Copulation Exerts Significant Effects on mRNA Expression of Cryptochrome Genes in a Moth

Abstract

It is recognized that the behavioral rhythms of organisms are controlled by the circadian clock, while the reverse direction, i.e., whether changes in physiology and behavior react to the internal rhythms, is unclear. Cryptochromes (CRYs) are photolyase-like flavoproteins with blue-light receptor function and other functions on circadian clock and migration in animals. Here, we cloned the full-length cDNA of CRY1 and CRY2 in Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae). Sl-CRYs show high similarity to orthologs from other insects, and their conserved regions contain a DNA photolyase domain and a FAD-binding seven domain. The expression levels of both genes were relatively low during the larval stage, which increased during the pupal stage and then peaked at the adult stage. The expression of Sl-CRY1 and Sl-CRY2 showed differences between males and females and between scotophase and photophase. Further, our study demonstrated that copulation has a significant effect on the expression of Sl-CRYs. More interestingly, the changes in the expression of Sl-CRY1 and Sl-CRY2 due to copulation showed the same trend in both sexes, in which the expression levels of both genes in copulated males and females decreased in the subsequent scotophase after copulation and then increased significantly in the following photophase. Considering the nature of the dramatic changes in reproductive behavior and physiology after copulation in S. litura, we propose that the changes in the expression of Sl-CRYs after copulation could have some function in the reproductive process.

Keywords: CRY1; CRY2; Spodoptera litura; copulation; cryptochrome.

Fig. 1.

Multiple sequence alignment of CRYs. S.L1 refers to Sl-CRY1; S.L2 refers to Sl-CRY2; A.S1 refers to A. segetum CRY1 (AUG44605.1); A.S2 refers to A. segetum CRY2 (AUG44606.1); A.G1 refers to A. gambiae CRY1 (ABB29886.1); A.G2 refers to A. gambiae CRY2 (ABB29887.1). Identical amino acids are highlighted in dark (100% identity), red (75% identity) and blue (50% identity). The green box indicates the DNA photolyase domain and the black box indicates the FAD-binding seven domain.

Fig. 2.

Phylogenetic analysis of CRYs. The phylogenetic tree was constructed using the Neighbor-Joining method based on amino acid sequences with 1,000 bootstrap replicates. The percentage bootstrap support was presented by the number above the branches. The scale bar indicates the amino acid substitution rate. The GenBank accession numbers for CRY1 sequences from 12 insect species are as follows: S. exigua (ADY17887.1), Mythimna separata (AFR54426.1), A. segetum (AUG44605.1), Antheraea pernyi (AAK11644.1), B. mori (NP_001182628.1), Amyelois transitella (XP_013199860.1), Chilo suppressalis (CDK02014.2), Papilio machaon (XP_014354985.1), Plutella xylostella (XP_011559253.1), A. gambiae (ABB29886.1), Bactrocera dorsalis (JAC52130.1), and Agrilus planipennis (XP_018322589.1). The GenBank accession numbers for CRY2 sequences from 9 insect species are as follows: H. armigera (ADN94465.2), M. separate (AFR54427.1), A. segetum (AUG44606.1), A. pernyi (ABO38435.1), B. mori (NP_001182627.1), P. machaon (XP_014360422.1), P. xuthus (XP_013166823.1), A. gambiae (ABB29887.1), and Bombus impatiens (NP_001267051.1).

Fig. 3.

The expression levels of Sl-CRY1 (a) and Sl-CRY2 (b) in the head of S. litura in relation to developmental stages. L1, L9 and L18 refer to 1-, 9- and 18-d-old larvae; P0, P6, and P11 refer to 0-, 6- and 11-d-old pupae; A0♀, A1♀ and A2♀ refer to 0-, 1- and 2-d-old adult virgin females; and A0♂, A1♂, and A2♂ refer to 0-, 1-, and 2-d-old adult virgin males. Calibrator: Sl-CRY1 of 2-d-old virgin females in scotophase.

Fig. 4.

Effect of copulation on the mRNA expression of Sl-CRY1 and Sl-CRY2 in the head of S. litura adults. (a) Sl-CRY1 in females; (b) Sl-CRY2 in females; (c) Sl-CRY1 in males; (d) Sl-CRY2 in males. Calibrator: Sl-CRY1 of virgin females in scotophase. For each parameter, bars with different letters are significantly different (P < 0.05).