The phenylpyrazole insecticide, fipronil, blocks insect RDL γ-aminobutyric acid (GABA) receptors, thereby impairs inhibitory neurotransmission. Some insect species, such as the diamondback moth (Plutella xylostella), possess more than one Rdl gene. The involvement of multiple Rdls in fipronil toxicity and resistance remain largely unknown. In this study, we investigated the roles of two Rdl genes, PxRdl1 and PxRdl2, from P. xylostella in the action of fipronil. Expressed in Xenopus oocytes, PxRDL2 receptors were 40-times less sensitive to fipronil than PxRDL1. PxRDL2 receptors were also less sensitive to GABA compared to PxRDL1. Knockout of the fipronil-sensitive PxRdl1 gene reduced the potency of fipronil by 10 fold, whereas knockout of the fipronil-resistant PxRdl2 gene enhanced the potency of fipronil by 4.4 fold. Furthermore, in two fipronil-resistant diamondback moth field populations, the expression of PxRdl2 was elevated by 3.7-fold and 4.1-fold, respectively compared to a susceptible strain, whereas the expression of PxRdl1 was comparable among the resistant and susceptible strains. Collectively, our results indicate antagonistic effects of PxRDL1 and PxRDL2 on the fipronil action in vivo and suggest enhanced expression of fipronil-resistant PxRdl2 potentially a new mechanism of fipronil resistance in insects.

BACKGROUND: Arylpyrazole insecticides display broad-spectrum insecticidal activity against insect pests. However, the high toxicity toward honeybees associated with fipronil prohibits its agronomic utility. To explore reducing the toxicity of aryl pyrazole analogs to bees, a series of new spiro-pyrazolo[1,5-a]quinazoline derivatives were designed and synthesized. RESULTS: Bioassay results showed that these compounds exhibited good insecticidal activity. In particular, the insecticidal activity of compound 5f against Plutella xylostella larvae (median lethal contentration, LC 50 = 1.43 mg L −1 ) was equivalent to that of fipronil. Moreover, some compounds also showed good insecticidal activity against Solenopsis invicta. Importantly, the bee toxicity study confirmed that compound 5f had much lower acute oral toxicity, with a median lethal dose (LD 50 ) = 1.15 ∼g bee −1 that was three to four orders of magnitude greater than that of fipronil (0.0012 ∼g bee −1 ). Electrophysiological studies were conducted using honeybee γ-aminobutyric acid receptor heterologously expressed in Xenopus oocytes to explain the reduced bee toxicity of compound 5f. The inhibitory effect of compound 5f (16.29 ∼mol L −1 ) was determined to be approximately 700-fold lower than that of fipronil (0.023 ∼mol L −1 ).CONCLUSION: These spiro-pyrazolo[1,5-a]quinazoline derivatives could be potential candidates and lead structures for the discovery of novel insecticides with low bee toxicity.

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