Expression pattern and pharmacological characterisation of two novel alternative splice variants of the glutamate-gated chloride channel in the small brown planthopper Laodelphax striatellus.

BACKGROUND: Glutamate-gated chloride channels (GluCl) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Although only one GluCl gene was presented in insects, it showed diverse alternative splicing that was speculated could affect channel function and pharmacology. RESULTS: In this study, we isolated GluCl cDNAs from adults of the small brown planthopper (SBPH) Laodelphax striatellus and showed that six L. striatellus GluCl variants (LsGluCl-AS, LsGluCl-BS, LsGluCl-CS, LsGluCl-AL, LsGluCl-BL, LsGluCl-CL) were present in the SBPH. The expression patterns of six variants differed among developmental stages (egg, first- to fifth-instar nymphs, male and female adults) and among the body parts (head, thorax, abdomen, leg) of the female adult SBPH. All the transcripts were abundant in the head of the adult. When expressed in African clawed frog, Xenopus laevis, oocytes, the two functional variants (LsGluCl-AS, LsGluCl-AL) had similar EC50 and IC50 values for L-glutamate and channel blockers picrotoxinin and fipronil. CONCLUSION: This study represents a comprehensive molecular, expression and pharmacological characterisation of GluCl in the SBPH. These findings should be useful in providing more opportunities to discover novel insect control chemicals. © 2016 Society of Chemical Industry.

MOLECULAR CLONING, EXPRESSION PATTERN OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN 1 (MRP1, ABCC1) GENE, AND THE SYNERGISTIC EFFECTS OF VERAPAMIL ON TOXICITY OF TWO INSECTICIDES IN THE BIRD CHERRY-OAT APHID.

The ATP-binding cassette (ABC) transporters are important transmembrane proteins encoded by a supergene family. The majority of ABC proteins are primary active transporters that bind and hydrolyze ATP to mediate the efflux of a diverse range of substrates across lipid membranes. In this study, we cloned and characterized a putative multidrug resistance associated protein 1 (MRP1) from Rhopalosiphum padi encoded by ABCC1. Structural analysis showed that this protein has structural features typical of the ABC transporter family. Phylogenetic analysis indicated that the amino acid sequence was highly similar that of the corresponding protein from Acyrthosiphon pisum. Real-time quantitative polymerase chain reaction (PCR) analysis showed that ABCC1 was expressed throughout all R. padi developmental stages, with the highest level of expression in the fourth larval instar. We also examined ABCC1 expression in four different tissue types and found that it was most highly expressed in the midgut. Exposing R. padi to imidacloprid and chlorpyrifos increased ABCC1 expression. Furthermore, ABCC1 expression was higher in the imidacloprid-resistant (IR) and chlorpyrifos-resistant (CR) strains than in an insecticide-susceptible strain (SS) of R. padi. Exposing R. padi to verapamil in combination with insecticides significantly increased the toxicity of the insecticides. The respective synergy factor of CR and IR R. padi strain was 1.33 and 1.26, which was lower than that (2.72 and 1.64, respectively) of the SS. Our results clarify the biological function of ABCC1 in R. padi, particularly its role in insecticide resistance, and suggest novel strategies for pest management that use ABC transporter inhibitors to increase the effectiveness of insecticides.

Molecular and functional characterization of cDNAs putatively encoding carboxylesterases from the migratory locust, Locusta migratoria.

Carboxylesterases (CarEs) belong to a superfamily of metabolic enzymes encoded by a number of genes and are widely distributed in microbes, plants and animals including insects. These enzymes play important roles in detoxification of insecticides and other xenobiotics, degradation of pheromones, regulation of neurodevelopment, and control of animal development. In this study, we characterized a total of 39 full-length cDNAs putatively encoding different CarEs from the migratory locust, Locusta migratoria, one of the most severe insect pests in many regions of the world, and evaluated the role of four CarE genes in insecticide detoxification. Our phylogenetic analysis grouped the 39 CarEs into five different clades including 20 CarEs in clade A, 3 in D, 13 in E, 1 in F and 2 in I. Four CarE genes (LmCesA3, LmCesA20, LmCesD1, LmCesE1), representing three different clades (A, D and E), were selected for further analyses. The transcripts of the four genes were detectable in all the developmental stages and tissues examined. LmCesA3 and LmCesE1 were mainly expressed in the fat bodies and Malpighian tubules, whereas LmCesA20 and LmCesD1 were predominately expressed in the muscles and hemolymph, respectively. The injection of double-stranded RNA (dsRNA) synthesized from each of the four CarE genes followed by the bioassay with each of four insecticides (chlorpyrifos, malathion, carbaryl and deltamethrin) increased the nymphal mortalities by 37.2 and 28.4% in response to malathion after LmCesA20 and LmCesE1 were silenced, respectively. Thus, we proposed that both LmCesA20 and LmCesE1 played an important role in detoxification of malathion in the locust. These results are expected to help researchers reveal the characteristics of diverse CarEs and assess the risk of insecticide resistance conferred by CarEs in the locust and other insect species.

Molecular cloning and characterization of a ryanodine receptor gene in brown planthopper (BPH), Nilaparvata lugens (Stål).

BACKGROUND: Ryanodine receptors (RyRs) are a distinct class of intracellular calcium (Ca(2+)) release channel. The recent discovery of diamide insecticides has prompted studies on insect RyRs. However, information about the structure and function of insect RyRs is still limited. In this study, we isolated and characterized a full-length RyR cDNA (named NlRyR) from the brown planthopper, Nilaparvata lugens (Stål) (Homoptera: Delphacidae), a serious rice pest throughout Asia. RESULTS: The composite NlRyR gene contains an open reading frame of 15 423 bp encoding a protein of 5140 amino acid residues, which shares high sequence identity (78-81%) with other insect homologues, except for two regions (IDR1: 4379-4732; IDR2: 1307-1529) with markedly low identity (44-48 and 38-41%, respectively). All hallmarks of the RyR proteins are conserved in the NlRyR protein, including the RyR domain as well as mannosyltransferase, IP3 R and RyR (pfam02815) (MIR) and RyR and IP3 R homology (pfam01365) (RIH) domains. Expression analysis of NlRyR revealed significant differences in mRNA expression levels among N. lugens developmental stages. Furthermore, three alternative splicing sites were identified in NlRyR, one of which forms the mutually exclusive exons A/B and is conserved in various insect species. Diagnostic PCR assays showed that the splice variant containing exon A was predominantly detected in all developmental stages. CONCLUSION: NlRyR may play an important role in the control of developmental processes of N. lugens. Alternative splicing may generate the functional diversity of NlRyR. The results provided the basis for further structural and functional characterization of NlRyR.

Effect of vaccination with a recombinant metalloprotease from Haemaphysalis longicornis.

We report the cloning, expression and characterization of an Haemaphysalis longicornis metalloprotease (named HLMP1). The gene encodes a predicted 550 aminoacid protein with similarity to metalloproteases of the reprolysin family. The protein sequence contains a signal sequence, the zinc-binding motif (HEXXHXXGXXH) common to metalloproteases and a cysteine-rich region. Reverse transcription-PCR expression analysis indicates the presence of mRNA in the salivary gland of larva, nymph and adult ticks. Rabbit repeatedly infested with H. longicornis recognized rHLMP1, suggesting that the immune-response against HLMP1 is naturally induced through the feeding of ticks. Vaccination of rabbit with rHLMP1 produced protective immunity against ticks, resulting in 15.6 and 14.6% mortality in nymph and adult ticks, respectively. This work provides information to understand the tick's defense system, and offers new insights to develop strategies to block this defense system with an anti-tick vaccine based on a metalloprotease.

Molecular dynamics of detoxification and toxin-tolerance genes in brown planthopper (Nilaparvata lugens Stål., Homoptera: Delphacidae) feeding on resistant rice plants.

To investigate the molecular response of brown planthopper, Nilaparvata lugens (BPH) to BPH-resistant rice plants, we isolated cDNA fragments of the genes encoding for carboxylesterase (CAR), trypsin (TRY), cytochrome P450 monooxygenase (P450), NADH-quinone oxidoreductase (NQO), acetylcholinesterase (ACE), and Glutathione S-transferase (GST). Expression profiles of the genes were monitored on fourth instar nymphs feeding on rice varieties with different resistance levels. Northern blot hybridization showed that, compared with BPH reared on susceptible rice TN1, expression of the genes for P450 and CAR was apparently up-regulated and TRY mRNA decreased in BPH feeding on a highly resistant rice line B5 and a moderately resistant rice variety MH63, respectively. Two transcripts of GST increased in BPH feeding on B5; but in BPH feeding on MH63, this gene was inducible and its expression reached a maximum level at 24 h, and then decreased slightly. The expression of NQO gene was enhanced in BPH on B5 plants but showed a constant expression in BPH on MH63 plants. No difference in ACE gene expression among BPH on different rice plants was detected by the RT-PCR method. The results suggest these genes may play important roles in the defense response of BPH to resistant rice.