A glutamate-gated chloride channel (GluCl) gene encodes a transmembrane domain protein that is an important target for insecticides. In insects, the GluCl genes of Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Drosophila melanogaster, Musca domestica, Lucilia cuprina, Aedes aegypti, Schistocerca gregaria, Plutella xylostella and Acyrthosiphon pisum were cloned, but not the corresponding gene of Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae), the small brown planthopper. In this study, the complete cDNA sequence of GluCl gene from L. striatellus was cloned and sequenced. The sequence includes 2,112 nucleotides, and the open reading frame ranges from 208 to 1,566 bp. The sequence encoded 452 amino acid residues. Alignment of the amino acid sequences of the GluCls between L. striatellus and other species indicated that the similarity of sequences was in the range of 48%–86%. The purified reverse transcription PCR product of GluCl coding sequence was cloned into a T-vector and then the sequence was analyzed. This analysis revealed polymorphism of GluCl gene among several strains, including a relatively susceptible strain, a fipronil resistant strain and a field strain of L. striatellus. Because the GluCls is an important target receptor, the GluCl gene in L. striatellus is worthy of further investigation.
The small brown planthopper (SBPH), Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae), has a wide distribution from south-east Asia to Siberia and Europe. As a pest of rice, wheat and corn, it causes serious damage to crops by the transmission of Rice stripe virus (RSV), Rice black streaked dwarf virus (RBSDV) and Maize rough dwarf virus (MRDV) (Kisimoto 1967; Fang et al. 2001). In China, the SBPH has been causing serious feeding damage or crop disease since 1999 and the density of the L. striatellus population increased dramatically from 1999 to 2008 (Liu et al. 2006). To control the pest, neonicotinoid and phenylpyrazole insecticides, such as imidacloprid and fipronil, have been widely used. Extensive use of fipronil against the rice planthopper, Nilaparvata lugens (Stål), has led to insecticide resistance problems in East and Southeast Asia (Matsumura & Otuka 2009; Matsumura et al. 2009; Zhao et al. 2011).
The glutamate-gated chloride channel (GluCl) is a member of the “Cys-loop” superfamily of ligand-gated ion channels. GluCls mediate synaptic inhibition in neurons and are expressed extra-junctionally in striated muscles. They may play key roles in regulating swallowing, locomotion, sensing, memory and juvenile hormone biosynthesis (Yates et al. 2003; Liu et al. 2005). Until now GluCl channels have been found only in invertebrates, and therefore they are ideal insecticide targets with high selectivity (Janssen et al. 2007). Most of the information about the GluCl channel has come from studies on model nematodes and model insects. All together several α subunits and β subunits of the channel are found in nematodes. One α subunit has been cloned from insects. The GluCl channels are most similar to GABA (γ-amino butyric acid) receptors with respect to their physiology and pharmacology. However, the amino acid sequences of GluCl channels are most similar to those of glycine receptor (Cully et al. 1996). The insecticides acting on GluCl channels are avermectin/milbemycin, fipronil, nodulisporic acid, a novelindole diterpene (Kane et al. 2000; Smith et al. 2000; Janssen et al. 2007).
Fipronil is a phenyl pyrazole insecticide introduced for pest control, and it is highly effective against both piercing sucking and chewing insects (Colliot et al. 1992; Moffat 1993). Fipronil is a potent blocker of the insect GABA-gated chloride channel or GABA receptor (Cole et al. 1993; Buckingham et al. 1994), and therefore it belongs to IRAC (Insecticide Resistance Action Committee) mode of action group 2B (IRAC 2006). Growing evidence indicates that fipronil may inhibit GluCls of invertebrates generally, because it does so in the nematode, Caenorhabditis elegans Maupas (Horoszok et al. 2001), in the American cockroach, Periplaneta americana (L.) (Ikeda et al. 2003) and the migratory locust, Locusta migratoria (L.) (Janssen et al. 2007).
Polymorphisms, abundant in insect genes, result in a permanent changes in gene activity that determine amino acid substitutions. Three amino acid (AA) substitutions, E114G, V235A and L256F, in the AVR-14B in C. oncophora have been associated with resistance to ivermectin (IVM), whose main site of action is glutamategated chloride (GluCl) channels (Njue & Prichard 2004; Njue et al. 2004). In this study, the cDNA sequence of glutamate-gated chloride channel gene from L. Striatellus was cloned by molecular technology and then analysed. Subsequently, single nucleotide polymorphisms sites were detected by direct sequencing. Our purpose was to provide a foundation of theory and findings for the further study of the mechanism of Fipronil resistance.
TABLE 1.
THE DEGENERATE PRIMERS FOR TARGETED FRAGEMENTS OF THE GLUCL SUBUNIT GENE.
MATERIALS AND METHODS
Insects
The relatively susceptible strain (WX) of L. striatellus, which had been maintained in the laboratory for 8 years without exposure to insecticides, was collected from Wuxi, Jiangsu, China in 2005. A fipronil resistant strain (WX-F) was derived from the field-collected strain (WX) by 90 generations of continuous selection with fipronil using the rice seedling dip method. A field-collected L. striatellus strain (CX) collected from Changxing, Zhejiang, China in 2013, which since 2010 was exposed several insecticides but not to fipronil. All strains were reared on rice seedlings, seedlings were kept at 25 ± 1 °C, 70 ± 10% RH and 16:8 (L: D) h.
RNA Preparation and cDNA Synthesis
Total cellular RNA was isolated from 30 mg of L. striatellus adult tissue using the RNeasy Midi kit (QIAGEN, Hilden, Germany) by following the manufacturer's instructions. Single-stranded cDNA was synthesized from the total RNA with reverse transcriptase M-MLV and oligo (dT) 18. 5′-and 3′-RACE-ready cDNA were prepared according to the instructions of the Gene Racer Kit (Invitrogen).
Degenerate PCR Amplification
Degenerate primers targeting conserved gene regions were determined by alignment of published GluCl sequences from related species. Primers were synthesized by Invitrogen (Shanghai, China). The 3 degenerate primers used are shown in Table 1. Polymerase chain reaction (PCR) contained 2 µL of cDNA, 5 µL of 10 × standard PCR buffer, 4 µL of 25 mM Mg2+, 10 pmol of each primer, 1 µL of 10 mM dNTP, and 1 U Taq DNA polymerase. The initial step of the amplification reaction denaturation at 94 °C for 3min, followed by 30 cycles of 94 
C for 30 s, 55 °C for 30 s, 72 °C for 1 kb/min, and a final extension at 72 °C for 10 min. PCR products of the expected size were excised and purified by using the DNA agarose Gel Pur Kit (BIOMIGA, Nanjing, China) and cloned by using the pUCm-T vector system (Sangon, Shanghai, China). The second round of polymerase chain reaction was the same as the above. Primer-F and primer-R2 were used.
Cloning of the Full Length Fragment of the GluCl Gene by the RACE Technique
In order to amplify the GluCl gene of L. striatellus by means of degenerate primers, specific primers were designed and used to amplify the full length sequence. Primers used are shown in Table 2.
Analysis of the GluCl Gene Sequence with Online Tools
The cDNA sequences were analyzed with BioEdit software (Thompson et al. 1997). Sequences were aligned with ClustalX software, and then viewed with the Bioedit software (Thompson et al. 1997). The identity of the cDNA as GluCl was confirmed by similarity searches of GenBank using Blastx NR ( http://www.ncbi.nlm.nih.gov/blast/). Multiple Sequences Alignment of the cDNA was accomplished by using bioedit and ClustalX softwares (Thompson et al. 1997). The trans-membrane helix was predicted using TMpred on-line tools ( http://www.cbs.dtu.dk/services/TMHMM-2.0/). A phylogenetic tree was constructed by the neighbor-joining method (1000 replicates; seed = 64,238) for 14 amino acid sequences of the GluCls of the various species using the MEGA 4.0 program.
Analysis of cDNA Polymorphism of GluCl from 3 Strains of L. striatellus
There is a phenomenon of single nucleotide polymorphism (SNP) in the open reading frame of GluCla of L. striatellus, moreover, some animo acids are changed because of SNP. To explore this phenomenon in-depth, the full-length GluCl coding sequence of individual L. striatellus adults from the original WX strain, the fipronil resistant WX-F strain and the field CX strain were cloned by the RNA prep MicroKit (BIOTEKE) in accordance with its instructions. Thereafter we used Phanta Super Fidelity DNA Polymerase (Vazyme) to minimize the probability of mismatches during PCR. We used 5′-CAGCGCAGCCCAACTAACCACC-3′ as the Specific Forward Primer (F5) and 5′-AGCTGTTGTGTTGCGAGTCGCCC-3′ as the Specific Reverse Primer (R5).
TABLE 2.
THE SPECIFIC PRIMERS FOR TARGETED FRAGEMENTS OF THE GLUCL SUBUNIT GENE.
RESULTS
Cloning the Full Length cDNA of the GluCl Gene of L. striatellus
The full-length cDNA of L. striatellus was amplified with the degenerate primers (Table 1) and the specific primers (Table 2). The cloned sequence included 2,112 nucleotides (GenBank accession JF430868), and the open reading frame ranged from 208 to 1566 bp, which encoded 452 amino acid residues. This gene was designated as LsGIuCl. This L. striatellus transcript was the most similar to the reported GluCl-like transcript of Lucilia cuprina (81%), followed by those of Musca domestica (80%), Drosophila melanogaster (80%–83%), Aedes aegypti (86%),Tribolium castaneum (84%–86%), Plutella xylostella (84%), Nasonia. vitripennis (83%–84%), Apis mellifera (82%), Acyrthosiphon pisum (79%), Tetranychus urticae (64%), Riphicephalus sanguineus (68%), Haemonchus contortus (45%–46%) and Caenorhabditis elegans (48%–51%) (Table 3). Furthermore, according to the analysis with TMpred on-line tools, the GluCl gene included 2 pairs of cysteine residues (enclosed in the black boxes Fig. 1) within the N-terminal region and the 4 proposed trans-membrane regions (M1–4: underlined in Fig. 1) in the C-terminal region.
Phylogenetic Relations of Various Invertebrates Based on Their GluCl Genes
A phylogenetic tree was generated using 14 amino acid sequences of GluCl genes from 14 invertebrate species (Fig. 2). From the phylogenetic tree branches, we may find that Class Insecta, Class Arachnida (mites, ticks, spiders, etc.) and Class Secernetea (nematodes) belong to 1 group, and that the GluCl genes of the various different species in these Classes share a high degree of similarity. This suggests that GluCl genes have had a long common evolutionary history. Thus, it is reasonable to deduce that the GluCl genes might have evolved from an ancient ancestral gene, and might be generally distributed in a wide range of organisms.
TABLE 3.
SIMILARITY ANALYSIS OF THE GLUCL GENE OF LAODELPHAX STRIATELLUS WITH THAT OF OTHER SPECIES.
Analysis of cDNA Polymorphism of GluCl from 3 Strains of L. striatellus
The ORF of the GluCl gene was cloned from the DNA of 10 L. striatellus individuals of each strain, i.e., the susceptible strain in laboratory, the fipronil-resistant strain, and the field strain . Upon comparing 30 sequences of the SS (the relatively fipronil-susceptible strain), the RS (the fipronil-resistant strain) and the field strain, we discovered notable polymorphisms involving 80 base substitutions, and these were predominantly C-T nucleotide substitutions. All single nucleotide substitutions (SNPs) led to 20 amino acid replacements within the GluCl subunit. These amino acid substitutions involved 20 loci of GluCl gene of the 3 strains, i.e., I31K, D63A, V68I, I73L, S79A, K80T, etc. By comparing and analyzing cDNA sequences of the GluCl gene of the 3 strains, we found 2 loci with amino acid substitutions (A294D ,G298D) located between the M2–M3 trans membrane domain, 3 loci with amino acid substitutions (R352C,A364V, K378S) located between the M3–M4 trans membrane domain, the remaining loci determining amino acid substitutions at the hydrophilic N-terminus extracellular region (Table 4).
DISCUSSION
GluCls were first identified in arthropods as extra-junctional glutamate receptors (H-receptors) that hyperpolarized the membrane potential of locust (S. gregaria) leg muscle (Gration et al. 1979; Patlak et al. 1979; Dudel et al. 1989), and later they were cloned from the soil nematode, Caenorhabditis elegans (Cully et al. 1994). GluCls are activated by the glutamate analog, ibotenic acid, and are inhibited weakly by the ligand-gated chloride channel blocker, picrotoxin (Smith et al. 1999; Raymond et al. 2000; Horoszok et al. 2001). Until now the glutamate-gated chloride channel has been described only in invertebrates, and it is an important target for the development of insecticides with high selective toxicity to arthopods (Cully et al. 1996; Raymond & Sattelle, 2002).
Insect glutamate-gated chloride channels, along with GABA-gated chloride channel, are important targets for the action of certain insecticides (Bloomquist 2001). In this study, the GluCl gene in the L. striatellus was cloned successfully, then submitted to GenBank and given the registration number, JF430868. Thus the GluCl gene of the L. striatellus probably will be of great fundamental importance for further research.
Fipronil was designated as a new GABA-gated chloride channel blocker and introduced into pest control, for instance against the Colorado potato beetle, Leptinotarsa decemlineata Say (Chrysomelidae), and against important crop and stored product pests in various Orders and families (Moffat 1993; Smith & Lockwood 2003). The blocking action of fipronil on GluCl channels was demonstrated in Xenopus sp. (Anura: Pipidae) oocytes transfected with GluCls (Horoszok et al. 2001), and a similar degree of blocking was observed on chloride currents induced by glutamate and ibotenate in the dorsal unpaired median (DUM) neurons of the cockroach, Periplaneta americana, and the grasshopper, Melanoplus sanguinipes (F.) (Smith et al. 1999; Raymond et al. 2000; Ikeda et al. 2003). The blocking effect of fipronil on GluCls was previously proposed to explain part of the toxicity of this insecticide, which is widely used in pest control to eradicate dieldrin-resistant insects (Horoszok et al. 2001; Smith & Lockwood 2003; Tingle et al. 2003; Zhao et al. 2004).
This study revealed substantial polymorphism of GluCl gene in L. striatellus. The relatively fipronil-susceptible strain (WX) of L. striatellus and the fipronil-resistant strain (WX-F), which had been continuously selected with fipronil for 90 generations, were collected in Wuxi, Jiangsu, China, they each have a uniform genetic background. By comparing and analyzing amino acid sequences of GluCla, we discovered 7 loci determining amino acid substitutions in the WX and WX-F strains, 2 loci unique to the WX-F strain, and 5 loci unique to the WX strain. In addition, we analyzed polymorphism in the L. striatellus field strain, which had a genetic background different from the SS (the relatively fipronil-susceptible) strain and the RS (fipronil-resistant) strain. There were 6 other amino acid replacements in the field strain besides the above loci, and no sharp contrast between the WX and the CX strains. Most of these polymorphism sites exist in an extracellular region. It was reported that 3 amino acid (AA) substitutions, E114G, V235A and L256F in the AVR-14B of GluClα of C. oncophora, which is associated with IVM resistance, also exist in an extracellular region (Njue & Prichard 2004; Njue et al. 2004). There is no published report linking fipronil-resistance to specific polymorphisms in the glutamate-gated chloride channel. Our comparison of the full-length GluCl coding sequence in the susceptible and resistant L. striatellus strains did not show definitive evidence linking nucleic acid sequences to fipronil-resistance in this study. This important problem awaits further study.
Fig. 1.
Alignment of the GluCla subunit amino acid sequences from Lucilia cuprina, Musca domestica, Drosophila melanogaster, Aedes aegypti, Plutella xylostella, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Acyrthosiphon pisum and Laodelphax striatellus. These include the signature pairs of cysteine residues (enclosed in the black boxes at the third row) within the N-terminal region and the 4 proposed transmembrane regions (M1–4: underlined) in the C-terminal region. A second pair of N-terminal cysteine residues is enclosed by the black boxes at the fourth row.
TABLE 4.
POLYMORPHISM SITES OF THREE STRAINS OF LAODELPHAX STRIATELLUS.
Fig. 2.
A phylogenetic tree constructed by neighbor-joining method (1000 replicates; seed = 64238) for amino acid sequences of GluCls from 14 species using MEGA 4.0 program. Bootstrap values are shown at the branching points. Scale bar indicates the number of changes inferred as having occurred along each branch. GenBank accession codes: LcGluCl (Lucilia cuprina; AF081674); MdGluCl (Musca domestica; AB177546); DmGluCl (Drosophila melanogaster, DQ665648); AaGluCl (Aedes aegypti, XM001662847); TcGluCl (Tribolium castaneum, EF545123); PxGluCl (Plutella xylostella, GQ221939); LsGluCl (Laodelphax Striatellus, JF430868); NvGluCl (Nasonia vitripennis, FJ851100), AmGluCl (Apis niellifera, DQ667186); ApGluCl (Acyrthosiphon pisum, XM 001943378); TuGluCl (Tetranychus urticae, JQ738193); RsGluCl (Rhipicephalus sanguineus, GQ215234); CeGluCl (Caenorhabditis elegans, CEU14524); HcGluCl (Haemonchus contortus, AF119791).
ACKNOWLEDGMENTS
We gratefully acknowledge that this work was funded by the Natural Science Foundation of China (31171184).
