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.

Fig. 1.

Schematic diagram of the organization of the TcLac2 gene. cDNA sequences of TcLac2A and TcLac2B were compared to the genomic sequences gleaned from the BeetleBase database to define the exons (including alternative exons) and introns. The boxes and lines indicate exons and introns, respectively. A translation start codon is located in exon 1. The arrowheads indicate the translation termination positions for TcLac2A and TcLac2B.

Fig. 2.

Developmental patterns of expression of phenoloxidase genes and dsRNA-mediated down-regulation. (A) The developmental profiles of expression for TcLac1, TcLac2A, TcLac2B, TcTyr1, and TcTyr2 from the prepupal stage through the adult stage were determined by using RT-PCR (24 cycles) and total RNA prepared from pools of three insects at each stage. Lane 1, prepupae; lane 2, 0- to 1-d-old pupae; lane 3, 1- to 2-d-old pupae; lane 4, 2- to 3-d-old pupae; lane 5, 3- to 4-d-old pupae (pharate adults); lane 6, 4- to 5-d-old pupae (pharate adults); lane 7, 0-d-old adults; lane 8, 5- to 6-d-old adults. (B) To analyze the knock-down levels of transcripts of TcLac1, TcLac2, and TcTyr, total RNA was isolated from 5- to 6-d-old pupae (pharate adults) (6–7 d after injection) for TcLac2 RNAi or from 0- to 1-d-old pupae (6–7 d after injection) for TcLac1 and TcTyr (TcTyr1) RNAi. The RT-PCR analysis of Tribolium polyubiquitin transcripts with the same cDNA templates served as an internal control for normalization of equal sample loading.

Fig. 3.

The effect of dsRNAs for TcLac2, TcLac1, and TcTyr on pupal and adult development and cuticle tanning of Tribolium. (A) dsRNAs for TcLac2, TcLac1, or TcTyr (200 ng per insect) were injected into last-instar larvae or prepupae as indicated in Fig. 2 (n = 40, two replicates of 20 insects each). All dsTcLac2-injected pupae developed without tanning, did not eclose normally, and died after several days. Two different terminal phenotypes observed after injection of dsRNA for TcLac2 are shown. Injection of dsLac1,dsTcTyr (dsTcTyr1), or buffer had no effect on cuticle tanning, with all pupae and adults developing normally. The red slash line indicates that the insect has died. (B) Exon-specific RNAi using dsRNAs for TcLac2A, TcLac2B (100 ng per insect), and TcLac2A/2B (mixture of dsLac2A and dsLac2B, 100 ng of each dsRNA per insect) were injected into prepupae (n = 40, two replicates of 20 insects each). All of the animals treated with dsRNA died within a week after eclosion. The red slash line indicates that the insect has died.

Fig. 4.

The larval and pupal phenotypes produced by injection of dsRNA for TcLac2. dsRNA for TcLac2 was injected into late larvae to observe the effect on larval and pupal cuticle tanning. (A) Last-instar, 1-d-old larvae, injected 3 d earlier with buffer or dsLac2. (B) One-day-old pupae injected 7 d earlier with buffer or dsLac2. No tanning was observed in the larval or pupal cuticle. (C) Dorsal view of pupal cuticle-specific gin traps (arrows) displayed in pupae shown in B magnified ≈3 times. No tanning was observed in the larval or pupal cuticle.

Fig. 5.

Inhibition of cuticle tanning is correlated with the concentration of TcLac2 dsRNA injected. Shown are phenotypes (ventral and dorsal views) produced by injection of 2–200 ng of dsRNA for TcLac2 into prepupae. In each panel, the individual on the left is a control injected with buffer only and the one on the right was injected with TcLac2 dsRNA. All of the insects (n = 20 per group) injected with 200 and 20 ng of dsRNA died at the last time point shown. The insects injected with 2 ng of dsRNA developed into slightly malformed adults with longevity and appearance more similar to control insects.