Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum

Abstract

Aspartate 1-decarboxylase (ADC) and 3,4-dihydroxyphenylalanine decarboxylase (DDC) provide beta-alanine and dopamine used in insect cuticle tanning. beta-Alanine is conjugated with dopamine to yield N-beta-alanyldopamine (NBAD), a substrate for the phenol oxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC double-stranded (ds) RNA (dsTcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. Injection of beta-alanine, the expected product of ADC, into dsTcADC-treated pupae rescued the pigmentation phenotype, resulting in normal rust-red color. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA were drastically reduced. Furthermore, from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype, and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark brown body color, but there was little mortality. Injection of dsTcDDC resulted in more than a 5-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA.

FIGURE 1.

Proposed cuticle pigmentation and sclerotization metabolic pathway in T. castaneum. TH, tyrosine hydroxylase; DOPA, 3,4-dihydroxyphenylalanine; Dopamine, 3,4-dihydroxyphenethylamine; Ebony, N-β-alanyldopamine synthase; NBADH, N-β-alanyldopamine hydrolase; DCE, dopachrome conversion enzyme; Lac2, laccase 2; CP, cuticle protein(s). The solid and dotted lines represent proposed major and minor reactions for cuticle tanning in Tribolium.

FIGURE 2.

Phylogenetic analysis of amino acid decarboxylases in Tribolium and Drosophila. The phylogenetic tree was constructed by MEGA 3.0 software using Unweighted Pair Group Method with Arithmetic Mean. Numbers by each branch indicate results of bootstrap analysis of 5,000 replications. See supplemental Fig. 1 for the accession numbers of protein sequences used here. For the expression profiles of amino acid decarboxylases in Tribolium by RT-PCR, total RNA was extracted from whole beetles (n = 5) at various developmental stages from embryos to adult. E, embryos; YL, young larvae; OL, old larvae; PP, pharate pupae; P, pupae; A, adults. The numbers in parentheses represent PCR cycles. un, unknown.

FIGURE 3.

Expression profiles of TcADC and TcDDC mRNAs during development. A, total RNA was extracted from whole beetles (n = 5) at late developmental stages (pharate pupa to young adult). PP, pharate pupae; P1, 0–1-day-old pupae; P2, 1–2-day-old pupae, P3, 2–3-day-old pupae; P4, 3–4-day-old pupae; P5, 4–5-day-old pupae; A1, 0-day adults; A2, 5–6-day-old adults. B, expression levels of TcADC gene in the wild-type and two black body color mutants. cDNA templates for RT-PCR and real time PCR were prepared from total RNA isolated from whole beetles (n = 5, duplicate for wild-type and b^Chr/b^ST, and triplicate for Bl) at pharate adult stage corresponding to P5 in A. Wt, wild type; b, black body color mutant; b^Chr/b^ST, balanced lethal black body color mutant. The numbers in parentheses represent PCR cycles. RT-PCR and real time PCR of Tribolium ribosomal protein 6 (rpS6) transcript with the same cDNA template served as an internal control. For real time PCR, expression levels for TcADC and TcDDC are presented relative to the wild-type level or the levels of expression in pharate pupae (PP). qPCR, quantitative PCR.

FIGURE 4.

Effect of dsRNA for TcADC on larval, pupal, and adult cuticle tanning. A, dsRNA (200 ng per insect) for TcADC (dsTcADC) or Tcv (dsTcv) were injected into penultimate instar or last instar larvae (n = 60, triplicate of 20 insects each). Left panel, 1-day-old last instar larvae; middle panel, 5-day-old pupae (pharate adults); right panel, 4-day-old adults. B, knockdown level of TcADC by RNAi. cDNAs for RT-PCR and real time PCR were prepared from total RNA isolated from whole beetles (n = 3) at 5 days (10-day post-injection). The numbers in parentheses represent PCR cycles. RT-PCR and real time PCR of Tribolium ribosomal protein 6 (rpS6) transcript with the same cDNA template served as an internal control. For real time PCR, expression levels of TcADC in dsTcADC-treated insects are presented relative to the levels in dsTcv control. qPCR, quantitative PCR.

FIGURE 5.

Effect of injection of β-alanine on adult cuticle pigmentation of dsTcADC-treated Tribolium pupae. dsRNAs for TcADC (dsADC) or Tcver (dsTcv) (200 ng per insect) were injected into 1-day-old pupae (1st injection), and then β-alanine (0.4 μmol per insect) or buffer were injected into 5-day-old pupae (2nd injection) (n = 40 for each treatment, duplicate of 20 insects each). Rescue of the black body adult phenotype produced by injection of dsADC was observed by injection of β-alanine in 100% of the resulting adults.

FIGURE 6.

Catechol content of T. castaneum pharate adults. GA-1 wild-type larvae or pupae were injected with dsRNA for TcADC or Tcv (tryptophan oxygenase) as a control. When these insects became pharate adults, catechols were extracted and analyzed by HPLC with electrochemical detection (A). Catechols of untreated prepupae from two black body color mutants (black and b^Chr/b^ST) were also analyzed (B). Bars indicate mean ± S.D. (n = 3). Values significantly different from the dsTcv controls (p < 0.05) are marked with an asterisk (determined by analysis of variance and Newman-Keuls multiple comparison test).

FIGURE 7.

Effect of dsRNA for TcLac2 or TcTyr on larval, pupal, and adult cuticle tanning of b^Chr/b^ST black body color mutant. dsRNA (200 ng per insect) for TcLac2 or TcTyr was injected into late larvae or pharate pupae of b^Chr/b^ST black body color strain. Little or no cuticle tanning was observed after injection of dsLac2 at each developmental stage (A), whereas dsTyr had no effect on cuticle tanning (B). dsRNA for Tcv was injected as a control.

FIGURE 8.

Lethal pupal phenotype produced by injection of dsRNA for TcDDC into late larvae. dsRNA (200 ng per insect) for TcDDC was injected into late larvae. The resulting pupal cuticle turned a grayish color and exhibited many minute black patches. The pupa died after 3–5 days. dsRNA for Tc vermilion (dsTcv) was injected as a control. A, whole body; B, head; C, dorsal view; D, side view; E, urogomphi.

FIGURE 9.

Adult phenotypes produced by injection of dsRNA for TcDDC. A, dsRNA (200 ng per insect) for TcDDC was injected into 1–2-day-old pupae (D, right pictures of each panel). The adult body color turned dark rust-red but not black after injection of dsDDC. dsRNA for vermilion (dsTcv) was injected as a control (V, left pictures of each panel). B, knockdown level of TcDDC by RNAi. cDNAs for RT-PCR and real time PCR were prepared from total RNA isolated from whole beetles (n = 3) at 5 days (3–4-day post-injection). The numbers in parentheses represent PCR cycles. RT-PCR and real time PCR of Tribolium ribosomal protein 6 (rpS6) transcript with the same cDNA template served as an internal control. For real time PCR, expression levels of TcDDC in dsTcDDC treated insects are presented relative to the levels in dsTcv control. qPCR, quantitative PCR.

FIGURE 10.

Dependence of the E′ of Tribolium elytral cuticle upon frequency of sinusoidally oscillating stress. Frequency sweeps of a single elytron were performed at 0.2% strain. Each curve is an average of four trials. The lesser frequency dependence displayed by elytra from dsTcv-injected control insects is indicative of a more effectively cross-linked structure than elytra from black or dsTcADC-injected insects.

FIGURE 11.

Dependence of the ratio of the E″ to the E′ of Tribolium elytral cuticle upon frequency of sinusoidally oscillating stress. Frequency sweeps of a single elytron were performed at 0.2% strain. Each curve is an average of four trials. Deficiency of ADC is anticipated to reduce cross-linking responsible for the elastic component, and instead to increase melanic pigment production, which would increase the viscous component. The higher E″/E′ ratio observed is consistent with that hypothesis.