3‐hydroxy‐3‐methyl glutaryl coenzyme A reductase: an essential actor in the biosynthesis of cantharidin in the blister beetle Epicauta chinensisLaporte

Abstract

Cantharidin (C(10)H(12)O(4)) is a monoterpene defensive toxin in insects involved in chemical defence as well as in courtship and mating behaviours. It is relatively well known in the medical literature because of its high anticancer activity and as an effective therapy for molluscum contagiosum. However, little is known about its biosynthesis pathway in vivo, and no enzyme involved in cantharidin biosynthesis has been identified. The purpose of this study was to identify the crucial enzyme that is involved in the biosynthesis of cantharidin. Using the homology cloning method, a 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) gene, the rate-limiting enzyme in the mevalonate pathway, was cloned from the blister beetle Epicauta chinensis. Quantitative reverse transcription PCR and gas chromatography methods revealed that the HMGR transcripts had a positive correlation with cantharidin production in the beetles (R = 0.891). RNA interference (RNAi) knockdown of HMGR mRNA expression was achieved by microinjection of a specific double-stranded RNA with more than 90% RNAi efficiency, and an apparent decrease of cantharidin production was observed. Furthermore, the HMGR mRNA was greatly upregulated by exogenous juvenile hormone III (JH III), and cantharidin production was also raised in males; however, when injecting the JH III with RNAi of HMGR mRNA at the same time, cantharidin production did not rise. These results demonstrate that HMGR is an essential enzyme in cantharidin biosynthesis in the blister beetle E. chinensis, which further verifies previous research results demonstrating that cantharidin is synthesized de novo by the mevalonate pathway in blister beetles.

Characterization of Juvenile Hormone Related Genes Regulating Cantharidin Biosynthesis in Epicauta chinensis

“…Further evidence indicates biosynthesis of cantharidin in a blister beetle is effectively inhibited by 6-fluoromevalonate, which can inhibit juvenile hormone III (JH III) biosynthesis presumably by blocking the utilization of mevalonate (MVA) 21 . Our recent research shows further evidence that cantharidin is biosynthesized via the MVA pathway that can also synthesize JH III 22 .…”

“…To clarify the function of EcMFE , EcJHEH and EcJHAMT on cantharidin production, we carried out RNA interference (RNAi) in just-mated male E. chinensis , the time window for rapid cantharidin production 22 . The efficiency of RNAi-mediated knockdown was detected by qRT-PCR.…”

“…Previous studies have well-demonstrated cantharidin is biosynthesized via the MVA pathway and regulated by 3-hydroxy-3-methylglutaryl coenzyme-A reductase in blister beetles 19–22 . Moreover, cantharidin production varies at different stages of development in the blister beetle 2, 13, 22 . Results of cantharidin content detection show that there is a synthesis period for cantharidin in male blister beetles after mating (Fig.…”

“…The quantification of cantharidin produced by the blister beetle was performed on a gas chromatography system essentially as described previously with slight modifications 22 . Cantharidin was quantified using GC-2010 Plus (SHIMADZU, Kyoto, Japan) with HP-5 capillary column (15 m × 0.53 mm × 1.5 μm) and flame ionization detector.…”

Non-target effects on soil microbial parameters of the synthetic pesticide carbendazim with the biopesticides cantharidin and norcantharidin

“…(2015) 61 . For norcantharidin concentration determination, 10 g soil samples from different incubation times were mixed with 40 ml acetone and extracted three times by a rotary shaker for 30 min.…”