Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning

Abstract

Cuticle tanning (or sclerotization and pigmentation) in invertebrates involves the oxidative conjugation of proteins, which renders them insoluble and hardens and darkens the color of the exoskeleton. Two kinds of phenoloxidases, laccase and tyrosinase, have been proposed to participate in tanning, but proof of the true identity of the enzyme(s) responsible for this process has been elusive. We report the cloning of cDNAs for laccases and tyrosinases from the red flour beetle, Tribolium castaneum, as well as their developmental patterns of expression. To test for the involvement of these types of enzymes in cuticle tanning, we performed RNA interference experiments to decrease the levels of individual phenoloxidases. Normal phenotypes were obtained after dsRNA-mediated transcript depletion for all phenoloxidases tested, with the exception of laccase 2. Insects injected with dsRNA for the laccase 2 gene failed to tan, were soft-bodied and deformed, and subsequently died in a dsRNA dose-dependent fashion. The results presented here support the hypothesis that two isoforms of laccase 2 generated by alternative splicing catalyze larval, pupal, and adult cuticle tanning in Tribolium.exoskeleton ͉ sclerotization ͉ pigmentation ͉ Tribolium castaneum ͉ RNA interference

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

“…For determining tissue specificity of expression, RNA was extracted from midgut, hindgut, and carcass (whole body without gut) of the last-instar feeding-stage larvae. cDNAs were synthesized from total RNA by using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) as described previously (17). RT-PCR was done to check the expression profile using the gene-specific primer pair (forward: 5′-AAAG-ATTTCCAAGTGTCGTGG-3′; reverse: 5′-CCAAATAGGCTTTAAAAGTCCG-3′).…”

Expression profile of a Laccase2 encoding gene during the metamorphic molt in Apis mellifera (Hymenoptera,Apidae)

“…In the lepidopteran Manduca sexta, the gene Mslac2 showed the higher expression in pharate pupae in comparison to the feeding/wandering larvae and 0-day pupae (Dittmer et al 2004). Similarly, in the coleopteran Tribolium castaneum, the highest levels of TcLac2 transcripts were detected in pharate pupae and pharate adults (Arakane et al 2005). In these studies, however, the expression profiles were not correlated with the respective ecdysteroid titers.…”

“…Its enzymatic activity has been characterized in the integument of Diptera (Barrett & Andersen 1981;Barrett 1987a,b;Binnington & Barrett 1988;Sugumaran et al 1992) and Lepidoptera (Dittmer et al 2009). In addition, the expression of genes encoding laccases has also been described in Diptera (Gorman et al 2008), Lepidoptera (Dittmer et al 2004;Yatsu & Asano 2009), Coleoptera (Arakane et al 2005;Niu et al 2008), and more recently, in Hymenoptera (Elias-Neto et al 2010) and Hemiptera (Futahashi et al 2010(Futahashi et al , 2011.…”

“…Laccases are supposed to be universally distributed in most various life domains (Claus 2004). They have aroused interest by their industrial applications (Durán et al 2002) and, in case of their studies in insects, by their biotechnological potential in agricultural pests and disease vectors control (Arakane et al 2005). …”

Genetic basis of natural variation in body pigmentation inDrosophila melanogaster

“…laccase2 is a multicopper oxidase that is essential for cuticular sclerotinization. 20,32,33 As with mgl, to the best of our knowledge, this is the first study to associate natural genetic variation in laccase2 with variation in female abdominal pigmentation in D. melanogaster. Similarly, on chromosome 2L, there was a SNP (P D 6.30 £ 10 ¡10 ) 67-bp downstream of Dopa decarboxylase (Ddc), which could affect regulation of Ddc expression.…”