Molecular Characterization and Expression Profiles of Cryptochrome Genes in a Long-Distance Migrant, Agrotis segetum (Lepidoptera: Noctuidae)

Abstract

Cryptochromes act as photoreceptors or integral components of the circadian clock that involved in the regulation of circadian clock and regulation of migratory activity in many animals, and they may also act as magnetoreceptors that sensed the direction of the Earth's magnetic field for the purpose of navigation during animals' migration. Light is a major environmental signal for insect circadian rhythms, and it is also necessary for magnetic orientation. We identified the full-length cDNA encoding As-CRY1 and As-CRY2 in Agrotis segetum Denis and Schiffermaller (turnip moth (Lepidoptera: Noctuidae)). The DNA photolyase domain and flavin adenine dinucleotide-binding domain were found in both cry genes, and multiple alignments showed that those domains that are important for the circadian clock and magnetosensing were highly conserved among different animals. Quantitative polymerase chain reaction showed that cry genes were expressed in all examined body parts, with higher expression in adults during the developmental stages of the moths. Under a 14:10 (L:D) h cycle, the expression of cry genes showed a daily biological rhythm, and light can affect the expression levels of As-cry genes. The expression levels of cry genes were higher in the migratory population than in the reared population and higher in the emigration population than in the immigration population. These findings suggest that the two cryptochrome genes characterized in the turnip moth might be associated with the circadian clock and magnetosensing. Their functions deserve further study, especially for potential control of the turnip moth.

Fig. 1.

Nucleotide sequence and deduced amino acid sequence of complete As-cry1 (A) and As-cry2 (B) cDNA. The predicted start ATG and stop TAA codons, and polyadenylation signal are shaded in gray. DNA-photolyase domains are shaded in yellow; flavin adenine dinucleotide (FAD)-binding-7 domains are shaded in blue.

Fig. 2.

Multiple alignment of predicted amino acid sequences of CRY1 (A) and CRY2 (B) from Agrotis segetum with CRY proteins from other insects. Highly conserved amino acids are shaded in black. (A) GenBank accessions: Agrotis ipsilon (JQ616846.1), Bombyx mori (NM_001195699.1), Helicoverpa armigera (JN997418.1), Danaus plexippus (AY860425.1), and Drosophila melanogaster (AF099734.1). (B) GenBank accessions: A. ipsilon (JQ616847.1), B. mori (NM_001195698.1), H. armigera (JQ713142.1), D. plexippus (DQ184682.1), and Nilaparvata lugens (KM108578.1).

Fig. 3.

Phylogenetic analysis based on As-CRY1 and As-CRY2 amino acid sequences using the neighbor-joining method. The numbers at the nodes indicate bootstrap values. GenBank accessions: Agrotis ipsilon-CRY1 (JQ616846.1), Bombyx mori-CRY1 (NM_001195699.1), Helicoverpa armigera-CRY1 (JN997418.1), Drosophila melanogaster-CRY1 (AF099734.1), Nilaparvata lugens-CRY1 (KM108579.1), Danaus plexippus-CRY1 (AY860425.1), Mythimna separata-CRY1 (JX077108.1), Spodoptera littoralis-CRY1 (EF364035.1), A. ipsilon-CRY2 (JQ616847.1), B. mori-CRY2 (NM_001195698.1), H. armigera-CRY2 (JQ713142.1), Apis mellifera-CRY2 (NM_001083630.1), N. lugens-CRY2 (KM108578.1), D. plexippus-CRY2 (DQ184682.1), M. separata-CRY2 (JX077109.1), and S. littoralis-CRY2 (EF396286.1).

Fig. 4.

Relative mRNA levels of As-cry1 (A) and As-cry2 (B) during development, and transcript levels of As-cry1 (C) and As-cry2 (D) in different organs of 3-d-old male and female of Agrotis segetum moths as determined by real-time quantitative PCR (Actin as internal standard). Each value is the mean ± SE of three collections. Lowercase letters above the bar indicate significant differences among the developmental stages according to Tukey’s honestly significant difference (HSD) tests (P < 0.05) in (A) and (B). Lowercase letters above the bar indicate significant differences among females according to Tukey’s HSD tests (P < 0.05), and capital lowercase letters above the bar indicate significant differences among males according to Tukey’s HSD tests (P < 0.05) in (C) and (D). Asterisks above bars indicate a significant difference between males and females according to a t-test (*P < 0.05, **P < 0.001).

Fig. 5.

Relative mRNA transcript levels of As-cry1 (A) and As-cry2 (B) as determined by real-time quantitative PCR of DNA from Agrotis segetum moths exposed to different photoperiods. Actin was used as an internal standard. Heads were collected at 4-h intervals for 24 h. Each value is the mean ± SE of three collections.

Fig. 6.

Nightly catches of Agrotis segetum in the searchlight trap on Beihuang Island from April to October in 2016 (A) and 2017 (B). Relative mRNA transcript levels of As-cry1 (C) and As-cry2 (D) in the whole body of migratory populations in 2016 and 2017 as determined by real-time quantitative PCR. Actin was used as an internal standard. Each value is the mean ± SE of three collections. Asterisks above bars indicate a significant difference between the immigration and the emigration moths according to a t-test (**P < 0.001).