Abstract
Insects require molting fluids to shed the old cuticle during molting. β-N-acetyl-D-hexosaminidase, known as Hex1, together with various chitinases, is responsible for degrading the chitin component of the old cuticle. This study showed that another β-N-acetyl-D-hexosaminidase, termed OfHex3, interacted with Hex1 and functioned in the molting fluid, although the homolog of OfHex3 was known as a sperm–plasma enzyme functioning in egg–sperm recognition. OfHex3 is an enzyme cloned from the insect Asian corn borer, Ostrinia furnacalis, which is one of the most destructive pests of maize. The enzymatic activity analysis indicated that OfHex3 was able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Because OfHex3 did not have substrate inhibition, we deduced that the presence of OfHex3 might help OfHex1 relieve substrate inhibition during chitin degradation during molting. The expression patterns of OfHex3 during O. furnacalis development were studied by real-time PCR as well as western blot. The results showed that both gene transcription and protein translation levels of OfHex3 were up-regulated during larval–larval molting. The tissue-specific expression pattern analysis indicated that OfHex3 was mostly localized in the fat body and testis. All these data further supported that Hex3 was involved in molting as well as in fertilization. This study may help to understand the complexity of cuticle degradation during insect molting, and may provide a possible target for pest control.
Highlights
Molting is an important process in insect growth and development [1]
The phylogenetic analysis of insect glycoside hydrolase family 20 (GH20) Hexes from four insect species, including B. mori, O. furnacalis, Drosophila melanogaster (Diptera) and T. castaneum, indicated these Hexes fell into four groups (Fig. 1A)
Insect molting fluid contains Hex, which participates in the degradation of old cuticle during molting
Summary
Molting is an important process in insect growth and development [1]. To molt, an insect synthesizes and secrets molting fluids into a lumen located between the old and new cuticle [2]. Insect genomes contain as many as seven genes encoding Hex that function during different life processes These genes fall into four phylogenetic classes [5]. Studies of the enzymatic properties of Hex from both Manduca sexta (Lepidoptera) and B. mori suggested that it could degrade (GlcNAc) to GlcNAc [9] and was more active toward the synthesized substrate pNP-b-GlcNAc (p-nitrophenyl-N-acetyl-b-Dglucosaminide) than toward pNP-b-(GlcNAc) (p-nitrophenyl -bD-chitobiose) [6]. In Choristoneura fumiferana (Lepidoptera), immunohistochemistry staining indicated that CfHex was localized in molting fluid, epidermis and trachea [12]. Taken together, these data demonstrated that Hex is an enzyme involved in chitin degradation
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