Three Melanin Pathway Genes, TH, yellow, and aaNAT, Regulate Pigmentation in the Twin-Spotted Assassin Bug, Platymeris biguttatus (Linnaeus)

Abstract

Pigmentation plays a vital role in insect survival and reproduction. Many melanin pathway genes have been studied in holometabolous insects; however, they have only been studied in two hemimetabolous insect genera, Oncopeltus and Periplaneta. Here we analyzed three melanin pathway genes (TH, yellow, and aaNAT) using RNA interference (RNAi) in another hemimetabolous insect, namely the twin-spotted assassin bug, Platymeris biguttatus. TH was highly expressed in freshly molted nymphs and adults. TH RNAi resulted in a complete loss of black pigment, with yellow coloration maintained. Therefore, black pigment in this assassin bug is solely generated from the melanin pathway, whereas yellow pigment is generated from other unknown pigmentation pathways. yellow and aaNAT were highly expressed in the white spot of the hemelytra. Downregulation of yellow caused a brown phenotype with high mortality, indicating an important role of yellow functions in cuticle formation and in the process of converting melanin from brown to black. Interestingly, aaNAT RNAi caused not only loss of white pigment, but also loss of yellow and red pigments. This phenotype of aaNAT has not been reported in other insects. Our results provide new information for understanding the melanin pathway in which aaNAT is essential for the formation of colorless patterns.

Keywords: Platymeris biguttatus; arylalkylamine-N-acetyltransferase (aaNAT); pigmentation; tyrosine hydroxylase (TH); yellow.

Figure 1

Expression profiles of TH, yellow, and aaNAT in P. biguttatus. (A,D,G) Relative expression levels of TH, yellow, and aaNAT were detected by quantitative real-time PCR at different developmental stages. All these nymphs and adults have completed the pigmentation process before RNA extraction. (B,E,H) Relative expression levels of these three genes at different pigmentation time points. (C,F,I) Relative expression levels of these three genes in body regions with different color patterns from different developmental stages. I–V, the first- to fifth-instar nymphs; A, adults; 0, 5, 10, and 48 h of the corresponding instar nymphs; Y, the yellow annulation of the leg; B, the black region of the leg; H-B, the black region of the hemelytra; H-W, the white spot of the hemelytra. Expression levels were normalized to the expression of EF1α of P. biguttatus. Data are shown as the mean values ± SE (error bars) (n = 3). Different letters (a, b, c, d, and e) on the error bars indicate statistically significant differences (one-way ANOVA analysis, p < 0.05).

Figure 2

Functions of TH in P. biguttatus nymphs. Nymphs treated with dsGFP showed normal pigmentation patterns (A), well-developed cuticles (B,D), and bristles (C). The dsTH nymphs displayed nearly a complete loss of black pigment, with yellow pigment not affected (E). The deformed compound eyes and white bristles on the head (F). The short and limp bristles on the tibia (G). The wrinkled wing discs and some dark brown markings on the cuticle (H). Scale bars, 5 mm (A,E), 500 μm (B,F), 250 μm (C,G), 1.25 mm (D,H).

Figure 3

Functions of yellow in P. biguttatus nymphs. Nymphs treated with dsGFP showed normal pigmentation patterns (A), well-developed cuticles (B,D) and bristles (C). Nymphs injected with dsyellow showed a brown phenotype (E), with intact cuticles (F,H) and bristles (G). Scale bars, 5 mm (A,E), 500 μm (B,F), 250 μm (C,G), 1.25 mm (D,H).

Figure 4

Functions of aaNAT in P. biguttatus nymphs. Nymphs treated with dsGFP showed normal pigmentation patterns of the dorsal (A) and ventral side (B), including the wing discs (C), the sternum (D), the head (E), and the leg (F). Nymphs treated with dsaaNAT showed darker pigmentation patterns than those treated with dsGFP (G–I,K), the yellow annulation of the leg turned black (L), while the color of sternum was not apparently changed (J). Scale bars, 5 mm (A,B,G,H), 2.5 mm (C,I), 1.25 mm (E,K), 1 mm (D,F,J,L).

Figure 5

Functions of aaNAT in P. biguttatus adults. Adults treated with dsGFP showed normal pigmentation patterns (A), including the brownish red of ocelli (B), the white of the hemelytra (C), the yellow of the annulation on the leg (D), the red of the dorsal abdomen (E), and the white of the fenestrate area on the hindwing (F). (G) Adults treated with dsaaNAT showed the expansion of black pigment in the head (H), the forewing and the hindwing (I,L), the leg (J), and the dorsal abdomen (K). The white arrowhead (H) indicates the ocelli. Scale bars, 5 mm (A,E,G,K), 2.5 mm (D,J), 1.25 mm (B,H), 1 mm (C,F,I,L).

Figure 6

A proposed melanin pathway in P. biguttatus. In this assassin bug, black melanin is solely generated from the melanin pathway, and dopamine melanin is the main precursor of black pigment. However, yellow functions in the process of converting brown melanin to black melanin (red arrow). The yellow pigment of this species is not synthesized by the NBAD branch, but by other pigmentation pathways. In the NADA branch, aaNAT acts as an “eraser” to maintain the white spot on the preliminary black background. Furthermore, aaNAT can inhibit yellow expression, thereby inhibiting the formation of black melanin (red suppression symbol).