Tyrosine hydroxylase is required for cuticle sclerotization and pigmentation in Tribolium castaneum

Abstract

Newly synthesized insect cuticle is soft and pale but becomes stronger (sclerotized) and often darker (pigmented) over several hours or days. The first step in the sclerotization and pigmentation pathways is the hydroxylation of tyrosine to produce 3,4-dihydroxyphenylalanine (DOPA). Tyrosine hydroxylase (TH) is known to catalyze this reaction during pigmentation, but a role for TH in sclerotization has not been documented. The goal of this study was to determine whether TH is required for cuticle sclerotization in the red flour beetle, Tribolium castaneum. We used quantitative RT-PCR to verify that TH expression occurs at the time of cuticle tanning and immunohistochemistry to confirm that TH is expressed in the epithelial cells underlying sclerotized cuticle. In addition, we found that a reduction in TH function (mediated by RNA interference) resulted in a decrease in cuticle pigmentation and a decrease in the hardness of both pigmented and colorless cuticle. These results demonstrate a requirement for TH in sclerotization as well as brown pigmentation of insect cuticle.

Figure 1

Amino acid alignment of TH sequences from T. castaneum, M. sexta and D. melanogaster. DmTH Ser32 (shaded) is phosphorylated by cAMP-dependent protein kinase and is conserved in TcTH. An acidic region with low sequence conservation is highlighted in black. The putative catalytic domain is indicated by bold type. The carboxyl-terminal region (in standard type) is predicted to be a tetramerization domain. (The putative domain boundaries were selected based on the experimentally determined domain boundaries of rat TH [Goodwill et al., 1997].)

Figure 2

Developmental expression profile of TH. The level of transcript abundance relative to that of rpS6 in whole insects was determined by quantitative RT-PCR. PP0, 0-1 d-old pharate pupae; PP1, 1-2 d-old pharate pupae; P0, 0 d-old pupae; P1, 1 d-old pupae; P2, 2 d-old pupae; P3, 3 d-old pupae; P4, 4 d-old pupae; P5, 5 d-old pupae; A0, 0 d-old adults and A1, 7 d-old adults. Expression levels for TcTH are presented relative to the levels of expression in the earliest developmental stage analyzed (PP0).

Figure 3

Characterization of the TH polyclonal antiserum. Protein extracts of elytra from dsV and dsTH insects were analyzed by SDS-PAGE and Coomassie staining (left panel) and western blotting (right panel). Commassie staining showed that similar amounts of protein were present in the dsV and dsTH samples. Western blotting demonstrated that the TH antiserum detected a single band of the expected mass (~60 kDa) in dsV but not dsTH protein extract.

Figure 4

Localization of TH protein. The location of TH protein in newly eclosed adults was determined by immunohistochemistry. The cryosection shown in panel 1 was incubated with polyclonal antiserum made against MsTH. The cryosection shown in panel 2 was incubated with preimmune serum. Anti-TH antibodies were detected with Alexa Fluor 488 conjugated anti-rabbit IgG antibodies (green). Nuclei were stained with DAPI (blue). Cuticle was stained with a Rhodamine-conjugated chitin-binding probe (red). Portions of the images in panels 1 and 2 (dashed lines) are shown without the DAPI signal to increase the visibility of any TH staining (boxed inset image). TH was detected in epithelial cells underlying the cuticle on the dorsal side of the elytra, which becomes highly sclerotized and pigmented after eclosion. TH was not detected in cells of the hind wing, which are less sclerotized and almost colorless, nor was it detected in the epithelial cells underlying the dorsal abdominal cuticle, which is flexible and colorless. E = elytron, H = hindwing, D = dorsal abdomen. Scale bar = 20 μm.

Figure 5

Phenotypes associated with loss of TH function. For each pair of insects, the insect on the left was injected with dsTH and the insect on the right was injected with dsEGFP (as a negative control). Pupae in the A panels were injected with 200 ng dsRNA during the last larval instar. The pupae in the first four panels were one day old, while the pupae in the last panel were five days old. Control pupae had a normal tanning pattern, with brown pigment in the posterior edge of the abdominal segments, urogomphi, bristles, gin traps, mandibles, and legs (orange arrows). The dsTH pupae did not develop this pigmentation pattern (white arrrows indicate bristles [middle, top panel] and gin traps [center]). In addition, the bristles of dsTH were limp and curved rather than stiff and straight (compare bristles of dsTH and dsEGFP insects in middle, top panel). The dsTH pupae developed black spots (middle, bottom panel) that were not seen in control pupae. The yellow line indicates that the insect was very weak and died before adult eclosion. Adults in panel B were injected with 2 ng of dsRNA in the pupal stage. The dsTH adults were very pale compared with dsEGFP controls.