Comparative characterization of two intracellular Ca2+-release channels from the red flour beetle, Tribolium castaneum

Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are members of a family of tetrameric intracellular Ca2+-release channels (CRCs). While it is well known in mammals that RyRs and IP3Rs modulate multiple physiological processes, the roles of these two CRCs in the development and physiology of insects remain poorly understood. In this study, we cloned and functionally characterized RyR and IP3R cDNAs (named TcRyR and TcIP3R) from the red flour beetle, Tribolium castaneum. The composite TcRyR gene contains an ORF of 15,285 bp encoding a protein of 5,094 amino acid residues. The TcIP3R contains an 8,175 bp ORF encoding a protein of 2,724 amino acids. Expression analysis of TcRyR and TcIP3R revealed significant differences in mRNA expression levels among T. castaneum during different developmental stages. When the transcript levels of TcRyR were suppressed by RNA interference (RNAi), an abnormal folding of the adult hind wings was observed, while the RNAi-mediated knockdown of TcIP3R resulted in defective larval–pupal and pupal–adult metamorphosis. These results suggested that TcRyR is required for muscle excitation-contraction (E-C) coupling in T. castaneum, and that calcium release via IP3R might play an important role in regulating ecdysone synthesis and release during molting and metamorphosis in insects.

C alcium (Ca 21 ) is a key second messenger that plays important physiological roles in various cells. There are two main Ca 21 mobilizing systems in eukaryotic organisms including Ca 21 influx through the plasma membrane and Ca 21 release from internal stores. Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are large tetrameric intracellular Ca 21 -release channels (CRCs) located in the endo/ sarcoplasmic reticulum (ER/SR) of cells. An increasing number of both RyR and IP 3 R functional genes have been identified in a variety of multicellular eukaryotes ranging from Caenorhabditis elegans to humans 1 , and recently, putative RyR/IP 3 R homologs have also been identified in unicellular organisms 2,3 . In mammals, three isoforms of RyRs (RyR1, RyR2 and RyR3) and IP 3 Rs (IP 3 R1, IP 3 R2 and IP 3 R3) have been identified, which are encoded by separate genes and show distinct cellular distribution patterns. While the IP 3 Rs are approximately half the size of the RyRs, these two receptors show similarities in their regulation, and a recent study indicated that RyRs and IP 3 Rs have co-evolved from an ancestral unicellular RyR/IP 3 R 1 .
In contrast to mammals, only one of each RyR (DmRyR) and IP 3 R (DmIP 3 R) gene was identified in Drosophila melanogaster [4][5][6] , which showed approximately 45% and 60% amino acid identity with the three mammalian RyRs and IP 3 Rs, respectively. Compared with IP 3 Rs, insect RyRs have attracted increasing attention due to the discovery of diamide insecticides including the compounds flubendiamide, chlorantraniliprole (Rynaxypyr TM ) and cyantraniliprole (Cyazypyr TM ) 7,8 . Functional expression studies of the recombinant silkworm RyR (sRyR) in HEK293 cells have suggested that the insecticide flubendiamide is mainly incorporated into the transmembrane domains (residues 4111-5084) of sRyR 9 . Recently, a short segment of the C-terminus transmembrane region of DmRyR (residues 4610-4655) was found to be critical to diamide insecticide sensitivity 10 . Additionally, it was reported that high levels of diamide cross-resistance in Plutella xylostella are associated with a target-site mutation (G4946E) in the COOH-terminal membrane-spanning domain of the RyR 11 . Beyond the recent characterization of RyRs in moths and fruit flies, little molecular characterization of insect IP 3 Rs has been performed.
It is well known in mammals that RyRs and IP 3 Rs modulate a wide variety of Ca 21 -dependent physiological processes 1,12 . However, information about the physiological processes affected by their function in insects is still limited. In the present study, we cloned RyR and IP 3 R cDNAs (named as TcRyR and TcIP 3 R) from the red flour beetle, Tribolium castaneum. We report the expression patterns of the TcRyR and TcIP 3 R transcripts. We also explored the roles of these two CRC genes in the development and physiology of T. castaneum by in vivo RNA interference (RNAi).

Results
cDNA Cloning and characterization of TcRyR and TcIP 3 R in Tribolium castaneum. RT-PCR was used to amplify the entire coding sequences of the RyR and IP 3 R cDNAs from T. castaneum. A total of 12 and 6 overlapping cDNA fragments were obtained for TcRyR and TcIP 3 R, respectively ( Table 1). Compilation of the cDNA clones resulted in a 15,308 bp contiguous sequence containing a 15,285 bp ORF for TcRyR and an 8,231 bp contiguous sequence containing an 8,175 bp ORF for TcIP 3 R. Amino acid sequence alignments showed that the encoded 5,094 amino acid residues of TcRyR and 2,724 amino acid residues of TcIP 3 R share 78% and 70% overall amino acid identity with the D. melanogaster DmRyR and DmIP 3 R, respectively. The overall amino acid identities of TcRyR with its human homologues, HsRyR1, HsRyR2 and HsRyR3, were 44%, 46% and 44%, respectively, while identities of TcIP 3 R with human homologues HsIP 3 R1, HsIP 3 R2 and HsIP 3 R3 were 61%, 58% and 53%, respectively. Phylogenetic analyses were consistent with these proteins representing RyR and IP 3 R homologues, respectively (Fig. 1).
The sequence alignments also revealed the conservation of critical amino acid residues within TcRyR and TcIP 3 R. For example, a glutamate residue proposed to be involved in the Ca 21 sensitivity of the rabbit RyR3 (E 3885 ) 13 and RyR1 (E 4032 ) 14 was detected in TcRyR (E 4140 ). Additionally, residues corresponding to I 4897 , R 4913 , and D 4917 of the rabbit RyR1, which were recently shown to play an important role in the activity and conductance of the Ca 21 release channel 15 , were also conserved in TcRyR (I 4950 , R 4966 , D 4970 ). Eleven amino acid residues known to be important for the strict recognition of IP 3 within the IP 3 -binding core domain of the mouse IP 3 R1 16 were conserved in TcIP 3 R (R 267 , T 268 , T 269 , G 270 , R 271 , R 496 , K 500 , R 503 , Y 560 , R 561 , K 562 ). Seven residues in the NH 2 -terminal suppression domain of the mouse IP 3 R1 critical for the suppression of IP 3 binding 17 were also found in TcIP 3 R (L 31 , L 33 , V 34 , D 35 , R 37 , R 55 , K 128 ).
The genomic structures of TcRyR and TcIP 3 R were predicted by comparing the composite cDNA sequences with the genomic sequences retrieved from contigs in the whole genome shotgun release for T. castaneum 18 (Fig. 2). The TcRyR comprises 55 exons ranging in size from 54 bp to 1462 bp including a pair of mutually exclusive exons (19a/19b, Fig. 3A), which were confirmed by multiple cDNA clone sequence alignment and were conserved in other insect RyRs 6,19-20 . The TcIP 3 R was split into 26 exons ranging in size from 71bp to 1269 bp. The 59 donor and 39 acceptor site sequences in both TcRyR and TcIP 3 R were in agreement with the GT/AG consensus sequence, except the 59 donor sequence (GC) for intron 7 in TcRyR. Additionally, the alignment of multiple cDNA clone sequences also revealed one alternative splice site in TcIP 3 R, which is located between amino acid residues 922-929 and forms the optional exon encoding GDSLLDER (Fig. 3B). This alternative splice site was first reported in the insect IP 3 Rs, but it was conserved in the human IP 3 R1 21 .
Conserved structural domains in TcRyR and TcIP 3 R. Similar to the mammalian RyR and IP 3 R proteins 22  Developmental expression of TcRyR and TcIP 3 R. To gain understanding of the developmental expression of TcRyR and TcIP 3 R in T. castanuem, the mRNA levels of these two CRC genes were analyzed using RT-qPCR at different developmental stages of T. castanuem insects, including 3-day-old eggs, 1-, 5-and 20-day-old larvae, 1-and 5-day-old pupae, 1-and 7-day-old female adults, and 1-and 7-day-old male adults. The developmental expression pattern revealed that the mRNA levels of TcRyR were highest in the 1-dayold female adults, while there was no significant difference among the egg, larval and pupal stages (Fig. 4A). The highest and lowest mRNA expression levels of TcIP 3 R were observed in the 1-day-old larvae and 3-day-old eggs, respectively (Fig. 4B).
RNAi of TcRyR and TcIP 3 R. We employed RNAi to investigate the putative function of TcRyR and TcIP 3 R. The silencing effects of dsTcRyR and dsTcIP 3 R were detected by qPCR on the sixth day after the dsRNA injection. The results showed that the transcript levels of TcRyR and TcIP 3 R in the injected larvae were significantly suppressed by 67.86% and 61.99%, respectively, compared with those in the uninjected wild-type larvae (Fig. 5). While the injected larvae with dsTcRyR underwent normal larval-larval and larval-pupal molts and developed into adults, the hind wings of 65.9% of the individual adults could not fold properly (Fig. 6A), and all individual adults lost their ability to crawl early in adulthood and died two weeks later. In the group treated with dsTcIP 3 R, 64.7% of the larvae were unable to cast their molts completely and could not undergo normal larval-pupal metamorphosis (Fig. 6B), and thus died entrapped in their larval cuticles during the pupal stage. While the rest of the larvae could develop into pupae, the pupae could not undergo normal pupal-adult metamorphosis (Fig. 6C).

Discussion
Developing insecticides that act on novel biochemical targets is important for crop protection due to the ability of insects to rapidly evolve insecticide resistance. It has been suggested that insect calcium channels would offer an excellent insecticide target for commercial exploitation [23][24] , and the recent discovery of diamide insecticides has prompted the studies on insect RyRs. However, no insecticidal compounds targeting IP 3 Rs have been reported so far in the literature, and the studies on insect IP 3 R are solely limited to Drosophila. In this study, we cloned and characterized RyR and IP 3 R genes from T. castaneum. As with other invertebrates, the sequencing data evidenced the existence of only a single RyR and IP 3 R gene, TcRyR and TcIP 3 R, in T. castaneum, which was supported by homology searches on the T. castaneum genomic database. The amino acid identities of TcRyR with human homologues (44-46%) were considerably lower than those observed with TcIP 3 R (53%-61%), which may suggest that RyRs are better targets for insecticidal molecules with lower mammalian toxicity. Despite the large difference in size and the low amino acid identity between TcRyR and TcIP 3 R, these two CRCs share a similar architecture consisting of NH 2 -terminal modular regulatory domains that contain an RIH-RIH-RIHA arrangement and a COOH-terminal transmembrane (TM) domain that contains the conserved GGGXGD motif. The RIH-RIH-RIHA arrangement is also found in many ''ancestral CRC'' eukaryotic proteins, but it is undetectable in any prokaryotic protein 25 . In both RyRs and IP 3 Rs, the conserved GGGXGD motif acts as the selectivity filter, which enables the channels to discriminate between ions. Mutagenesis of residues in this region of both RyR and IP 3 R alters the channel conductance [26][27][28] . Recently, it was found that an IP 3 R in which the COOH-terminal transmembrane region was replaced with that from the RyR1 was blocked by ryanodine, indicating that activation mechanisms were conserved between IP 3 R and RyR 29 . These conserved structural features and activation mechanisms suggested that an ancient duplication event probably gave rise to these two classes of intracellular CRC genes. A recent study revealed that RyRs might arise from pre-existing, ancestral IP 3 R-like channels present in prokaryotes by incorporating promiscuous 'RyR' and 'SPRY' domains via horizontal gene transfer 25 .  Both RyRs and IP 3 Rs contribute to Ca 21 signals and play important roles in a vast array of physiological processes, as has been investigated in knockout mouse models. RyR1 knockout mice die perinatally due to respiratory failure caused by defective excitationcontraction (E-C) coupling in the diaphragm 30 , and RyR2 knockout mice died at approximately embryonic day 10 with morphological abnormalities in the heart tube 31 . In contrast, RyR3 knockout mice are viable but exhibited impairments in memory functions and social interaction [32][33][34] . IP 3 R-knockout studies have revealed that IP 3 R1deficient mice die in utero or by the weaning period, and the survivors have severe behavioral abnormalities in the form of ataxia and epileptic seizures 35 , whereas IP 3 R2 and IP 3 R3 double knock-out mice exhibit hypoglycemia and deficits of olfactory mucus secretion, suggesting that these two isoforms play key roles in the exocrine physiology and perception of odors [36][37] .
While knockout studies of the mammalian RyR and IP 3 R have demonstrated their critical role in development and physiology, the functional characterization of the insect RyR and IP 3 R is still limited. In this study, the contribution of TcRyR and TcIP 3 R to the developmental and physiological outcomes was assessed by in vivo RNAi.    Our data show that the suppression of the TcRyR transcript in the late larval stage leads to abnormalities in the folding of the hind wings and crawling behavior in adults. It has been reported in the neopterous insects that the third axillary sclerite and muscle were involved in the folding of the wing into the rest position [38][39] . On the other hand, it has been reported in Drosophila that crawling movements were powered by muscle contractions 40 . Thus, the abnormal phenotype observed in this study might be due to the impairment of muscle EC-coupling. This is consistent with previous findings showing that mutant fruit flies lacking RYR expression (Ryr 16 ) display impairment of muscle EC-coupling in the larval development 41 , and mutant Caenorhabditis elegans with a defective RyR gene (unc-68) exhibit diminished muscle function and decreased movement 42 . On the other hand, when the mRNA expression levels of TcIP 3 R were suppressed in the late larval stage, defects were observed in larval-pupal and pupal-adult metamorphosis. A similar result was also observed in Drosophila that disruption of the Drosophila IP 3 R gene leads to lowered levels of ecdysone and delayed larval molting 43 . These results suggest that calcium release via IP 3 R might play an important role in regulating ecdysone synthesis and release during molting and metamorphosis in insects. Further research is needed to confirm this hypothesis.
Total RNA isolation and reverse transcription. Total RNAs were extracted using the SV total RNA isolation system (Promega, Madison, WI) according to the manufacturer's instructions. First-strand cDNA was synthesized from 5 mg of total RNA using the Primescript TM First-Strand cDNA Synthesis kit (TaKaRa, Dalian, China), according to the manufacturer's instructions.
Polymerase chain reaction. The amino acid sequences of RyR and IP 3 R from D. melanogaster (GenBank: BAA41471 and AAN13240) were searched against BeetleBase (http://www.bioinformatics.ksu.edu/blast/bblast.html), and the regions with significant hits were manually annotated to identify the putative transcript and translation products. The ClustalW algorithm 45 was used to align protein sequences to further support annotation predictions. Specific primer pairs were designed based on the sequences identified above (Table 1). PCR reactions were performed with LA Taq TM DNA polymerase (TaKaRa, Dalian, China).
Reverse transcription quantitative PCR (RT-qPCR). RT-qPCR reactions were performed on the Bio-Rad CFX 96 Real-time PCR system using SYBRH PrimeScript TM RT-PCR Kit II (Takara, Dalian, China) and gene specific primers ( Table 1). The procedures for RT-qPCR were the same as those described by Zhu et al 46 . Ribosomal protein S3 (rps3, GenBank: CB335975) was used as an internal control 47 . The PCR reaction volume was 20 mL containing 2 mL of diluted cDNA, 0.4 mM of each primer, 10.0 mL SYBR Premix EX Taq TM II(23)and 0.4 mLROX Reference Dye II(503). Two types of negative controls were set up including a notemplate control and a reverse transcription negative control. Thermocycling conditions were set as an initial incubation of 95uC for 30 s and 40 cycles of 95uC for 10 s and 60uC for 15 s. Afterwards, a dissociation protocol with a gradient from 57uC to 95uC was used for each primer pair to verify the specificity of the RT-qPCR reaction and the absence of primer dimer. The mRNA levels were normalized to rps3 with the DDC T method using Bio-Rad CFX Manager 2.1 software. The means and standard errors for each time point were obtained from the average of three independent sample sets.
Cloning and sequence analysis. RT-PCR products were cloned 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 45 with the default settings. 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 and IP 3 Rs.
RNAi. Double-stranded RNAs (dsRNAs) were synthesized using the MEGAclear TM Kit (Ambion, Austin, TX) based on nucleotides 502-1118 (617 bp) and 1136-1646 (511 bp) of the ORF region of the TcRyR and TcIP 3 R, respectively. Each 20-day-old larva was injected with 200 nL of a solution containing approximately 200 ng of dsRNA. On the sixth day after the dsRNA injection, the insects were used to detect the suppression of the TcRyR and TcIP 3 R transcript by RT-qPCR. Afterwards, the insects were reared under the standard conditions mentioned above, and the phenotypes were visually observed. The buffer-injected larvae (IB group) and the uninjected wildtype larvae (WT group) were set as controls in all injection experiments. Three replications were carried out with at least 30 insects in each control or treatment.
Database entries. The entire coding sequences of TcRyR and TcIP 3 R have been deposited in the GenBank and the accession numbers are KM216386 and KM216387, respectively.