Shun-Fan Wu

Knockdown of the ionotropic γ-aminobutyric acid receptor (GABAR) RDL gene decreases fipronil susceptibility of the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae)

Insect γ-aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil-susceptible and fipronil-resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance-conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27-82%, and greatly decreased fipronil-induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth-instar nymphs, and female adults than in third-instar and fourth-instar nymphs. Lsrdl was predominantly expressed in the heads of 2-day-old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.

Susceptibility of the Rice Stem Borer, Chilo suppressalis (Lepidoptera: Crambidae), to Flubendiamide in China

ABSTRACT The rice stem borer, Chilo suppressalis (Walker), is an important rice pest in China, and has evolved resistance to several classes of insecticides. Flubendiamide is a phthalic diamide insecticide that shows selective insecticidal activity against lepidopterous insects. The susceptibility of 40 field populations of C. suppressalis, collected in 2011 and 2012 in seven provinces of south-eastern China, to flubendiamide was determined through rice seedling dipping bioassay method. Of these 40 populations, seven populations that were seldom exposed to flubendiamide were used to set up the baseline sensitivity, and the LC50 value was 0.092 mg/L. Variation in susceptibility among the 40 field populations was high (34-fold). The range of mean lethal concentration (LC50) values in response to this chemical was between 0.032 mg/L (FS11) and 1.090 mg/L (JH12) across the populations. Substantial variations of the susceptibility to flubendiamide were detected among different geographic populations. There was no significant difference observed between years for most populations, except for populations from Jinhua and Lujiang. Resistance ratios to the chemical ranged from 0.8 to 11.8, indicating that most colonies remained susceptible or showed certain decrease in susceptibility. It was found that 16 of the 40 populations had some level of resistance. However, moderate level of resistance was discovered in only one population from JH12 from Zhejiang province (11.8-fold). Other 15 populations showed low level of resistance (5.1–9.3-fold) to flubendiamide. These data are useful in future monitoring programs for detecting any changes in susceptibility as a result of using flubendiamide.

Monitoring and mechanisms of insecticide resistance in Chilo suppressalis (Lepidoptera: Crambidae), with special reference to diamides.

BACKGROUND The rice stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae), is one of the most economically important pests of rice in Asia. Chemical control remains the most efficient primary means for controlling this pest. RESULTS Significant variations among field populations in their resistance to seven insecticides were observed. The populations exhibited LC50 values that ranged between 0.605 and 108.088 mg AI L-1 for chlorantraniliprole and between 0.046 and 3.919 mg AI L-1 for flubendiamide. The YY14 population collected from Yuyao in Zhejiang Province in 2014 showed a moderate resistance level to the two diamides, i.e. up to 77.6-fold and 42.6-fold for chlorantraniliprole and flubendiamide respectively. Synergism tests and biochemical assays showed no obvious correlations between diamide resistance and three detoxifying enzymes. Sequence comparison of the ryanodine receptor gene between the YY14 resistant population and susceptible population revealed that a glycine to glutamic acid substitution (G4910E) was presented in the YY14 population. CONCLUSION G4910E mutation might be involved in the resistance evolution of C. suppressalis to the diamides. An appropriate insecticide resistance management programme should be established to maintain the effectiveness of the insecticides and to ensure sustainable management.

Resistance monitoring and cross-resistance patterns of three rice planthoppers, Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus to dinotefuran in China

Three rice planthoppers, brown planthopper, Nilaparvata lugens, white-backed planthopper, Sogatella furcifera and small brown planthopper, Laodelphax striatellus, are important pests of cultivated rice in tropical and temperate Asia. They have caused severe economic loss and developed resistance to insecticides from most chemical classes. Dinotefuran is the third neonicotinoid which possesses a broad spectrum and systemic insecticidal activity. We determined the susceptibility of dinotefuran to field populations from major rice production areas in China from 2013 to 2015. All the populations of S. furcifera and L. striatellus were kept susceptible to dinotefuran (0.7 to 1.4-fold of S. furcifera and 1.1-to 3.4-fold of L. striatellus) However, most strains of N. lugens (except FQ15) collected in 2015 had developed moderate resistance to dinotefuran, with resistance ratios (RR) ranging from 23.1 to 100.0 folds. Cross-resistance studies showed that chlorpyrifos-resistant and buprofezin-resistant Sogatella furcifera, chlorpyrifos-resistant and fipronil-resistant L. striatellus, imidacloprid-resistant and buprofezin-resistant Nilaparvata lugens exhibited negligible or no cross-resistance to dinotefuran. Synergism tests showed that piperonyl butoxide (PBO) produced a high synergism of dinotefuran effects in the DY15 and JS15 populations (2.14 and 2.52-fold, respectively). The obvious increase in resistance to dinotefuran in N. lugens indicates that insecticide resistance management strategies are urgently needed to prevent or delay further increase of insecticide resistance in N. lugens.

Comparison of Insecticide Susceptibilities of Empoasca vitis (Hemiptera: Cicadellidae) from Three Main Tea-Growing Regions in China

ABSTRACT Empoasca vitis (Göthe) is an important insect pest in tea-growing areas of China, and chemical control is the main tactic for the management of this pest. Due to the pressure of increasing insecticide resistance and more stringent food safety regulations, development of sound IPM strategies for E. vitis is an urgent matter. This study comparatively evaluated four field populations of E. vitis from three different tea-growing regions in China for their susceptibilities to eight insecticides using a simple leaf-dip methodology. E. vitis was found to be most sensitive to indoxacarb (LC50 <0.5 mg/liter) and least sensitive to isoprocarb (LC50>5 mg/liter) and sophocarpidine (LC50>95 mg/liter, a botanical pesticide) regardless of populations. Population (geographical) variations were higher for indoxacarb and imidacloprid than other compounds. Judging by the 95% fiducial limits of LC50 values, all populations had similar susceptibilities to chlorfenapyr, bifenthrin, and acetamiprid or imidacloprid. Correlation analysis suggested that chlorfenapyr and indoxacarb or isoprocarb may have a high risk of cross resistance. Considering potency (LC50) and maximum residual levels, chlorfenapyr and bifenthrin are good insecticide options followed by acetamiprid and indoxacarb. These results provide valuable information to intelligently select insecticides for IPM programs that are efficacious against E. vitis while also managing insecticide resistance and maximum residual levels for tea production in China.

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.

The evolution of insecticide resistance in the brown planthopper ( Nilaparvata lugens Stål) of China in the period 2012–2016

The brown planthopper, Nilaparvata lugens, is an economically important pest on rice in Asia. Chemical control is still the most efficient primary way for rice planthopper control. However, due to the intensive use of insecticides to control this pest over many years, resistance to most of the classes of chemical insecticides has been reported. In this article, we report on the status of eight insecticides resistance in Nilaparvata lugens (Stål) collected from China over the period 2012–2016. All of the field populations collected in 2016 had developed extremely high resistance to imidacloprid, thiamethoxam, and buprofezin. Synergism tests showed that piperonyl butoxide (PBO) produced a high synergism of imidacloprid, thiamethoxam, and buprofezin effects in the three field populations, YA2016, HX2016, and YC2016. Functional studies using both double-strand RNA (dsRNA)-mediated knockdown in the expression of CYP6ER1 and transgenic expression of CYP6ER1 in Drosophila melanogaster showed that CYP6ER1 confers imidacloprid, thiamethoxam and buprofezin resistance. These results will be beneficial for effective insecticide resistance management strategies to prevent or delay the development of insecticide resistance in brown planthopper populations.

Molecular characterization and expression pattern of three GABA receptor-like subunits in the small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae)

Ionotropic γ-aminobutyric acid (GABA)-gated chloride channel receptors mediate rapid inhibitory neurotransmission in vertebrates and invertebrates. GABA receptors are well known to be the molecular targets of synthetic insecticides or parasiticides. Three GABA receptor-like subunits, LsLCCH3, LsGRD and LS8916, of the small brown planthopper, Laodelphax striatellus (Fallén), a major insect pest of crop systems in East Asia, had been identified and characterized in this study. All three genes were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). They shared common structural features with known Cys-loop ligand-gated ion channels (LGICs): the well-conserved dicysteine-loop structures, an extracellular N-terminal domain containing six distinct regions (loops A-F) that form the ligand binding sites and four transmembrane regions (TM1-4). Additionally, temporal and spatial transcriptional profiling analysis indicated that Lslcch3 was significantly higher than the other two genes. All of them were expressed at higher levels in fifth-instar nymph and adults than in eggs and from first- to fourth-instar nymph. They were predominantly expressed in the heads of 2-d old female adults. These findings enhanced our understanding of cys-loop LGIC functional characterization in Hemiptera and provided a useful basis for the development of improved insecticides that targeting this important agricultural pest.

Molecular characterization and expression profiling of ryanodine receptor gene in the pink stem borer, Sesamia inferens (Walker)

The susceptibilities of three field populations of pink stem borer (PSB), Sesamia inferens (walker) to diamide insecticides, chlorantraniliprole and flubendiamide, were evaluated in this study. The results showed that these PSB field populations were still sensitive to the two diamide insecticides after many years of exposure. To further understand PSB and diamide insecticide, the full-length ryanodine receptor (RyR) cDNA (named as SiRyR), the molecular target of diamide insecticides was cloned from PSB and characterized. The SiRyR gene contains an open reading frame of 15,420 nucleotides, encoding 5140 amino acid residues, which shares 77% to 98% sequence identity with RyR homologous of other insects. All hallmarks of RyR proteins are conserved in the SiRyR protein, including the conserved C-terminal domain with the consensus calcium-biding EF-hands (calcium-binding motif), the six transmembrane domains, as well as mannosyltransferase, IP3R and RyR (pfam02815) (MIR) domains. Real-time qPCR analysis revealed that the highest mRNA expression levels of SiRyR were observed in pupa and adults, especially in males. SiRyR was expressed at the highest level in thorax, and the lowest level in wing. The full genetic characterization of SiRyR could provide useful information for future functional expression studies and for discovery of new insecticides with selective insecticidal activity.

Transferrin Family Genes in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) in Response to Three Insecticides

Abstract Transferrins are involved in iron metabolism, immunity, xenobiotics tolerance, and development in eukaryotic organisms including insects. However, little is known about the relationship between transferrins and insecticide toxicology and resistance. Three transferrin family genes, NlTsf1, NlTsf2, and NlTsf3, of the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)a major insect pest of rice field in Asia, had been identified and characterized in this study. Quantitative polymerase chain reaction results demonstrated that NlTsf1 was significantly higher than the other two genes in different tissues. All of them were expressed at higher levels in abdomen and head than in antenna, leg, stylet, and thorax. Compared with the control, the expression of three N. lugens transferrin family genes decreased dramatically 24 h after treatment with buprofezin, pymetrozine and imidacloprid.