Identification of LmUAP1 as a 20-hydroxyecdysone response gene in the chitin biosynthesis pathway from the migratory locust, Locusta migratoria

In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N-acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene Lm- CHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20-hydroxyecdysone （20E） is still largely unclear. Here, we showed the developmental expression pattern ofLmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference （RNAi） directed toward a common region of the two isoforms of LmEcR （LmEcRcom） reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection-based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late-response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi-based pest control.

RNA interference to reveal roles of β-N-acetylglucosaminidase gene during molting process in Locusta migratoria

β-N-acetylglucosaminidases are crucial enzymes involved in chitin degrada- tion in insects. We identified a β-N-acetylglucosaminidase gene （LmNAG1） from Locusta migratoria. The full-length complementary DNA （cDNA） of LmNAG1 consists of 2 667 nucleotides, including an open reading frame （ORF） of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5＇- and 3＇- ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized β-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real- time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA （dsRNA）. As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAGl-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process ofL. migratoria.

Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria

Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate （4MU-（GlcNAe）3）, and higher Km value for the longer substrate （CM-Chitin- RBV） compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain （CBD） tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5- I and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-（GlcNAc）3, whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBE These findings are helpful for further research to clarit}g their different roles in insect growth and development.