRMKYVGYVFGGDVLRFFHG-GDECLTIPSSWSNEGQHNIVVYEGGSVMSQARSLWR EVSIVNASFHVTHWSVQPYGTGISRMKYVGYVFGGDVLRFFHG-GDECLTIPSTWTRDGGQNIVVYEGGSVMSQARSLWR DLSIVNASFHVTHWSVQPYGTGISRMKYVGYVFGGDVLRFFHG-GDECLTIPSTWSEAPGQNIVVYEGGSVMSQARSLWR EQSIVNASFHVTHWSVQPYGTGISRMKYVGYVFGGDVLRFFHG-GDECLTIPSTWGREAGQNIVIYEGGVVMAQARSLWR GELQVDASFMQTLWNMNPICS--C--CEEGYVTGGHVLRLFHGHMDECLTISAADS-DDQRRLVYYEGGAVCTHARSLWR GSLHVDAAFQQTLWSVAPISSGSE--AAQGYLIGGDVLRLLHGHMDECLTVPSGEHGEEQRRTVHYEGGAVSVHARSLWR GNIQVDASFMQTLWNVHPTCSGSS--IEEGYLLGGHVVRLFHG-HDECLTIPSTDQNDSQHRRIFYEAGGAGTRARSLWR LELARTKWAGGFINWYHPMRIRHITTGRY LELARTKWAGGFINWYHPMRIRHITTGRY LELARTKWAGGFINWYHPMRIRHITTGRY LELARTKWAGGFINWYHPMRIRHITTGRY LELARTKWAGGFINWYHPMRIRHLTTGRY LELARTKWTGGFINWYHPMRIRHITTGRY LEPLRISWSGSHLRWGQPLRIRHVTTGRY LETLRVAWSGSHIRWGQPFRLRHVTTGKY VEPLRISWSGSNIRWGQAFRLRHLTTGHY

4632 4579 4659 4579 4611 4592 4523 4461 4370

AIEAESKKAVQG----PAP-SALSQVDLSQYTRRAVSFLARNFYNLKYVA AIEAESKKAVSG----PAP-SALAQVDLSHYTSRAVSFLARNFYNLKYVA AIEAESKKAVQA----PAP-SALSQVDLSQYTRRAVSFLARKFYTLKYVA AIEAESKKAVQG----PASSSALSQVDLSQYTRRAVSFLARNFYNLKYVA AVEAEAKHDVVA----EPS--AFSQIDFNGYTHRAVSFLARNFYNLKYVA SIEAEAKKSSSA----PQETPAVHQIDFSQYTHRAVSFLARNFYNLKYVA PSPPAKKEEAGG-------AGMEFWGELEVQRVKFLNYLSRNFYTLRFLA KLRQLHTHRYGE----PEVPESAFWKKIIAYQQKLLNYFARNFYNMRMLA EVTKKKKRRRGQKVEKPEAFMANFFKGLEIYQTKLLHYLARNFYNLRFLA

Fig. 4. Amino acid sequence alignments of the N-terminal region (A) and the short segment of the C-terminal transmembrane region (B) of HaRyR and other representative insect and mammal RyRs. Identical amino acids are shown in black boxes and similar amino acids are highlighted in gray boxes. Gaps have been introduced to permit alignment. Abbreviations and GenBank entries for the RyR isoforms are described in Figs. 1 and 3.

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Fig. 6. Relative mRNA expression levels of HaRyR in different body parts of the third instar larvae of Helicoverpa armigera. The relative expression level was expressed as mean ± SE (N = 3), with the abdomen as the calibrator. Different lowercase letters above the columns indicate the significant differences at the P < 0.05 level.

insect RyRs compared to mammalian RyRs, the basis of this selectivity is not yet well understood until recently. In the study of the interaction of flubendiamide with the recombinant sRyR, Kato et al. [22] found that the transmembrane domain (residues 4111–5084) plays an important role in the formation of an action site for flubendiamide, while the N-terminus (residues 183–290) is a structural requirement for flubendiamide-induced activation of the sRyR. Subsequently, it was reported that amino acid residues at positions analogous to CmRyR residues N4922, N4924, N4935, L4950, L4981, N5013 and T5064 are unique in lepdopteran RyR homologues, whereas corresponding residues are highly conserved in non-lepidopteran insect RyRs and other invertebrate as well as vertebrate RyRs, suggesting that these residues might be involved in the differences in channel properties between lepidopteran and non-lepidopteran insect RyRs and in the species selective toxicity of diamide insecticides [25]. Recently, a short segment of the C-terminal transmembrane region of DmRyR (residues 4610–4655) was found to be critical to diamide insecticide sensitivity [41]. Additionally, it was reported that high levels of diamide cross-resistance in P. xylostella strains collected from the Philippines and Thailand are associated with a target-site mutation (G4946E) in the C-terminal membrane-spanning domain of PxRyR [27]. In the present study, we found that the seven lepdopteran-specific RyR residues were also conserved in HaRyR, among which four residues (N4977, N4979, N4990 and L5005) are clustered near the poreforming segment (residues 4994–5003), the residue L5036 is located in predicted transmembrane helix 6 (TM6, 5022–5041) and corresponds to I4862 in the mouse RyR2, which plays a crucial role in RyR channel activation and gating [44], and the last two residues (N5068 and T5119) are located in predicted cytosolic side of the reticulum membrane. Multiple sequence alignment showed that the N-terminal region of HaRyR (residues 188–295) shared high sequence identity with NlRyR (94%) and DmRyR (92%), and moderate sequence identity (47–50%) with three rabbit RyR isoforms. In contrast, the short segment of C-terminal transmembrane region of HaRyR (residues 4632–4676) only exhibited moderate sequence identity with NlRyR (69%) and DmRyR (67%), and low sequence identity (19–28%) with three rabbit RyR isoforms. This segment also falls within the insect divergent region 1 (IDR1) identified by Wang et al. [26]. These results indicated that species selective toxicity of diamide insecticides might be associated with several residues and/or regions in insect RyRs, which may either physically

interact with diamide insecticides or be critical for the RyR conformation required for the binding of diamide insecticides. Considering that G4946E mutation (corresponding to G4924 in HaRyR) in PxRyR is associated with diamide insensitivity to P. xylostella [27], we can expect that the C-terminal transmembrane domain is critical for the selective toxicity of diamide insecticides. Most likely, the leipdopteran-specific RyR residues may play an important role in the selective toxicity of diamide insecticides against lepidopteran and non-lepidopteran insects, and the short segment (residues 4632–4676 in HaRyR) in the C-terminal transmembrane region might contribute to the selective toxicity of diamide insecticides against insect and mammal RyRs. Further studies, including mutagenesis and a heterologous expression of various mutagenized RyRs, are needed to address the exact binding sites of diamide insecticides in insect RyRs. While the primary function of RyRs is to mediate excitation– contraction coupling in muscle, the nonmuscle roles of RyRs in insect development are not yet clearly understood. In this study, expression analysis of HaRyR revealed that the mRNA expression level in eggs was significantly lower than in other developmental stages, and the head exhibited the highest expression level of HaRyR compared with thorax and abdomen of third instar larvae. These results are inconsistent with previous study of PxRyR in the P. xylostella, which showed no significant difference of the mRNA expression levels between eggs and other developmental stages as well as head and other tissues [23]. It has been reported in D. melanogaster that expression of RyR varied among different stages of embryonic development [45]. The discrepancy of expression levels of RyR in eggs of H. armigera and P. xylostella might be due to the different collection time of the eggs. In the present study, eggs were collected within 24 h after oviposition and thus showed low expression levels. The high expression levels of HaRyR in head of third instar larvae suggested that RyR-mediated Ca2+ signals may play an important role in nervous system development in H. armigera, however, mechanisms determining different expression profiling of RyRs in heads of H. armigera and P. xylostella remain uncertain and warrant further investigation. Acknowledgements We thank the editor John Marshall Clark and three anonymous reviewers for their helpful comments on the manuscript. This work was supported by the National Natural Science Foundation of China under grant no. 31171841. References [1] M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signaling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11–21. [2] G.P. Lahm, T.P. Selby, J.H. Freudenberger, T.M. Stevenson, B.J. Myers, G. Seburyamo, B.K. Smith, L. Flexner, C.E. Clark, D. Cordova, Insecticidal anthranilic diamides: a new class of potent ryanodine receptor activators, Bioorg. Med. Chem. Lett. 15 (2005) 4898–4906. [3] M. Tohnishi, H. Nakao, T. Furuya, A. Seo, H. Kodama, K. Tsubata, S. Fujioka, H. Kodama, T. Hirooka, T. Nishimatsu, Flubendiamide, a novel insecticide highly active against lepidopterous insect pests, J. Pestic. Sci. 30 (2005) 354–360. [4] T. Masaki, N. Yasokawa, M. Tohnishi, T. Nishimatsu, K. Tsubata, K. Inoue, K. Motoba, T. Hirooka, Flubendiamide, a novel Ca2+ channel modulator, reveals evidence for functional cooperation between Ca2+ pumps and Ca2+ release, Mol. Pharmacol. 69 (2006) 1733–1739. [5] D. Cordova, E.A. Benner, M.D. Sacher, J.J. Rauh, J.S. Sopa, G.P. Lahm, T.P. Selby, T.M. Stevenson, L. Flexner, S. Gutteridge, D.F. Rhoades, L. Wu, R.M. Smith, Y. Tao, Anthranilic diamides: a new class of insecticides with a novel mode of action, ryanodine receptor activation, Pestic. Biochem. Physiol. 84 (2006) 196– 214. [6] G.P. Lahm, T.M. Stevenson, T.P. Selby, J.H. Freudenberger, C.M. Dubas, B.K. Smith, D. Cordova, L. Flexner, C.E. Clark, C.A. Bellin, J.G. Hollingshaus, Rynaxypyr™: a new anthranilic diamide insecticide acting at the ryanodine receptor, in: H. Ohkawa, H. Miyagawa, P.W. Lee (Eds.), Pesticide Chemistry: Crop Protection, Public Health, Environmental Safety, Wiley-VCH, Weinheim, 2007, pp. 111–120.

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