The expression of three opsin genes from the compound eye of Helicoverpa armigera (Lepidoptera: Noctuidae) is regulated by a circadian clock, light conditions and nutritional status

Abstract

Visual genes may become inactive in species that inhabit poor light environments, and the function and regulation of opsin components in nocturnal moths are interesting topics. In this study, we cloned the ultraviolet (UV), blue (BL) and long-wavelength-sensitive (LW) opsin genes from the compound eye of the cotton bollworm and then measured their mRNA levels using quantitative real-time PCR. The mRNA levels fluctuated over a daily cycle, which might be an adaptation of a nocturnal lifestyle, and were dependent on a circadian clock. Cycling of opsin mRNA levels was disturbed by constant light or constant darkness, and the UV opsin gene was up-regulated after light exposure. Furthermore, the opsin genes tended to be down-regulated upon starvation. Thus, this study illustrates that opsin gene expression is determined by multiple endogenous and exogenous factors and is adapted to the need for nocturnal vision, suggesting that color vision may play an important role in the sensory ecology of nocturnal moths.

Figure 1. Evolutionary origin of opsins in H. armigera.

The phylogenetic tree is based on aligned and full-length amino acid sequences. Numbers at the nodes indicate the bootstrap, and all nodes are supported by more than 50%. The various insect opsin lineages are colored in purple (SW = short-wavelength clade), blue (MW = middle-wavelength clade) and green (LW = long-wavelength clade). The wavelength of peak sensitivity (λ_max) of the respective visual pigment is given , , . H. armigera is highlighted in red and bold. Moths and butterflies are highlighted in red and black boxes, respectively.

Figure 2. Diurnal changes in opsin gene levels under 14L:10D (A) and DD (B).

RNA samples were collected from compound eyes at 2 h intervals. Open bar: day. Filled bar: night. Filled and open circles represent female and male moths, respectively. ZT indicates zeitgeber time; CT indicates circadian time. Each value is the mean ± SE of three collections.

Figure 3. Cycling of opsin mRNA levels ceased under constant DD (A) and LL (B).

RNA samples were collected from compound eyes at 2 h intervals. Open bar: day. Filled bar: night. Filled and open circles represent female and male moths, respectively. CT indicates circadian time. Each value is the mean ± SE of three collections.

Figure 4. Temporal expression analysis of Ha-CRY1 and Ha-CRY2 levels in compound eyes under 14L:10D (A), DD (B), and constant DD (C).

RNA samples were collected from compound eyes at 2 h intervals. Open bar: day. Filled bar: night. Filled and open circles represent female and male moths, respectively. ZT indicates zeitgeber time; CT indicates circadian time. Each value is the mean ± SE of three collections.

Figure 5. Effects of light exposure on opsin gene expression in female (A) and male (B) moths.

RNA samples were collected from the compound eyes of 2-day-old moths after 6 h of UV, blue (BL) or green (LW) light irradiation at ZT20. RNA samples were collected at ZT20 from the compound eyes of moths reared under 14L:10D as CK. Each value is the mean ± SE of three collections. The letters above each bar indicate significant differences according to a Tukey HSD test (P<0.05).

Figure 6. Effects of starvation on opsin gene expression in female (A) and male (B) moths.

RNA samples were collected from the compound eyes of 2-day-old moths that were starved after adult emergence at ZT1. RNA samples were collected at ZT1 from the compound eyes of moths reared under 14L:10D as CK. Each value is the mean ± SE of three collections. The “*” and “**” indicate significant differences in opsin gene expression at P<0.05 and P<0.001, respectively, according to independent t-tests.