Tyrosine hydroxylase plays crucial roles in larval cuticle formation and larval-pupal tanning in the rice stem borer, Chilo suppressalis.

The striped stem borer, Chilo suppressalis (Walker), a notorious pest infesting rice, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH), which is required for larval development and cuticle tanning in many insects, could be a potential target for the control of C. suppressalis. We identified and characterized the full-length cDNA (CsTH) of C. suppressalis. The complete open reading frame of CsTH (MW690914) was 1683 bp in length, encoding a protein of 560 amino acids. Within the first to the sixth larval instars, CsTH was high in the first day just after molting, and lower in the ensuing days. From the wandering stage to the adult stage, levels of CSTH began to rise and reached a peak at the pupal stage. These patterns suggested a role for the gene in larval development and larval-pupal cuticle tanning. When we injected dsCsTH or 3-iodotyrosine (3-IT) as a TH inhibitor or fed a larva diet supplemented with 3-IT, there were significant impairments in larval development and larval-pupal cuticle tanning. Adult emergence was severely impaired, and most adults died. These results suggest that CsTH might play a critical role in larval development as well as larval-pupal tanning and immunity in C. suppressalis, and this gene could form a potential novel target for pest control.

Cloning of Two Acetylcholinesterase Genes and Analysis of Point Mutations Putatively Associated with Triazophos Resistance in Chilo auricilius (Lepidoptera: Pyralidae).

Acetylcholinesterase (AChE) is the target of organophosphate (OP) and carbamate insecticides. Mutations in the AChE gene (ace) leading to decreased insecticide susceptibility is the main resistance mechanism in insects. In this study, two Chilo auricilius acetylcholinesterase genes, designated as Caace1 and Caace2, were cloned using RT-PCR and RACE. Caace1 cDNA is 2534 bp, with ORF of 2082 bp, and it encodes an acetylcholinesterase 1 (CaAChE1) protein comprising a calculated 693 amino acid (aa) residues. Caace2 cDNA contains 2280 bp, with a full-length ORF of 1917 bp, encoding acetylcholinesterase 2 (CaAChE2) comprising a calculated 638 aa residues. At the aa level, CaAChE1 displays the highest similarity (97%) with the Chilo suppressalis AChE1, and CaAChE2 shows the highest similarity with the C. suppressalis AChE2 (99%). From the restriction fragment length polymorphism (RFLP) PCR (RFLP-PCR) analysis, one mutation in Caace1, similar to the ace1 mutation associated with triazophos resistance in C. suppressalis, was detected. Detailed examination of field populations of C. auricilius indicated this resistance mutation in C. auricilius is still quite infrequent. Based on the assay of AChE activity and RFLP-PCR testing, an individual that contains resistance mutation has lower AChE activities, while the individual that does not contain the resistance mutation has higher AChE activities. This study provides a basis for future investigations into the mechanism of OP resistance in C. auricilius, as well as a guidance for C. auricilius control with reasonable choice of pesticides.

Identification of polymorphisms in Cyrtorhinus lividipennis RDL subunit contributing to fipronil sensitivity.

As one of the most important predatory enemies, the miridbug, Cyrtorhinus lividipennis, plays an important role in rice planthoppers control, such as Nilaparvata lugens (brown planthopper). In order to compare insecticide selectivity between C. lividipennis and N. lugens, the contact acute toxicities of six insecticides (diazoxon, paraoxon, carbaryl, fenobucarb, fipronil and ethofenprox) were monitored. The results showed that all tested insecticides were more toxic to C. lividipennis than to N. lugens and fipronil had the biggest difference. The RDL subunit (Cl-RDL) was cloned from C. lividipennis and a RDL isoform (Cl-RDL-In) was also found with 31 amino acids insertion in RDL intracellular region. In order to understand the role of the insertion on insecticide sensitivities, three subunits (Nl-RDL, Cl-RDL and Cl-RDL-In) were constructed to obtain the functional receptors in Xenopus oocytes and the fipronil sensitivities were detected by the voltage-clamp technique. Nl-RDL (IC50=32.36 ± 4.07 µM) was more insensitive to fipronil than Cl-RDL (IC50=6.47 ± 1.12 µM). The insertion in Cl-RDL significantly reduced fipronil sensitivity with IC50 value in Cl-RDL-In of 16.83 ± 2.30 µM. Interestingly, after the elution of fipronil, the current response of Cl-RDL-In appeared obvious recovery, which were not observed in Cl-RDL and Nl-RDL. It might imply that the insertion played a special role in fipronil sensitivity.

Recent progress of highly efficient in vivo biological screening for novel agrochemicals in China.

This paper describes the recent progress of in vivo biological screening for pesticides in China. According to the criteria, including the severity of damage caused by pests and the economic value of the crops, the investigated insects, pathogens, herbs and other species in the agricultural field were selected as the main screening targets for pesticides. Corresponding in vivo microscreening methods have been established and applied in the pesticide screening procedure, which has higher reproducibility, a shorter time and greater efficiency that offset the drawbacks of conventional methods for pesticide screening.