Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle, Leptinotarsa decemlineata

Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate ofL. decemlineata during flight and proline dehydrogenase （ProDH） catalyzes the first step in proline catabolism. Here we identified a putative LdProDHgene; it had three cDNA isoforms which shared the same 5＇UTR and coding region, but differed in the lengths of 3＇UTRs （515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively）. LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDHwas expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH- dsRNA by adults significantly decreased its messenger RNA （mRNA） level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals （AATAAA） were tandemly located in the 3＇UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3＇UTR to control proline catabolism for the season-dependent activity ofL. decemlineata adults.

Ecdysone receptor isoforms play distinct roles in larval–pupal–adult transition in Leptinotarsa decemlineata

A heterodimer of two nuclear receptors,ecdysone receptor(EcR)and ultraspiracle,mediates 20‐hydroxyecdysone(20E)signaling to modulate many aspects in insect life,such as molting and metamorphosis,reproduction,diapause and innate immunity.In the present paper,we intended to determine the isoform‐specific roles of EcR during larval–pupal–adult transition in the Colorado potato beetle.Double‐stranded RNAs(dsRNAs)were prepared using the common(dsEcR)or isoform‐specific(dsEcRA,dsEcRB1)regions of EcR as templates.Ingestion of either dsEcR or dsEcRA,rather than dsEcRB1,by the penultimate(3rd)and final(4th)instar larvae caused failure of larval–pupal and pupal–adult ecdysis.The RNA interference(RNAi)larvae remained as prepupae,or became deformed pupae and adults.Determination of messenger RNA(mRNA)levels of EcR isoforms found that LdEcRA regulates the expression of LdEcRB1.Moreover,silencing the two EcR transcripts,LdEcRA or LdEcRB1 reduced the mRNA levels of Ldspo and Ldsad,and lowered 20E titer.In contrast,the expression levels of HR3,HR4,E74 and E75 were significantly decreased in the LdEcR or LdEcRA RNAi larvae,but not in LdEcRB1 depleted specimens.Dietary supplement with 20E did not restore the expression of five 20E signaling genes(USP,HR3,HR4,E74 and E75),and only partially alleviated the pupation defects in dsEcR‐or dsEcRA‐fed beetles.These data suggest that EcR plays isoform‐specific roles in the regulation of ecdysteroidogenesis and the transduction of 20E signal in L.decemlineata.